WO2004050711A2 - Gp120 specific antigens, conjugates thereof, methods for their preparation and uses thereof - Google Patents

Gp120 specific antigens, conjugates thereof, methods for their preparation and uses thereof Download PDF

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WO2004050711A2
WO2004050711A2 PCT/US2003/038471 US0338471W WO2004050711A2 WO 2004050711 A2 WO2004050711 A2 WO 2004050711A2 US 0338471 W US0338471 W US 0338471W WO 2004050711 A2 WO2004050711 A2 WO 2004050711A2
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occurrence
independently
alkyl
moiety
aryl
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PCT/US2003/038471
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French (fr)
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WO2004050711A3 (en
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Samuel J. Danishefsky
Vadim Dudkin
Xudong Geng
Mihirbaran Mandal
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Sloan-Kettering Institute For Cancer Research
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Priority to EP03810054A priority Critical patent/EP1569955A2/en
Priority to JP2004571002A priority patent/JP2006514981A/en
Priority to AU2003302235A priority patent/AU2003302235A1/en
Priority to CA002508539A priority patent/CA2508539A1/en
Publication of WO2004050711A2 publication Critical patent/WO2004050711A2/en
Publication of WO2004050711A3 publication Critical patent/WO2004050711A3/en
Priority to US11/145,084 priority patent/US7531181B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/375—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Basidiomycetes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6018—Lipids, e.g. in lipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • One problem stems from a very low immunogenicity of the viral surface. Pairs of the envelope spike proteins (gpl20 and gp41) form a trimer, inside of which much of the potentially antigenic surface of the unprocessed precursor protein (gpl60) is buried. Moreover, the "outer" face of gpl20 is extensively glycosylated (and therefore unavailable for peptide - recognizing antibodies), further complicating the problem.
  • the mature envelope oligomer is itself a very weak antigen.
  • Many explanations have been proposed to explain the unusually low antigenicity of the viral envelope spikes.
  • the "glycan shield" concept implies that steric hindrance created by N-linked carbohydrates of gpl20 prevents the immune system form generating antibodies with a broadly neutralizing action.
  • Another hypothesis states that binding of neutralizing antibodies to the CD4 site of gpl20 leads to conformational changes and is entropically disfavored, thereby allowing for HIV neutralization escape.
  • a very strong initial immune response to gpl60 which does not lead to broadly neutralizing antibody production (vide supra) suppresses response to the mature oligomer, which is expressed in much lower concentrations.
  • Gpl20 surface carbohydrates can be seen as an attractive target for such design.
  • the dendritic cell receptor DC-SIGN has been demonstrated to recognize the internal tri-mannose segment of the N-linked oligosaccharides.
  • a bacterial protein cyanovirin-N efficiently binds high-mannose type gpl20 carbohydrates.
  • the 2gl2 has been shown to have a carbohydrate epitope.
  • the present invention in one aspect, provides novel gpl20 glycans and glycoconjugates thereof, and methods for the synthesis and use thereof. [0011] In one aspect, the present invention provides compounds having formula
  • the invention provides a compound of formula (II) having the structure:
  • the invention provides a compound of formula (III) having the structure:
  • the invention provides multi-antigenic constructs comprising one or more carbohydrate domains having the structure:
  • each occurrence of R 1 is independently hydrogen or an oxygen protecting group; each occurrence of R 2A and R 2B is independently hydrogen or a nitrogen protecting group; each occurrence of R is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure: wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R 5 , R 6 and R 7 is independently hydrogen, OH, OR', NR H R m , NHCOR', F, CH 2 OH, CH 2 OR', or a substituted or unsubstituted linear or branched chain alkyl,
  • W , W and W are independently optionally substituted mannose, galactose or lactosamine moieties; wherein each carbohydrate domain is independently covalently bound to a linker system, said linker system being a peptide or non-peptide nature, and wherein the linker system may be cyclic or acyclic.
  • the invention provides clustered glycoconjugates comprising a cyclic or acyclic backbone made up of two or more amino acids or other structural units, wherein one or more of said amino acids or structural units is/are independently substituted with a glycosidic moiety having the structure:
  • each occurrence of L 1 is independently a substituted or unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated aliphatic or heteroaliphatic moiety; and and each occurrence of A is independently a carbohydrate domain having the structure:
  • Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R 5 , R 6 and R 7 is independently hydrogen, OH, OR 1 , NR a R , NHCOR', F, CH 2 OH, CH 2 OR', or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R', R" and R" 1 is independently hydrogen, a protecting group, a si
  • W 1 , W 2 and W 3 are independently optionally substituted mannose, galactose or lactosamine moieties.
  • the invention encompasses clustered multi-antigenic constructs having the structure: wherein q is 0 or 1 ; each occurrence of s is independently an integer from 2-20; t is an integer from 1-6;
  • R X1 is hydrogen, alkyl, acyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl) or a nitrogen protecting group; or R X1 is covalently bound to a substituent on the last occurrence of the spacer, thereby forming a cyclic backbone;
  • R is hydrogen or an immunogenic carrier; each occurrence of the structural unit SU is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; each occurrence of the spacer is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; the linker is either a free carboxylic acid, -O-, (carboxamido)alkyl carboxamide, MBS, primary carboxamide, mono- or dialkyl carboxamide, mono- or diarylcarboxamide, linear or branched chain (carboxy)alkyl carboxamide, linear or branched chain (alkoxycarbonyl)alkyl-carboxamide, linear or branched chain
  • each occurrence of L 1 is independently a substituted or unsubstituted aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydrate domain of formula (I det ), ⁇ i det ) or (IH det )-
  • the invention provides dimeric glycopeptides having the structure: wherein each peptide may be the same or different; each occurrence of L 1 may be the same or different and is as defined above; each occurrence of A is independently a carbohydrate domain of formula (I det ), (II det ) or (III de ). [0018] In another aspect, the invention provides compositions of any of the compounds, glycopeptides and/or constructs disclosed herein. [0019] In another aspect, the invention provides an antibody or antibody fragment which is specific to one or more of the inventive gpl20 glycans and/or glycoconjugates thereof described herein, said antibody being a purified polyclonal antibody or a monoclonal antibody.
  • the invention provides methods for the use thereof in the treatment of HIV, methods for the prevention of HIV, and methods for inducing antibodies in a subject, comprising administering to a subject in need thereof, an effective amount of any of the inventive compounds as disclosed herein, either in conjugated form or unconjugated and in combination with a suitable immunogenic carrier.
  • any of the inventive compounds may be conjugated to generate a glycoconjugate, and may be administered alone, with an immunogenic carrier (for example, a carrier protein, peptide or lipid), or with an immunological adjuvant or any combination thereof for the treatment of HIV infection and/or for preventing HIV infection, or may be administered alone or with an immunological adjuvant to induce antibodies in a subject.
  • an immunogenic carrier for example, a carrier protein, peptide or lipid
  • the invention provides kits for conveniently and effectively carrying out the methods in accordance with the present invention.
  • the compounds, as described herein, may be substituted with any number of substituents or functional moieties.
  • substituted whether preceded by the term “optionally” or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent.
  • substituents When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position unless otherwise indicated.
  • substituted is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.
  • this invention is not intended to be limited in any manner by the permissible substituents of organic compounds.
  • Combinations of substituents and variables envisioned by this invention are preferably those that result in the formation of stable compounds useful in the treatment and or prevention of HIV, or in the inducement of antibodies, as described herein.
  • stable as used herein, preferably refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein.
  • aliphatic includes both saturated and unsaturated, straight chain (i.e., unbranched) or branched aliphatic hydrocarbons, which are optionally substituted with one or more functional groups.
  • aliphatic is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl moieties.
  • alkyl includes straight and branched alkyl groups.
  • alkyl encompass both substituted and unsubstituted groups.
  • lower alkyl is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-6 carbon atoms.
  • the alkyl, alkenyl and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-10 aliphatic carbon atoms.
  • the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-4 carbon atoms.
  • Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n- propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, n-hexyl, sec-hexyl, moieties and the like, which again, may bear one or more substituents.
  • Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, and the like.
  • Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl and the like.
  • alicyclic refers to compounds which combine the properties of aliphatic and cyclic compounds and include but are not limited to cyclic, or polycyclic aliphatic hydrocarbons and bridged cycloalkyl compounds, which are optionally substituted with one or more functional groups.
  • alicyclic is intended herein to include, but is not limited to, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties, which are optionally substituted with one or more functional groups.
  • Illustrative alicyclic groups thus include, but are not limited to, for example, cyclopropyl, -CH 2 - cyclopropyl, cyclobutyl, -CH 2 -cyclobutyl, cyclopentyl, -CH -cyclopentyl-n, cyclohexyl, -CH 2 -cyclohexyl, cyclohexenylethyl, cyclohexanylethyl, norborbyl moieties and the like, which again, may bear one or more substituents.
  • alkoxy refers to an alkyl group, as previously defined, attached to the parent molecular moiety through an oxygen atom or through a sulfur atom.
  • the alkyl group contains 1-20 aliphatic carbon atoms.
  • the alkyl group contains 1-10 aliphatic carbon atoms.
  • the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms.
  • the alkyl group contains 1-6 aliphatic carbon atoms.
  • the alkyl group contains 1-4 aliphatic carbon atoms.
  • alkoxy include but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy and n-hexoxy.
  • thioalkyl include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.
  • alkylamino refers to a group having the structure - NHR'wherein R' is alkyl, as defined herein.
  • aminoalkyl refers to a group having the structure NH 2 R'-, wherein R' is alkyl, as defined herein.
  • the alkyl group contains 1-20 aliphatic carbon atoms.
  • the alkyl group contains 1-10 aliphatic carbon atoms.
  • the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms.
  • the alkyl group contains 1-6 aliphatic carbon atoms.
  • the alkyl group contains 1-4 aliphatic carbon atoms.
  • alkylamino include, but are not limited to, methylamino, ethylamino, iso-propylamino and the like.
  • substituents of the above-described aliphatic (and other) moieties of compounds of the invention include, but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; CI ; Br; I; -OH; -NO 2 ; -CN; -CF 3 ; -CH 2 CF 3 ; -CHC1 2 ; -CH 2 OH; - CH 2 CH 2 OH; -CH 2 NH 2 ; -CH 2 SO 2 CH 3 ; -C(O)R x ; -CO 2 (R x ); -CON(R x ) 2 ; -OC(O)R x ; - OCO 2 R x ; -OCON
  • aryl and heteroaryl refer to stable mono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having preferably 3-14 carbon atoms, each of which may be substituted or unsubstituted. It will also be appreciated that aryl and heteroaryl moieties, as defined herein may be attached via an aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, alkyl or heteroalkyl moiety and thus also include - (aliphatic)aryl, -(heteroaliphatic)aryl, -(aliphatic)heteroaryl,
  • heteroaliphatic heteroaryl, -(alkyl)aryl, -(heteroalkyl)aryl, -(heteroalkyl)aryl, and - (heteroalkyl)heteroaryl moieties.
  • aryl or heteroaryl and “aryl, heteroaryl, -(aliphatic)aryl, -(heteroaliphatic)aryl, - (aliphatic)heteroaryl, -(heteroaliphatic)heteroaryl, -(alkyl)aryl, -(heteroalkyl)aryl, - (heteroalkyl)aryl, and -(heteroalkyl)heteroaryl” are interchangeable.
  • Substituents include, but are not limited to, any of the previously mentioned substitutents, i.e., the substituents recited for aliphatic moieties, or for other moieties as disclosed herein, resulting in the formation of a stable compound.
  • aryl refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like.
  • heteroaryl refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from S, O and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.
  • aryl and heteroaryl groups can be unsubstituted or substituted, wherein substitution includes replacement of one, two or three of the hydrogen atoms thereon independently with any one or more of the following moieties including, but not limited to: aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; CI; Br; I; -OH; -NO 2 ; -CN; -CF 3 ; -CH 2 CF 3 ; -CHC1 2 ; -CH 2 OH; -CH 2 CH 2 OH; - CH 2 NH 2 ; -CH 2 SO 2 CH 3 ; -C(O)R x ; -CO 2 (R x );
  • cycloalkyl refers specifically to groups having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the case of aliphatic, heteroaliphatic or heterocyclic moieties, may optionally be substituted with substituents including, but not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; CI; Br; I; -OH; -NO 2 ; -CN; -CF 3 ; -CH 2 CF 3 ; -CHC1 2
  • heteroaliphatic refers to aliphatic moieties in which one or more carbon atoms in the main chain have been substituted with a heteroatom.
  • a heteroaliphatic group refers to an aliphatic chain which contains one or more oxygen, sulfur, nitrogen, phosphorus or silicon atoms, e.g., in place of carbon atoms.
  • Heteroaliphatic moieties may be branched or linear unbranched.
  • heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to aliphatic; alicyclic; heteroaliphatic; heteroalicyclic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; CI; Br; I; -OH; -NO 2 ; -CN; -CF 3 ; -CH 2 CF 3 ; -CHC1 2 ; -CH 2 OH; -CH 2 CH 2 OH; - CH 2 NH 2 ; -CH 2 SO 2 CH 3 ; -C(O)R x ; -CO 2 (R x ); -CON(R x ) 2 ; -OC(O)R x ; ;
  • heteroalicyclic refers to compounds which combine the properties of heteroaliphatic and cyclic compounds and include but are not limited to saturated and unsaturated mono- or polycyclic heterocycles such as morpholino, pyrrolidinyl, furanyl, thiofuranyl, pyrrolyl etc., which are optionally substituted with one or more functional groups, as defined herein.
  • any of the alicyclic or heteroalicyclic moieties described above and herein may comprise an aryl or heteroaryl moiety fused thereto. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.
  • haloalkyl denotes an alkyl group, as defined above, having one, two, or three halogen atoms attached thereto and is exemplified by such groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.
  • heterocycloalkyl refers to a non-aromatic 5-, 6- or 7- membered ring or a polycyclic group, including, but not limited to a bi- or tri-cyclic group comprising fused six-membered rings having between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 1 double bonds and each 6- membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring.
  • heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.
  • a "substituted heterocycloalkyl or heterocycle” group refers to a heterocycloalkyl or heterocycle group, as defined above, substituted by the independent replacement of one, two or three of the hydrogen atoms thereon with but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; CI; Br; I; - OH; -NO 2 ; -CN; -CF 3 ; -CH 2 CF 3 ; - CHC1 2 ; -CH 2 OH; -CH 2 CH 2 OH; -CH 2 NH 2 ; -CH 2 SO 2 CH 3 ; -C(O)R x ; -CO 2 (R x
  • aliphatic As used herein, the terms “aliphatic”, “heteroaliphatic”, “alkyl”, “alkenyl”, “alkynyl”, “heteroalkyl”, “heteroalkenyl”, “heteroalkynyl”, and the like encompass substituted and unsubstituted, saturated and unsaturated, and linear and branched groups. Similarly, the terms “alicyclic”, “heteroalicyclic”,
  • heterocycloalkyl encompass substituted and unsubstituted, and saturated and unsaturated groups. Additionally, the terms “cycloalkyl”, “cycloalkenyl”, “cycloalkynyl”, “heterocycloalkyl”,
  • heterocycloalkenyl encompass both substituted and unsubstituted groups.
  • a pro-drug is a derivative of a compound, usually with significantly reduced pharmacological activity, which contains an additional moiety, which is susceptible to removal in vivo yielding the parent molecule as the pharmacologically active species.
  • An example of a pro-drug is an ester, which is cleaved in vivo to yield a compound of interest.
  • Pro-drugs of a variety of compounds, and materials and methods for derivatizing the parent compounds to create the pro-drugs are known and may be adapted to the present invention. Certain exemplary pharmaceutical compositions and pharmaceutically acceptable derivatives will be discussed in more detail herein below.
  • protecting group By the term “protecting group”, has used herein, it is meant that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound.
  • a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group must be selectively removed in good yield by readily available, preferably nontoxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction.
  • oxygen, sulfur, nitrogen and carbon protecting groups may be utilized.
  • oxygen protecting groups include, but are not limited to methyl ethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM (methylthiomethyl ether), BOM (benzyloxymethyl ether), PMBM or MPM (p- methoxybenzyloxymethyl ether), to name a few), substituted ethyl ethers, substituted benzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilylether), TIPS (triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzyl silyl ether, TBDPS (t-butyldiphenyl silyl ether), to name a few), esters (e.g., formate, acetate, benzoate (Bz),
  • nitrogen protecting groups are utilized. These nitrogen protecting groups include, but are not limited to, carbamates (including methyl, ethyl and substituted ethyl carbamates (e.g., Troc), to name a few) amides, cyclic imide derivatives, N-Alkyl and N-Aryl amines, imine derivatives, and enamine derivatives, to name a few. Certain other exemplary protecting groups are detailed herein, however, it will be appreciated that the present invention is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the present invention. Additionally, a variety of protecting groups are described in "Protective Groups in Organic Synthesis" Third Ed. Greene, T.W. and Wuts, P.G., Eds., John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference.
  • the term "adjuvant” or “immunogenic stimulant” refers to a moiety, which, when co-administered with an immunogen, enhances the immunogenicity of the immunogen.
  • immunogenicity of the inventive gpl20 compounds can be significantly improved if the immunizing agent(s) (e.g., gpl20 glycan(s) and/or construct(s) thereof) and/or composition thereof is, regardless of administration format, co-immunized with an adjuvant.
  • adjuvants are used as an 0.05 to 1.0 percent solution in phosphate-buffered saline.
  • Adjuvants enhance the immunogenicity of an immunogen but are not necessarily immunogenic themselves.
  • Adjuvants may act by retaining the immunogen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of immunogen to cells of the immune system.
  • Adjuvants can also attract cells of the immune system to an immunogen depot and stimulate such cells to elicit immune responses.
  • embodiments of this invention encompass compositions further comprising adjuvants.
  • Adjuvants have been used for many years to improve the host immune responses to, for example, vaccines.
  • Intrinsic adjuvants (such as lipopolysaccharides) normally are the components of killed or attenuated bacteria used as vaccines.
  • Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune responses. Thus, adjuvants have been identified that enhance the immune response to antigens delivered parenterally. Some of these adjuvants are toxic, however, and can cause undesirable side-effects making them unsuitable for use in humans and many animals. Indeed, aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established. Notwithstanding, it does have limitations.
  • alum is ineffective for influenza vaccination and inconsistently elicits a cell mediated immune response with other immunogens.
  • the antibodies elicited by alum-adjuvanted antigens are mainly of the IgGl isotype in the mouse, which may not be optimal for protection by some vaccinal agents.
  • adjuvants used for therapeutic purposes e.g., vaccines
  • other adjuvants may be used for raising antibodies in animals, which antibodies may be used, for example, in diagnostic and immunoassays.
  • adjuvants include, but are not limited to, bacteria or liposomes.
  • suitable adjuvants include but are not limited to, saponin adjuvants (e.g., GPI-0100), Salmonella minnesota cells, bacille Calmette-Guerin or QS21.
  • extrinsic adjuvants can provoke potent immune responses to immunogens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria and mineral oil, Freund's complete adjuvant, bacterial products such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes.
  • MDP muramyl dipeptide
  • LPS lipopolysaccharide
  • natural amino acid refers to any one of the common, naturally occurring L-amino acids found in naturally occurring proteins: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (He), lysine (Lys), arginine (Arg), histidine (His), proline (Pro), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Tip), aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine (Gin), cysteine (Cys) and methionine (Met).
  • unnatural amino acid refers to all amino acids which are not natural amino acids. This includes, for example, o , ⁇ -, D-, L- amino acids
  • amino acid encompasses natural amino acids and unnatural amino acids.
  • biological sample includes, without limitation, cell cultures or extracts thereof; biopsied material obtained from an animal (e.g., mammal) or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof; or purified versions thereof.
  • biological sample refers to any solid or fluid sample obtained from, excreted by or secreted by any living organism, including single-celled micro-organisms (such as bacteria and yeasts) and multicellular organisms (such as plants and animals, for instance a vertebrate or a mammal, and in particular a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated).
  • the biological sample can be in any form, including a solid material such as a tissue, cells, a cell pellet, a cell extract, cell homogenates, or cell fractions; or a biopsy, or a biological fluid.
  • the biological fluid may be obtained from any site (e.g. blood, saliva (or a mouth wash containing buccal cells), tears, plasma, serum, urine, bile, seminal fluid, cerebrospinal fluid, amniotic fluid, peritoneal fluid, and pleural fluid, or cells therefrom, aqueous or vitreous humor, or any bodily secretion), a transudate, an exudate (e.g. fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (e.g.
  • the biological sample can be obtained from any organ or tissue (including a biopsy or autopsy specimen) or may comprise cells (whether primary cells or cultured cells) or medium conditioned by any cell, tissue or organ.
  • the biological sample is obtained from the prostate epithelium.
  • Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.
  • Biological samples also include mixtures of biological molecules including proteins, lipids, carbohydrates and nucleic acids generated by partial or complete fractionation of cell or tissue homogenates.
  • biological samples may be from any animal, plant, bacteria, virus, yeast, etc.
  • the term animal refers to humans as well as non-human animals, at any stage of development, including, for example, mammals, birds, reptiles, amphibians, fish, worms and single cells. Cell cultures and live tissue samples are considered to be pluralities of animals.
  • the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig).
  • An animal may be a transgenic animal or a human clone.
  • the biological sample may be subjected to preliminary processing, including preliminary separation techniques.
  • the biological sample is taken from a male human subject.
  • the biological sample has been processed so that the gpl20 glycan concentration out of the total glycan concentration in the original sample is increased.
  • the sample may be purified serum gpl20, purified gpl20 glycoprotein, purified gpl20 glycoprotein that has undergone sialidase digestion, purified gpl20 glycans obtained from deglycosylated gpl20 glycoprotein, or any combination thereof.
  • biological sample encompasses any combination of gpl20 materials obtained from any biological sources (e.g., as detailed above) or by any processes that may be used to obtain gpl20 glycan from the original sample (e.g., extraction, purification, glycoprotein deglycosylation, sialidase digestion, etc.).
  • isolated when applied to the compounds of the present invention, refers to such compounds that are (i) separated from at least some components with which they are associated in nature or when they are made and/or (ii) produced, prepared or manufactured by the hand of man. In certain embodiments, isolated compounds of the invention are not substantially contaminated with, or otherwise in contact with any other compound.
  • the present invention provides compounds of formula (I) and/or (II) in substantially pure form, i.e., in a purity of greater than about 95% by weight (not including H 2 O or salt content, which is to be expected, for example, from lyophilized peptides and glycopeptides), preferably greater than about 98%, and more preferably greater than about 99% by weight.
  • the impurity in contact with a compound of formula (I) and/or (II) of the invention is an organic chemical, e.g., an organic solvent.
  • the impurity in contact with a compound of formula (I) and/or (II) is another compound of formula (I) and/or (II).
  • the present invention provides a compound of formula (I) and/or (II) that is pure in that it is not in contact with another compound of formula (I) and/or (II).
  • the term "glycoconjugate” refers to one or more glycans covalently linked to a peptidic or non-peptidic backbone.
  • gpl20 glycan refers to a carbohydrate domain present on gpl20. More specifically, gpl20 glycan designates the carbohydrate portion of compounds of formula (I), (II) and/or (III) described herein. In certain embodiments, the term refers to compounds of formula (I), (II) and/or (III) where R 4 is a moiety other than a peptide, protein or other polymeric construct.
  • glycopeptide refers to compounds of formula (I), (II) and/or (III) where R 4 comprises a peptide moiety covalently linked to the rest of the construct either directly (e.g., through N or O) or through a crosslinker.
  • the term "eliciting an immune response” is defined as initiating, triggering, causing, enhancing, improving or augmenting any response of the immune system, for example, of either a humoral or cellular nature.
  • the initiation or enhancement of an immune response can be assessed using assays known to those skilled in the art including, but not limited to, antibody assays (for example ELISA assays).
  • the inventive gpl20 glycans and/or glycoconjugates thereof, and the methods of the present invention essentially trigger or enhance primarily a humoral immune response.
  • Figure 1 depicts structures of gpl20 glycopeptides 1-2.
  • the present invention provides novel methodologies for the synthesis of complex carbohydrates and related therapeutic compounds (e.g., glycans and/or glycoconjugates thereof).
  • complex carbohydrates and related therapeutic compounds e.g., glycans and/or glycoconjugates thereof.
  • generalized methodologies were developed for the improved synthesis of complex carbohydrate structures. This general synthetic method encompasses the realization that the incorporation of an amino group at the reducing end of a carbohydrate acceptor allows for accessibility to complex N-linked carbohydrate conjugates.
  • the present invention also provides the recognition that for certain protected carbohydrates, the amino carbohydrate moieties can serve as useful precursors that can be utilized ultimately for the synthesis of complex N-linked glycopeptides or other glycoconjugates.
  • the amino carbohydrate moieties can serve as useful precursors that can be utilized ultimately for the synthesis of complex N-linked glycopeptides or other glycoconjugates.
  • Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R 5 , R 6 and R 7 is independently hydrogen, OH, OR 1 , NR 11 ⁇ 11 , NHCOR 1 , F, CH 2 OH, CH ⁇ R 1 , or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R', R" and R" 1 is independently hydrogen, a protecting group,
  • W , W and W are independently optionally substituted mannose, galactose or lactosamine moieties; and wherein R 4 is -OR 4A or -NHR 4A ; wherein R 4A is hydrogen, aliphatic, heteroaliphatic, aryl, heteroaryl, an amino acyl moiety, an amino acyl residue of a peptide, an amino acyl residue of a protein, or R 4A comprises a protein, peptide or lipid moiety covalently linked to the rest of the construct, or to the N or O atom to which it is attached, either directly or through a crosslinker.
  • W 3 is R 1 , R 3 , as defined above, or a moiety having the structure:
  • R H wherein X is -OR or -NR R ; and each occurrence of R is independently R 1 or a sialic acid moiety.
  • W and W are independently R , R or a moiety having the structure:
  • each occurrence of R 8 is independently R 1 or a sialic acid moiety.
  • a compound of formula (II) having the structure as shown below is provided: wherein R 1 , R 2A , R 2B , R 3 and R 4 are as defined above.
  • R 1 , R 2A , R 2B , R 3 and R 4 are as defined above and X is OR 1 or
  • compounds of formula (I), (II) or (III) exclude naturally occurring gpl20 (e.g., a glycan domain found on naturally occurring gpl20 glycoprotein).
  • R 4 comprises a peptide
  • the peptide is either identical to or closely related to that of gpl20 near an N-glycosylation site.
  • the peptide has the structure:
  • truncated refers to a peptide fragment comprising no fewer than about 6 amino acid residues
  • elongated refers to a peptide comprising no more than about 60 amino acid residues
  • derivatized refers to a peptide in which at least one, but not more than about 2 out of every 10, amino acid residues have been added and/or deleted; and/or in which at least one amino acid residue has been substituted with a natural or non-natural amino acid residue so that the resulting peptide has a sequence identity equal or greater to about 70% with the original peptide.
  • each occurrence of R 1 is independently an oxygen protecting group.
  • R 1C and R 1D are independently hydrogen, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety.
  • each occurrence of R 1 is independently hydrogen, Bn or Bz. In certain other exemplary embodiments, each occurrence of R is independently hydrogen.
  • each occurrence of -NR 2A R 2B at least one occurrence of R 2A or R is independently a nitrogen protecting group.
  • at least one occurrence of R or R is independently acyl, -SO 2 Ph or R 2A and R 2B , taken together with the nitrogen atom to which they are attached, form an azide or a substituted or unsubstituted phthalimide moiety.
  • each occurrence of -NR 2A R 2B is -NHAc.
  • X is -OR 1 , wherein R 1 is as defined generally above and in classes and subclasses herein.
  • each occurrence of R 3 is independently R 1 , wherein R 1 is as defined generally above and in classes and subclasses herein.
  • each occurrence of R and R is independently hydrogen, Bn or Bz. In certain other exemplary embodiments, each occurrence of R 1 is Bn and each occurrence of R 3 is Bz. In certain other exemplary embodiments, each occurrence of R 1 and R 3 is independently hydrogen.
  • R 4 is -OR 4A and the saccharide unit bearing R 4 has the structure:
  • R 1 , R 2A and R 2B are as defined generally above and in classes and subclasses herein;
  • R A is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, an amino acyl moiety, an amino acyl residue of a peptide, an amino acyl residue of a protein, -
  • R 4A is -Si(R 4B ) 3 , wherein R 4B is as defined above.
  • R 4A is TBS.
  • R 4A comprises a serine (ser) amino acyl residue.
  • R 4A comprises a threonine (Thr) amino acyl residue.
  • R 4A comprises a peptide attached to O through a serine (Ser) residue.
  • R 4A comprises a peptide attached to O through a Threonine (Thr) residue.
  • R 4 is -NHR 4A and the saccharide unit bearing R 4 has the structure:
  • R 1 , R 2A and R 2B are as defined generally above and in classes and subclasses herein; and R is hydrogen, aliphatic, heteroaliphatic, aryl, heteroaryl, an amino acyl moiety, an amino acyl residue of a peptide, an amino acyl residue of a protein, or R 4A comprises a protein, peptide or lipid moiety covalently linked to the rest of the construct, or to the N atom to which it is attached, either directly or through a crosslinker.
  • R 4A is hydrogen.
  • R 4A comprises an amino acyl residue of a peptide whose structure is either identical or closely related to that of gpl20 near an N-glycosylation site.
  • R 4A comprises an Asparagine residue (Asn) of a peptide whose structure is either identical or closely related to that of gpl20 near an N-glycosylation site.
  • a peptide whose structure is "closely related to that of gpl20 near an N-glycosylation site" designates a gpl20 peptide fragment, or truncated, elongated or derivatized version thereof, comprising ⁇ about 60 amino acid residues, wherein one amino acid residue bears an N-glycosylation site, at least one amino acid residue has been added, deleted and/or substituted with a natural or non-natural amino acid residue, so that the resulting peptide has a sequence identity greater or equal to about 70% with the original gpl20 peptide fragment.
  • the peptide comprises ⁇ about 55 amino acid residues. In certain embodiments, the peptide comprises ⁇ about 50 amino acid residues. In certain embodiments, the peptide comprises ⁇ about 45 amino acid residues. In certain embodiments, the peptide comprises ⁇ about 40 amino acid residues. In certain embodiments, the peptide comprises ⁇ about 35 amino acid residues. In certain embodiments, the peptide comprises ⁇ about 30 amino acid residues. In certain embodiments, the peptide comprises ⁇ about 25 amino acid residues. In certain embodiments, the peptide comprises ⁇ about 20 amino acid residues. In certain embodiments, the peptide has a sequence identity greater or equal to about 75% with the original gpl20 peptide fragment.
  • the peptide has a sequence identity greater or equal to about 80% with the original gpl20 peptide fragment. In certain other embodiments, the peptide has a sequence identity greater or equal to about 85% with the original gpl20 peptide fragment. In certain other embodiments, the peptide has a sequence identity greater or equal to about 90% with the original gpl20 peptide fragment. In certain other embodiments, the peptide has a sequence identity greater or equal to about 95% with the original gpl20 peptide fragment.
  • a peptide whose structure is "identical to that of gpl20 near an N- glycosylation site” designates a gpl20 peptide fragment of a naturally occurring gpl20 glycoprotein, comprising ⁇ about 60 amino acid residues, wherein one amino acid residue bears an N-glycosylation site.
  • the peptide comprises ⁇ about 55 amino acid residues.
  • the peptide comprises ⁇ about 50 amino acid residues.
  • the peptide comprises ⁇ about 45 amino acid residues.
  • the peptide comprises ⁇ about 40 amino acid residues.
  • the peptide comprises ⁇ about 35 amino acid residues.
  • the peptide comprises ⁇ about 30 amino acid residues.
  • the peptide comprises ⁇ about 25 amino acid residues.
  • the peptide comprises ⁇ about 20 amino acid residues.
  • R 4 is -NHR 4A wherein R 4A comprises an Asparagine residue (Asn) of a peptide whose structure is either identical or closely related to that of gpl20 near an N-glycosylation site and the saccharide unit bearing R 4 has the structure:
  • the saccharide unit bearing R 4 has the structure:
  • the saccharide unit bearing R has the structure:
  • R 1 , R 2A and R 2B are as defined generally above and in classes and subclasses herein.
  • any of the isolated compounds, glycopeptides and/or glycoconjugates described herein may be further conjugated to an immunogenic carrier.
  • the carrier is a protein, a peptide or a lipid.
  • the carrier is Bovine Serum Albumin (BSA), Keyhole Limpet Hemocyanin (KLH) or polylysine.
  • BSA Bovine Serum Albumin
  • KLH Keyhole Limpet Hemocyanin
  • the carrier is is a lipid carrier having the structure:
  • m, n and p are each independently integers between about 8 and 20; and Ry is hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl.
  • m', n' and p' are each 14 and the lipid is tripalmitoyl-S-glycerylcysteinylserine (e.g., PamCys).
  • R 4 encompasses proteins, peptides, and lipids, as well as (crosslinker-protein), (crosslinker-peptide) and (crosslinker-lipid) moieties.
  • the crosslinker is MMCCH (4-(maleimidomethyl) cyclohexane-1- carboxyl hydrazide).
  • the crosslinker is MBS (m-maleimidobenzoyl acid N-Hydroxysuccinimidyl ester).
  • the crosslinker is a fragment having the structure:
  • an antigenic construct comprising one or more carbohydrate domains having the structure: d det ) wherein each occurrence of R 1 is independently hydrogen or an oxygen protecting group; each occurrence of R 2 ⁇ and R 2B is independently hydrogen or a nitrogen protecting group; each occurrence of R is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
  • Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R 5 , R 6 and R 7 is independently hydrogen, OH, OR', NR H R ia , NHCOR 1 , F, CH 2 OH, CH 2 OR i , or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R', R" and R 1 " is independently hydrogen, a protecting group
  • W 1 , W 2 and W 3 are independently optionally substituted mannose, galactose or lactosamine moieties; wherein each carbohydrate domain is independently covalently bound to a linker system, said linker system being a peptide or non-peptide nature, and wherein the linker system may be cyclic or acyclic.
  • W is R , R , as defined above, or a moiety having the structure:
  • X is -OR 1 or -NR 2A R 2B ; and each occurrence of R 8 is independently R 1 or a sialic acid moiety.
  • W and W are independently R , R or a moiety having the structure:
  • inventive constructs comprise one or more carbohydrate domains having the structure:
  • inventive constructs comprise one or more carbohydrate domains having the structure:
  • carbohydrate domains are O- linked to the linker system. In certain other embodiments, some or all of carbohydrate domains are N-linked to the linker system. In yet other embodiments, the linker system is a peptide. In certain embodiments, the linker system is a cyclic peptide. In certain other embodiments, the linker system is cyclodextrin.
  • the linker system is a peptide and comprises two or more carbohydrate domains covalently attached thereto, wherein the peptide sequence between each point of attachment of the carbohydrate domains comprises a cysteine residue.
  • the multi-glycan construct is prepared by Native Chemical Ligation.
  • the inventive constructs are symmetrical, nonsymmetrical and mixed (N-linked and O-linked carbohydrates).
  • the linker system is designed to approximate the spatial position(s) of carbohydrate(s) in gpl20.
  • the linker system is further attached to a carrier immunostimulant.
  • inventive constructs comprising one or more carbohydrate domains of the formula (I det ), (II det ) or (III det ) are similar to multi- antigenic constructs described in U.S.S.N. 09/083,776 filed March 25, 1998, 09/276,595 filed March 25, 1999, 10/600,012 filed June 19, 2003, 09/641,742 filed August 18, 2000, 10/ 209,618 filed July 31, 2002 and 10/430,822, filed December 3, 2003 and entitled "Clustered Multi-Antigenic Carbohydrate Constructs, Methods for their Preparation, and Uses Thereof; each of the above applications is hereby incorporated by reference in its entirety. Guidance for preparing such constructs can be found, inter alia, in the above-cited applications.
  • the present invention encompasses clustered glycoconjugates comprising a cyclic or acyclic backbone made up of two or more amino acids or other structural units, wherein one or more of said amino acids or structural units is/are independently substituted with a glycosidic moiety having the structure:
  • each occurrence of L 1 is independently a substituted or unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydrate domain of formula (I det ), (II det ) or (IH det ).
  • the invention encompasses clustered multi- antigenic constructs having the structure: wherein q is 0 or 1 ; each occunence of s is independently an integer from 2-20; t is an integer from 1-6;
  • R X1 is hydrogen, alkyl, acyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl) or a nitrogen protecting group; or R is covalently bound to a substituent on the last occurrence of the spacer, thereby forming a cyclic backbone;
  • R is hydrogen or an immunogenic carrier; each occurrence of the structural unit SU is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; each occurrence of the spacer is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; the linker is either a free carboxylic acid, -O-, (carboxamido)alkyl carboxamide, MBS, primary carboxamide, mono- or dialkyl carboxamide, mono- or diarylcarboxamide, linear or branched chain (carboxy)alkyl carboxamide, linear or branched chain (alkoxycarbonyl)alkyl-carboxamide, linear or branched chain (carboxy)arylalkylcarboxamide, linear or branched chain
  • each occurrence of L 1 is independently a substituted or unsubstituted aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydrate domain of formula (I det ), (H det ) or (III det ).
  • each occurrence of n is 1 and each occurrence of R 33 is hydrogen or methyl.
  • each occurrence of L 1 is independently a moiety having the structure -O(CH 2 ) consult- wherein n is an integer from 1-10 and each occurrence of A is O-linked to the construct backbone.
  • the structural unit SU for each occurrence, is independently an amino acid residue, a peptidyl moiety, a bivalent aryl or heteroaryl moiety or a substituted or unsubstituted C ⁇ - 6 alkylidene or C -6 alkenylidene chain wherein up to two non-adjacent methylene units are independently optionally replaced by CO, CO 2 , COCO, CONR zl , OCONR 21 , NR Z1 NR Z2 , NR zl NR z2 CO,
  • each occurrence of R ZI and R Z2 is independently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl.
  • each occurrence of the structural unit SU is an amino acid residue, and the clustered multi-antigenic construct has the structure:
  • q is 0 or 1 ; each occurrence of s is independently an integer from 2-20; t is an integer from 1-6;
  • R X1 is hydrogen, alkyl, acyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl) or a nitrogen protecting group;
  • R is hydrogen or an immunogenic canier; each occurrence of the spacer is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; the linker is either a free carboxylic acid, -O-, (carboxamido)alkyl carboxamide, MBS, primary carboxamide, mono- or dialkyl carboxamide, mono- or diarylcarboxamide, linear or branched chain (carboxy)alkyl carboxamide, linear or branched chain (alkoxycarbonyl)alkyl-carboxamide, linear or branched chain (carboxy)arylalkylcarboxamide, linear or branched chain
  • each occurrence of L 1 is independently a substituted or unsubstituted aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydrate domain of formula (I det ), (H det ) or (III det ).
  • each occurrence of A is the same. In certain embodiments, occurrences of A from one bracketed structure s to the next may be the same or different. In certain embodiments, occurrences of A from one bracketed structure s to the next are different. In certain other embodiments, each occurrence of A is independently O or N-linked to the construct backbone. In certain other embodiments, each occurrence of A is independently ⁇ - or ⁇ -linked to the construct backbone. [0101] In certain embodiments, for the clustered multi-antigenic construct ⁇
  • R is an amino acid residue
  • the spacer for each occurrence, is independently a substituted or unsubstituted C ⁇ -6 alkylidene or C 2-6 alkenylidene chain wherein up to two non-adjacent methylene units are independently optionally replaced by CO, CO 2 , COCO, CONR zl , OCONR 21 , NR Z1 NR Z2 , NR zl NR Z2 CO, NR zl CO, NR zl CO 2 , NR zl CONR Z2 , SO, SO 2 , NR zl SO 2 , SO 2 NR zl , NR zl SO 2 NR Z2 , O, S, or NR Z1 ; wherein each occurrence of R Z1 and R Z2 is independently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl; a peptidyl moiety or a
  • the spacer for each occurrence, is independently -(CHR sp ) n -, where n is 1-8 and each occurrence of R sp is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, -alkyl(aryl), - alkyl(heteroaryl), -OR spl , -SR sp l or -NR spl R sp2 where R spl and R spl are independently hydrogen or lower alkyl; a peptidyl moiety comprising one or more ⁇ -amino acid residues, or a bivalent aryl moiety having the structure:
  • each occurrence of the spacer is independently a dipeptidyl moiety.
  • each occurrence of L 1 is independently a natural amino acid side chain.
  • each occunence of L 1 is independently an unnatural amino acid side chain.
  • each occunence of n is 1 and each occunence of R aa is hydrogen or methyl.
  • each occunence of L 1 is independently a moiety having the structure -O(CH ) n - wherein n is an integer from 1-10 and each occunence of A is O-linked to the construct backbone.
  • the clustered multi-antigenic constructs described directly above have the following structure:
  • peptide backbone may be linear, as shown, above, or cyclic (e.g., the two occurrences of Rsp at the N- and C-termini, taken together, form a cyclic moiety); wherein L 1 and R sp are as defined above; si, s2 and s3 are independently integers from 2-5; Aj-A are independently a carbohydrate domain of formula (I det ), (H det ) or (III det ), and are different from each other; and R ⁇ is hydrogen, alkyl, acyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl) or a nitrogen protecting group.
  • each occunence of n is independently an integer from 1-10; and each occunence of R aa is hydrogen, lower alkyl, aryl, heteroaryl, -alkyl(aryl) or -alkyl(heteroaryl).
  • each occunence of n is 1 and each occunence of R aa is hydrogen.
  • each occunence of R sp is independently a natural amino acid side chain.
  • each occunence ofR sp is hydrogen.
  • the clustered multi-antigenic construct is attached to a suitable immunogenic carrier via a linker and the construct has the structure:
  • each occunence of SU is an amino acid residue and the clustered multi-antigenic construct a glycopeptide having the structure:
  • R is a protein, peptide or lipid immunogenic canier.
  • each occunence of A, A , A and A is independently a carbohydrate domain having one of the following structures:
  • R is a protein, peptide or lipid immunogenic carrier.
  • R is NHR
  • the canier R' is KLH or Bovine Serum Albumin.
  • R is NHR'
  • the canier R' is a lipid having the structure:
  • m', n' and p' are each independently integers between about 8 and 20; and Ry is hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl.
  • m', n' and p ' are each 14 and the lipid is tripalmitoyl-S-glycerylcysteinylserine (e.g., PamCys).
  • the protein or lipid can be linked to N or the rest of the construct either directly or through a crosslinker and thus R'" incorporates proteins, peptides and lipids, as well as (crosslinker-protein), (crosslinker-peptide) and (crosslinker-lipid) moieties.
  • the crosslinker is MMCCH (4-(maleimidomethyl) cyclohexane-1 -carboxyl hydrazide).
  • the invention encompasses multi-antigenic constructs having the structure:
  • linker is -O-, -NR G -, -NRG(CR H Rj) NR ⁇ -, -(CR H Rj)kNR K -, -O(CR H Rj)kNR K -, an oligoester fragment comprising from 2 to about 20 hydroxy acyl residues, a peptidic fragment comprising from 2 to about 20 amino acyl residues, or a linear or branched chain alkyl or aryl carboxylic ester, wherein each occunence of k is independently 1-5; wherein each occurrence of RQ, R H> R J or R K is independently hydrogen, a linear or branched, substituted or unsubstituted, cyclic or acyclic alkyl moiety, or a substituted or unsubstituted aryl moiety; wherein the crosslinker is a moiety derived from a crosslinking reagent capable of conjugating the canier with the linker; wherein the canier is a
  • the carrier is a protein, peptide or lipid immunogenic canier.
  • the carrier is NHR'", and R'" is KLH or Bovine Serum Albumin.
  • the canier is NHR'", and R'" is a lipid having the structure:
  • m', n' and p' are each independently integers between about 8 and 20; and Ry is hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl.
  • m', n' and p' are each 14 and the lipid is tripalmitoyl-S-glycerylcysteinylserine (e.g., PamCys).
  • the protein or lipid can be linked to N or the rest of the construct either directly or through a crosslinker and thus R'" incorporates proteins, peptides and lipids, as well as (crosslinker-protein), (crosslinker-peptide) and (crosslinker-lipid) moieties.
  • the crosslinker is MMCCH (4-(maleimidomethyl) cyclohexane-1 -carboxyl hydrazide).
  • the crosslinker is MMCCH (4-(maleimidomethyl) cyclohexane-1- carboxyl hydrazide).
  • the crosslinker is MBS (m-maleimidobenzoyl acid N-Hydroxysuccinimidyl ester).
  • q is 1 and the crosslinker is a fragment having the structure:
  • the invention encompasses multi- antigenic constructs having the structure:
  • n', n' and p' are integers between about 8 and 20; j is an integer between 1 and about 8;
  • Ry, R A , R B and Re are independently hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl;
  • R D , R E and R F are each independently a carbohydrate domain of formula (I det ), (II det ) or (III det ).
  • j is 3.
  • the invention encompasses multi- antigenic constructs having the structure:
  • n and p are each independently an integer from 1-6; m', n' and p' are independently integers between about 8 and 20; j is an integer between 1 and about 8;
  • R is a nitrogen protecting group
  • Ry, and R A , R B , R C , RE and R F are independently hydrogen, substituted or unsubstituted linear or branched lower alkyl or substituted or unsubstituted phenyl; each occunence of R D is independently a carbohydrate domain of formula (I det ), (II det ) or (III det ). In certain exemplary embodiments, j is 3. [0119] In certain embodiments, for the clustered multi-antigenic constructs described above and herein, each occunence of R D , R E and R F is independently a carbohydrate domain having one of the following structures:
  • the invention provides glycopeptides comprising one occunence of a carbohydrate domain of the formula (I det ), (II det ) or (III det ), whereby the glycopeptide structure allows for dimerization.
  • the glycopeptide comprises one cysteine residue and the glycopeptide has the structure: wherein L 1 is as defined above; A is a carbohydrate domain of the formula (I det ), (II det ) or (III det ); R x is hydrogen or a thiol protecting group.
  • inventive constructs comprising one or more carbohydrate domains of the formula (I det ), (H det ) or (HI det ) are dimers of the above glycopeptides, and the constructs have the structure:
  • each peptide may be the same or different; and each occunence of A is independently a carbohydrate domain of the formula (I det ), (II det ) or (III det ).
  • each occunence of L 1 is independently a natural amino acid side chain.
  • each occunence of L is independently an unnatural amino acid side chain.
  • each occunence of n is 1 and each occunence of R 33 is hydrogen or methyl.
  • each occunence of L 1 is independently a moiety having the structure -O(CH 2 ) n - wherein n is an integer from 1-10 and each occunence of A is O-linked to the construct backbone.
  • each occunence of L 1 is an aspartyl side chain.
  • the peptide has a structure that is either identical or closely related to that of gpl20 near an N-glycosylation site.
  • the peptide comprises the amino acid sequence: Cys-Asn-Ile-Ser-Arg, wherein any one or more of the amino acid residues may bear one or more protecting groups.
  • the peptide comprises the amino acid sequence: Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His-Cys- Asn-Ile-Ser-Arg, wherein any one or more of the amino acid residues may bear one or more protecting groups.
  • the invention provides dimeric constructs having the structure:
  • each occunence of A is independently a carbohydrate domain having one of the structures:
  • dimeric constructs having the following structure are provided:
  • R 1 , R 2A , R 2B , R 3 , R 4 and W'-W 3 are as defined generally above and in classes and subclasses herein.
  • R 4 is -NHR 4A ; wherein R 4A is an amino acyl residue of a peptide and the invention provides a method for preparing homogeneous N-linked gpl20-derived glycopeptides.
  • Glycan Synthesis generally suffers from the stereochemical diversity of its targets and therefore of its building blocks, as well. The advent of a new target often requires a reworked, if not entirely different synthetic plan, based on varying protecting groups, coupling strategies, and starting materials.
  • the present invention provides a method allowing access to a number of gpl20-derived saccharides using only a small set of building blocks and the same general procedure for each glycan.
  • trisaccharide 3 in Scheme 1 embodies the protected core structure reported for the glycoforms expressed in gpl20.
  • Scheme 1 Proposed methodology for glycan synthesis.
  • trisaccharide 3 may be elaborated to give a pentasaccharide either by deprotection of the 6-position followed by simultaneous ⁇ -mannosylation at the free 3- and 6-positions or by sequential mannosylation at the 3-and 6-positions with an intermediate deprotection step. Simultaneous mannosylation with equivalently protected mannosyl donors would yield a "symmetrically" substituted pentasaccharide; further deprotections and glycosylations could be achieved in a synchronous fashion at both nonreducing termini. Sequential mannosylation would allow the inclusion of differentially protected mannose building blocks, permitting independent elaboration of the 3- and 6-substituted antennae.
  • the high-mannose pentasaccharide core (which is conserved in most natural N-linked glycans) may be synthesized in large quantities and used as a starting point for all of the gpl20 targets.
  • hybrid- type gp!20 differs from high-mannose type gpl20 in its degree of branching beyond the core pentasaccharide, this synthetic scheme would provide easy access to the multi-antennary glycoforms expressed in gpl20.
  • the synthetic approach includes: synthesis of protected oligasaccaride (undecassaccharide), global deprotection to prepare free glycan, animation, coupling with peptide acid and deprotection (Scheme 2). [0143] Scheme 2. Exemplary synthetic strategy
  • Scheme 3 a synthetic plan for the preparation of the undecasaccharide is shown in Scheme 3. For example, starting from a trisaccharide intermediate (e.g., trisacchraide 3), 1 two successive glycosylations will give pentasaccharide, then two consecutive triple glycosylation would furnish the undecasaccharide . [0145] Scheme 3. Exemplary retrosynthesis of undesaccharide 1.
  • the desired undecasaccharide could be synthesized by a 3+3 glycosylation (trisaccharide couples with another trisaccharide) followed by a 6+5 coupling.
  • This synthetic plan is much shorter and more convergent than the first strategy.
  • trisaccharide 3 first underwent glycosylation with trisaccharide donor 13 using MeOTf as promoter to afford hexasaccharide in 70% yield. Then reductive ring-opening of the benzylidene ring gave saccharide 15 in 87% yield.
  • the pentasaccharide which is the precursor for the upper-left portion of the final compound (1) was synthesized as shown in scheme 8. For example, double-glycosylation of mannose derivative 16 using chloro donor 17 and promoter silver triflate gave trisaccharide 18. After cleavage of the two acetyl groups, another double-glycosylation provided pentasaccharide 20 in 87% yield.
  • protected undecasaccharide 12b was treated with sodium methoxide and HF-pyridine to remove the acetyl groups and TBS group, respectively.
  • the resulting oligosaccharide 21 was then subjected to global Birch deprotection followed by selective acetylation using acetyl anhydride in saturated sodium bicarbonate solution to give free glycan in high yield. 5 Following Kochetkov amination 6 furnished free glycosylamine (Scheme 10).
  • Scheme 10 Scheme 10
  • 20-mer peptide acid 34 which was made through applied biosynthesis synthesizer, was activate using HATU and coupled directly with glycosylamine 23.
  • the Fmoc and ivDde protecting groups were removed by treatment with hydrazine and piperidine to give glycopeptide fragment 25 in 16% two steps yield (Scheme 11).
  • inventive glycopeptides may be divided logically into two sections: glycan synthesis (top) and glycopeptide assembly (bottom).
  • inventive method would extend the method of Wang, et al. (Wang, Z. G.; Zhang, X. F.; Visser, M.; Live, D.; Zatorski, A.; Iserloh, U.; Lloyd, K. O.; Danishefsky, S. J. "Toward fully synthetic homogeneous glycoproteins: A high mannose core containing glycopeptide carrying full H-type2 human flood group specificity.”
  • NCL native chemical ligation
  • the lysine residue is differentially protected with respect to Fmoc removal during peptide synthesis, and remains protected through the peptide glycosylation step (due to its free amine side chain).
  • Suitably protected Lys derivatives have been designed (See, for example, Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft, B. W.; Chan, W. C. "An appraisal of new variants of Dde amine protecting group for solid phase peptide synthesis.” Tetrahedron Lett.
  • the model thioester is a C-terminal glycine thioester, which is locally achiral and cannot be epimerized, and is therefore easy to synthesize.
  • the desired gpl20 thioester contains an epimerization-prone C-terminal histidine (His) residue, such thioesters have been synthesized previously and have in fact been shown to modulate favorably the rate of NCL (See, for example, Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology.” Proc. Natl Acad. Sci. U. S. A. 1999, 96, 10068-10073).
  • each occunence of R is independently hydrogen or an oxygen protecting group; eeaacchh ooccccunence of R 2A and R 2B is independently hydrogen or a nitrogen protecting group; each occunence of R 3 is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
  • Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occunence of R 5 , R 6 and R 7 is independently hydrogen, OH, OR 1 , NR' ⁇ R"', NHCOR', F, CH 2 OH, CH OR', or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occunence of R 1 , R" and R 1 " is independently hydrogen, a protecting
  • W 1 , W 2 and W 3 are independently optionally substituted mannose, galactose or lactosamine moieties; said method comprising steps of:
  • R > 4A . is hydrogen or a suitable oxygen protecting group; (b) reacting the construct of step (a) under suitable conditions to form a ⁇ - amino carbohydrate construct having the structure:
  • the peptide comprises a cysteine residue and thus, the peptide may be dimerized under suitable oxidization conditions to form the conesponding disulfide dimer.
  • the disulfide dimer has the structure:
  • each peptide may be the same or different; each occunence of L 1 may be the same or different and is as defined above; and each occunence of A is independently a carbohydrate domain as defined above.
  • step of reacting the carbohydrate construct of step (a) under suitable conditions to form the ⁇ -amino carbohydrate construct Kochetkov animation conditions are used.
  • step of reacting the carbohydrate construct of step (a) under suitable conditions to form the ⁇ -amino carbohydrate construct NH 4 HCO 3 /H 2 O is used.
  • each occunence of R 1 and R 3 is hydrogen and each occunence of-NR 2A R 2B is -NHAc.
  • the reaction conditions comprise HATU and H ⁇ nig's base in a suitable solvent.
  • the solvent is DMSO.
  • the peptide has the following structure:
  • each occunence of R and R is hydrogen
  • each occunence of-NR 2A R 2B is -NHAc.
  • the ⁇ -O-protected carbohydrate construct of step (a) has the structure:
  • the glycopeptide formed in step (c) has the structure:
  • the ⁇ -O-protected carbohydrate construct of step (a) has the structure:
  • the glycopeptide formed in step (c) has the structure:
  • the ⁇ -O-protected carbohydrate construct of step (a) has the structure:
  • the glycopeptide formed in step (c) has the structure:
  • the ⁇ -O-protected carbohydrate construct of step (a) has the structure:
  • the glycopeptide formed in step (c) has the structure:
  • the method further comprises a step of subjecting the glycopeptide formed in step (c) to Native Chemical Ligation conditions in the presence of a suitable polypeptide to form a glycopolypeptide having the structure:
  • the peptide is either identical to or closely related to that of gpl20 near an N-glycosylation site and comprises the amino acid sequence: Cys- Asn-lle-Ser-Arg wherein any one or more of the amino acid residues may bear one or more protecting groups.
  • the carbohydrate construct is attached to an Asparagine residue (Asn) on the peptide via an amide linkage.
  • the peptide is either identical to or closely related to that of gpl20 near an N-glycosylation site and comprises the amino acid sequence:
  • polypeptide comprises the amino acid sequence: Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-
  • amino acid residues may bear one or more protecting groups or a moiety suitable for Native Chemical
  • the polypeptide comprises a moiety suitable for
  • NCL moiety comprises a thioester
  • the synthetic methodology is easily applicable to the generation of significantly longer (or shorter) segments of gpl20.
  • Both the peptide to be glycosylated and the thioester utilized for NCL can more closely approach the -60 residue limit for linear synthesis; the resulting peptide can thus extend entirely to the N-terminus of gpl20. If the peptide to be glycosylated is extended significantly towards the C-terminus of gpl20 the glycosylation yield might suffer due to secondary structure formation of the longer peptide (See, for example, (1) Kent, S. B. H. "Chemical Synthesis of Peptides and Proteins.” Annu. Rev. Biochem. 1988, 57, 957- 989; and (2) Tarn, J.
  • the polypeptide has the structure:
  • R in the polypeptide used for native chemical ligation, is -(CH 2 ) 2 C(-O)NH 2 .
  • polypeptide has the structure:
  • the invention provides a method of preparing an ⁇ -
  • each occunence of R is independently Bz or Ac; said method comprising steps of:
  • step (b) partially deprotecting the protected tetrasaccharide formed in step (a) under suitable conditions to form a partially deprotected tetrasaccharide having the structure:
  • step (c) coupling the partially deprotected tetrasaccharide formed in step (b) with a monosaccharide having the structure:
  • R 10 is lower alkyl or aryl; in the presence of an activating agent under suitable conditions to form a protected pentasaccharide having the structure:
  • step (d) partially deprotecting the pentasaccharide formed in step (c) under suitable conditions to form a partially deprotected pentasaccharide having the structure:
  • step (e) coupling the partially deprotected pentasaccharide formed in step (d) with a monosaccharide having the structure:
  • R 10 is lower alkyl or aryl; in the presence of an activating agent under suitable conditions to form an octasaccharide having the structure:
  • step (f) partially deprotecting the octasaccharide formed in step (e) under suitable conditions to form a partially deprotected octasaccharide having the structure:
  • step (g) coupling the partially deprotected octasaccharide formed in step (f) with a monosaccharide having the structure:
  • the activating agent used in cteps is the activating agent used in cteps
  • step (a), (c), (e) and (g) comprises (BrC 6 H 4 ) 3 NSbCl 6 .
  • step (b) in the step of partially deprotecting the protected tetrasaccharide (step (b)), the protected tetrasaccharide formed in step (a) is subjected to reductive reaction conditions comprising Bu 2 BOTf, BH .
  • step (d) in the step of partially deprotecting the protected pentasaccharide (step (d)
  • the protected pentasaccharide formed in step (c) is subjected to reaction conditions comprising NaOMe.
  • step (f) in the step of partially deprotecting the protected octasaccharide (step (f)
  • the protected octasaccharide formed in step (e) is subjected to reaction conditions comprising
  • the invention provides a method of preparing an ⁇ -
  • said method comprising steps of:
  • R 10 is lower alkyl or aryl
  • step (b) partially deprotecting the protected tetrasaccharide formed in step (a) under suitable conditions to form a partially deprotected tetrasaccharide having the structure:
  • step (c) coupling the partially deprotected tetrasaccharide formed in step (b) with an ethylthioglycoside having the structure:
  • step (d) partially deprotecting the hexasaccharide formed in step (c) under suitable conditions to form a partially deprotected hexasaccharide having the structure:
  • step (e) coupling the partially deprotected hexasaccharide formed in step (d) with a monosaccharide having the structure:
  • R 10 is lower alkyl or aryl; in the presence of an activating agent under suitable conditions to form an heptasaccharide having the structure:
  • step (f) partially deprotecting the heptasaccharide formed in step (e) under suitable conditions to form a partially deprotected heptasaccharide having the structure:
  • step (g) coupling the partially deprotected heptasaccharide formed in step (f) with a monosaccharide having the structure: in the presence of an activating agent under suitable conditions to the ⁇ -O- protected carbohydrate construct.
  • R 10 is ethyl or phenyl.
  • step (a), (e) and (g) comprises (BrC H 4 ) 3 NSbCl 6 .
  • the protected hexasaccharide formed in step (c) is subjected to reductive reaction conditions comprising Bu 2 BOTf, BH 3 .
  • the protected tetrasaccharide formed in step (a) is subjected to reaction conditions comprising NaOMe.
  • the protected heptasaccharide formed in step (e) is subjected to reaction conditions comprising NaOMe.
  • suitable protecting groups utilized herein include, but are not limited to, Bn (benzyl), TIPS (triisopropylsilyl), and Ac (acetate).
  • coupling of glycoside moieties are effected under MeOTf promotion, as described herein. It will be appreciated by one of ordinary skill in the art however, that a variety of conditions known in the art of organic synthesis can be utilized to effect coupling of glycoside moieties. [0207] The skilled practitioner will know how to adapt the synthetic methods detailed in the present invention to access a variety of other multi-branched gpl20 glycans and constucts thereof.
  • the construct should be so functionalized as to anticipate the need for its conjugation to an immunogenic canier (e.g., protein or lipid) in anticipation of the need to stimulate an immune response.
  • an immunogenic canier e.g., protein or lipid
  • such constructs may be used to generate antibodies for use in HIV vaccine.
  • the present invention provides improvements in total synthesis and HIV therapy.
  • the present invention provides novel glycopeptide synthetic methodology that allows access to complex glycans linked to various backbones.
  • the inventive compounds can be conjugated either directly or through a crosslinker to an appropriate canier (e.g., KLH) to generate a synthetic tumor antigen.
  • an appropriate canier e.g., KLH
  • Methods of conjugation are well known in the art.
  • a conjugation strategy may be employed that involves a reductive coupling of an aldehyde (CHO) functionality on the antigenic compound, with the intended protein canier, or lipid, presumably at the ⁇ -amino acid residues of exposed lysines.
  • CHO aldehyde
  • the present invention provides synthetic constructs, whereby novel antigenic structures, as described herein, are conjugated to immunogenic caniers (e.g., proteins, peptides or lipids).
  • the present invention provides compositions comprising any one or more of the inventive gpl20 glycans and/or constructs thereof.
  • the inventive compositions may comprise an adjuvant.
  • the adjuvant is a saponin adjuvant (see, e.g., Marciani et al, Vaccine, 2000, 18, 3141, US Patent No.: 6,080,725 and 5,977,081, the entire contents of which are hereby incorporated by reference).
  • prefened saponin adjuvant includes, but is not limited to, GPI-OlOO, (Galenica Pharmaceuticals, Inc., Frederick, MD) which is a semi-synthetic adjuvant derived by modifying selected natural saponins.
  • Saponins isolated from Quillaja soponaria Molina contain two acyl moieties, a normonoterpene carboxylic acid and a normonoterpene carboxylic acid glycoside, which are linked linearly to a fucosyl residue attached at position C-28. It has been hypothesized that these lipophilic acyl groups may be responsible for these saponins' toxicity and their ability to stimulate cytotoxic T cells against exogenous antigens. The linkage between the fucosyl residue and the acyl group is unstable and hydrolyzes under mild conditions (pH>6) with concomittant loss of saponins capability to stimulate cell-mediated immune response.
  • GPI-OlOO adjuvants comprise a stable non-toxic lipophilic moiety in the saponin' s glucuronic residue.
  • Methods for preparing these semi-synthetic adjuvants are well-known in the art.
  • GPI-OlOO adjuvants may be prepared by hydrolizing quillaja saponins (which are commercially available) under basic conditions to yield the conesponding deacylated product. The deacylated intermediate may then be reacted with a suitable amine reagent using standard carboxylic acid moiety activation methodology to give the desired compounds.
  • a wide variety of procedures are effective for extrating saponin compounds.
  • preliminary fractionation may be canied out using conventional open column chromatography or flash chromatography on silica gel, in combination with a more sophisticated chromatographic technique such as High Pressure Liquid Chromatography (HPLC), droplet counter-cunent liquid chromatography (DCCC) or centrifugal Liquid Chromatography (RLCC).
  • HPLC High Pressure Liquid Chromatography
  • DCCC droplet counter-cunent liquid chromatography
  • RLCC centrifugal Liquid Chromatography
  • the adjuvant is bacteria or liposomes.
  • the adjuvant includes but is not limited to, Salmonella minnesota cells, bacille Calmette-Guerin or QS21.
  • the present invention provides compounds and synthetic methodologies useful in the development of novel therapeutic agents, particularly for fully synthetic HIV vaccines and/or therapeutics.
  • the compounds e.g., gpl20 glycans, glycopeptides thereof and other constructs thereof
  • the compounds can be conjugated to a protein canier or a lipid to generate useful glycoconjugates for the treatment and/or prevention of HIV in a subject suffering therefrom.
  • glycoconjugates prepared by processes disclosed herein are useful in adjuvant therapies as vaccines capable of inducing a potent and broad neutralizing antibody response. Such adjuvant therapies may reduce the rate of progression of HIV and/or prevent the onset of HIV.
  • the present invention provides pharmaceutical compositions for treating HIV, and for preventing the onset or progression of HIV, comprising any of the compounds of the present invention disclosed herein, as an active ingredient, optionally, though typically in combination with a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions of the present invention may further comprise other therapeutically active ingredients (e.g., anti-HIV and/or palliative agents).
  • the term "Palliative" refers to treatment that is focused on the relief of symptoms of a disease and/or side effects of a therapeutic regimen, but is not curative.
  • palliative treatment encompasses painkillers, antinausea medications and anti-sickness drugs.
  • compositions include those suitable for oral, rectal, topical (including transdermal devices, aerosols, creams, ointments, lotions and dusting powders), parenteral (including subcutaneous, intramuscular, and intravenous), ocular (opthalmic), pulmonary (nasal or buccal inhalation) or nasal administration.
  • parenteral including subcutaneous, intramuscular, and intravenous
  • ocular optical
  • pulmonary nasal or buccal inhalation
  • nasal administration including those suitable for oral, rectal, topical (including transdermal devices, aerosols, creams, ointments, lotions and dusting powders), parenteral (including subcutaneous, intramuscular, and intravenous), ocular (opthalmic), pulmonary (nasal or buccal inhalation) or nasal administration.
  • parenteral including subcutaneous, intramuscular, and intravenous
  • ocular optical
  • pulmonary nasal or buccal inhalation
  • any of the unusual pharmaceutical media may be used, such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (e.g., suspensions, elixers and solutions); or earners such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disinterating agents, etc., in the case of oral solid preparations are prefened over liquid oral preparations such as powders, capsules and tablets. If desired, capsules may be coated by standard aqueous or non-aqueous techniques.
  • the compounds of the invention may be administered by controlled release means and devices.
  • compositions of the present invention suitable for oral administration may be prepared as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient in powder or granular form or as a solution or suspension in an aqueous or nonaqueous liquid or in an oil-in-water or water-in-oil emulsion.
  • Such compositions may be prepared by any of the methods known in the art of pharmacy. In general, compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers, finely divided solid carriers, or both and then, if necessary, shaping the product into the desired form.
  • a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granule optionally mixed with a binder, lubricant, inert diluent or surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. [0221] 4) Pharmaceutical Uses and Methods of Treatment
  • the present invention provides gpl20 glycans and constructs thereof for use as active pharmaceutical agent useful for preventing or reducing the rate of infection with HIV in subjects.
  • inventive gpl20 glycans and constructs thereof may be used to raise antibodies specific to HIV virus.
  • the invention provides an antibody which is specific to one or more gpl20 glycans and/or constructs thereof described herein.
  • an antibody or antibody fragment which is specific to one or more of the inventive gpl20 glycans and/or glycoconjugates thereof described herein, said antibody being a purified polyclonal antibody or a monoclonal antibody.
  • antibody fragment is generally intended to mean any antibody fragment having conserved the specificity of the antibody of origin, and in particular fragments of the Fab and F(ab') type. Unless otherwise indicated, the term “antibody” also subsequently denotes antibody fragments when appropriate.
  • antibody which binds specifically to gpl20 antigen or "antibody which is specific to gpl20 antigen” is intended to denote, an antibody which binds to one or more gpl20 glycans described herein, with high specificity.
  • the product which is bound to the antibody consists of at least 80% and preferably of at least 90%, of said gpl20 antigen.
  • the invention provides an antibody or antibody fragment which is specific to any one of the inventive antigens (independently of the others) present on a multi-antigenic construct comprising one or more carbohydrate domains having the structure:
  • each occunence of R 1 is independently hydrogen or an oxygen protecting group; each occunence of R 2A and R 2B is independently hydrogen or a nitrogen protecting group; each occunence of R 3 is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
  • Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occunence of R 5 , R 6 and R 7 is independently hydrogen, OH, OR 1 , NR 1 ⁇ 111 , NHCOR 1 , F, CH 2 OH, CH ⁇ OR 1 , or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occunence of R', R" and R'" is independently hydrogen,
  • W 1 , W 2 and W 3 are independently optionally substituted mannose, galactose or lactosamine moieties; wherein each carbohydrate domain is independently covalently bound to a linker system, said linker system being a peptide or non-peptide nature; and wherein the linker system may be cyclic or acyclic; and and wherein said antibody is a purified polyclonal antibody or a monoclonal antibody. In certain embodiments, the antibody is a monoclonal antibody. [0227] In certain embodiments, W is R , R , as defined above, or a moiety having the structure
  • X is -OR or -NR >2A D R2B ; and each occunence of R is independently R 1 or a sialic acid moiety.
  • W and W 2 a shall «re, : i whondJepend je concentraten use + tuly.
  • the antigen comprises a carbohydrate domain having the structure:
  • the antigen comprises a carbohydrate domain having the structure:
  • the invention provides an antibody or antibody fragment which is specific to any one or more of the inventive antigens present on a multi-antigenic construct comprising a cyclic or acyclic peptidic or non- peptidic backbone made up of two or more structural units, wherein one or more of said structural units is/are independently substituted with a glycosidic moiety having the structure:
  • each occunence of L is independently a substituted or unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated aliphatic or heteroaliphatic moiety; and each occunence of A is independently a carbohydrate domain of formula:
  • each occunence of R 1 is independently hydrogen or an oxygen protecting group; each occunence of R 2A and R 2B is independently hydrogen or a nitrogen protecting group; each occunence of R is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
  • Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R 5 , R 6 and R 7 is independently hydrogen, OH, OR 1 , NR' 1 ⁇ ", NHCOR 1 , F, CH 2 OH, CH 2 OR 1 , or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occunence of R', R" and R'" is independently hydrogen, a protecting group
  • W 1 , W 2 and W 3 are independently optionally substituted mannose, galactose or lactosamine moieties.
  • the invention provides an antibody or antibody fragment which is specific to any one or more of the inventive antigens present on a dimeric glycopeptide having the structure:
  • each peptide may be the same or different; and each occunence of A is independently a carbohydrate domain of formula (I det ), (II det ) or (III det ).
  • the antigen has the structure:
  • each occunence of A is independently a carbohydrate domain having one of the structures:
  • the antigen has the structure:
  • the antigen comprises a carbohydrate antigen having the structure:
  • the invention provides an antibody or antibody fragment which is specific to a compound of formula (II A ) having the structure:
  • each occunence of R 1 is independently hydrogen or an oxygen protecting group; each occunence of R 2A and R 2B is independently hydrogen or a nitrogen protecting group; and each occunence of R 3 is independently hydrogen or a protecting group; wherein the peptide has a structure either identical to or closely related to that of gpl20 near an N-glycosylation site; and wherein said antibody is a purified polyclonal antibody or a monoclonal antibody.
  • the invention provides an antibody or antibody fragment which is specific to a compound of formula (III A ) having the structure:
  • each occunence of R 1 is independently hydrogen or an oxygen protecting group; each occunence of R 2A and R 2B is independently hydrogen or a nitrogen protecting group; and each occunence of R is independently hydrogen or a protecting group; wherein the peptide has a structure either identical to or closely related to that of gpl20 near an N-glycosylation site; and wherein said antibody is a purified polyclonal antibody or a monoclonal antibody.
  • the antibody is a monoclonal antibody.
  • the compounds of the invention may be used to prepare monoclonal or polyclonal antibodies. Conventional methods can be used to prepare the antibodies. As to the details relating to the preparation of monoclonal antibodies reference can be made to Goding, J. W., Monoclonal Antibodies: Principles and Practice, 2nd Ed., Academic Press, London, 1986. [0239]
  • the compounds, as well as antibodies specific for the inventive gpl20 glycans and/or constructs thereof may be labelled using conventional methods with various enzymes, fluorescent materials, luminescent materials and radioactive material.
  • an antibody or an antibody fragment to a label, whether it is a radioactive, enzymatic or colored label or any other type of label commonly used in immunological techniques, is well known and described in the literature. Suitable enzymes, fluorescent materials, luminescent materials, and radioactive material are well known to the skilled artisan.
  • glycopeptide may not have enough native structure to develop appropriately specific antibodies.
  • the glycopeptide might not itself be immunogenic, and could therefore require the use of an adjuvant to stimulate an immune response.
  • suitable adjuvants include, but are not limited to, saponin adjuvants (e.g., GPI-OlOO), Salmonella minnesota cells, bacille Calmette-Guerin and or QS21.
  • a lack of immune response with any length glycopeptide would call for the use of a canier protein such as keyhole limpet hemocyanin (KLH), 34"36 an adjuvant such as covalently bound Pam 3 Cys, or coadministered QS21.
  • KLH keyhole limpet hemocyanin
  • Such immunostimulants have been used alone or in concert 40"42 to generate antibodies from small glycopeptide haptens, 43"45 and should prove effective here, as well. Though the first two systems require covalent conjugation, the synthetic design allows late-stage conjugation as demonstrated previously for other glycopeptides.
  • carbohydrate epitopes are generally not readily available by isolation from natural sources.
  • the immense difficulties associated with their purification from natural sources render them virtually nonavailable as homogeneous starting materials for a clinical program.
  • the incorporation of these naturally occu ing epitopes into canier proteins/peptides or any favorable molecular context via conjugation for eliciting a therapeutically useful immunological response is inefficient at best, and often virtually impossible. Therefore, to effectively study vaccines as therapeutic agents, sufficient material can only be obtained by chemical synthesis.
  • the present invention provides a variety of synthetic glycoforms of gpl20 (glycans glycopeptide conjugates and/or other constructs thereof), and methods for preparing them.
  • a method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of any of the gpl20 glycans and or glyconjugates thereof disclosed herein (e.g., glycopeptides, which may additionally be conjugated to a protein, peptide or lipid carrier, either directly or through a crosslinker), optionally in combination with a pharmaceutically acceptable canier.
  • a method for preventing the infection with HIV comprising administering to a subject in need thereof a therapeutically effective amount of any of the gpl20 glycans and/or glyconjugates thereof disclosed herein, optionally in combination with an adjuvant.
  • a method for the treatment of HIV comprising administering to a subject in need thereof a therapeutically effective amount of any of the g ⁇ l20 glycans and or glyconjugates thereof disclosed herein, optionally in combination with an adjuvant.
  • a method for inducing antibodies in a human subject, wherein the antibodies are specific to a carbohydrate antigen expressed on the surface of gpl20 which comprises administering to the subject an amount of any of the glycans and/or glycoconjugates disclosed above effective to induce antibodies.
  • the method utilized any one or more of the gpl20 glycans and/or glycoconjugates thereof disclosed herein, where the glycan(s) and/or glycoconjugate(s) is/are linked to an immunogenic carrier either directly or through a crosslinker, which canier is a protein, peptide or lipid.
  • the canier is Bovine Serum Albumin, polylysine or KLH.
  • the carrier is a lipid having the structure:
  • m', n' and p' are each independently integers between about 8 and 20; and Ry is hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl.
  • m', n' and p' are each 14 and the lipid is tripalmitoyl-S-glycerylcysteinylserine (e.g., PamCys).
  • the method comprises administering to a subject in need thereof a therapeutically effective amount of any of the compounds and/or glycoconjugates disclosed herein, in combination with an immunogenic carrier, optionally in combination with a pharmaceutically acceptable canier.
  • the method comprises administering a gpl20 glycan and or glycoconjugate thereof additionally conjugated to an immunogenic carrier.
  • the method comprises administering to the subject a therapeutically effective amount of any one or more of the glyconjugates disclosed herein (e.g., glycopeptides, which may additionally be conjugated to a protein, peptide or lipid canier, either directly or through a crosslinker), in combination with an immunogenic canier, optionally in combination with a pharmaceutically acceptable canier.
  • the method comprises administering one or more gpl20 glycans and/or glycoconjugates and an immunogenic carrier that have not been conjugated. Rather, they are administered concunently, or successively, as separate entities.
  • the method comprises administering one or more gpl20 glycans and/or glycoconjugates of the invention conjugated (i.e., covalently linked) to an immunogenic carrier.
  • the method comprises administering any one or more inventive gpl20 glycans and/or glycoconjugates thereof disclosed herein that have not been conjugated to an immunogenic canier. Rather, the gpl20 glycan(s) and/or glycoconjugate(s) thereof and the immunogenic carrier are administered concunently, or successively, as separate entities.
  • the immunogenic canier is a protein, peptide or lipid.
  • the canier is Bovine Serum Albumin, polylysine or KLH. In certain other embodiments, the canier is PamCys.
  • a compound/glycoconjugate and a canier are said to be administered concunently when they are administered (i) as a single composition containing the compound/glycoconjugate and the carrier, (ii) as two separate compositions or (iii) are delivered by separate routes within a short enough period of time that the effective result is equivalent to that obatined when both compound/ glycoconjugate and canier are administered as a single composition.
  • the present invention provides the related method of inducing antibodies which further comprises co-administering an immunological adjuvant, or a combination of immunological adjuvants.
  • inventive gpl20 glycans and glycoconjugates thereof comprise carbohydrate domains, or truncated or elongated versions thereof, that are found on the surface of gpl20.
  • inventive glycoconjugates comprise peptidic domains, or truncated or elongated versions thereof, that are found near an N-glycosylation site of naturally occurring gpl20.
  • embodiments of this invention encompass methods of eliciting immune responses in animals comprising administering effective amounts of inventive gpl20 glycans and/or glycoconjugate(s) thereof and/or compositions of the invention wherein the immune response is directed against on eor more carbohydrates expressed on the surface of gpl20.
  • a further embodiment of this invention encompasses a use of effective amounts of inventive gpl20 glycans and or glycoconjugate(s) thereof and/or a composition of the present invention to elicit an immune response in an animal preferably to treat and or prevent HIV.
  • the present invention further includes a use of effective amounts of inventive gpl20 glycans and/or glycoconjugate(s) thereof and/or a composition of the present invention to prepare a medicament to elicit an immune response in animal, preferably to treat and/or prevent HIV.
  • the magnitude of the therapeutic dose of the compounds of the invention will vary with the nature and severity of the condition to be treated and with the particular compound and its route of administration. In general, the daily dose range for antiHIV activity lies in the range of 0.0001 to 1.0 mg/kg of body weight in a mammal, although the present invention is not intended to be limited by this range.
  • Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dosage of a compound disclosed herein.
  • oral, rectal, topical, parenteral, ocular, pulmonary, nasal, etc. routes may be employed.
  • Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, etc.
  • the effective dosage is employed using a syringe injection.
  • the most suitable route for administration will depend largely on the nature and severity of the condition being treated and on the nature of the active ingredient.
  • inventive therapeutics may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.
  • mouse immunological studies can be performed to assess the potency and/or specificity of the novel HIV vaccines.
  • the present invention relates to a kit for conveniently and effectively carrying out the methods in accordance with the present invention.
  • the pharmaceutical pack or kit comprises one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • kits are especially suited for the delivery of solid oral forms such as tablets or capsules.
  • Such a kit preferably includes a number of unit dosages, and may also include a card having the dosages oriented in the order of their intended use.
  • a memory aid can be provided, for example in the form of numbers, letters, or other markings or with a calendar insert, designating the days in the treatment schedule in which the dosages can be administered.
  • placebo dosages, or calcium dietary supplements can be included to provide a kit in which a dosage is taken every day.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • Tetrasaccharide 3 A mixture of trisacchride l 1 (106 mg, 0.074 mmol), thiomannoside 2 (133 mg, 0.222 mmol) and molecular sieves in CH 3 CN (2 mL) was stined for 2 h at r.t. and tris(4-bromophenyl)aminium hexachloroantimonate (199 mg, 0.244 mmol) was added at 15 °C. The solution was stined for 4 h at r.t. and then quenched by triethylamine. The mixture was filtered through celite, concentrated, dissolved in EtOAc, filtered through silica gel and concentrated.
  • Tetrasaccharide 4 To a solution of 3 (200 mg, 0.101 mmol) in borane tetrahydrofuran etherate (1.1 mL, 1.0 M in THF, 1.01 mmol) was added dibutylboron triflate (0.334 mL, 1.0 M in CH 2 C1 2 , 0.333 mmol) at 0 °C. The reaction mixture was stined for 7 h at 0 °C and quenched with triethylamine and methanol and concentrated. The residue was purified by PTLC using pentane/ether (1/2) as the eluent to afford 4 as a white solid (172 mg, 90%).
  • Pentasaccharide triol 7 To a solution of 6 (80 mg, 0.032 mmol) in
  • Octasaccharide 8 8 was prepared following the same protocol as used for 3 using thiol mannoside donor 2 as excess (10 eq.) White solid: (61 mg, 55%). [ ⁇ ] D 25 32.8 (c 0.15, CHC1 3 ). ⁇ NMR (400 MHz, CDC1 3 ) selected signals: ⁇ 0.00 (s, 3 H), 0.05 (s, 3 H), 0.88 (s, 9 H), 4.79 (s, 1 H), 4.88 (s, 1 H), 5.01 (s, 1 H), 5.06 (s, 1 H), 5.23 (s, 1 H), 5.58 (s, 1 H), 5.62 (s, 1 H), 5.66 (s, 1 H).
  • Octasaccharide triol 9 The synthesis of 9 follows the synthetic procedure of 7. White solid (46 mg, 87%). [ ⁇ ] D 25 280.0 (c 0.12, CHCI 3 ). ⁇ NMR (400 MHz, CDCI 3 ) selected signals: ⁇ -0.08 (s, 3 H), -0.03 (s, 3 H), 0.80 (s, 9 H), 4.92 (s, 1 H), 4.94 (s, 1 H), 4.97 (s, 1 H), 5.03 (s, 1 H), 5.07 (s, 1 H).
  • Hexasaccharide 12 To a mixture of 1 (35 mg, 0.024 mmol), 11 (51 mg, 0.037 mmol) and molecular sieves in CH 2 C1 2 (2 mL) was added di-tert- butylpyridine (DTBP) (0.019 mL, 0.085 mmol) at -40 °C and stined for 1 h at -40 °C. MeOTf (0.011 mL, 0.096 mmol) was added and the reaction mixture was warmed up to r.t.
  • DTBP di-tert- butylpyridine
  • Hexasaccharide 13:13 was prepared using the same procedure as the one for 4. 13, white solid (542 mg, 86%). [ ⁇ ] D 25 91.5 (c 0.54, CHC1 3 ). ⁇ NMR (400 MHz, CDC1 3 ) selected signals: ⁇ 0.02 (s, 3 H), 0.04 (s, 3 H), 0.87 (s, 9 H), 5.01 (s, 1 H), 5.06 (s, 1 H), 5.09 (s, 1 H), 5.15 (s, 1 H), 5.49 (s, 1 H).
  • Tirsaccharide 16 To a 25 mL flask containing donor 15 (169 mg,
  • the solution was stined in dark with warming up to room temperature over 18 hr.
  • the reaction mixture was diluted with ethyl acetate and was added aqueous saturated NaHCO 3 solution. After stirring for 10 minutes, the reaction mixture was filtered through bed of Celite and the filtrate was washed with water, brine, dried over MgSO 4 and evaporated in vacuo.
  • the crude product was purified by silica gel column chromatography (10% ethyl acetate/toluene) to afford diacetate 16. This diacetate was used for next step without further purification.
  • Pentasaccharide 18 To a mixture of 17 (208 mg, 0.158 mmol), 15
  • Undecasaccharide 10b The preparation of 10b from 18 and 13 follows the same procedure as the one used for 3. 10b, white solid (529 mg, 63% yield, 85% based on recovered starting material). [ ⁇ ] D 25 214.3 (c 0.23, CHCI 3 ).
  • Undecasaccharide triol 24 24 was prepared using the same procedure as described for 7. 24, white solide (468 mg, 96%). [ ⁇ ] D 25 214.3 (c 0.23, CHC1 3 ). 1H NMR (400 MHz, CDC1 3 ) selected signals: ⁇ 0.03 (s, 3 H), 0.05 (s, 3 H), 0.90 (s, 9 H), 5.07 (s, 1 H), 5.08 (s, 1 H), 5.13 (s, 1 H), 5.18 (s, 1 H), 5.21 (s, 1 H), 5.30 (s, 1 H).
  • Glycan 20 To a solution of sodium (101 mg, 4.391 mmol) in 15 mL liquid ammonia was added 19 (95 mg, 0.020 mmol) in THF (4 mL) at -78 °C and the reaction mixture was stined for 2 h at -78 °C. The reaction was quenched with solid NH 4 C1 at -78 °C and then warmed up to r.t. while argon was blowing through the reaction flask to evaporate all liquid. The residue was dried on vaccum for 2 h and dissolved in saturated NaHCO 3 aqueous solution (2 mL) and cooled to 0 °C.
  • Glycosylamine 21 A solution of 20 (33 mg, 0.018 mmol), NH 4 C1
  • Gpl20 glycopeptide 23 A solution of peptide acid 32 (21 mg, 0.008 mmol), HATU (6 mg, 0.016 mmol), diehtylpropylamine (DIEPA) (2 ⁇ L, 0.011 mmol) in DMSO (150 ⁇ L) was stined for 5 min and transfened to the flask containing 21 (5 mg, 0.002 mmol) and the reaction mixture was stined for 2 h. Additional DIEPA was added (0.6 ⁇ L at 4 h and 0.6 ⁇ L at 6 h).
  • DIEPA diehtylpropylamine
  • the flask containing the AgOTf/ DTBP was cooled to - 10 °C and the solution containing mixture of donor and acceptor was added over 5 minutes. The solution was stined in dark with gradual warming up to room temperature over 24 hr.
  • the reaction mixture was diluted with ethyl acetate and was added aqueous saturated NaHCO 3 . After stining for 10 minutes, the reaction mixture was filtered through bed of Celite and the filtrate was washed with water, then with brine, dried over MgSO and evaporated in vacuo.
  • the crude product was purified by silica gel column chromatography (10% ethyl acetate / toluene) to afford semi pure trimer diacetate.
  • This diacetate was dried azeotropically with toluene and dissolved in 2 mL of anhydrous methanol under argon.
  • Sodium methoxide, 25% by weight in methanol (100 ⁇ L) was added and the reaction mixture was stined for 12h.
  • Solid ammonium chloride was added and the resulting solution was stined for 20 min. The reaction mixture was carefully evaporated to solid residues, and the solid residues were washed with ethyl acetate.
  • the flask containing donor was cooled to 15 °C and 0.091 gm of tris (4-bromophenyl) aminium hexachloroantimonate [(BrC 6 H 4 ) 3 NSbCl 6 ] (promoter) was added followed by the solution of acceptor.
  • the reaction mixture was stined at 15 °C for 20 min and then additional 0.031 gm of promoter was added.
  • the cooling bath was removed and the reaction mixture was stined for 3 hr.
  • the reaction mixture was cooled to 0 °C and triethyl amine (1 mL) was added.
  • reaction mixture was cooled to 0 °C and 2 mL triethyl amine was added and stined for 30 minutes with gradual warming up to room temperature.
  • the reaction mixture was filtered through a pad of Celite and concentrated to provide crude material, which was purified by preparative TLC (20x20cm x 1 mm thickness PK6F plates) using 40% ethyl acetate in hexanes to yield 27.
  • Free glycan (10 mg) in 15 mL of saturated ammonium bicarbonate was heated at 40 °C. Additional ammoniumhydrogen carbonate was added time to time to keep the solution saturated. After two days of stirring the content of the flask was shell frozen, lyophilized, dissolved in water (10 mL), lyophilized; this process was repeated until the white solid residue reached constant mass of 10 mg, which was used directly in the next step.
  • Glycopeptide 34 [0299] A solution of acid 33 (6 mg, 0.007 mmol), HATU (5 mg, 0.013 mmol), DIEPA (1.7 ⁇ L, 0.012 mmol) in DMSO (0.1 mL) was stined for 10 min and transfened to a falcon tube (25 mL) containing 4.2 mg of 21. The solution was stined for 2 h and additional DIEPA (1.2 ⁇ L) was added. The reaction mixture was purified by semiprep HPLC column (30 to 50%B over 20 min) to afford Fmoc- protected glycopeptide (3.6 mg, 60%).
  • the conical tube was rinsed with additional 0.1 mL of DMSO and transfened to the flask containing glycosyl amine using the same 0.5 mL syringe. Monitoring by LCMS showed that no additional product formation after 6 hr. Purification of the reaction mixture by size exclusion chromatography provided the 37. To this Fmoc protected 38 was added a 1 : 3 : 16 mixture of hydrazine : piperidine : DMF (200 ⁇ L). The resulting yellowish solution was stined for 30 min before addition of a solution of TFA to bring the pH to 3. The reaction mixture was purified by semiprep HPLC column (5 to 25%B over 25 min) to afford the Fmoc deprotected 38 in 30% yield.
  • ACT ⁇ l-antichymotrypsin
  • BPH benign prostatic hyperplasia
  • HATU 7-azahydroxybenzotriazolyl tetramethyluronium hexafluorophosphate
  • KLH keyhole limpet hemocyanin
  • MES-Na 2-mercaptoethanesulfonic acid, sodium salt
  • MHC major histocompatibility complex
  • ⁇ CL native chemical ligation
  • ⁇ -terminus peptide amine terminus
  • PBS phosphate-buffered saline
  • Gpl20 prostate specific antigen
  • TBAF tetra-H-butylammonium fluoride
  • TBS tert-butyldimethylsilyl
  • Tf trifluoromethanesulfonate
  • t-Gpl20 total prostate specific antigen
  • membrane-impermeable cross- linkers To ensure cell-surface specific cross-linking for identification of surface receptors or their ligands, it is best to use membrane-impermeable cross- linkers. In the past, researchers used water-insoluble cross-linkers and carefully controlled the amount of cross-linker and the cross-linking duration. This prevented penetration of the membrane by the cross-linker and subsequent reaction with membrane proteins. Many references cite the use of membrane-permeable cross-linkers for cell surface cross-linking. Staros developed water-soluble sulfo-NHS analogs as alternatives to membrane permeable, homobifu ⁇ ctional NHS-ester and imi- doester cross-linkers.' 8 The sulfo-NHS-ester, ho- mobifu ⁇ ctio ⁇ a!
  • cross-linker BS 3 (Product #21579) is very useful for cell surface cross-linking of ligands to receptors through primary amines on each.
  • the sulfonyl groups attached to the succin- imidyl rings of sulfo-NHS cross-linkers make them membrane-impermeable and non-reactive with inner membrane proteins. Therefore, cross-linking time and quantity of cross-linker are less critical when using sulfo-NHS-esters.
  • Pierce offers a variety of sulfo-NHS-ester cross-linkers, both homobi- functional and heterobifunctional.
  • Homobifunc- tional sulfo-NHS-esters, heterobifunctional sulfo-NHS-esters and photoreactive phenyl azides are good choices for cross-linking on the surface of a cell. See Tables 3, 5 and 9 for specific characteristics and selection of cross-linkers for cell surface applications.
  • Reagent " Water- H+-ATP” insoluble, thiol- • Producing interactions between protein components cleavable— can be of the chemotaxis mechanism in E coli" cleaved with 10-50 • Chemical cross-linking of a-CPI" mM DTT at 37 0 C • Identifying cross-linked cytochrome P-450 in rat liver for 30 minutes or microsomes* with 5% ⁇ -mercap- • Studying the influence of metal ions on prothrombin toethanol in SDS- self-association"
  • DTSSP [3,3'- 21577 60851 12 A Water-soluble analog Cross-linking the extracytoplasmic domain of the
  • Sulfo-DST 20591 54834 64 A Water-soluble analog of Disulfosucci ⁇ imidyl tartarate DST 100,110-112
  • SMCC 22320 33433 11 6 A Water-insoluble, • Conjugation of glucose oxidase from Aspergillus
  • MBS 22310 314 2 99 A Water-insoluble, • Preparing an insulin ⁇ -galactos ⁇ dase conjugate"' m Maleimidobenzoyl-N- noncleavable cross- • Conjugating hen egg ovalbumin with thiolated synthetic hydroxysucci ⁇ imide ester linker copolymers of D-glutamic acid and D-lys ⁇ ne"'
  • GMBS 22314 280.24 10.2 A Water-insoluble, Acylation of antibody to introduce maleimide groups
  • succini ide ester N-(g-maleimidobutyryloxy) noncleavable. succini ide ester
  • N-(g-maleimidobutyryloxy) of GMBS N-(g-maleimidobutyryloxy) of GMBS; noncleavable, sulfosuccinimide ester membrane impermeable.
  • ANB-NOS 21551 30521 77 A NHS-ester Cross-linking cobra venom
  • APG 20107 19316 93 A Phenylazide Inhibiting bovine heart lactic dehydrogenase, p-Azidophenyl Phenyf glyoxal eggwhite lysozyme, horse liver alcohol glyoxal monohydrate dehydrogenase, and yeast alcohol dehydro genase'"
  • PNP-DTP 20669 27615 • Diazo • Photoaffinity labeling of thyroid hormone nup-Nitrophenyl- clear receptors in intact cells'" '"
  • Telephone 800.874.3723 or 815.968.0747 Fax 800.842.5007 or 815.968.7316 Internet, http://www.piercenet.com E-mail TA @ piercenet.com Pierce Chemical Technical Library
  • Telephone 800.874.3723 or 815.968.0747 Fax 800.842.5007 or 815.968.7316 Internet http://www.piercenet.com E-mail TA@piercenet.com Pierce Chemical Technical Library
  • a ⁇ ti-Thy 1.2 monoclonal antibody linked to ricin is a potent cell-type-specific toxin. P ⁇ MS77(9), 5483-5486.
  • Anti-peptide antibodies detect oncogene-related proteins in urine. Proc. Natl. Acad. Sci. USA 82, 7924-7928.

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Abstract

The present invention provides compounds having formula (I): wherein Rl, R2A, R2B, R3, R4, W1 , W2 and W3 are as defined herein; and additionally provides methods for the synthesis thereof, compositions thereof, and methods of use thereof in the treatment of HIV, methods for the prevention of HIV, and methods for inducing HIV-specific antibodies in a subject, comprising administering to a subject in need thereof, an effective amount of any of the inventive compounds as disclosed herein, either in conjugated form or unconjugated and in combination with a suitable immunogenic carrier. In another aspect, the invention provides an antibody or antibody fragment which binds specifically to a gp 120 glycan or glycopeptide of the invention.

Description

GP120 SPECIFIC ANTIGENS, CONJUGATES THEREOF, METHODS FOR THEIR PREPARATION AND USES THEREOF
PRIORITY [0001] This application claims priority to U.S. Provisional Application Nos.:
60/500,708, filed September 5, 2003; 60/500,161, filed September 4, 2003; and 60/430,822, filed December 3, 2002; each of the above applications is hereby incorporated by reference in its entirety.
GOVERNMENT SUPPORT [0002] The invention was supported in part by Grant Nos.: BC020513 and
BC022120 from the US Army (DOD) Breast Cancer Research Foundation. The U.S. government may have certain rights in this invention.
BACKGROUND OF THE INVENTION [0003] Despite enormous scientific effort, the development of a vaccine against HIV has proven to be a largely elusive goal. There are several major factors complicating the creation of such vaccine.
[0004] One problem stems from a very low immunogenicity of the viral surface. Pairs of the envelope spike proteins (gpl20 and gp41) form a trimer, inside of which much of the potentially antigenic surface of the unprocessed precursor protein (gpl60) is buried. Moreover, the "outer" face of gpl20 is extensively glycosylated (and therefore unavailable for peptide - recognizing antibodies), further complicating the problem.
[0005] Secondly, the mature envelope oligomer is itself a very weak antigen. Many explanations have been proposed to explain the unusually low antigenicity of the viral envelope spikes. The "glycan shield" concept implies that steric hindrance created by N-linked carbohydrates of gpl20 prevents the immune system form generating antibodies with a broadly neutralizing action. Another hypothesis states that binding of neutralizing antibodies to the CD4 site of gpl20 leads to conformational changes and is entropically disfavored, thereby allowing for HIV neutralization escape. It has also been suggested that a very strong initial immune response to gpl60, which does not lead to broadly neutralizing antibody production (vide supra) suppresses response to the mature oligomer, which is expressed in much lower concentrations.
[0006] In addition, extremely high degree and rate of viral variation provide a powerful mechanism for HIV to escape immune defense.
[0007] Accordingly, commonly utilized vaccine formulations have been unable to elicit a potent and broadly neutralizing antibody response. Administration of the whole virus in attenuated or inactivated form presents safety issues as well as the problem of low antigenicity. Immunization with a part of HIV DNA in a carrier is more promising, however it requires a very careful choice of the carrier virus. Also, low envelope antigenicity still remains a serious obstacle to the success of this method. A solution may lie in the use of artificial HIV antigens based on the epitopes of known broadly neutralizing antibodies. A highly focused immune response may be developed with this approach, potentially circumventing the problem of low antigenicity. The biggest challenge in this case is the design and synthesis of the antigens.
[0008] Gpl20 surface carbohydrates can be seen as an attractive target for such design. There are a number of molecules that can efficiently bind to HIV envelope glycans. Among them, the dendritic cell receptor DC-SIGN has been demonstrated to recognize the internal tri-mannose segment of the N-linked oligosaccharides. A bacterial protein cyanovirin-N efficiently binds high-mannose type gpl20 carbohydrates. Also, one of the most potent broadly neutralizing antibodies known to date, the 2gl2, has been shown to have a carbohydrate epitope. Administering synthetic antigens containing one or more glycans on a part of gpl20 peptide backbone or appropriately chosen linker system and further conjugated to an antigenic carrier could elicit strong immune response ultimately aimed at the real viral envelope. Some of the N-linked carbohydrates of gpl20 appear to be conserved in most of HIV primary isolates. Since the glycans recognized by these molecules are located on the outer, "silent" face of the oligomer, they are easily accessible for antibody binding. Entropically disfavored interaction does not present a problem since the epitope does not overlap with the CD4-binding site. Finally, an extensive glycosylation of the envelope is an advantage, rather than a problem for such antigen design. [0009] Accordingly, there remains a need for novel synthetic methods leading to the preparation of gpl20 glycans and conjugates thereof, and their evaluation in immunologic and therapeutic studies.
SUMMARY OF THE INVENTION [0010] In recognition of the need to provide access to synthetically unavailable gpl20 glycans and glycoconjugates thereof, the present invention, in one aspect, provides novel gpl20 glycans and glycoconjugates thereof, and methods for the synthesis and use thereof. [0011] In one aspect, the present invention provides compounds having formula
(I):
Figure imgf000005_0001
(I) wherein R1, R2A, R2B, R3, R4, W1 , W2 and W3 are as defined herein.
[0012] In certain embodiments, the invention provides a compound of formula (II) having the structure:
Figure imgf000005_0002
(II) wherein R1, R2A, R2B, R3 and R4 are as defined herein.
[0013] In certain embodiments, the invention provides a compound of formula (III) having the structure:
Figure imgf000006_0001
(HI) wherein X, R1, R2A, R2B, R3 and R4 are as defined herein. [0014] In another aspect, the invention provides multi-antigenic constructs comprising one or more carbohydrate domains having the structure:
Figure imgf000006_0002
wherein each occurrence of R1 is independently hydrogen or an oxygen protecting group; each occurrence of R2A and R2B is independently hydrogen or a nitrogen protecting group; each occurrence of R is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000007_0001
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R5, R6 and R7 is independently hydrogen, OH, OR', NRHRm, NHCOR', F, CH2OH, CH2OR', or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R1, R" and R"' is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COOR'v, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R'", taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occurrence of R1V is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group;
W , W and W are independently optionally substituted mannose, galactose or lactosamine moieties; wherein each carbohydrate domain is independently covalently bound to a linker system, said linker system being a peptide or non-peptide nature, and wherein the linker system may be cyclic or acyclic.
[0015] In another aspect, the invention provides clustered glycoconjugates comprising a cyclic or acyclic backbone made up of two or more amino acids or other structural units, wherein one or more of said amino acids or structural units is/are independently substituted with a glycosidic moiety having the structure:
→H wherein each occurrence of L1 is independently a substituted or unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated aliphatic or heteroaliphatic moiety; and and each occurrence of A is independently a carbohydrate domain having the structure:
Figure imgf000008_0001
(Idet)
Figure imgf000008_0002
(IIdet) or
Figure imgf000008_0003
(IIIdet)
wherein each occurrence of R1 is independently hydrogen or an oxygen protecting group; each occurrence of R2A and R2B is independently hydrogen or a nitrogen protecting group; each occurrence of R3 is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000009_0001
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R5, R6 and R7 is independently hydrogen, OH, OR1, NRaR , NHCOR', F, CH2OH, CH2OR', or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R', R" and R"1 is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COORlv, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R'", taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occurrence of Rιv is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group;
W1, W2 and W3 are independently optionally substituted mannose, galactose or lactosamine moieties.
[0016] In another aspect, the invention encompasses clustered multi-antigenic constructs having the structure:
Figure imgf000010_0001
wherein q is 0 or 1 ; each occurrence of s is independently an integer from 2-20; t is an integer from 1-6;
RX1 is hydrogen, alkyl, acyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl) or a nitrogen protecting group; or RX1 is covalently bound to a substituent on the last occurrence of the spacer, thereby forming a cyclic backbone;
R is hydrogen or an immunogenic carrier; each occurrence of the structural unit SU is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; each occurrence of the spacer is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; the linker is either a free carboxylic acid, -O-, (carboxamido)alkyl carboxamide, MBS, primary carboxamide, mono- or dialkyl carboxamide, mono- or diarylcarboxamide, linear or branched chain (carboxy)alkyl carboxamide, linear or branched chain (alkoxycarbonyl)alkyl-carboxamide, linear or branched chain
(carboxy)arylalkylcarboxamide, linear or branched chain
(alkoxycarbonyl)alkylcarboxamide, an oligoester fragment comprising from 2 to about 20 hydroxy acyl residues, a peptidic fragment comprising from 2 to about 20 amino acyl residues, or a linear or branched chain alkyl or aryl carboxylic ester; each occurrence of L1 is independently a substituted or unsubstituted aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydrate domain of formula (Idet), σidet) or (IHdet)-
[0017] In yet another aspect, the invention provides dimeric glycopeptides having the structure:
Figure imgf000011_0001
wherein each peptide may be the same or different; each occurrence of L1 may be the same or different and is as defined above; each occurrence of A is independently a carbohydrate domain of formula (Idet), (IIdet) or (IIIde ). [0018] In another aspect, the invention provides compositions of any of the compounds, glycopeptides and/or constructs disclosed herein. [0019] In another aspect, the invention provides an antibody or antibody fragment which is specific to one or more of the inventive gpl20 glycans and/or glycoconjugates thereof described herein, said antibody being a purified polyclonal antibody or a monoclonal antibody.
[0020] In another aspect, the invention provides methods for the use thereof in the treatment of HIV, methods for the prevention of HIV, and methods for inducing antibodies in a subject, comprising administering to a subject in need thereof, an effective amount of any of the inventive compounds as disclosed herein, either in conjugated form or unconjugated and in combination with a suitable immunogenic carrier.
[0021] As detailed herein, in another aspect of the present invention, any of the inventive compounds may be conjugated to generate a glycoconjugate, and may be administered alone, with an immunogenic carrier (for example, a carrier protein, peptide or lipid), or with an immunological adjuvant or any combination thereof for the treatment of HIV infection and/or for preventing HIV infection, or may be administered alone or with an immunological adjuvant to induce antibodies in a subject.
[0022] In yet another aspect, the invention provides kits for conveniently and effectively carrying out the methods in accordance with the present invention. DEFINITIONS
[0023] Certain compounds of the present invention, and definitions of specific functional groups are also described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein by reference.
[0024] It will be appreciated that the compounds, as described herein, may be substituted with any number of substituents or functional moieties. In general, the term "substituted" whether preceded by the term "optionally" or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position unless otherwise indicated. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, this invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Combinations of substituents and variables envisioned by this invention are preferably those that result in the formation of stable compounds useful in the treatment and or prevention of HIV, or in the inducement of antibodies, as described herein. The term "stable", as used herein, preferably refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein.
[0025] The term "aliphatic", as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched) or branched aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl moieties. Thus, as used herein, the term "alkyl" includes straight and branched alkyl groups. An analogous convention applies to other generic terms such as "alkenyl", "alkynyl" and the like. Furthermore, as used herein, the terms "alkyl", "alkenyl", "alkynyl" and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "lower alkyl" is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-6 carbon atoms. [0026] In certain embodiments, the alkyl, alkenyl and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-4 carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n- propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, n-hexyl, sec-hexyl, moieties and the like, which again, may bear one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl and the like.
[0027] The term "alicyclic", as used herein, refers to compounds which combine the properties of aliphatic and cyclic compounds and include but are not limited to cyclic, or polycyclic aliphatic hydrocarbons and bridged cycloalkyl compounds, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "alicyclic" is intended herein to include, but is not limited to, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties, which are optionally substituted with one or more functional groups. Illustrative alicyclic groups thus include, but are not limited to, for example, cyclopropyl, -CH2- cyclopropyl, cyclobutyl, -CH2-cyclobutyl, cyclopentyl, -CH -cyclopentyl-n, cyclohexyl, -CH2-cyclohexyl, cyclohexenylethyl, cyclohexanylethyl, norborbyl moieties and the like, which again, may bear one or more substituents. [0028] The term "alkoxy" (or "alkyloxy"), or "thioalkyl" as used herein refers to an alkyl group, as previously defined, attached to the parent molecular moiety through an oxygen atom or through a sulfur atom. In certain embodiments, the alkyl group contains 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl group contains 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl group contains 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl group contains 1-4 aliphatic carbon atoms. Examples of alkoxy, include but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy and n-hexoxy. Examples of thioalkyl include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.
[0029] The term "alkylamino" refers to a group having the structure - NHR'wherein R' is alkyl, as defined herein. The term "aminoalkyl" refers to a group having the structure NH2R'-, wherein R' is alkyl, as defined herein. In certain embodiments, the alkyl group contains 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl group contains 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl group contains 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl group contains 1-4 aliphatic carbon atoms. Examples of alkylamino include, but are not limited to, methylamino, ethylamino, iso-propylamino and the like. [0030] Some examples of substituents of the above-described aliphatic (and other) moieties of compounds of the invention include, but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; CI ; Br; I; -OH; -NO2; -CN; -CF3; -CH2CF3; -CHC12; -CH2OH; - CH2CH2OH; -CH2NH2; -CH2SO2CH3; -C(O)Rx; -CO2(Rx); -CON(Rx)2; -OC(O)Rx; - OCO2Rx; -OCON(Rx)2; -N(RX)2; -S(O)2Rx; -NRx(CO)Rx wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl, wherein any of the aliphatic, heteroaliphatic, alkylaryl, or alkylheteroaryl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.
[0031] In general, the terms "aryl" and "heteroaryl", as used herein, refer to stable mono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclic unsaturated moieties having preferably 3-14 carbon atoms, each of which may be substituted or unsubstituted. It will also be appreciated that aryl and heteroaryl moieties, as defined herein may be attached via an aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, alkyl or heteroalkyl moiety and thus also include - (aliphatic)aryl, -(heteroaliphatic)aryl, -(aliphatic)heteroaryl,
(heteroaliphatic)heteroaryl, -(alkyl)aryl, -(heteroalkyl)aryl, -(heteroalkyl)aryl, and - (heteroalkyl)heteroaryl moieties. Thus, as used herein, the phrases "aryl or heteroaryl" and "aryl, heteroaryl, -(aliphatic)aryl, -(heteroaliphatic)aryl, - (aliphatic)heteroaryl, -(heteroaliphatic)heteroaryl, -(alkyl)aryl, -(heteroalkyl)aryl, - (heteroalkyl)aryl, and -(heteroalkyl)heteroaryl" are interchangeable. Substituents include, but are not limited to, any of the previously mentioned substitutents, i.e., the substituents recited for aliphatic moieties, or for other moieties as disclosed herein, resulting in the formation of a stable compound. In certain embodiments of the present invention, "aryl" refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like. In certain embodiments of the present invention, the term "heteroaryl", as used herein, refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from S, O and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.
[0032] It will be appreciated that aryl and heteroaryl groups (including bicyclic aryl groups) can be unsubstituted or substituted, wherein substitution includes replacement of one, two or three of the hydrogen atoms thereon independently with any one or more of the following moieties including, but not limited to: aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; CI; Br; I; -OH; -NO2; -CN; -CF3; -CH2CF3; -CHC12; -CH2OH; -CH2CH2OH; - CH2NH2; -CH2SO2CH3; -C(O)Rx; -CO2(Rx); -CON(Rx)2; -OC(O)Rx; -OCO2Rx; - OCON(Rx)2; -N(RX)2; -S(O)2Rx; -NRx(CO)Rx wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl, wherein any of the aliphatic, heteroaliphatic, alkylaryl, or alkylheteroaryl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.
[0033] The term "cycloalkyl", as used herein, refers specifically to groups having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the case of aliphatic, heteroaliphatic or heterocyclic moieties, may optionally be substituted with substituents including, but not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; CI; Br; I; -OH; -NO2; -CN; -CF3; -CH2CF3; -CHC12; -CH2OH; - CH2CH2OH; -CH2NH2; -CH2SO2CH3; -C(O)Rx; -CO2(Rx); -CON(Rx)2; -OC(O)Rx; - OCO2Rx; -OCON(Rx)2; -N(RX)2; -S(O)2Rx; -NRx(CO)Rx wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl, wherein any of the aliphatic, heteroaliphatic, alkylaryl, or alkylheteroaryl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.
[0034] The term "heteroaliphatic", as used herein, refers to aliphatic moieties in which one or more carbon atoms in the main chain have been substituted with a heteroatom. Thus, a heteroaliphatic group refers to an aliphatic chain which contains one or more oxygen, sulfur, nitrogen, phosphorus or silicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moieties may be branched or linear unbranched. In certain embodiments, heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to aliphatic; alicyclic; heteroaliphatic; heteroalicyclic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; CI; Br; I; -OH; -NO2; -CN; -CF3; -CH2CF3; -CHC12; -CH2OH; -CH2CH2OH; - CH2NH2; -CH2SO2CH3; -C(O)Rx; -CO2(Rx); -CON(Rx)2; -OC(O)Rx; -OCO2Rx; - OCON(Rx)2; -N(RX)2; -S(O)2Rx; -NRx(CO)Rx wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl, wherein any of the aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, alkylaryl, or alkylheteroaryl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein. [0035] The term "heteroalicyclic", as used herein, refers to compounds which combine the properties of heteroaliphatic and cyclic compounds and include but are not limited to saturated and unsaturated mono- or polycyclic heterocycles such as morpholino, pyrrolidinyl, furanyl, thiofuranyl, pyrrolyl etc., which are optionally substituted with one or more functional groups, as defined herein. [0036] Additionally, it will be appreciated that any of the alicyclic or heteroalicyclic moieties described above and herein may comprise an aryl or heteroaryl moiety fused thereto. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.
[0037] The terms "halo" and "halogen" as used herein refer to an atom selected from fluorine, chlorine, bromine and iodine.
[0038] The term "haloalkyl" denotes an alkyl group, as defined above, having one, two, or three halogen atoms attached thereto and is exemplified by such groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.
[0039] The term "heterocycloalkyl" or "heterocycle", as used herein, refers to a non-aromatic 5-, 6- or 7- membered ring or a polycyclic group, including, but not limited to a bi- or tri-cyclic group comprising fused six-membered rings having between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 1 double bonds and each 6- membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may be optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring. Representative heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. In certain embodiments, a "substituted heterocycloalkyl or heterocycle" group is utilized and as used herein, refers to a heterocycloalkyl or heterocycle group, as defined above, substituted by the independent replacement of one, two or three of the hydrogen atoms thereon with but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; CI; Br; I; - OH; -NO2; -CN; -CF3; -CH2CF3; - CHC12; -CH2OH; -CH2CH2OH; -CH2NH2; -CH2SO2CH3; -C(O)Rx; -CO2(Rx); - CON(Rx)2; -OC(O)Rx; -OCO2Rx; -OCON(Rx)2; -N(RX)2; -S(O)2Rx; -NRx(CO)Rx wherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl, wherein any of the aliphatic, heteroaliphatic, alkylaryl, or alkylheteroaryl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substitutents described above and herein may be substituted or unsubstituted. Additional examples or generally applicable substituents are illustrated by the specific embodiments shown in the Examples, which are described herein.
[0040] As used herein, the terms "aliphatic", "heteroaliphatic", "alkyl", "alkenyl", "alkynyl", "heteroalkyl", "heteroalkenyl", "heteroalkynyl", and the like encompass substituted and unsubstituted, saturated and unsaturated, and linear and branched groups. Similarly, the terms "alicyclic", "heteroalicyclic",
"heterocycloalkyl", "heterocycle" and the like encompass substituted and unsubstituted, and saturated and unsaturated groups. Additionally, the terms "cycloalkyl", "cycloalkenyl", "cycloalkynyl", "heterocycloalkyl",
"heterocycloalkenyl", "heterocycloalkynyl", "aryl", "heteroaryl" and the like encompass both substituted and unsubstituted groups.
[0041] It will be appreciated that additional examples of generally applicable substitutents are illustrated by the specific embodiments shown in the Examples which are described herein, but are not limited to these Examples. [0042] The phrase, "pharmaceutically acceptable derivative", as used herein, denotes any pharmaceutically acceptable salt, ester, or salt of such ester, of such compound, or any other adduct or derivative which, upon administration to a patient, is capable of providing (directly or indirectly) a compound as otherwise described herein, or a metabolite or residue thereof. Pharmaceutically acceptable derivatives thus include among others pro-drugs. A pro-drug is a derivative of a compound, usually with significantly reduced pharmacological activity, which contains an additional moiety, which is susceptible to removal in vivo yielding the parent molecule as the pharmacologically active species. An example of a pro-drug is an ester, which is cleaved in vivo to yield a compound of interest. Pro-drugs of a variety of compounds, and materials and methods for derivatizing the parent compounds to create the pro-drugs, are known and may be adapted to the present invention. Certain exemplary pharmaceutical compositions and pharmaceutically acceptable derivatives will be discussed in more detail herein below. [0043] By the term "protecting group", has used herein, it is meant that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. In preferred embodiments, a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group must be selectively removed in good yield by readily available, preferably nontoxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction. As detailed herein, oxygen, sulfur, nitrogen and carbon protecting groups may be utilized. For example, in certain embodiments, as detailed herein, certain exemplary oxygen protecting groups are utilized. These oxygen protecting groups include, but are not limited to methyl ethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM (methylthiomethyl ether), BOM (benzyloxymethyl ether), PMBM or MPM (p- methoxybenzyloxymethyl ether), to name a few), substituted ethyl ethers, substituted benzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilylether), TIPS (triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzyl silyl ether, TBDPS (t-butyldiphenyl silyl ether), to name a few), esters (e.g., formate, acetate, benzoate (Bz), trifluoroacetate, dichloroacetate, to name a few), carbonates, cyclic acetals and ketals. In certain other exemplary embodiments, nitrogen protecting groups are utilized. These nitrogen protecting groups include, but are not limited to, carbamates (including methyl, ethyl and substituted ethyl carbamates (e.g., Troc), to name a few) amides, cyclic imide derivatives, N-Alkyl and N-Aryl amines, imine derivatives, and enamine derivatives, to name a few. Certain other exemplary protecting groups are detailed herein, however, it will be appreciated that the present invention is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the present invention. Additionally, a variety of protecting groups are described in "Protective Groups in Organic Synthesis" Third Ed. Greene, T.W. and Wuts, P.G., Eds., John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference.
[0044] As used herein, the term "adjuvant" or "immunogenic stimulant" refers to a moiety, which, when co-administered with an immunogen, enhances the immunogenicity of the immunogen. Specifically, in certain embodiments, immunogenicity of the inventive gpl20 compounds can be significantly improved if the immunizing agent(s) (e.g., gpl20 glycan(s) and/or construct(s) thereof) and/or composition thereof is, regardless of administration format, co-immunized with an adjuvant. Commonly, adjuvants are used as an 0.05 to 1.0 percent solution in phosphate-buffered saline. Adjuvants enhance the immunogenicity of an immunogen but are not necessarily immunogenic themselves. Adjuvants may act by retaining the immunogen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of immunogen to cells of the immune system. Adjuvants can also attract cells of the immune system to an immunogen depot and stimulate such cells to elicit immune responses. As such, embodiments of this invention encompass compositions further comprising adjuvants. [0045] Adjuvants have been used for many years to improve the host immune responses to, for example, vaccines. Intrinsic adjuvants (such as lipopolysaccharides) normally are the components of killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune responses. Thus, adjuvants have been identified that enhance the immune response to antigens delivered parenterally. Some of these adjuvants are toxic, however, and can cause undesirable side-effects making them unsuitable for use in humans and many animals. Indeed, aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established. Notwithstanding, it does have limitations. For example, alum is ineffective for influenza vaccination and inconsistently elicits a cell mediated immune response with other immunogens. The antibodies elicited by alum-adjuvanted antigens are mainly of the IgGl isotype in the mouse, which may not be optimal for protection by some vaccinal agents. In addition to adjuvants used for therapeutic purposes (e.g., vaccines), other adjuvants may be used for raising antibodies in animals, which antibodies may be used, for example, in diagnostic and immunoassays. Examples of such adjuvants include, but are not limited to, bacteria or liposomes. For example, suitable adjuvants include but are not limited to, saponin adjuvants (e.g., GPI-0100), Salmonella minnesota cells, bacille Calmette-Guerin or QS21.
[0046] A wide range of extrinsic adjuvants can provoke potent immune responses to immunogens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria and mineral oil, Freund's complete adjuvant, bacterial products such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes.
[0047] The term "natural amino acid" as used herein refers to any one of the common, naturally occurring L-amino acids found in naturally occurring proteins: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (He), lysine (Lys), arginine (Arg), histidine (His), proline (Pro), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Tip), aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine (Gin), cysteine (Cys) and methionine (Met). [0048] The term "unnatural amino acid" as used herein refers to all amino acids which are not natural amino acids. This includes, for example, o , β-, D-, L- amino
acid residues, and compounds of the general formula o wherein the side chain R is other than the amino acid side chains occurring in nature.
[0049] More generally, the term "amino acid", as used herein, encompasses natural amino acids and unnatural amino acids.
[0050] As used herein the term "biological sample " includes, without limitation, cell cultures or extracts thereof; biopsied material obtained from an animal (e.g., mammal) or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof; or purified versions thereof. For example, the term "biological sample" refers to any solid or fluid sample obtained from, excreted by or secreted by any living organism, including single-celled micro-organisms (such as bacteria and yeasts) and multicellular organisms (such as plants and animals, for instance a vertebrate or a mammal, and in particular a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated). The biological sample can be in any form, including a solid material such as a tissue, cells, a cell pellet, a cell extract, cell homogenates, or cell fractions; or a biopsy, or a biological fluid. The biological fluid may be obtained from any site (e.g. blood, saliva (or a mouth wash containing buccal cells), tears, plasma, serum, urine, bile, seminal fluid, cerebrospinal fluid, amniotic fluid, peritoneal fluid, and pleural fluid, or cells therefrom, aqueous or vitreous humor, or any bodily secretion), a transudate, an exudate (e.g. fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (e.g. a normal joint or a joint affected by disease such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis). The biological sample can be obtained from any organ or tissue (including a biopsy or autopsy specimen) or may comprise cells (whether primary cells or cultured cells) or medium conditioned by any cell, tissue or organ. In certain embodiments, the biological sample is obtained from the prostate epithelium. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. Biological samples also include mixtures of biological molecules including proteins, lipids, carbohydrates and nucleic acids generated by partial or complete fractionation of cell or tissue homogenates. Although the sample is preferably taken from a human subject, biological samples may be from any animal, plant, bacteria, virus, yeast, etc. The term animal, as used herein, refers to humans as well as non-human animals, at any stage of development, including, for example, mammals, birds, reptiles, amphibians, fish, worms and single cells. Cell cultures and live tissue samples are considered to be pluralities of animals. In certain exemplary embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). An animal may be a transgenic animal or a human clone. If desired, the biological sample may be subjected to preliminary processing, including preliminary separation techniques. In certain emboidments, the biological sample is taken from a male human subject. In certain exemplary embodiment, the biological sample has been processed so that the gpl20 glycan concentration out of the total glycan concentration in the original sample is increased. In certain exemplary embodiments, the sample may be purified serum gpl20, purified gpl20 glycoprotein, purified gpl20 glycoprotein that has undergone sialidase digestion, purified gpl20 glycans obtained from deglycosylated gpl20 glycoprotein, or any combination thereof. It will be appreciated that the term "biological sample", as used herein, encompasses any combination of gpl20 materials obtained from any biological sources (e.g., as detailed above) or by any processes that may be used to obtain gpl20 glycan from the original sample (e.g., extraction, purification, glycoprotein deglycosylation, sialidase digestion, etc.).
[0051] As used herein, the term "isolated" when applied to the compounds of the present invention, refers to such compounds that are (i) separated from at least some components with which they are associated in nature or when they are made and/or (ii) produced, prepared or manufactured by the hand of man. In certain embodiments, isolated compounds of the invention are not substantially contaminated with, or otherwise in contact with any other compound. Accordingly, the present invention provides compounds of formula (I) and/or (II) in substantially pure form, i.e., in a purity of greater than about 95% by weight (not including H2O or salt content, which is to be expected, for example, from lyophilized peptides and glycopeptides), preferably greater than about 98%, and more preferably greater than about 99% by weight. In one aspect, the impurity in contact with a compound of formula (I) and/or (II) of the invention is an organic chemical, e.g., an organic solvent. In another aspect, the impurity in contact with a compound of formula (I) and/or (II) is another compound of formula (I) and/or (II). Thus, in one aspect, the present invention provides a compound of formula (I) and/or (II) that is pure in that it is not in contact with another compound of formula (I) and/or (II). [0052] As used herein, the term "glycoconjugate" refers to one or more glycans covalently linked to a peptidic or non-peptidic backbone.
[0053] As used herein, the term "gpl20 glycan" refers to a carbohydrate domain present on gpl20. More specifically, gpl20 glycan designates the carbohydrate portion of compounds of formula (I), (II) and/or (III) described herein. In certain embodiments, the term refers to compounds of formula (I), (II) and/or (III) where R4 is a moiety other than a peptide, protein or other polymeric construct. [0054] As used herein, the term "gpl20 glycopeptide" refers to compounds of formula (I), (II) and/or (III) where R4 comprises a peptide moiety covalently linked to the rest of the construct either directly (e.g., through N or O) or through a crosslinker.
[0055] As used herein, the term "eliciting an immune response" is defined as initiating, triggering, causing, enhancing, improving or augmenting any response of the immune system, for example, of either a humoral or cellular nature. The initiation or enhancement of an immune response can be assessed using assays known to those skilled in the art including, but not limited to, antibody assays (for example ELISA assays). In certain exemplary embodiments, the inventive gpl20 glycans and/or glycoconjugates thereof, and the methods of the present invention essentially trigger or enhance primarily a humoral immune response.
BRIEF DESCRIPTION OF THE DRAWING [0056] Figure 1 depicts structures of gpl20 glycopeptides 1-2.
DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE
INVENTION
[0057] As discussed above, the desire to develop improved methods for the preparation of synthetic vaccines has led to increased research efforts directed toward the synthesis of naturally occurring complex carbohydrate antigens, as well as novel complex structures (e.g., glycopeptides or other glycoconjugates) incorporating these antigenic structures. As is often the case during the course of any such large synthetic undertaking, improved synthetic methods are often developed that can be applied universally. In particular, synthetic studies of naturally occurring antigenic structures has led to the development of novel methodologies enabling the development of heretofore unavailable synthetic carbohydrate-based vaccines. For a review, see Danishefsky, S.J.; Allen, J.R., Angew. Chem. Int. Ed. Engl 2000, 39, 836-863, and references cited therein. [0058] Significantly, the present invention provides novel methodologies for the synthesis of complex carbohydrates and related therapeutic compounds (e.g., glycans and/or glycoconjugates thereof). In particular, in the context of synthetic studies developed for the total synthesis of glycosylated fragments of gpl20 and conjugates thereof, generalized methodologies were developed for the improved synthesis of complex carbohydrate structures. This general synthetic method encompasses the realization that the incorporation of an amino group at the reducing end of a carbohydrate acceptor allows for accessibility to complex N-linked carbohydrate conjugates. In yet another aspect, the present invention also provides the recognition that for certain protected carbohydrates, the amino carbohydrate moieties can serve as useful precursors that can be utilized ultimately for the synthesis of complex N-linked glycopeptides or other glycoconjugates. [0059] Specific examples, particularly with respect to the total synthesis of N- acetyllactosamine-type glycans and their incorporation into gpl20 glycopeptide fragments and other non-peptidic glycoconjugates are described in more detail below, along with certain general methodologies developed during the course of these syntheses. It will be appreciated by one of ordinary skill in the art that these examples are not intended to be limiting; rather all equivalents are intended to be incorporated into the scope of the present invention. [0060] 1) Inventive Compounds
[0061] As mentioned above, the total synthesis of complex antigenic structures has led to significant development in methodologies for complex carbohydrate synthesis. Of particular recent interest is the naturally occurring antigenic gpl20 glycans; e.g., "high-mannose"- and "hybrid"-type glycoforms thereof (See constructs 1-2 in Figure 1) which heretofore had not yet been synthesized. [0062] Thus, in one aspect of the present invention, the synthesis of the complex gpl20 carbohydrate domains has been achieved and an isolated compound of formula (I) having the structure as shown below is provided:
Figure imgf000027_0001
wherein each occurrence of R1 is independently hydrogen or an oxygen protecting group; each occurrence of R2A and R2B is independently hydrogen or a nitrogen protecting group; each occurrence of R3 is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000027_0002
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R5, R6 and R7 is independently hydrogen, OH, OR1, NR11^11, NHCOR1, F, CH2OH, CH^R1, or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R', R" and R"1 is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COOR'v, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R"1, taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occurrence of R'v is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group;
1 9 "
W , W and W are independently optionally substituted mannose, galactose or lactosamine moieties; and wherein R4 is -OR4A or -NHR4A; wherein R4A is hydrogen, aliphatic, heteroaliphatic, aryl, heteroaryl, an amino acyl moiety, an amino acyl residue of a peptide, an amino acyl residue of a protein, or R4A comprises a protein, peptide or lipid moiety covalently linked to the rest of the construct, or to the N or O atom to which it is attached, either directly or through a crosslinker.
[0063] In certain embodiments, W3 is R1, R3, as defined above, or a moiety having the structure:
Figure imgf000028_0001
1 Δ R H wherein X is -OR or -NR R ; and each occurrence of R is independently R1 or a sialic acid moiety.
1 9 I T
[0064] In certain embodiments, W and W are independently R , R or a moiety having the structure:
Figure imgf000028_0002
wherein each occurrence of R8 is independently R1 or a sialic acid moiety. [0065] In certain embodiments, a compound of formula (II) having the structure as shown below is provided:
Figure imgf000029_0001
wherein R1, R2A, R2B, R3 and R4 are as defined above.
[0066] In certain embodiments, a compound of formula (III) having the structure as shown below is provided:
Figure imgf000029_0002
n) wherein R1, R2A, R2B, R3 and R4 are as defined above and X is OR1 or
NR2AR2B.
[0067] In certain embodiments, compounds of formula (I), (II) or (III) exclude naturally occurring gpl20 (e.g., a glycan domain found on naturally occurring gpl20 glycoprotein). [0068] In certain embodiments, when R4 comprises a peptide, the peptide is either identical to or closely related to that of gpl20 near an N-glycosylation site. In certain exemplary embodiments, the peptide has the structure:
1^ Lys-lle-Gly-Asn- et-Arg-Gln-Ala-His-Cys— Asn — lle-Ser-Arg
Cys— Asn — lle-Ser-Arg or Gly-lle-Thr-Val-Phe-Ala or truncated, elongated or derivatized version thereof; wherein any one or more of the amino acid residues may bear one or more protecting groups. For the purpose of the invention, "truncated", refers to a peptide fragment comprising no fewer than about 6 amino acid residues; "elongated", refers to a peptide comprising no more than about 60 amino acid residues; and "derivatized" refers to a peptide in which at least one, but not more than about 2 out of every 10, amino acid residues have been added and/or deleted; and/or in which at least one amino acid residue has been substituted with a natural or non-natural amino acid residue so that the resulting peptide has a sequence identity equal or greater to about 70% with the original peptide.
[0069] In certain exemplary embodiments, for compounds of formula (I), (II) and (III) above, each occurrence of R1 is independently an oxygen protecting group. In certain other exemplary embodiments, each occurrence of R1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, -Si(R1A)3, -C(=O)R1A, -C(=S)R1A, - C(=NR1A)R1B, -SO2R1A, wherein R1A and R1B are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl,
1 C heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, -C(=O)R or - ZR1C, wherein Z is -O-, -S-, -NR1D, wherein each occurrence of R1C and R1D is independently hydrogen, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety. In yet other exemplary embodiments, each occurrence of R1 is independently hydrogen, alkylaryl, -Si(R1A)3 or -C(=O)R1A, wherein R1A is as defined above. In yet other exemplary embodiments, each occurrence of R1 is independently hydrogen, Bn or Bz. In certain other exemplary embodiments, each occurrence of R is independently hydrogen.
[0070] In certain other exemplary embodiments, for compounds of formula (I), (II) and (III) above, for each occurrence of -NR2AR2B, at least one occurrence of R2A or R is independently a nitrogen protecting group. In certain other exemplary embodiments, each occurrence of -NR R , R and R is independently hydrogen, alkyl, alkenyl, -C(=O)R2C, -C(=O)OR2C, -SR2C, SO2R2C, or R2A and R2B, taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; wherein each occurrence of R is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, -C(=O)R2D or -ZR2D, wherein Z is -O-, -S-, -NR2E, wherein each occurrence of R2D and R2E is independently hydrogen, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety. In certain exemplary embodiments, for each occurrence of -NR R , at least one occurrence of R or R is independently -C(=O)R2A or SO2R2A; or R2A and R2B, taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety. In yet other exemplary embodiments, for each occurrence of - NR2AR2B, at least one occurrence of R2A or R2B is independently -C(=O)R2C or SO2R wherein R is as defined above, or R and R , taken together with the nitrogen atom to which they are attached, form an azide or a substituted or unsubstituted phthalimide moiety. In yet other exemplary embodiments, for each occurrence of -NR R , at least one occurrence of R or R is independently acyl, -SO2Ph or R2A and R2B, taken together with the nitrogen atom to which they are attached, form an azide or a substituted or unsubstituted phthalimide moiety. In certain other exemplary embodiments, each occurrence of -NR2AR2B is -NHAc. [0071] In certain other embodiments, for compounds of formula (III) above, X is -OR1, wherein R1 is as defined generally above and in classes and subclasses herein. [0072] In certain other embodiments, for compounds of formula (I), (II) and (III) above, each occurrence of R3 is independently R1, wherein R1 is as defined generally above and in classes and subclasses herein. In certain embodiments, each occurrence of R3 is independently hydrogen, alkylaryl, -Si(R3A)3 or -C(=O)R3A, wherein R3A is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, -C(=O)R3B or -ZR3B, wherein Z is -O-, -S-, -NR3C, wherein each occurrence of R3B and R3 is independently hydrogen, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety. In yet other exemplary embodiments, each occurrence of R is independently hydrogen, Bn or Bz. In certain other exemplary embodiments, each occurrence of R3 is independently hydrogen. [0073] In certain other embodiments, for compounds of formula (I), (II) and (III) above, each occurrence of R1 and R3 is independently hydrogen, alkylaryl, - Si(R3A) or -C(=O)R3A, wherein R3A is as defined above. In yet other exemplary
1 T embodiments, each occurrence of R and R is independently hydrogen, Bn or Bz. In certain other exemplary embodiments, each occurrence of R1 is Bn and each occurrence of R3 is Bz. In certain other exemplary embodiments, each occurrence of R1 and R3 is independently hydrogen.
[0074] In certain embodiments, for compounds of formula (I), (II) and (III) above, R4 is -OR4A and the saccharide unit bearing R4 has the structure:
Figure imgf000032_0001
wherein R1, R2A and R2B are as defined generally above and in classes and subclasses herein; R A is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, an amino acyl moiety, an amino acyl residue of a peptide, an amino acyl residue of a protein, -
Si(R4B)3, -C(=O)R4B, -C(=S)R4B, -C(=NR4B)R4C, -SO2R4B, wherein R4B and R4C are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, -C(=O)R4D or -ZR4D, wherein Z is -O-, -S-, -NR4E, wherein each occurrence of R4D and R4E is independently hydrogen, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety; or R4A comprises a protein, peptide or lipid moiety covalently linked to the O atom to which it is attached, either directly or through a crosslinker. In yet other exemplary embodiments, R4A is -Si(R4B)3, wherein R4B is as defined above. In yet other exemplary embodiments, R4A is TBS. In yet other exemplary embodiments R4A comprises a serine (ser) amino acyl residue. In yet other exemplary embodiments R4A comprises a threonine (Thr) amino acyl residue. In yet other exemplary embodiments R4A comprises a peptide attached to O through a serine (Ser) residue. In yet other exemplary embodiments R4A comprises a peptide attached to O through a Threonine (Thr) residue. [0075] In certain embodiments, for compounds of formula (I), (II) and (III) above, R4 is -NHR4A and the saccharide unit bearing R4 has the structure:
Figure imgf000033_0001
wherein R1, R2A and R2B are as defined generally above and in classes and subclasses herein; and R is hydrogen, aliphatic, heteroaliphatic, aryl, heteroaryl, an amino acyl moiety, an amino acyl residue of a peptide, an amino acyl residue of a protein, or R4A comprises a protein, peptide or lipid moiety covalently linked to the rest of the construct, or to the N atom to which it is attached, either directly or through a crosslinker.
[0076] In certain exemplary embodiments, R4A is hydrogen. [0077] In certain other exemplary embodiments, R4A comprises an amino acyl residue of a peptide whose structure is either identical or closely related to that of gpl20 near an N-glycosylation site.
[0078] In certain other exemplary embodiments, R4A comprises an Asparagine residue (Asn) of a peptide whose structure is either identical or closely related to that of gpl20 near an N-glycosylation site. [0079] For the purpose of the invention, a peptide whose structure is "closely related to that of gpl20 near an N-glycosylation site" designates a gpl20 peptide fragment, or truncated, elongated or derivatized version thereof, comprising < about 60 amino acid residues, wherein one amino acid residue bears an N-glycosylation site, at least one amino acid residue has been added, deleted and/or substituted with a natural or non-natural amino acid residue, so that the resulting peptide has a sequence identity greater or equal to about 70% with the original gpl20 peptide fragment. In certain embodiments, the peptide comprises < about 55 amino acid residues. In certain embodiments, the peptide comprises < about 50 amino acid residues. In certain embodiments, the peptide comprises < about 45 amino acid residues. In certain embodiments, the peptide comprises < about 40 amino acid residues. In certain embodiments, the peptide comprises < about 35 amino acid residues. In certain embodiments, the peptide comprises < about 30 amino acid residues. In certain embodiments, the peptide comprises < about 25 amino acid residues. In certain embodiments, the peptide comprises < about 20 amino acid residues. In certain embodiments, the peptide has a sequence identity greater or equal to about 75% with the original gpl20 peptide fragment. In certain other embodiments, the peptide has a sequence identity greater or equal to about 80% with the original gpl20 peptide fragment. In certain other embodiments, the peptide has a sequence identity greater or equal to about 85% with the original gpl20 peptide fragment. In certain other embodiments, the peptide has a sequence identity greater or equal to about 90% with the original gpl20 peptide fragment. In certain other embodiments, the peptide has a sequence identity greater or equal to about 95% with the original gpl20 peptide fragment.
[0080] A peptide whose structure is "identical to that of gpl20 near an N- glycosylation site" designates a gpl20 peptide fragment of a naturally occurring gpl20 glycoprotein, comprising < about 60 amino acid residues, wherein one amino acid residue bears an N-glycosylation site. In certain embodiments, the peptide comprises < about 55 amino acid residues. In certain embodiments, the peptide comprises < about 50 amino acid residues. In certain embodiments, the peptide comprises < about 45 amino acid residues. In certain embodiments, the peptide comprises < about 40 amino acid residues. In certain embodiments, the peptide comprises < about 35 amino acid residues. In certain embodiments, the peptide comprises < about 30 amino acid residues. In certain embodiments, the peptide comprises < about 25 amino acid residues. In certain embodiments, the peptide comprises < about 20 amino acid residues.
[0081] In certain embodiments, for compounds of formula (I), (II) and (III) above, R4 is -NHR4A wherein R4A comprises an Asparagine residue (Asn) of a peptide whose structure is either identical or closely related to that of gpl20 near an N-glycosylation site and the saccharide unit bearing R4 has the structure:
Figure imgf000035_0001
wherein R1, R2A and R2B are as defined generally above and in classes and subclasses herein and wherein any of the amino acid residues may bear one or more protecting groups. [0082] In certain exemplary embodiments, the saccharide unit bearing R4 has the structure:
Figure imgf000035_0002
wherein R1, R2A and R2B are as defined generally above and in classes and subclasses herein. [0083] In certain other exemplary embodiments, the saccharide unit bearing R has the structure:
Figure imgf000036_0001
wherein R1, R2A and R2B are as defined generally above and in classes and subclasses herein.
[0084] In certain embodiments, any of the isolated compounds, glycopeptides and/or glycoconjugates described herein may be further conjugated to an immunogenic carrier. In certain exemplary embodiments, the carrier is a protein, a peptide or a lipid. In certain other exemplary embodiments, the carrier is Bovine Serum Albumin (BSA), Keyhole Limpet Hemocyanin (KLH) or polylysine. In certain other embodiments, the carrier is is a lipid carrier having the structure:
Figure imgf000036_0002
wherein m, n and p are each independently integers between about 8 and 20; and Ry is hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl. In certain exemplary embodiments, m', n' and p' are each 14 and the lipid is tripalmitoyl-S-glycerylcysteinylserine (e.g., PamCys).
[0085] It will be appreciated that the carrier can be linked to the rest of the construct either directly or through a crosslinker, and thus R4 encompasses proteins, peptides, and lipids, as well as (crosslinker-protein), (crosslinker-peptide) and (crosslinker-lipid) moieties.
[0086] Crosslinkers suited to the invention are widely known in the art (see, for example, 1994 Pierce Technical Handbook: cross-linking (Appendix A), which is available at http://www.piercenet.com/ resources/browse.cfm?fldID=184), including bromoacetic NHS ester, 6-(iodoacetamido)caproic acid NHS ester, maleimidoacetic acid NHS ester, maleimidobenzoic acide NHS ester, etc. In certain preferred embodiments, the crosslinker is MMCCH (4-(maleimidomethyl) cyclohexane-1- carboxyl hydrazide). In certain other preferred embodiments, the crosslinker is MBS (m-maleimidobenzoyl acid N-Hydroxysuccinimidyl ester). In certain embodiments, the crosslinker is a fragment having the structure:
Figure imgf000037_0001
whereby said structure is generated upon conjugation of a maleimidobenzoic acid N-hydroxy succinimide ester with a suitable functionality on R4. [0087] Recently, crystallographic studies revealed that the broadly neutralizing human antibody 2G12, which binds with nanomolar affinity to gpl20, contains multiple, distinct binding sites for carbohydrates (e.g., glycans expressed on gpl20) [see, Calarese et al, "Antibody domain exchange is an immunological solution to carbohydrate cluster recognition", Science, 300:2065-2071, 2003; which is incorporated herein by reference in its entirety]. It was proposed that these multiple binding sites of 2G12 were important for high-affinity interaction of the antibody with the dense array of oligomannose sugars on the surface of gpl20. [0088] Therefore, without wishing to be bound to any particular theory, Applicant proposes that constructs comprising several carbohydrate domains present on the surface of gpl20, or analogs or derivatives thereof, could therefore elicit a humoral immune response comprising antibodies with enhanced binding affinity for gpl20, and therefore have greater potential in the development of HIV vaccines. [0089] Thus, in certain embodiments, there is provided an antigenic construct comprising one or more carbohydrate domains having the structure:
Figure imgf000038_0001
ddet) wherein each occurrence of R1 is independently hydrogen or an oxygen protecting group; each occurrence of R2Λ and R2B is independently hydrogen or a nitrogen protecting group; each occurrence of R is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000038_0002
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R5, R6 and R7 is independently hydrogen, OH, OR', NRHRia, NHCOR1, F, CH2OH, CH2ORi, or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R', R" and R1" is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COOR'v, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R1", taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occurrence of R'v is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group;
W1, W2 and W3 are independently optionally substituted mannose, galactose or lactosamine moieties; wherein each carbohydrate domain is independently covalently bound to a linker system, said linker system being a peptide or non-peptide nature, and wherein the linker system may be cyclic or acyclic.
[0090] In certain embodiments, W is R , R , as defined above, or a moiety having the structure:
Figure imgf000039_0001
wherein X is -OR1 or -NR2AR2B; and each occurrence of R8 is independently R1 or a sialic acid moiety.
1 9 I T
[0091] In certain other embodiments, W and W are independently R , R or a moiety having the structure:
Figure imgf000039_0002
wherein each occurrence of R is independently R or a sialic acid moiety. [0092] In yet other embodiments, inventive constructs comprise one or more carbohydrate domains having the structure:
Figure imgf000040_0001
(IIdet) wherein R1,
Figure imgf000040_0002
R r>2ZAA and i r R>2BB are as d ie rfi:ned J a „Ibove r f-or (πTrde"K). [0093] In yet other embodiments, inventive constructs comprise one or more carbohydrate domains having the structure:
Figure imgf000040_0003
(IIIdet) wherein R1, R3, R2A and R2B are as defined above for (Idet). [0094] In certain embodiments, some or all of carbohydrate domains are O- linked to the linker system. In certain other embodiments, some or all of carbohydrate domains are N-linked to the linker system. In yet other embodiments, the linker system is a peptide. In certain embodiments, the linker system is a cyclic peptide. In certain other embodiments, the linker system is cyclodextrin. In certain embodiments, the linker system is a peptide and comprises two or more carbohydrate domains covalently attached thereto, wherein the peptide sequence between each point of attachment of the carbohydrate domains comprises a cysteine residue. In certain embodiments, the multi-glycan construct is prepared by Native Chemical Ligation. In certain embodiments, the inventive constructs are symmetrical, nonsymmetrical and mixed (N-linked and O-linked carbohydrates). In certain embodiments, the linker system is designed to approximate the spatial position(s) of carbohydrate(s) in gpl20. In yet other embodiments, the linker system is further attached to a carrier immunostimulant.
[0095] In certain embodiments, inventive constructs comprising one or more carbohydrate domains of the formula (Idet), (IIdet) or (IIIdet) are similar to multi- antigenic constructs described in U.S.S.N. 09/083,776 filed March 25, 1998, 09/276,595 filed March 25, 1999, 10/600,012 filed June 19, 2003, 09/641,742 filed August 18, 2000, 10/ 209,618 filed July 31, 2002 and 10/430,822, filed December 3, 2003 and entitled "Clustered Multi-Antigenic Carbohydrate Constructs, Methods for their Preparation, and Uses Thereof; each of the above applications is hereby incorporated by reference in its entirety. Guidance for preparing such constructs can be found, inter alia, in the above-cited applications.
[0096] For example, the present invention encompasses clustered glycoconjugates comprising a cyclic or acyclic backbone made up of two or more amino acids or other structural units, wherein one or more of said amino acids or structural units is/are independently substituted with a glycosidic moiety having the structure:
→ wherein each occurrence of L1 is independently a substituted or unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydrate domain of formula (Idet), (IIdet) or (IHdet).
[0097] In certain embodiments, the invention encompasses clustered multi- antigenic constructs having the structure:
Figure imgf000042_0001
wherein q is 0 or 1 ; each occunence of s is independently an integer from 2-20; t is an integer from 1-6;
RX1 is hydrogen, alkyl, acyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl) or a nitrogen protecting group; or R is covalently bound to a substituent on the last occurrence of the spacer, thereby forming a cyclic backbone;
R is hydrogen or an immunogenic carrier; each occurrence of the structural unit SU is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; each occurrence of the spacer is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; the linker is either a free carboxylic acid, -O-, (carboxamido)alkyl carboxamide, MBS, primary carboxamide, mono- or dialkyl carboxamide, mono- or diarylcarboxamide, linear or branched chain (carboxy)alkyl carboxamide, linear or branched chain (alkoxycarbonyl)alkyl-carboxamide, linear or branched chain (carboxy)arylalkylcarboxamide, linear or branched chain
(alkoxycarbonyl)alkylcarboxamide, an oligoester fragment comprising from 2 to about 20 hydroxy acyl residues, a peptidic fragment comprising from 2 to about 20 amino acyl residues, or a linear or branched chain alkyl or aryl carboxylic ester; each occurrence of L1 is independently a substituted or unsubstituted aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydrate domain of formula (Idet), (Hdet) or (IIIdet).
[0098] In certain other embodiments, for the clustered multi-antigenic construct described directly above, each occurrence of L1 is independently -O(CHRaa)n- or - NHC(=O)(CHRaa)n- wherein each occurrence of n is independently an integer from 1-10; and each occurrence of Raa is hydrogen, lower alkyl, aryl, heteroaryl, - alkyl(aryl) or -alkyl(heteroaryl). In certain exemplary embodiments, each occurrence of n is 1 and each occurrence of R33 is hydrogen or methyl. In certain embodiments, each occurrence of L1 is independently a moiety having the structure -O(CH2)„- wherein n is an integer from 1-10 and each occurrence of A is O-linked to the construct backbone. In certain embodiments, each occurrence of L1 is independently a moiety having the structure -NHC(=O)(CH2)n- wherein n is an integer from 1-10 and each occurrence of A is N-linked to the construct backbone. [0099] In certain embodiments, for the clustered multi-antigenic constructs described directly above, the structural unit SU, for each occurrence, is independently an amino acid residue, a peptidyl moiety, a bivalent aryl or heteroaryl moiety or a substituted or unsubstituted Cι-6alkylidene or C -6alkenylidene chain wherein up to two non-adjacent methylene units are independently optionally replaced by CO, CO2, COCO, CONRzl, OCONR21, NRZ1NRZ2, NRzlNRz2CO,
NRzlCO, NRzlCO2, NRzlCONRZ2, SO, SO2, NRzlSO2, SO2NR ,zΛιl,
Figure imgf000043_0001
O, S, or NRzl; wherein each occurrence of RZI and RZ2 is independently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl. In certain embodiments, each occurrence of the structural unit SU is an amino acid residue, and the clustered multi-antigenic construct has the structure:
Figure imgf000043_0002
wherein q is 0 or 1 ; each occurrence of s is independently an integer from 2-20; t is an integer from 1-6;
RX1 is hydrogen, alkyl, acyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl) or a nitrogen protecting group;
R is hydrogen or an immunogenic canier; each occurrence of the spacer is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; the linker is either a free carboxylic acid, -O-, (carboxamido)alkyl carboxamide, MBS, primary carboxamide, mono- or dialkyl carboxamide, mono- or diarylcarboxamide, linear or branched chain (carboxy)alkyl carboxamide, linear or branched chain (alkoxycarbonyl)alkyl-carboxamide, linear or branched chain (carboxy)arylalkylcarboxamide, linear or branched chain
(alkoxycarbonyl)alkylcarboxamide, an oligoester fragment comprising from 2 to about 20 hydroxy acyl residues, a peptidic fragment comprising from 2 to about 20 amino acyl residues, or a linear or branched chain alkyl or aryl carboxylic ester; each occurrence of L1 is independently a substituted or unsubstituted aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydrate domain of formula (Idet), (Hdet) or (IIIdet).
[0100] In certain other embodiments, for the two clustered multi-antigenic constructs described directly above, t is > 2 and within each bracketed structure s, independently, each occurrence of A is the same. In certain embodiments, occurrences of A from one bracketed structure s to the next may be the same or different. In certain embodiments, occurrences of A from one bracketed structure s to the next are different. In certain other embodiments, each occurrence of A is independently O or N-linked to the construct backbone. In certain other embodiments, each occurrence of A is independently α- or β-linked to the construct backbone. [0101] In certain embodiments, for the clustered multi-antigenic construct γ| described directly above, R is an acyl moiety. In certain exemplary embodiments,
R is an amino acid residue.
[0102] In certain embodiments, for the two clustered multi-antigenic constructs described directly above, the spacer, for each occurrence, is independently a substituted or unsubstituted Cι-6alkylidene or C2-6alkenylidene chain wherein up to two non-adjacent methylene units are independently optionally replaced by CO, CO2, COCO, CONRzl, OCONR21, NRZ1NRZ2, NRzlNRZ2CO, NRzlCO, NRzlCO2, NRzlCONRZ2, SO, SO2, NRzlSO2, SO2NRzl, NRzlSO2NRZ2, O, S, or NRZ1; wherein each occurrence of RZ1 and RZ2 is independently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl; a peptidyl moiety or a bivalent aryl or heteroaryl moiety. In certain exemplary embodiments, the spacer, for each occurrence, is independently -(CHRsp)n-, where n is 1-8 and each occurrence of Rsp is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, -alkyl(aryl), - alkyl(heteroaryl), -ORspl, -SRspl or -NRsplRsp2 where Rspl and Rspl are independently hydrogen or lower alkyl; a peptidyl moiety comprising one or more α-amino acid residues, or a bivalent aryl moiety having the structure:
Figure imgf000045_0001
In certain exemplary embodiments, each occurrence of the spacer is independently a dipeptidyl moiety.
[0103] In certain other embodiments, for the clustered multi-antigenic constructs described directly above, each occurrence of L1 is independently a natural amino acid side chain. In certain embodiments, each occunence of L1 is independently an unnatural amino acid side chain. In certain embodiments, each occunence of L1 is independently -O(CHRaa)n- or -NHC(=O)(CHRaa)n- wherein each occunence of n is independently an integer from 1-10; and each occunence of Raa is hydrogen, lower alkyl, aryl, heteroaryl, -alkyl(aryl) or -alkyl(heteroaryl). In certain exemplary embodiments, each occunence of n is 1 and each occunence of Raa is hydrogen or methyl. In certain embodiments, each occunence of L1 is independently a moiety having the structure -O(CH )n- wherein n is an integer from 1-10 and each occunence of A is O-linked to the construct backbone. In certain embodiments, each occunence of L1 is independently a moiety having the structure -NHC(=O)(CH2)n- wherein n is an integer from 1-10 and each occunence of A is N- linked to the construct backbone.
[0104] In certain embodiments, the clustered multi-antigenic constructs described directly above have the following structure:
Figure imgf000046_0001
X = < 1 LINKER H CROSSLINKER jj — F
where the peptide backbone may be linear, as shown, above, or cyclic (e.g., the two occurrences of Rsp at the N- and C-termini, taken together, form a cyclic moiety); wherein L1 and Rsp are as defined above; si, s2 and s3 are independently integers from 2-5; Aj-A are independently a carbohydrate domain of formula (Idet), (Hdet) or (IIIdet), and are different from each other; and R^ is hydrogen, alkyl, acyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl) or a nitrogen protecting group. In certain exemplary embodiments, each occunence of L1 is independently a natural amino acid side chain. In certain embodiments, each occunence of L is independently an unnatural amino acid side chain. In certain other embodiments, each occunence of L1 is independently -NHC(=O)(CHRaa)n- and the glycopeptide has the structure:
Figure imgf000046_0002
wherein R, R 2, Rsp, si, s2 and s3 and A[-A3 are as defined above; each occunence of n is independently an integer from 1-10; and each occunence of Raa is hydrogen, lower alkyl, aryl, heteroaryl, -alkyl(aryl) or -alkyl(heteroaryl). In certain exemplary embodiments, each occunence of n is 1 and each occunence of Raa is hydrogen. In certain embodiments, each occunence of Rsp is independently a natural amino acid side chain. In certain exemplary embodiments, each occunence ofRsp is hydrogen.
[0105] In certain embodiments, the clustered multi-antigenic construct is attached to a suitable immunogenic carrier via a linker and the construct has the structure:
Figure imgf000047_0001
[0106] In certain embodiments, each occunence of SU is an amino acid residue and the clustered multi-antigenic construct a glycopeptide having the structure:
Figure imgf000047_0002
In certain embodiments, for the clusters glycopeptides described above and herein, R is a protein, peptide or lipid immunogenic canier.
[0107] In certain embodiments, for the clustered multi-antigenic constructs
1 "λ described above and herein, each occunence of A, A , A and A is independently a carbohydrate domain having one of the following structures:
Figure imgf000048_0001
[0108] In certain embodiments, for the clustered multi-antigenic constructs described above and herein, R is a protein, peptide or lipid immunogenic carrier. In certain other embodiments of the present invention, R is NHR", and the canier R'" is KLH or Bovine Serum Albumin. In still other embodiments of the present invention, R is NHR'", and the canier R'" is a lipid having the structure:
Figure imgf000048_0002
wherein m', n' and p' are each independently integers between about 8 and 20; and Ry is hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl. In certain exemplary embodiments, m', n' and p' are each 14 and the lipid is tripalmitoyl-S-glycerylcysteinylserine (e.g., PamCys). It will be appreciated that the protein or lipid can be linked to N or the rest of the construct either directly or through a crosslinker and thus R'" incorporates proteins, peptides and lipids, as well as (crosslinker-protein), (crosslinker-peptide) and (crosslinker-lipid) moieties. In certain prefened embodiments, the crosslinker is MMCCH (4-(maleimidomethyl) cyclohexane-1 -carboxyl hydrazide). [0109] In certain embodiments, the invention encompasses multi-antigenic constructs having the structure:
Figure imgf000049_0001
wherein the linker is -O-, -NRG-, -NRG(CRHRj) NRκ-,
Figure imgf000049_0002
-(CRHRj)kNRK-, -O(CRHRj)kNRK-, an oligoester fragment comprising from 2 to about 20 hydroxy acyl residues, a peptidic fragment comprising from 2 to about 20 amino acyl residues, or a linear or branched chain alkyl or aryl carboxylic ester, wherein each occunence of k is independently 1-5; wherein each occurrence of RQ, RH> RJ or RK is independently hydrogen, a linear or branched, substituted or unsubstituted, cyclic or acyclic alkyl moiety, or a substituted or unsubstituted aryl moiety; wherein the crosslinker is a moiety derived from a crosslinking reagent capable of conjugating the canier with the linker; wherein the canier is a peptide, protein or lipid; wherein n is 1, 2, 3 or 4; wherein q is 0 or 1 ; wherein each occunence of RA, RB and Re is independently hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl; and wherein each occunence of RQ, RE and RF are each independently a carbohydrate domain of formula (Idet), (IIdet) or (IHdet).
[0110] In certain embodiments, for the multi-antigenic constructs described herein, the linker is -O-, -NRG-, -NRo(aliphatic)NRj-, -NRG(heteroaliphatic)NRj-, - (aliphatic)NRj-, -(heteroaliphatic)NRj-, -O(aliphatic)NRj-, -O(heteroaliphatic)NRj-, -NRG(aliphatic)NRj(C=O)(CRHR kS-, -NRG(heteroaliphatic)NRj(C=O)(CRHRι)kS-, -(aliphatic)NRj(C=O)(CRHRι)kS-, -(heteroaliphatic)NRj(C=O)(CRHRι)kS-, O(aliphatic)NRj(C=O)(CRHRι)kS-, -O(heteroaliphatic)NRj(C=O)(CRHRι)kS-, an oligoester fragment comprising from 2 to about 20 hydroxy acyl residues, a peptidic fragment comprising from 2 to about 20 amino acyl residues, or a linear or branched chain alkyl or aryl carboxylic ester, wherein each occunence of k is independently 1-5; wherein each occunence of RG, RH, RI or Rj is independently hydrogen, a linear or branched, substituted or unsubstituted, cyclic or acyclic moiety, or a substituted or unsubstituted aryl moiety, and wherein each aliphatic or heteroaliphatic moiety is independently substituted or unsubstituted, linear or branched, cyclic or acyclic. [0111] In certain embodiments, for the multi-antigenic constructs described herein, the linker is -O-, -NRG(CRHRι)kNRj-, -NRG(CRHRι)kNRj(C=O)(CRHRι)kS-, - NRG-, -(CRHRJ NRΓ, -O(CRH ι)kNRj, an oligoester fragment comprising from 2 to about 20 hydroxy acyl residues, a peptidic fragment comprising from 2 to about 20 amino acyl residues, or a linear or branched chain alkyl or aryl carboxylic ester, wherein each occunence of k is independently 1-5, wherein each occunence of RG, RH, RI or Rj is independently hydrogen, a linear or branched, substituted or unsubstituted, cyclic or acyclic moiety, or a substituted or unsubstituted aryl moiety. [0112] In certain embodiments, for the multi-antigenic constructs described herein, the linker is a moiety having the structure -NH(CH )tNHC(=O)(CH )vS- wherein t and v are each integers from 1-6. In certain exemplary embodiments, t is 3 and v is 1.
[0113] In certain embodiments, for the multi-antigenic constructs described above, the carrier is a protein, peptide or lipid immunogenic canier. In certain other embodiments of the present invention, the carrier is NHR'", and R'" is KLH or Bovine Serum Albumin. In still other embodiments of the present invention, the canier is NHR'", and R'" is a lipid having the structure:
Figure imgf000051_0001
wherein m', n' and p' are each independently integers between about 8 and 20; and Ry is hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl. In certain exemplary embodiments, m', n' and p' are each 14 and the lipid is tripalmitoyl-S-glycerylcysteinylserine (e.g., PamCys). It will be appreciated that the protein or lipid can be linked to N or the rest of the construct either directly or through a crosslinker and thus R'" incorporates proteins, peptides and lipids, as well as (crosslinker-protein), (crosslinker-peptide) and (crosslinker-lipid) moieties. In certain prefened embodiments, the crosslinker is MMCCH (4-(maleimidomethyl) cyclohexane-1 -carboxyl hydrazide). [0114] Crosslinkers suited to the invention are widely known in the art (see, for example, Appendix A: 1994 Pierce Technical Handbook: cross-linking, also available at www.piercenet.com resources/browse.cfm?fldID=184), including bromoacetic NHS ester, 6-(iodoacetamido)caproic acid NHS ester, maleimidoacetic acid NHS ester, maleimidobenzoic acide NHS ester, etc. In certain prefened embodiments, the crosslinker is MMCCH (4-(maleimidomethyl) cyclohexane-1- carboxyl hydrazide). In certain other prefened embodiments, the crosslinker is MBS (m-maleimidobenzoyl acid N-Hydroxysuccinimidyl ester). [0115] In certain embodiments, for the multi-antigenic constructs described herein, q is 1 and the crosslinker is a fragment having the structure:
Figure imgf000051_0002
[0116] whereby said structure is generated upon conjugation of a maleimidobenzoic acid N-hydroxy succinimide ester with a linker. [0117] In certain other embodiments, the invention encompasses multi- antigenic constructs having the structure:
Figure imgf000052_0001
wherein m', n' and p' are integers between about 8 and 20; j is an integer between 1 and about 8;
Ry, RA, RB and Re are independently hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl; and
RD, RE and RF are each independently a carbohydrate domain of formula (Idet), (IIdet) or (IIIdet). In certain exemplary embodiments, j is 3. [0118] In certain other embodiments, the invention encompasses multi- antigenic constructs having the structure:
Figure imgf000052_0002
wherein n and p are each independently an integer from 1-6; m', n' and p' are independently integers between about 8 and 20; j is an integer between 1 and about 8;
R is a nitrogen protecting group;
Ry, and RA, RB, RC, RE and RF, for each occunence, are independently hydrogen, substituted or unsubstituted linear or branched lower alkyl or substituted or unsubstituted phenyl; each occunence of RD is independently a carbohydrate domain of formula (Idet), (IIdet) or (IIIdet). In certain exemplary embodiments, j is 3. [0119] In certain embodiments, for the clustered multi-antigenic constructs described above and herein, each occunence of RD, RE and RF is independently a carbohydrate domain having one of the following structures:
Figure imgf000053_0001
[0120] In certain embodiments, the invention provides glycopeptides comprising one occunence of a carbohydrate domain of the formula (Idet), (IIdet) or (IIIdet), whereby the glycopeptide structure allows for dimerization. In certain exemplary embodiments, the glycopeptide comprises one cysteine residue and the glycopeptide has the structure:
Figure imgf000054_0001
wherein L1 is as defined above; A is a carbohydrate domain of the formula (Idet), (IIdet) or (IIIdet); Rx is hydrogen or a thiol protecting group.
[0121] The skilled practitioner will know how to select a thiol protecting group to allow protection/deprotection of the cysteine thiol without negatively affecting other protecting groups that might be present on the construct (e.g., on carbohydrate A). Guidance can be found, for example, in "Protective Groups in Organic Synthesis" , Chapter 6, Third Ed. Greene, T.W. and Wuts, P.G., Eds., John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference. In certain exemplary embodiments, Rx is-StBu. [0122] In certain embodiments, inventive constructs comprising one or more carbohydrate domains of the formula (Idet), (Hdet) or (HIdet) are dimers of the above glycopeptides, and the constructs have the structure:
Figure imgf000054_0002
wherein each peptide may be the same or different; and each occunence of A is independently a carbohydrate domain of the formula (Idet), (IIdet) or (IIIdet). [0123] In certain other embodiments, for the two glycopeptidic constructs described above, each occunence of L1 is independently a natural amino acid side chain. In certain embodiments, each occunence of L is independently an unnatural amino acid side chain. In certain embodiments, each occunence of L1 is independently -O CHR33),,- or -NHC(=O)(CHRaa)n- wherein each occunence of n is independently an integer from 1-10; and each occunence of Raa is hydrogen, lower alkyl, aryl, heteroaryl, -alkyl(aryl) or -alkyl(heteroaryl). In certain exemplary embodiments, each occunence of n is 1 and each occunence of R33 is hydrogen or methyl. In certain embodiments, each occunence of L1 is independently a moiety having the structure -O(CH2)n- wherein n is an integer from 1-10 and each occunence of A is O-linked to the construct backbone. In certain embodiments, each occunence of L1 is independently a moiety having the structure - NHC(=O)(CH2)n- wherein n is an integer from 1-10 and each occunence of A is N- linked to the construct backbone. In certain embodiments, each occunence of L1 is an aspartyl side chain.
[0124] In certain embodiments, for the two glycopeptidic constructs described above, the peptide has a structure that is either identical or closely related to that of gpl20 near an N-glycosylation site. In certain embodiments, for the two glycopeptidic constructs described above, the peptide comprises the amino acid sequence: Cys-Asn-Ile-Ser-Arg, wherein any one or more of the amino acid residues may bear one or more protecting groups. In certain embodiments, for the two glycopeptidic constructs described above, the peptide comprises the amino acid sequence: Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His-Cys- Asn-Ile-Ser-Arg, wherein any one or more of the amino acid residues may bear one or more protecting groups.
[0125] In certain embodiments, the invention provides dimeric constructs having the structure:
Figure imgf000055_0001
wherein each occunence of A is independently a carbohydrate domain having one of the structures:
Figure imgf000056_0001
[0126] In certain embodiments, dimeric constructs having the following structure are provided:
Figure imgf000056_0002
wherein A is as defined above. [0127] 2) Synthetic Methodology
[0128] The practitioner has a a well-established literature of carbohydrate chemistry to draw upon, in combination with the information contained herein, for guidance on synthetic strategies, protecting groups, and other materials and methods useful for the synthesis of the compounds of this invention, and conjugates thereof. [0129] The various patent documents and other references cited herein provide helpful background information on preparing compounds similar to the inventive compounds described herein or relevant intermediates, as well as information on formulation, uses, and administration of such compounds which may be of interest.
[0130] Moreover, the practitioner is directed to the specific guidance and examples provided in this document relating to various exemplary compounds and intermediates thereof.
[0131] In one aspect of the invention, there is provided a method for preparing isolated an compound of formula (I):
Figure imgf000057_0001
(I)
[0132] wherein R1, R2A, R2B, R3, R4 and W'-W3 are as defined generally above and in classes and subclasses herein.
[0133] In one aspect of the invention, there is provided a method for preparing an isolated compound of formula (II):
Figure imgf000058_0001
(H) wherein R , R , R , R and R are as defined generally above and in classes and subclasses herein.
[0134] In another aspect of the invention, there is provided a method for preparing isolated an compound of formula (III):
Figure imgf000058_0002
(III)
[0135] wherein X, R , 1', r R>2AΛ, r R»2/BB, π R3J and R are as defined generally above and in classes and subclasses herein.
[0136] In certain exemplary embodiments, R4 is -NHR4A; wherein R4A is an amino acyl residue of a peptide and the invention provides a method for preparing homogeneous N-linked gpl20-derived glycopeptides.
[0137] Glycan Synthesis [0138] Glycan synthesis generally suffers from the stereochemical diversity of its targets and therefore of its building blocks, as well. The advent of a new target often requires a reworked, if not entirely different synthetic plan, based on varying protecting groups, coupling strategies, and starting materials. The present invention provides a method allowing access to a number of gpl20-derived saccharides using only a small set of building blocks and the same general procedure for each glycan. [0139] In certain embodiments, trisaccharide 3 in Scheme 1 embodies the protected core structure reported for the glycoforms expressed in gpl20. [0140] Scheme 1. Proposed methodology for glycan synthesis.
Figure imgf000059_0001
[0141] In certain exemplary embodiments, trisaccharide 3 may be elaborated to give a pentasaccharide either by deprotection of the 6-position followed by simultaneous α-mannosylation at the free 3- and 6-positions or by sequential mannosylation at the 3-and 6-positions with an intermediate deprotection step. Simultaneous mannosylation with equivalently protected mannosyl donors would yield a "symmetrically" substituted pentasaccharide; further deprotections and glycosylations could be achieved in a synchronous fashion at both nonreducing termini. Sequential mannosylation would allow the inclusion of differentially protected mannose building blocks, permitting independent elaboration of the 3- and 6-substituted antennae. Thus the high-mannose pentasaccharide core (which is conserved in most natural N-linked glycans) may be synthesized in large quantities and used as a starting point for all of the gpl20 targets. Moreover, because hybrid- type gp!20 differs from high-mannose type gpl20 in its degree of branching beyond the core pentasaccharide, this synthetic scheme would provide easy access to the multi-antennary glycoforms expressed in gpl20.
[0142] In certain embodiments, the synthetic approach includes: synthesis of protected oligasaccaride (undecassaccharide), global deprotection to prepare free glycan, animation, coupling with peptide acid and deprotection (Scheme 2). [0143] Scheme 2. Exemplary synthetic strategy
gi> SSt«Stt-OH m,nat,on.
Figure imgf000060_0001
OH
Global Deprotection Amide Formation Deprotection
Bolypeptide
Protected" OligosaccharidH tpiigosaccharid — NH
Chemical Synthesis £&b£B I-l- -d
Figure imgf000060_0002
[0144] In certain embodiments, a synthesis for the high-mannose type glycopeptide having the structure:
Figure imgf000060_0003
is provided. In certain embodiments, a synthetic plan for the preparation of the undecasaccharide is shown in Scheme 3. For example, starting from a trisaccharide intermediate (e.g., trisacchraide 3),1 two successive glycosylations will give pentasaccharide, then two consecutive triple glycosylation would furnish the undecasaccharide . [0145] Scheme 3. Exemplary retrosynthesis of undesaccharide 1.
Figure imgf000061_0001
[0146] An exemplary synthesis using this route is shown in Scheme 4. For example, mannosylation of trisaccharide 3 using thiomannoside 4 and Sinay radical
9 "\ cation activation ' gave tetresaccharide in 78% yield. The benzylidene ring was reductively opened by borane and the resulting free alcohol 5 underwent mannosylation to give pentasaccharide 8 in 74% yield. After Zemplen reaction, the newly generated three free OH were mannosylated to afford octasaccharide 10 using
9 ~ same Sinay conditions ' . The same triple-glycosylation sequence was repeated to synthesize the undecsaccharide 12a in 55% yield (Scheme 5). [0147] Scheme 4
Figure imgf000061_0002
[0148] Scheme 5
Figure imgf000062_0001
[0149] For example, as shown in Scheme 6, the desired undecasaccharide could be synthesized by a 3+3 glycosylation (trisaccharide couples with another trisaccharide) followed by a 6+5 coupling. This synthetic plan is much shorter and more convergent than the first strategy. [0150] Scheme 6
Figure imgf000062_0002
[0151] In certain embodiments, as shown in scheme 7, trisaccharide 3 first underwent glycosylation with trisaccharide donor 13 using MeOTf as promoter to afford hexasaccharide in 70% yield. Then reductive ring-opening of the benzylidene ring gave saccharide 15 in 87% yield. [0152] Scheme 7
Figure imgf000063_0001
[0153] In certain embodiments, the pentasaccharide which is the precursor for the upper-left portion of the final compound (1) was synthesized as shown in scheme 8. For example, double-glycosylation of mannose derivative 16 using chloro donor 17 and promoter silver triflate gave trisaccharide 18. After cleavage of the two acetyl groups, another double-glycosylation provided pentasaccharide 20 in 87% yield.
[0154] Scheme 8
Figure imgf000064_0001
[0155] In certain embodiments, the 6+5 glycosylation using Sinay radical cation activation2'3 proceeded smoothly giving the desired undecasaccharide 12b in 85% yield (Scheme 9). [0156] Scheme 9
Figure imgf000064_0002
[0157] In certain embodiments, protected undecasaccharide 12b was treated with sodium methoxide and HF-pyridine to remove the acetyl groups and TBS group, respectively. The resulting oligosaccharide 21 was then subjected to global Birch deprotection followed by selective acetylation using acetyl anhydride in saturated sodium bicarbonate solution to give free glycan in high yield.5 Following Kochetkov amination6 furnished free glycosylamine (Scheme 10). [0158] Scheme 10
Figure imgf000065_0001
[0159] In certain embodiments, 20-mer peptide acid 34, which was made through applied biosynthesis synthesizer, was activate using HATU and coupled directly with glycosylamine 23. The Fmoc and ivDde protecting groups were removed by treatment with hydrazine and piperidine to give glycopeptide fragment 25 in 16% two steps yield (Scheme 11). [0160] Scheme 11
Fmoc-A-F-V-T-I-G-K-I-G-N-M-R-Q-A-H-C-D-I-S-R-NH,
I
IvDde
34
OH
1) HATU, DIPEA, 23, DMSO, 7 h
2) Piperidine, NH2NH2, DMF
16% for two steps
Figure imgf000065_0002
25 [0161] Methods of preparing trisaccharide 3 are known in the art. For example, guidance may be found in U.S. Provisional Patent Application No.:
60/500,161 filed September 4, 2003; and International Application No.: PCT US03/
, filed December 3, 2003 entitled "Prostate Specific Antigens, Conjugates Thereof,
Methods for their Preparation and Uses Thereof; the entire contents of each of the above applications are hereby incorporated by reference herein.
[0162] References ("Glycan synthesis" section)
[0163] 1. Dudkin, V. Y.; Miller, J. S.; Danishefsky, S. J. Tetrahedron Letters
2003, 44, 1791-1793.
[0164] 2. Zhang, Y.-M.; Mallet, J.-M.; Sinay, P. Carbohydrate Research
1992, 236, 73-88.
[0165] 3. Marra, A.; Mallet, J. M.; Amatore, C; Sinay, P. Synlett 1990, 572-
574.
[0166] 4. Matsuo, I.; Wada, M.; Manabe, S.; Yamaguchi, Y.; Otake, K.;
Kato, K.; Ito, Y. Journal of the American Chemical Society 2003, 125, 3402-3403.
[0167] 5. Calarese, D. A.; Scanlan, C. N.; Zwick, M. B.; Deechongkit, S.;
Mimura, Y.; Kunert, R.; Zhu, P.; Wormald, M. R.; Stanfield, R. L.; Roux, K. H.;
Kelly, J. W.; Rudd, P. M.; Dwek, R. A.; Katinger, H.; Burton, D. R.; Wilson, I. A.
Science (Washington, DC, United States) 2003, 300, 2065-2071.
[0168] 6. Likhosherstov, L. M.; Novikova, O. S.; Derevitskaya, V. A.;
Kochetkov, N. K. Carbohydrate Research 1986, 146, C1-C5.
[0169] Glycopeptides
[0170] Automated peptide synthesis is reliable for sequences up to about 60 amino acid residues in length, but saccharide moieties contained in glycopeptides render their solid phase synthesis less practical. Unlike peptide synthesis, complex glycan and glycoconjugate synthesis remains readily accessible only to a few select laboratories (See, for example, Hang, H. C; Bertozzi, C. R. "Chemoselective approaches to glycoprotein assembly." Ace. Chem. Res. 2001, 34, 727-736). Syntheses of several natural O-linked glycopeptides containing simple glycans have been reported (See, for example, (1) Arsequell, G.; Haurum, J. S.; Elliott, T.; Dwek, R. A.; Lellouch, A. C. "Synthesis of Major Histocompatibility Complex Class-I Binding Glycopeptides." J Chem. Soc.-Perkin Trans. 1 1995, 1739-1745, (2) Chen, X. T.; Sanies, D.; Danishefsky, S. J. "Exploration of modalities in building alpha-O- linked systems through glycal assembly: A total synthesis of the mucin-related Fl alpha antigen." J. Am. Chem. Soc. 1998, 120, 7760-7769; (3) Macmillan, D.; Bertozzi, C. R. "New directions in glycoprotein engineering." Tetrahedron 2000, 56, 9515-9525; (4) Koeller, K. M.; Smith, M. E. B.; Huang, R. F.; Wong, C. H. "Chemoenzymatic synthesis of a PSGL- 1 N-terminal glycopeptide containing tyrosine sulfate and alpha-O-linked sialyl Lewis X." J Am. Chem. Soc. 2000, 122, 4241-4242; (5) Ajisaka, K.; Miyasato, M.; Ishii-Karakasa, I. "Efficient synthesis of O-linked glycopeptide by a transglycosylation using endo alpha-N- acetylgalactosaminidase from Streptomyces sp." Biosci.
Biotechnol Biochem. 2001, 65, 1240-1243; and (6) Marcaurelle, L. A.; Mizoue, L. S.; Wilken, J.; Oldham, L.; Kent, S. B. H.; Handel, T. M.; Bertozzi, C. R. "Chemical synthesis of lymphotactin: A glycosylated chemokine with a C-terminal mucin-like domain." Chem. Eur. J. 2001, 7, 1129-1132), as have examples of mimetics for N- linked glycopeptides (See, for example, Hang, H. C; Bertozzi, C. R. "Chemoselective approaches to glycoprotein assembly." Ace. Chem. Res. 2001, 34, 727-736), and a chemoenzymatic synthesis of an N-linked glycopeptide (See, for example, Inazu, T.; Haneda, K.; Mizuno, M. "Synthetic study on Ν-glycopeptides." J Syn. Org. Chem. Jpn. 1998, 56, 210-220), but no chemical synthesis has been reported for a natural N-linked glycopeptide with complex glycan and peptide structure. The state of the art for chemically synthesized N-linked glycopeptides is exemplified by the pentadecasaccharide N-linked to a pentapeptide reported by Wang and coworkers, which was recognized by appropriate antibodies to the H-type blood group antigens present at the glycan nonreducing termini (See, for example, Wang, Z. G.; Zhang, X. F.; Visser, M.; Live, D.; Zatorski, A.; Iserloh, U.; Lloyd, K. O.; Danishefsky, S. j. "Toward fully synthetic homogeneous glycoproteins: A high mannose core containing glycopeptide carrying full H-type2 human flood group specificity." Angew. Chem. Int. Ed. 2001, 40, 1728-1732).
[0171] Scheme 12. Exemplary synthetic approach for the preparation of gpl20 glycopeptides.
Figure imgf000068_0001
[0172] In certain embodiments, as shown in Scheme 12, the chemical synthesis of inventive glycopeptides may be divided logically into two sections: glycan synthesis (top) and glycopeptide assembly (bottom). At its core, the inventive method would extend the method of Wang, et al. (Wang, Z. G.; Zhang, X. F.; Visser, M.; Live, D.; Zatorski, A.; Iserloh, U.; Lloyd, K. O.; Danishefsky, S. J. "Toward fully synthetic homogeneous glycoproteins: A high mannose core containing glycopeptide carrying full H-type2 human flood group specificity." Angew. Chem. Int. Ed. 2001, 40, 1728-1732) to include one or more peptide elongation steps after synthesis of a short glycopeptide, allowing entry into the realm of fully elaborated, naturally derived glycoproteins (See, for example, Dawson, P. E.; Kent, S. B. H. "Synthesis of native proteins by chemical ligation." Annu. Rev. Biochem. 2000, 69, 923-960). In an inventive and important improvement, the glycan is fashioned here in a more convergent manner than previously realized, allowing the strategy to be adjusted in its late stage to accommodate the synthesis of various glycoforms, as illustrated in the next section. [0173] Glycopeptide Assembly
[0174] Guidance for glycopeptide assembly may be found, inter alia, in U.S.
Provisional Patent Application No.: 60/500,161 entitled "Prostate Specific Antigens, Conjugates Thereof, Methods for their Preparation and Uses Thereof, filed September 4, 2003; the entire contents of which are hereby incorporated by reference herein. For example, a glycopeptide assembly strategy, as outlined in Scheme 12, involves peptide glycosylation followed by elongation of the peptide backbone, was examined, as illustrated in Scheme 13, using a model peptide and glycan (Miller, J. S. et al, Angew. Chemie Int. Ed., 2003, 42, 431). To prepare free glycan 38 for coupling, its anomeric hydroxyl was first aminated to give β- aminoglycoside 39 as described by Kochetkov (See, for example, Likhosherstov, L. M.; Novikova, O. S.; Derevitskaja, V. A.; Kochetkov, N. K. "A New Simple Synthesis of Amino Sugar Beta-D-Glycosylamines." Carbohydr. Res. 1986, 146, C1-C5). Glycosylamine 39 and the aspartate free acid of peptide 40 were coupled in peptidic fashion according to the procedure of Lansbury and coworkers ((1) Cohen- Anisfeld, S. T.; Lansbury, P. T. "A Practical, Convergent Method for Glycopeptide Synthesis." J. Am. Chem. Soc. 1993, 115, 10531-10537; and (2) Anisfeld, S. T.; Lansbury, P. T. "A Convergent Approach to the Chemical Synthesis of Asparagine- Linked Glycopeptides." J Org. Chem. 1990, 55, 5560-5562) with certain modifications: the reported peptide glycosylations involved excess or equimolar amounts of glycosylamine relative to peptide, and their isolated yields (50 - 60%) are reported based on peptide starting material (Cohen-Anisfeld, S. T.; Lansbury, P. T. "A Practical, Convergent Method for Glycopeptide Synthesis." J. Am. Chem. Soc. 1993, 115, 10531-10537). As is often the case, however, the saccharide here is the more precious material entering glycosylation because its preparation involves multistep, solution phase synthesis in relatively low overall yield compared to that of the peptide. A trial glycosylation of model pentapeptide 40 with pentasaccharide 39 indicates that under the appropriate reaction conditions, an excess of peptide produces a significantly greater yield of coupled product (over 70% based on valuable glycosylamine) [Miller, J. S. et al, Angew. Chemie Int. Ed., 2003, 42, 431. Subsequent Fmoc (Fmoc = 9-fluorenylmethyloxy-carbonyl) removal with piperidine afforded glycopeptide 41.
[0175] Scheme 13. Exemplary glycopeptide assembly route with a model peptide and glycan. Manα
Manα
Figure imgf000070_0001
43
[0176] The final step toward completion of a model glycopeptide involved native chemical ligation (NCL) [See, for example, Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. "Synthesis of Proteins by Native Chemical Ligation." Science 1994, 266, 776-779], as indicated in Scheme 13. In situ deprotection of cysteine disulfide 41 and transthioesterification (See, for example, Dawson, P. E.; Churchill, M. J.; Ghadiri, M. R.; Kent, S. B. H. "Modulation of reactivity in native chemical ligation through the use of thiol additives." J. Am. Chem. Soc. 1997, 119, 4325-4329) of peptide thioester 42 with sodium 2-mercaptoethanesulfonate (43) in phosphate-buffered saline (PBS) at neutral pH led to a second thioester exchange with the (now free) cysteine thiol and subsequent reanangement to give fully unprotected glycopeptide 44. gpl20-derived glycopeptides obtained using the strategy detailed in Scheme 13 will require no additional manipulation other than purification before they can be examined for the generation of antibodies. The synthetic strategy thus requires only four assembly steps starting from free glycans to obtain homogeneous glycopeptides.
[0177] In certain embodiments, the lysine residue is differentially protected with respect to Fmoc removal during peptide synthesis, and remains protected through the peptide glycosylation step (due to its free amine side chain). Suitably protected Lys derivatives have been designed (See, for example, Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft, B. W.; Chan, W. C. "An appraisal of new variants of Dde amine protecting group for solid phase peptide synthesis." Tetrahedron Lett. 1998, 39, 1603-1606), and can be deprotected in the presence of N-linked saccharides along with the Ν-terminal Fmoc amine in minutes using hydrazine at room temperature. [0178] Peptide Thioester Synthesis [0179] Several methods have been developed for peptide thioester synthesis, including the original "Boc chemistry" (Boc = tert-butyloxycarbonyl) method (See, for example, (1) Canne, L. E.; Walker, S. M.; Kent, S. B. H. "A General Method for the Synthesis of Thioester Resin Linkers for Use in the Solid-Phase Synthesis of Peptide Alpha-Thioacids." Tetrahedron Lett. 1995, 36, 1217-1220; and (2) Hojo, H.; Aimoto, S. "Polypeptide Synthesis Using the S-Alkyl Thioester of a Partially Protected Peptide Segment—Synthesis of the DNA- Binding Domain of C-Myb Protein ( 142-193 )-NH2." Bull Chem. Soc. Jpn. 1991, 64, 111-117) and several Fmoc-compatible systems (See, for example, (1) Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B. H; Ellman, J. A.; Bertozzi, C. R. "Fmoc-based synthesis of peptide-(alpha)thioesters: Application to the total chemical synthesis of a glycoprotein by native chemical ligation." J. Am. Chem. Soc. 1999, 121, 11684- 11689; (2) Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. "Solid phase synthesis of peptide C-terminal thioesters by Fmoc/t-Bu chemistry." J. Am. Chem. Soc. 1999, 121, 11369-11374; (3) Li, X. Q.; Kawakami, T.; Aimoto, S. "Direct preparation of peptide thioesters using an Fmoc solidphase method." Tetrahedron Lett. 1998, 39, 8669-8672; (4) Clippingdale, A. B.; Banow, C. J.; Wade, J. D. "Peptide thioester preparation by Fmoc solid phase peptide synthesis for use in native chemical ligation." J. Pept. Sci. 2000, 6, 225-234; and (5) Bu, X. Z.; Xie, G. Y.; Law, C. W.; Guo, Z. H. "An improved deblocking agent for direct Fmoc solidphase synthesis of peptide thioesters." Tetrahedron Lett. 2002, 43, 2419-2422). In ceratin embodiments, the model thioester is a C-terminal glycine thioester, which is locally achiral and cannot be epimerized, and is therefore easy to synthesize. Though the desired gpl20 thioester contains an epimerization-prone C-terminal histidine (His) residue, such thioesters have been synthesized previously and have in fact been shown to modulate favorably the rate of NCL (See, for example, Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology." Proc. Natl Acad. Sci. U. S. A. 1999, 96, 10068-10073).
[0180] In another aspect of the present invention, a method of preparing an isolated compound having the structure:
Figure imgf000072_0001
wherein each occunence of R is independently hydrogen or an oxygen protecting group; eeaacchh ooccccunence of R2A and R2B is independently hydrogen or a nitrogen protecting group; each occunence of R3 is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000072_0002
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occunence of R5, R6 and R7 is independently hydrogen, OH, OR1, NR'^R"', NHCOR', F, CH2OH, CH OR', or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occunence of R1, R" and R1" is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COORlv, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R1", taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occunence of R'v is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group;
W1, W2 and W3 are independently optionally substituted mannose, galactose or lactosamine moieties; said method comprising steps of:
(a) providing an α-O-protected carbohydrate construct having the structure:
Figure imgf000073_0001
wherein R > 4A . is hydrogen or a suitable oxygen protecting group; (b) reacting the construct of step (a) under suitable conditions to form a β- amino carbohydrate construct having the structure:
Figure imgf000073_0002
(c) reacting said β-amino carbohydrate construct under suitable conditions with a peptide whose structure is either identical or closely related to that of gpl20 near an N-glycosylation site and which comprises a -CH2CO2H moiety, to form a glycopeptide having the structure:
Figure imgf000074_0001
[0181] In certain embodiments, the peptide comprises a cysteine residue and thus, the peptide may be dimerized under suitable oxidization conditions to form the conesponding disulfide dimer. In certain exemplary embodiments, the disulfide dimer has the structure:
Figure imgf000074_0002
wherein each peptide may be the same or different; each occunence of L1 may be the same or different and is as defined above; and each occunence of A is independently a carbohydrate domain as defined above.
[0182] In certain exemplary embodiments, in the step of reacting the carbohydrate construct of step (a) under suitable conditions to form the β-amino carbohydrate construct, Kochetkov animation conditions are used. In certain exemplary embodiments, in the step of reacting the carbohydrate construct of step (a) under suitable conditions to form the β-amino carbohydrate construct, NH4HCO3/H2O is used. In certain exemplary embodiments, in the β-amino carbohydrate construct of step (b), each occunence of R1 and R3 is hydrogen and each occunence of-NR2AR2B is -NHAc. [0183] In certain other exemplary embodiments, in the step of reacting the β- amino carbohydrate construct under suitable conditions with a peptide whose structure is either identical or closely related to that of gpl20 near an N- glycosylation site, the reaction conditions comprise HATU and Hϋnig's base in a suitable solvent. In certain embodiments, the solvent is DMSO. In certain embodiments, the peptide has the following structure:
Figure imgf000075_0001
[0184] In certain exemplary embodiments, in the β-amino carbohydrate construct formed in step (b), each occunence of R and R is hydrogen, each occunence of-NR2AR2B is -NHAc.
[0185] In certain other exemplary embodiments, the α-O-protected carbohydrate construct of step (a) has the structure:
Figure imgf000076_0001
[0186] In certain other exemplary embodiments, the glycopeptide formed in step (c) has the structure:
Figure imgf000076_0002
[0187] In certain other exemplary embodiments, the α-O-protected carbohydrate construct of step (a) has the structure:
Figure imgf000077_0001
[0188] In certain other exemplary embodiments, the glycopeptide formed in step (c) has the structure:
Figure imgf000077_0002
[0189] In certain other exemplary embodiments, the α-O-protected carbohydrate construct of step (a) has the structure:
Figure imgf000078_0001
[0190] In certain other exemplary embodiments, the glycopeptide formed in step (c) has the structure:
Figure imgf000078_0002
[0191] In certain other exemplary embodiments, the α-O-protected carbohydrate construct of step (a) has the structure:
Figure imgf000079_0001
; wherein MCA represent monochloroacetate.
[0192] In certain other exemplary embodiments, the glycopeptide formed in step (c) has the structure:
Figure imgf000079_0002
[0193] In certain other embodiments, the method further comprises a step of subjecting the glycopeptide formed in step (c) to Native Chemical Ligation conditions in the presence of a suitable polypeptide to form a glycopolypeptide having the structure:
Figure imgf000080_0001
[0194] In certain embodiments, the peptide is either identical to or closely related to that of gpl20 near an N-glycosylation site and comprises the amino acid sequence: Cys- Asn-lle-Ser-Arg wherein any one or more of the amino acid residues may bear one or more protecting groups. In certain exemplary embodiments, the carbohydrate construct is attached to an Asparagine residue (Asn) on the peptide via an amide linkage. In certain other exemplary embodiments, the peptide is either identical to or closely related to that of gpl20 near an N-glycosylation site and comprises the amino acid sequence:
Figure imgf000080_0002
[0195] In certain other embodiments, when the glycopeptide formed in step
(c) is further subjected to Native Chemical Ligation, the polypeptide comprises the amino acid sequence: Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-
His-Cys- Asn-lle-Ser-Arg, wherein any one or more of the amino acid residues may bear one or more protecting groups or a moiety suitable for Native Chemical
Ligation. In certain embodiments, the polypeptide comprises a moiety suitable for
Native Chemical Ligation, wherein the NCL moiety comprises a thioester.
[0196] The synthetic methodology is easily applicable to the generation of significantly longer (or shorter) segments of gpl20. Both the peptide to be glycosylated and the thioester utilized for NCL can more closely approach the -60 residue limit for linear synthesis; the resulting peptide can thus extend entirely to the N-terminus of gpl20. If the peptide to be glycosylated is extended significantly towards the C-terminus of gpl20 the glycosylation yield might suffer due to secondary structure formation of the longer peptide (See, for example, (1) Kent, S. B. H. "Chemical Synthesis of Peptides and Proteins." Annu. Rev. Biochem. 1988, 57, 957- 989; and (2) Tarn, J. P.; Lu, Y. A. "Coupling Difficulty Associated with Interchain Clustering and Phase- Transition in Solid-Phase Peptide-Synthesis." J. Am. Chem. Soc. 1995, 117, 12058-12063), but reaction conditions involving chaotropic salts have been devised to overcome issues of aggregation (See, for example, Thaler, A.; Seebach, D.; Cardinaux, F. "Lithium Salt Effects in Peptide Synthesis. 2. Improvement of Degree of Resin Swelling and of Efficiency of Coupling in Solid-Phase Synthesis." Helv. Chim. Ada 1991, 74, 628-643). [0197] In certain exemplary embodiments, the polypeptide has the structure:
Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His-SR; where R is a functional group suitable for effecting chemical ligation; and the resulting glycopeptide has the structure:
Figure imgf000081_0001
[0198] In certain embodiments, R, in the polypeptide used for native chemical ligation, is -(CH2)2C(-O)NH2.
[0199] In certain exemplary embodiments, the polypeptide has the structure:
Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His-SR; where R is a functional group suitable for effecting chemical ligation; and the resulting glycopeptide has the structure:
Figure imgf000082_0001
[0200] In certain embodiments, R, in the polypeptide used for native chemical ligation, is -(CH2)2C(=O)NH2.
[0201] In another aspect, the invention provides a method of preparing an α-
O-protected carbohydrate construct having the structure:
Figure imgf000082_0002
wherein each occunence of R is independently Bz or Ac; said method comprising steps of:
(a) coupling a trisaccharide having the structure:
Figure imgf000082_0003
with a monosaccharide having the structure:
Figure imgf000083_0001
wherein R10 is lower alkyl or aryl; in the presence of an activating agent under suitable conditions to form a protected tetrasaccharide having the structure:
Figure imgf000083_0002
(b) partially deprotecting the protected tetrasaccharide formed in step (a) under suitable conditions to form a partially deprotected tetrasaccharide having the structure:
Figure imgf000083_0003
(c) coupling the partially deprotected tetrasaccharide formed in step (b) with a monosaccharide having the structure:
Figure imgf000083_0004
wherein R10 is lower alkyl or aryl; in the presence of an activating agent under suitable conditions to form a protected pentasaccharide having the structure:
Figure imgf000084_0001
(d) partially deprotecting the pentasaccharide formed in step (c) under suitable conditions to form a partially deprotected pentasaccharide having the structure:
Figure imgf000084_0002
(e) coupling the partially deprotected pentasaccharide formed in step (d) with a monosaccharide having the structure:
Figure imgf000084_0003
wherein R10 is lower alkyl or aryl; in the presence of an activating agent under suitable conditions to form an octasaccharide having the structure:
Figure imgf000085_0001
and
(f) partially deprotecting the octasaccharide formed in step (e) under suitable conditions to form a partially deprotected octasaccharide having the structure:
Figure imgf000085_0002
(g) coupling the partially deprotected octasaccharide formed in step (f) with a monosaccharide having the structure:
Figure imgf000085_0003
in the presence of an activating agent under suitable conditions to the α-O- protected carbohydrate construct.
[0202] In certain exemplary embodiments, the activating agent used in cteps
(a), (c), (e) and (g) comprises (BrC6H4)3NSbCl6. In certain other exemplary embodiments, in the step of partially deprotecting the protected tetrasaccharide (step (b)), the protected tetrasaccharide formed in step (a) is subjected to reductive reaction conditions comprising Bu2BOTf, BH . In certain other exemplary embodiments, in the step of partially deprotecting the protected pentasaccharide (step (d)), the protected pentasaccharide formed in step (c) is subjected to reaction conditions comprising NaOMe. In certain other exemplary embodiments, in the step of partially deprotecting the protected octasaccharide (step (f)), the protected octasaccharide formed in step (e) is subjected to reaction conditions comprising
NaOMe.
[0203] In another aspect, the invention provides a method of preparing an α-
O-protected carbohydrate construct having the structure:
Figure imgf000086_0001
said method comprising steps of:
(a) coupling a trisaccharide having the structure:
Figure imgf000086_0002
with a monosaccharide having the structure:
Figure imgf000086_0003
wherein R10 is lower alkyl or aryl;
in the presence of an activating agent under suitable conditions to form a protected tetrasaccharide having the structure: 0
Figure imgf000087_0001
(b) partially deprotecting the protected tetrasaccharide formed in step (a) under suitable conditions to form a partially deprotected tetrasaccharide having the structure:
Figure imgf000087_0002
(c) coupling the partially deprotected tetrasaccharide formed in step (b) with an ethylthioglycoside having the structure:
Figure imgf000087_0003
under suitable conditions to form a protected hexasaccharide having the structure:
Figure imgf000087_0004
(d) partially deprotecting the hexasaccharide formed in step (c) under suitable conditions to form a partially deprotected hexasaccharide having the structure:
Figure imgf000088_0001
(e) coupling the partially deprotected hexasaccharide formed in step (d) with a monosaccharide having the structure:
Figure imgf000088_0002
wherein R10 is lower alkyl or aryl; in the presence of an activating agent under suitable conditions to form an heptasaccharide having the structure:
Figure imgf000088_0003
(f) partially deprotecting the heptasaccharide formed in step (e) under suitable conditions to form a partially deprotected heptasaccharide having the structure:
Figure imgf000088_0004
(g) coupling the partially deprotected heptasaccharide formed in step (f) with a monosaccharide having the structure:
Figure imgf000089_0001
in the presence of an activating agent under suitable conditions to the α-O- protected carbohydrate construct.
[0204] In certain embodiments, R10 is ethyl or phenyl.
[0205] In certain exemplary embodiments, the activating agent used in steps
(a), (e) and (g) comprises (BrC H4)3NSbCl6. In certain other exemplary embodiments, in the step of partially deprotecting the protected hexasaccharide (step (d)), the protected hexasaccharide formed in step (c) is subjected to reductive reaction conditions comprising Bu2BOTf, BH3. In certain other exemplary embodiments, in the step of partially deprotecting the protected tetrasaccharide (step (b)), the protected tetrasaccharide formed in step (a) is subjected to reaction conditions comprising NaOMe. In certain other exemplary embodiments, in the step of partially deprotecting the protected heptasaccharide (step (f)), the protected heptasaccharide formed in step (e) is subjected to reaction conditions comprising NaOMe.
[0206] It will be appreciated that for each of the methods as detailed herein, the full arsenal of protecting groups known in the art of organic synthesis can be utilized, for example, as set forth in "Activating Agents and Protecting Groups: Handbook of Reagents for Organic Synthesis" Roush, W.R. and Pearson, A.J., Eds., John Wiley & Sons: 1999; and "Protective Groups in Organic Synthesis" Greene, T.W. and Wuts, P.G., John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference. In but a few examples, suitable protecting groups utilized herein include, but are not limited to, Bn (benzyl), TIPS (triisopropylsilyl), and Ac (acetate). In a certain exemplary embodiments of the present invention, coupling of glycoside moieties are effected under MeOTf promotion, as described herein. It will be appreciated by one of ordinary skill in the art however, that a variety of conditions known in the art of organic synthesis can be utilized to effect coupling of glycoside moieties. [0207] The skilled practitioner will know how to adapt the synthetic methods detailed in the present invention to access a variety of other multi-branched gpl20 glycans and constucts thereof.
[0208] In certain other exemplary embodiments, the construct should be so functionalized as to anticipate the need for its conjugation to an immunogenic canier (e.g., protein or lipid) in anticipation of the need to stimulate an immune response. As discussed above, such constructs may be used to generate antibodies for use in HIV vaccine. The present invention provides improvements in total synthesis and HIV therapy. For example, as discussed extensively herein, the present invention provides novel glycopeptide synthetic methodology that allows access to complex glycans linked to various backbones.
[0209] As discussed above, in one embodiment of the present invention, the inventive compounds can be conjugated either directly or through a crosslinker to an appropriate canier (e.g., KLH) to generate a synthetic tumor antigen. Methods of conjugation are well known in the art. For example, a conjugation strategy may be employed that involves a reductive coupling of an aldehyde (CHO) functionality on the antigenic compound, with the intended protein canier, or lipid, presumably at the ε-amino acid residues of exposed lysines. (M.A. Bernstein; L.D. Hall, Carbohydr. Res. 1980, 78, CI; R.V. Lemieux Chem. Soc. Rev. 1978, 7, 423). Thus, in another aspect, the present invention provides synthetic constructs, whereby novel antigenic structures, as described herein, are conjugated to immunogenic caniers (e.g., proteins, peptides or lipids).
[0210] In summary, there is provided a method for gpl20 glycan synthesis that is easily modified to incorporate higher degrees of carbohydrate branching. In addition, the inventive synthetic method allows the incorporation of synthetic glycans into relatively long gpl20 peptides using a fast, high-yielding strategy that remains synthetically flexible. Accordingly, the glycopeptide structures may be optimized based on their abilities to generate antibodies for use in an HIV vaccine. [0211] 3) Compositions
[0212] In another aspect, the present invention provides compositions comprising any one or more of the inventive gpl20 glycans and/or constructs thereof. [0213] In certain embodiments, the inventive compositions may comprise an adjuvant. In certain embodiments, the adjuvant is a saponin adjuvant (see, e.g., Marciani et al, Vaccine, 2000, 18, 3141, US Patent No.: 6,080,725 and 5,977,081, the entire contents of which are hereby incorporated by reference). One example of a prefened saponin adjuvant includes, but is not limited to, GPI-OlOO, (Galenica Pharmaceuticals, Inc., Frederick, MD) which is a semi-synthetic adjuvant derived by modifying selected natural saponins.
Figure imgf000091_0001
GPI-0100 [0214] Saponins isolated from Quillaja soponaria Molina contain two acyl moieties, a normonoterpene carboxylic acid and a normonoterpene carboxylic acid glycoside, which are linked linearly to a fucosyl residue attached at position C-28. It has been hypothesized that these lipophilic acyl groups may be responsible for these saponins' toxicity and their ability to stimulate cytotoxic T cells against exogenous antigens. The linkage between the fucosyl residue and the acyl group is unstable and hydrolyzes under mild conditions (pH>6) with concomittant loss of saponins capability to stimulate cell-mediated immune response. Unlike their saponin precursors, GPI-OlOO adjuvants comprise a stable non-toxic lipophilic moiety in the saponin' s glucuronic residue. Methods for preparing these semi-synthetic adjuvants are well-known in the art. For example, GPI-OlOO adjuvants may be prepared by hydrolizing quillaja saponins (which are commercially available) under basic conditions to yield the conesponding deacylated product. The deacylated intermediate may then be reacted with a suitable amine reagent using standard carboxylic acid moiety activation methodology to give the desired compounds. A wide variety of procedures are effective for extrating saponin compounds. They are generalized as follows: (i) defatting of the organic matter with a hydrophobic organic solvent such as petroleum ether; (ii) extraction with a suitable alcohol (e.g. , methanol or ethanol) or alcohol-water mixture; (iii) evaporation of the carinol solvent; and (iv) partitioning of the dried alcohol extract between water and n- butanol saturated with water, followed by precipitation of the crude saponins from the n-butanol/water with a suitable organic solvent (e.g., diethyl ether). Purification of the saponin extract may require multiple separation steps. For example, preliminary fractionation may be canied out using conventional open column chromatography or flash chromatography on silica gel, in combination with a more sophisticated chromatographic technique such as High Pressure Liquid Chromatography (HPLC), droplet counter-cunent liquid chromatography (DCCC) or centrifugal Liquid Chromatography (RLCC). The integration of these techniques with preparative TLC typically affords separated and purified saponins. [0215] In certain other prefened embodiments, the adjuvant is bacteria or liposomes. In certain examples, the adjuvant includes but is not limited to, Salmonella minnesota cells, bacille Calmette-Guerin or QS21. [0216] As described above, the present invention provides compounds and synthetic methodologies useful in the development of novel therapeutic agents, particularly for fully synthetic HIV vaccines and/or therapeutics. In general, the compounds (e.g., gpl20 glycans, glycopeptides thereof and other constructs thereof) prepared as disclosed herein can be conjugated to a protein canier or a lipid to generate useful glycoconjugates for the treatment and/or prevention of HIV in a subject suffering therefrom. In addition, glycoconjugates prepared by processes disclosed herein are useful in adjuvant therapies as vaccines capable of inducing a potent and broad neutralizing antibody response. Such adjuvant therapies may reduce the rate of progression of HIV and/or prevent the onset of HIV. [0217] Thus, the present invention provides pharmaceutical compositions for treating HIV, and for preventing the onset or progression of HIV, comprising any of the compounds of the present invention disclosed herein, as an active ingredient, optionally, though typically in combination with a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may further comprise other therapeutically active ingredients (e.g., anti-HIV and/or palliative agents). For purposes of the invention, the term "Palliative " refers to treatment that is focused on the relief of symptoms of a disease and/or side effects of a therapeutic regimen, but is not curative. For example, palliative treatment encompasses painkillers, antinausea medications and anti-sickness drugs.
[0218] The inventive compositions include those suitable for oral, rectal, topical (including transdermal devices, aerosols, creams, ointments, lotions and dusting powders), parenteral (including subcutaneous, intramuscular, and intravenous), ocular (opthalmic), pulmonary (nasal or buccal inhalation) or nasal administration. Although the most suitable route in any given case will depend largely on the nature and severity of the condition being treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. In certain embodiments, the compositions are suitable for parenteral administration. In certain exemplary embodiments, the compositions are suitable for intravenous administration.
[0219] In preparing oral dosage forms, any of the unusual pharmaceutical media may be used, such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (e.g., suspensions, elixers and solutions); or earners such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disinterating agents, etc., in the case of oral solid preparations are prefened over liquid oral preparations such as powders, capsules and tablets. If desired, capsules may be coated by standard aqueous or non-aqueous techniques. In addition to the dosage forms described above, the compounds of the invention may be administered by controlled release means and devices. [0220] Pharmaceutical compositions of the present invention suitable for oral administration may be prepared as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient in powder or granular form or as a solution or suspension in an aqueous or nonaqueous liquid or in an oil-in-water or water-in-oil emulsion. Such compositions may be prepared by any of the methods known in the art of pharmacy. In general, compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers, finely divided solid carriers, or both and then, if necessary, shaping the product into the desired form. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granule optionally mixed with a binder, lubricant, inert diluent or surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. [0221] 4) Pharmaceutical Uses and Methods of Treatment
[0222] Pharmaceutical Uses
[0223] In one aspect, the present invention provides gpl20 glycans and constructs thereof for use as active pharmaceutical agent useful for preventing or reducing the rate of infection with HIV in subjects.
[0224] In another aspect, the inventive gpl20 glycans and constructs thereof may be used to raise antibodies specific to HIV virus. In another aspect, the invention provides an antibody which is specific to one or more gpl20 glycans and/or constructs thereof described herein.
[0225] Accordingly, in one aspect of the invention, there is provided an antibody or antibody fragment which is specific to one or more of the inventive gpl20 glycans and/or glycoconjugates thereof described herein, said antibody being a purified polyclonal antibody or a monoclonal antibody. As used herein, the term "antibody fragment" is generally intended to mean any antibody fragment having conserved the specificity of the antibody of origin, and in particular fragments of the Fab and F(ab') type. Unless otherwise indicated, the term "antibody" also subsequently denotes antibody fragments when appropriate. The expression "antibody which binds specifically to gpl20 antigen" or "antibody which is specific to gpl20 antigen" is intended to denote, an antibody which binds to one or more gpl20 glycans described herein, with high specificity. For example, in certain embodiments, the product which is bound to the antibody consists of at least 80% and preferably of at least 90%, of said gpl20 antigen.
[0226] Thus, in one aspect, the invention provides an antibody or antibody fragment which is specific to any one of the inventive antigens (independently of the others) present on a multi-antigenic construct comprising one or more carbohydrate domains having the structure:
Figure imgf000095_0001
wherein each occunence of R1 is independently hydrogen or an oxygen protecting group; each occunence of R2A and R2B is independently hydrogen or a nitrogen protecting group; each occunence of R3 is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000095_0002
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occunence of R5, R6 and R7 is independently hydrogen, OH, OR1, NR1^111, NHCOR1, F, CH2OH, CH^OR1, or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occunence of R', R" and R'" is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COOR'v, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R'", taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occunence of R'v is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group;
W1, W2 and W3 are independently optionally substituted mannose, galactose or lactosamine moieties; wherein each carbohydrate domain is independently covalently bound to a linker system, said linker system being a peptide or non-peptide nature; and wherein the linker system may be cyclic or acyclic; and and wherein said antibody is a purified polyclonal antibody or a monoclonal antibody. In certain embodiments, the antibody is a monoclonal antibody. [0227] In certain embodiments, W is R , R , as defined above, or a moiety having the structure
Figure imgf000096_0001
wherein X is -OR or -NR >2AD R2B ; and each occunence of R is independently R1 or a sialic acid moiety.
[0228] In certain embodiments, W and W 2 a „«re, : i„ndJepend je„n„+tuly. D R1 , R r>3 or a moiety having ÷.he structure:
Figure imgf000096_0002
wherein each occunence of R is independently R or a sialic acid moiety. [0229] In certain other embodiments, the antigen comprises a carbohydrate domain having the structure:
Figure imgf000097_0001
In certain other embodiments, the antigen comprises a carbohydrate domain having the structure:
Figure imgf000097_0002
[0230] In certain embodiments, the invention provides an antibody or antibody fragment which is specific to any one or more of the inventive antigens present on a multi-antigenic construct comprising a cyclic or acyclic peptidic or non- peptidic backbone made up of two or more structural units, wherein one or more of said structural units is/are independently substituted with a glycosidic moiety having the structure:
→H wherein each occunence of L is independently a substituted or unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated aliphatic or heteroaliphatic moiety; and each occunence of A is independently a carbohydrate domain of formula:
Figure imgf000098_0001
ddet)
Figure imgf000098_0002
ιdet) or
Figure imgf000099_0001
(IIIdet)
wherein each occunence of R1 is independently hydrogen or an oxygen protecting group; each occunence of R2A and R2B is independently hydrogen or a nitrogen protecting group; each occunence of R is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000099_0002
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R5, R6 and R7 is independently hydrogen, OH, OR1, NR'1^", NHCOR1, F, CH2OH, CH2OR1, or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occunence of R', R" and R'" is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COOR'v, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R'", taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occunence of R'v is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group; and
W1, W2 and W3 are independently optionally substituted mannose, galactose or lactosamine moieties.
[0231] In certain embodiments, the invention provides an antibody or antibody fragment which is specific to any one or more of the inventive antigens present on a dimeric glycopeptide having the structure:
Figure imgf000100_0001
wherein each peptide may be the same or different; and each occunence of A is independently a carbohydrate domain of formula (Idet), (IIdet) or (IIIdet).
[0232] In certain embodiments, the antigen has the structure:
Figure imgf000100_0002
wherein each occunence of A is independently a carbohydrate domain having one of the structures:
Figure imgf000101_0001
[0233] In certain embodiments, the antigen has the structure:
Figure imgf000101_0002
wherein A is as defined above. [0234] In yet other embodiments, the antigen comprises a carbohydrate antigen having the structure:
(iA) wherein the peptide has a structure either identical to or closely related to that of gpl20 near an N-glycosylation site.
[0235] In certain embodiments, the invention provides an antibody or antibody fragment which is specific to a compound of formula (IIA) having the structure:
Figure imgf000102_0002
σιA) wherein each occunence of R1 is independently hydrogen or an oxygen protecting group; each occunence of R2A and R2B is independently hydrogen or a nitrogen protecting group; and each occunence of R3 is independently hydrogen or a protecting group; wherein the peptide has a structure either identical to or closely related to that of gpl20 near an N-glycosylation site; and wherein said antibody is a purified polyclonal antibody or a monoclonal antibody.
[0236] In certain embodiments, the invention provides an antibody or antibody fragment which is specific to a compound of formula (IIIA) having the structure:
Figure imgf000103_0001
(IIIA) wherein each occunence of R1 is independently hydrogen or an oxygen protecting group; each occunence of R2A and R2B is independently hydrogen or a nitrogen protecting group; and each occunence of R is independently hydrogen or a protecting group; wherein the peptide has a structure either identical to or closely related to that of gpl20 near an N-glycosylation site; and wherein said antibody is a purified polyclonal antibody or a monoclonal antibody.
[0237] In certain exemplary embodiments, the antibody is a monoclonal antibody.
[0238] The compounds of the invention may be used to prepare monoclonal or polyclonal antibodies. Conventional methods can be used to prepare the antibodies. As to the details relating to the preparation of monoclonal antibodies reference can be made to Goding, J. W., Monoclonal Antibodies: Principles and Practice, 2nd Ed., Academic Press, London, 1986. [0239] The compounds, as well as antibodies specific for the inventive gpl20 glycans and/or constructs thereof may be labelled using conventional methods with various enzymes, fluorescent materials, luminescent materials and radioactive material. Linking an antibody or an antibody fragment to a label, whether it is a radioactive, enzymatic or colored label or any other type of label commonly used in immunological techniques, is well known and described in the literature. Suitable enzymes, fluorescent materials, luminescent materials, and radioactive material are well known to the skilled artisan.
[0240] It is presently unknown, however, how large a segment of gpl20 is required to generate appropriate antibodies; e.g., the glycopeptide may not have enough native structure to develop appropriately specific antibodies. The glycopeptide might not itself be immunogenic, and could therefore require the use of an adjuvant to stimulate an immune response. Examples of suitable adjuvants include, but are not limited to, saponin adjuvants (e.g., GPI-OlOO), Salmonella minnesota cells, bacille Calmette-Guerin and or QS21.
[0241] A lack of immune response with any length glycopeptide would call for the use of a canier protein such as keyhole limpet hemocyanin (KLH), 34"36 an adjuvant such as covalently bound Pam3Cys, or coadministered QS21. Such immunostimulants have been used alone or in concert40"42 to generate antibodies from small glycopeptide haptens, 43"45 and should prove effective here, as well. Though the first two systems require covalent conjugation, the synthetic design allows late-stage conjugation as demonstrated previously for other glycopeptides.46 [0242] References
34. Ragupathi, G. "Carbohydrate antigens as targets for active specific immunotherapy." Cancer Immunol. Immun. 1996, 43, 152-157.
35. Helling, F.; Shang, A.; Calves, M.; Zhang, S. L.; Ren, S. L.; Yu, R. K.; Oettgen, H. F.; Livingston, P. O. "G(D3) Vaccines for Melanoma - Superior Immunogenicity of Keyhole Limpet Hemocyanin Conjugate Vaccines." Cancer Res. 1994, 54, 197- 203.
36. Hanis, J. R.; Markl, J. "Keyhole limpet hemocyanin (KLH): A biomedical review." Micron 1999, 30, 597-623. 37. Kellner, J.; Erhard, M.; Schranner, I.; Losch, U. "The Influence of Various Adjuvants on Antibody Synthesis Following Immunization with an Hapten." Biol Chem. Hoppe-Seyler 1992, 373, 51-55.
38. Metzger, J.; Wiesmuller, K. H; Schaude, R.; Bessler, W. G.; Jung, G. "Synthesis of Novel Immunologically Active Tripalmitoyl-S- Glycerylcysteinyl Lipopeptides as Useful Intermediates for Immunogen Preparations." Int. J. Pept. Protein Res. 1991, 37, 46-57.
39. Kensil, C. R.; Patel, U.; Lennick, M.; Marciani, D. "Separation and Characterization of Saponins with Adjuvant Activity from Quillaja-Saponaria Molina Cortex." J. Immunol 1991, 146, 431-437.
40. Livingston, P. O.; Adluri, S.; Helling, F.; Yao, T. J.; Kensil, C. R.; Newman, M. J.; Marciani, D. "Phase-1 Trial of Immunological Adjuvant QS-21 with a GM2 Ganglioside-Keyhole Limpet Hemocyanin Conjugate Vaccine in Patients with Malignant-Melanoma." Vaccine 1994, 12, 1275-1280.
41. Zhang, S. L.; Graeber, L. A.; Helling, F.; Ragupathi, G.; Adluri, S.; Lloyd, K. O.; Livingston, P. O. "Augmenting the immunogenicity of synthetic MUC1 peptide vaccines in mice." Cancer Res. 1996, 56, 3315-3319.
42. Musselli, C; Livingston, P. O.; Ragupathi, G. "Keyhole limpet hemocyanin conjugate vaccines against cancer: the Memorial Sloan Kettering experience." J. Cancer Res. Clin. Oncol 2001, 127, R20- R26.
43. Adluri, S.; Helling, F.; Ogata, S.; Zhang, S. L.; Itzkowitz, S. H.; Lloyd, K. O.; Livingston, P. O. "Immunogenicity of Synthetic TF-KLH (Keyhole Limpet Hemocyanin) and STn-KLH Conjugates in Colorectal Carcinoma Patients." Cancer Immunol. Immun. 1995, 41, 185-192.
44. Kudryashov, V.; Glunz, P. W.; Williams, L. J.; Hintermann, S.; Danishefsky, S. J.; Lloyd, K. O. "Toward optimized carbohydrate-based anticancer vaccines: Epitope clustering, canier structure, and adjuvant all influence antibody responses to Lewis(y) conjugates in mice." Proc. Natl Acad. Sci. U. S A. 2001, 98, 3264-3269.
45. Ragupathi, G; Howard, L.; Cappello, S.; Koganty, R. R.; Qiu, D. X.; Longenecker, B. M.; Reddish, M. A.; Lloyd, K. O.; Livingston, P. O. "Vaccines prepared with sialyl-Tn and sialyl-Tn trimers using the 4-(4- maleimidomethyl)cyclohexane-l -carboxyl hydrazide linker group result in optimal antibody titers against ovine submaxillary mucin and sialyl-Tn-positive tumor cells." Cancer Immunol. Immun. 1999, 48, 1-8.
46. Glunz, P. W.; Hintermann, S.; Williams, L. J.; Schwarz, J. B.; Kuduk, S. D.; Kudryashov, V.; Lloyd, K. O.; Danishefsky, S. J. "Design and synthesis of Le(y)- bearing glycopeptides that mimic cell surface Le(y) mucin glycoprotein architecture." J. Am. Chem. Soc. 2000, 122, 7273-7279.
[0243] Methods of Treatment
[0244] As detailed above, a major drawback in using carbohydrate epitopes, is that they are generally not readily available by isolation from natural sources. For example, the immense difficulties associated with their purification from natural sources render them virtually nonavailable as homogeneous starting materials for a clinical program. Thus, the incorporation of these naturally occu ing epitopes into canier proteins/peptides or any favorable molecular context via conjugation for eliciting a therapeutically useful immunological response is inefficient at best, and often virtually impossible. Therefore, to effectively study vaccines as therapeutic agents, sufficient material can only be obtained by chemical synthesis. As discussed above, the present invention provides a variety of synthetic glycoforms of gpl20 (glycans glycopeptide conjugates and/or other constructs thereof), and methods for preparing them.
[0245] Accordingly, in another aspect of the invention, a method of treatment is provided comprising administering to a subject in need thereof a therapeutically effective amount of any of the gpl20 glycans and or glyconjugates thereof disclosed herein (e.g., glycopeptides, which may additionally be conjugated to a protein, peptide or lipid carrier, either directly or through a crosslinker), optionally in combination with a pharmaceutically acceptable canier. In certain embodiments, a method for preventing the infection with HIV is provided comprising administering to a subject in need thereof a therapeutically effective amount of any of the gpl20 glycans and/or glyconjugates thereof disclosed herein, optionally in combination with an adjuvant. In certain embodiments, a method for the treatment of HIV is provided comprising administering to a subject in need thereof a therapeutically effective amount of any of the gρl20 glycans and or glyconjugates thereof disclosed herein, optionally in combination with an adjuvant. In certain embodiments, a method for inducing antibodies in a human subject, wherein the antibodies are specific to a carbohydrate antigen expressed on the surface of gpl20, which comprises administering to the subject an amount of any of the glycans and/or glycoconjugates disclosed above effective to induce antibodies. In certain embodiments, the method utilized any one or more of the gpl20 glycans and/or glycoconjugates thereof disclosed herein, where the glycan(s) and/or glycoconjugate(s) is/are linked to an immunogenic carrier either directly or through a crosslinker, which canier is a protein, peptide or lipid. In certain embodiments, the canier is Bovine Serum Albumin, polylysine or KLH. In certain other embodiments, the carrier is a lipid having the structure:
Figure imgf000107_0001
wherein m', n' and p' are each independently integers between about 8 and 20; and Ry is hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl. In certain exemplary embodiments, m', n' and p' are each 14 and the lipid is tripalmitoyl-S-glycerylcysteinylserine (e.g., PamCys).
[0246] In certain other embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of any of the compounds and/or glycoconjugates disclosed herein, in combination with an immunogenic carrier, optionally in combination with a pharmaceutically acceptable canier. Specifically, in certain exemplary embodiments, the method comprises administering a gpl20 glycan and or glycoconjugate thereof additionally conjugated to an immunogenic carrier. In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of any one or more of the glyconjugates disclosed herein (e.g., glycopeptides, which may additionally be conjugated to a protein, peptide or lipid canier, either directly or through a crosslinker), in combination with an immunogenic canier, optionally in combination with a pharmaceutically acceptable canier. In certain embodiments, the method comprises administering one or more gpl20 glycans and/or glycoconjugates and an immunogenic carrier that have not been conjugated. Rather, they are administered concunently, or successively, as separate entities. In certain other exemplary embodiments, the method comprises administering one or more gpl20 glycans and/or glycoconjugates of the invention conjugated (i.e., covalently linked) to an immunogenic carrier. In certain embodiments, the method comprises administering any one or more inventive gpl20 glycans and/or glycoconjugates thereof disclosed herein that have not been conjugated to an immunogenic canier. Rather, the gpl20 glycan(s) and/or glycoconjugate(s) thereof and the immunogenic carrier are administered concunently, or successively, as separate entities. In certain embodiments, the immunogenic canier is a protein, peptide or lipid. In certain exemplary embodiments, the canier is Bovine Serum Albumin, polylysine or KLH. In certain other embodiments, the canier is PamCys. For the purpose of the invention, a compound/glycoconjugate and a canier are said to be administered concunently when they are administered (i) as a single composition containing the compound/glycoconjugate and the carrier, (ii) as two separate compositions or (iii) are delivered by separate routes within a short enough period of time that the effective result is equivalent to that obatined when both compound/ glycoconjugate and canier are administered as a single composition.
[0247] In still other embodiments, the present invention provides the related method of inducing antibodies which further comprises co-administering an immunological adjuvant, or a combination of immunological adjuvants. [0248] In certain exemplary embodiments, the inventive gpl20 glycans and glycoconjugates thereof comprise carbohydrate domains, or truncated or elongated versions thereof, that are found on the surface of gpl20. In certain exemplary embodiments, the inventive glycoconjugates comprise peptidic domains, or truncated or elongated versions thereof, that are found near an N-glycosylation site of naturally occurring gpl20.
[0249] Accordingly, embodiments of this invention encompass methods of eliciting immune responses in animals comprising administering effective amounts of inventive gpl20 glycans and/or glycoconjugate(s) thereof and/or compositions of the invention wherein the immune response is directed against on eor more carbohydrates expressed on the surface of gpl20.
[0250] A further embodiment of this invention encompasses a use of effective amounts of inventive gpl20 glycans and or glycoconjugate(s) thereof and/or a composition of the present invention to elicit an immune response in an animal preferably to treat and or prevent HIV. The present invention further includes a use of effective amounts of inventive gpl20 glycans and/or glycoconjugate(s) thereof and/or a composition of the present invention to prepare a medicament to elicit an immune response in animal, preferably to treat and/or prevent HIV. [0251] It will be appreciated that the magnitude of the therapeutic dose of the compounds of the invention will vary with the nature and severity of the condition to be treated and with the particular compound and its route of administration. In general, the daily dose range for antiHIV activity lies in the range of 0.0001 to 1.0 mg/kg of body weight in a mammal, although the present invention is not intended to be limited by this range.
[0252] Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dosage of a compound disclosed herein. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, etc. routes may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, etc. In prefened embodiments, the effective dosage is employed using a syringe injection. [0253] It will be appreciated by one of ordinary skill in the art, however, that the most suitable route for administration will depend largely on the nature and severity of the condition being treated and on the nature of the active ingredient. As discussed above, the inventive therapeutics may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. [0254] Additionally, once a synthetic vaccine has been derivatized and characterized, mouse immunological studies can be performed to assess the potency and/or specificity of the novel HIV vaccines.
KITS OF THE INVENTION [0255] In other embodiments, the present invention relates to a kit for conveniently and effectively carrying out the methods in accordance with the present invention. In general, the pharmaceutical pack or kit comprises one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Such kits are especially suited for the delivery of solid oral forms such as tablets or capsules. Such a kit preferably includes a number of unit dosages, and may also include a card having the dosages oriented in the order of their intended use. If desired, a memory aid can be provided, for example in the form of numbers, letters, or other markings or with a calendar insert, designating the days in the treatment schedule in which the dosages can be administered. Alternatively, placebo dosages, or calcium dietary supplements, either in a form similar to or distinct from the dosages of the pharmaceutical compositions, can be included to provide a kit in which a dosage is taken every day. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
EQUIVALENTS [0256] The representative examples which follow are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein. In but one illustrative example, protecting groups play an important role in the synthesis of the carbohydrate domains and synthetic conjugates, as described herein; however it will be appreciated by one of ordinary skill in the art that the present invention encompasses the use of various alternate protecting groups known in the art. Those protecting groups used in the disclosure including the Examples below are merely illustrative. [0257] It should further be appreciated that, uless otherwise indicated, the contents of those cited references are incorporated herein by reference to help illustrate the state of the art. The following examples contain important additional information, exemplification and guidance which can be adapted to the practice of this invention in its various embodiments and the equivalents thereof.
EXEMPLIFICATION [0258] Gpl20 glycans and glycopeptides
[0259] General Methods: Reagents obtained from commercial suppliers were used without further purification unless otherwise noted. THF, toluene, and methylene chloride was obtained from a dry solvent system (passed through a prepacked column of alumina) and used without further drying. All air and water sensitive reactions were performed in flame-dried glassware under a positive
1 11 pressure of prepurified argon gas. NMR ( H and C) spectra were recorded on Bruker AMX-400 MHz or Bruker Advance DRX-500 MHz as noted individually, referenced to CDC13 (7.27 ppm for 1H and 77.0 ppm for 13C) or CD3COCD3 (2.09 ppm for Η and 30.6 and 205.9 ppm for 13C). Optical rotations were obtained on a JASCO model DIP-370 digital ,polarimeter. Analytical thin-layer chromatography was performed on E. Merck silica gel 60 F254 plates. Compounds which were not UV active were visualized by dipping the plates in para-anisaldehyde solution and heating. Preparative thin layer chromatography was performed using the indicated solvent on Whatman® (LK6F Silica gel 60 A 250 μM or Pk6F Silica Gel 60 A 1000 μM) TLC plate.
Figure imgf000111_0001
[0260] Tetrasaccharide 3: A mixture of trisacchride l1 (106 mg, 0.074 mmol), thiomannoside 2 (133 mg, 0.222 mmol) and molecular sieves in CH3CN (2 mL) was stined for 2 h at r.t. and tris(4-bromophenyl)aminium hexachloroantimonate (199 mg, 0.244 mmol) was added at 15 °C. The solution was stined for 4 h at r.t. and then quenched by triethylamine. The mixture was filtered through celite, concentrated, dissolved in EtOAc, filtered through silica gel and concentrated. The residue was purified by preparative TLC (PTLC) using pentane/ether (1/2) as the eluent to afford 3 as a white solid (113 mg, 78%). [α]D 25 - 205.0 (c 0.14, CHC ). 1H NMR (400 MHz, CDC13) selected signals: δ 0.00 (s, 3 H), 0.06 (s, 3 H), 0.87 (s, 9 H), 5.07 (s, 1 H), 5.30 (s, 1 H), 5.36 (s, 1 H), 5.74 (s, 3 H). 13C NMR (100 MHz, CDC13) δ -5.8, -4.6, 13.9, 17.8, 20.8, 25.6, 57.8, 58.6, 60.1, 66.8, 67.5, 67.8, 68.2, 68.4, 68.9, 69.5, 71.0, 72.3, 73.1, 73.3, 73.5, 73.9, 74.3, 74.9, 75.1, 75.3, 75.8, 77.3, 77.6, 77.9, 78.2, 79.9, 92.6, 98.4, 100.7, 100.9, 125.6, 126.7, (126.8-129.5), 129.6, 136.9, 137.4, 137.6, 138.0, 138.1, 138.2, 138.3, 140.4, 141.3, 165.1. LRMS (ESI) calcd for Ci12H122N2O24S2SiNa+ [M+Na]+ 1994.76, found 1994.8.
Figure imgf000112_0001
[0261] Tetrasaccharide 4: To a solution of 3 (200 mg, 0.101 mmol) in borane tetrahydrofuran etherate (1.1 mL, 1.0 M in THF, 1.01 mmol) was added dibutylboron triflate (0.334 mL, 1.0 M in CH2C12, 0.333 mmol) at 0 °C. The reaction mixture was stined for 7 h at 0 °C and quenched with triethylamine and methanol and concentrated. The residue was purified by PTLC using pentane/ether (1/2) as the eluent to afford 4 as a white solid (172 mg, 90%). [α]D 25 -187.0 (c 0.13, CHC13). 1H NMR (400 MHz, CDC13) selected signals: δ -0.08 (s, 3 H), -0.04 (s, 3 H), 0.80 (s, 9 H), 4.96 (d, J = 2.6 Hz, 1 H), 5.15 (s, 1 H), 5.55 (s, 1 H). 13C NMR (100 MHz, CDC13) δ -5.7, -4.6, 17.9, 25.7, 57.9, 58.3, 67.6, 68.9, 69.8, 71.3, 72.4, 73.2, 73.4, 73.9, 74.3, 74.5, 75.0, 76.0, 77.3, 78.1, 79.6, 79.9, 92.7, 99.3, 100.6, 101.0, 126.8- 128.7, 129.8, 137.6, 137.7, 138.2, 138.3, 138.4, 140.5, 141.0, 165.2. LRMS (ESI) calcd for Cn2Hι24N2O24S2SiNa+ [M+Na]+ 1995.8, found 1995.8.
Figure imgf000113_0001
[0262] Pentasaccharide 6: 6 was prepared using same procedure as the synthesis of 3. White solid (80 mg, 74%). [α]D 25 51.0 (c 0.13, CHCI3). Η NMR (400 MHz, CDCI3) selected signals: δ -0.07 (s, 3 H), -0.02 (s, 3 H), 0.80 (s, 9 H), 4.95 (s, 1 H), 4.99 (s, 1 H), 5.25 (s, 1 H), 5.54 (dd, J= 9.5, 2.5 Hz, 1 H), 5.58 (s, 1 H). 13C NMR (100 MHz, CDC13) δ -5.8, -4.6, 14.0, 17.9, 20.9, 22.5, 25.7, 31.4, 57.8, 58.6, 60.2, 67.7, 68.9, 69.6, 70.1, 71.5, 72.5, 72.9, 73.2, 73.9, 74.4, 74.8, 75.2, 75.9, 76.4, 77.3, 79.1, 92.7, 97.9, 99.4, 101.1, 126.9-129.5, 129.6, 137.3, 137.7, 138.2, 138.4, 141.1, 165.2, 165.5, 166.1. LRMS (ESI) calcd for C146H154N2O31S2SiNa+ [M+Na]+ 2546.0, found 2545.9.
Figure imgf000113_0002
[0263] Pentasaccharide triol 7: To a solution of 6 (80 mg, 0.032 mmol) in
MeOH (2 mL) was added sodium methoxide in MeOH (25%, 0.1 mL) and stined for 12 h and quenched with NH4C1 saturated aqueous solution and concentrated. The residue was dissolved in EtOAc and washed with water and brine. The organic layer was dried with anhydrous MgSO4, filtered and concentrated. The residue was purified by PTLC using pentane/ether (1/3) as the eluent to afford 7 as a white solid (64 mg, 91%). [α]D 25 121.8 (c 0.16, CHC13). 1H NMR (400 MHz, CDC13) selected signals: δ 0.00 (s, 3 H), 0.05 (s, 3 H), 0.93 (s, 9 H), 4.92 (s, 1 H), 5.06 (d, J= 1.8 Hz, 1 H), 5.14 (s, 1 H). I3C NMR (100 MHz, CDC13) δ -5.8, -4.6, 14.0, 17.9, 20.9, 25.7, 57.8, 58.4, 60.2, 65.9, 68.4, 69.6, 71.6, 71.9, 72.4, 72.6, 73.1, 73.2, 73.3, 73.9, 74.1, 74.4, 74.7, 74.8, 74.9, 75.3, 75.8, 76.1, 76.4, 77.3, 78.3, 79.1, 79.6, 80.6, 92.7, 97.2, 100.5, 101.2, 101.3, 126.8-128.6, 137.6, 137.8, 137.9, 138.2, 138.3, 138.6, 140.5, 141.1. LRMS (ESI) calcd for Ci25H142N2O28S2SiNa+ [M+Na+] 2233.9, found 2233.9.
Figure imgf000114_0001
[0264] Octasaccharide 8: 8 was prepared following the same protocol as used for 3 using thiol mannoside donor 2 as excess (10 eq.) White solid: (61 mg, 55%). [α]D 25 32.8 (c 0.15, CHC13). Η NMR (400 MHz, CDC13) selected signals: δ 0.00 (s, 3 H), 0.05 (s, 3 H), 0.88 (s, 9 H), 4.79 (s, 1 H), 4.88 (s, 1 H), 5.01 (s, 1 H), 5.06 (s, 1 H), 5.23 (s, 1 H), 5.58 (s, 1 H), 5.62 (s, 1 H), 5.66 (s, 1 H). 13C NMR (100 MHz, CDCI3) δ -5.1, -3.9, 14.7, 18.5, 21.5, 26.3, 30.2, 58.4, 59.1, 60.9, 66.5, 69.0, 69.3, 69.4, 70.3, 71.3, 72.0, 72.2, 72.6, 72.8, 73.6, 73.7, 73.8, 74.0, 74.6, 75.1, 75.3, 75.5, 75.7, 78.4, 78.9, 80.4, 82.2, 93.3, 98.2, 98.9, 99.7, 101.2, 101.8, 102.8, 126.9- 130.5, 138.5, 138.6, 138.8, 139.0, 139.1, 139.6, 141.2, 165.8, 165.9. LRMS (ESI) calcd for C227H238N2O46S2SiNa2 [M+2Na]2+ 1932.8, found 1933.0.
Figure imgf000114_0002
[0265] Octasaccharide triol 9: The synthesis of 9 follows the synthetic procedure of 7. White solid (46 mg, 87%). [α]D 25 280.0 (c 0.12, CHCI3). Η NMR (400 MHz, CDCI3) selected signals: δ -0.08 (s, 3 H), -0.03 (s, 3 H), 0.80 (s, 9 H), 4.92 (s, 1 H), 4.94 (s, 1 H), 4.97 (s, 1 H), 5.03 (s, 1 H), 5.07 (s, 1 H). 13C NMR (100 MHz, CDCI3) δ -5.7, -4.4, 0.0, 14.1, 18.0, 22.7, 25.8, 29.3, 29.7, 31.9, 58.0, 58.6, 65.5, 66.3, 67.7, 68.4, 68.5, 68.8, 71.1, 71.2, 71.7, 71.8, 72.0, 72.3, 72.9, 73.2, 73.3, 73.5, 74.1, 74.2, 74.3, 74.5, 74.8, 74.9, 75.0, 79.4, 81.8, 92.8, 97.4, 100.0, 100.7, 100.9, 101.3, 102.9, 127.1-128.8, 138.0-138.5, 140.7, 141.3. LRMS (ESI) calcd for C206H226N2O43S2SiNa2 [M+2Na]2+ 1776.7, found 1776.7.
Figure imgf000115_0001
[0266] Undecasaccharide 10a: The synthesis of 10a follows same synthetic procedure as 8. 10a, white solid (81 mg, 51%). [α]D 25 73.8 (c 0.09, CHC13). 1H NMR (400 MHz, CDC13) selected signals: δ -0.05 (s, 3 H), -0.00 (s, 3 H), 0.82 (s, 9 H), 5.00-5.20 (m, 7 H), 5.65-5.68 (m, 3 H). LRMS (ESI) calcd for C308H322N2O61S2SiNa2 [M+2Na]2+ 2581.1, found 2581.3.
Figure imgf000115_0002
[0267] Trisaccharide donor 11: Trisaccharide donor 11 was prepared from the known chloride and thiomannoside monosaccharides according to standard coupling procedures. l H NMR (CDC13, 400 MHz) δ: 1.21 (t, J= 7.5 Hz, 3H), 2.16 (s, 3H), 2.47-2.57 (m, 2H), 3.57 (d, J = 10.7 Hz, 1H), 3.67-3.75 (m, 4H), 3.78-3.85 (m, 4H), 3.89-3.97 (m, 3H), 3.99-4.04 (m, 2H), 4.08-4.12 (m, 3H), 4.36 (d, J= 12.2 Hz, IH), 4.43-4.73 (m, 14H), 4.82-4.88 (m, 3H), 5.08 (d, J = 2.0 Hz, IH), 5.20 (d, J = 2.0 Hz, IH), 5.46 (d, J = 1.3 Hz, IH), 5.56 (dd, J = 3.0, 1.9 Hz, IH), 7.14-7.38 (m, 45H).
Figure imgf000116_0001
[0268] Hexasaccharide 12: To a mixture of 1 (35 mg, 0.024 mmol), 11 (51 mg, 0.037 mmol) and molecular sieves in CH2C12 (2 mL) was added di-tert- butylpyridine (DTBP) (0.019 mL, 0.085 mmol) at -40 °C and stined for 1 h at -40 °C. MeOTf (0.011 mL, 0.096 mmol) was added and the reaction mixture was warmed up to r.t. and stined for 12 h before quenched with triethylamine, filtered through celite, washed with NaHCO3 saturated aqueous solution, brine, dried over anhydrous MgSO4 and filtered. The organic layer was concentrated and residue purified by PTLC using pentane/ether (1/1.3) as the eluent to afford 12 as a white solid (47 mg, 47%). [α]D 25 41.4 (c 0.65, CHC13). Η NMR (400 MHz, CDC13) selected signals: δ 0.91 (s, 9 H), 4.97 (s, 1 H), 5.11 (s, 1 H), 5.21 (s, 1 H), 5.25 (s, 1 H), 5.28 (s, 1 H), 5.52 (s, 1 H). 13C NMR (100 MHz, CDC13) δ -5.7, -4.4, 0.0, 14.2, 18.0, 21.0, 21.1, 25.8, 58.0, 58.8, 60.4, 67.0, 68.6, 68.7, 69.8, 71.6, 71.9, 72.1, 72.9, 73.3, 73.7, 73.8, 74.4, 74.6, 75.1, 75.4, 75.5, 76.0, 77.4, 78.3, 78.8, 80.2, 92.9, 99.5, 99.9, 100.2, 100.8, 101.0, 101.1, 125.9, 127.1-128.5, 137.9, 138.1, 138.4, 138.6, 138.8, 140.7, 170.0, 171.1. LRMS (ESI) calcd for Ci6iH176N2O34S2SiNa2 [M+2Na]2+ 1409.6, found 1409.4.
Figure imgf000116_0002
[0269] Hexasaccharide 13:13 was prepared using the same procedure as the one for 4. 13, white solid (542 mg, 86%). [α]D 25 91.5 (c 0.54, CHC13). Η NMR (400 MHz, CDC13) selected signals: δ 0.02 (s, 3 H), 0.04 (s, 3 H), 0.87 (s, 9 H), 5.01 (s, 1 H), 5.06 (s, 1 H), 5.09 (s, 1 H), 5.15 (s, 1 H), 5.49 (s, 1 H). 13C NMR (100 MHz, CDC13) δ -5.7, -4.4, 0.0, 14.2, 18.0, 21.0, 21.1, 21.4, 25.8, 58.0, 58.3, 60.4, 61.3, 67.6, 68.6, 69.9, 71.8, 72.0, 72.1, 73.1, 73.3, 73.5, 74.1, 74.5, 74.6, 74.8, 75.1, 76.1, 76.2, 78.2, 78.7, 79.8, 81.1, 92.8, 99.4, 100.5, 100.7, 101.0, 101.3, 125.3, 127.0- 128.5, 137.9, 138.0, 138.4, 138.5, 138.6, 138.7, 140.7, 170.1. LRMS (ESI) calcd for Cι6ιH178N2O34S2SiNa2 [M+2Na]2+ 1410.6, found 1410.4.
Figure imgf000117_0001
[0270] Tirsaccharide 16: To a 25 mL flask containing donor 15 (169 mg,
0.332 mmol) and acceptors 14 (37 mg, 0.083 mmol) (dried azeotropically with toluene) in 1.5 mL dichloromethane was added activated MS 4 A and the mixture was stined for 1 h at room temperature. In a separate flask, AgOTf (0.087 gm, 0.332 mmol) and DTBP (0.078 mL, 0.347 mmol) in 1.5 mL dichloromethane were stined with MS 4A. After one hour the flask containing the AgOTf/ DTBP was cooled to - 10 °C and the solution containing mixture of donor and acceptor was added over 5 minutes. The solution was stined in dark with warming up to room temperature over 18 hr. The reaction mixture was diluted with ethyl acetate and was added aqueous saturated NaHCO3 solution. After stirring for 10 minutes, the reaction mixture was filtered through bed of Celite and the filtrate was washed with water, brine, dried over MgSO4 and evaporated in vacuo. The crude product was purified by silica gel column chromatography (10% ethyl acetate/toluene) to afford diacetate 16. This diacetate was used for next step without further purification.
Figure imgf000118_0001
[0271] Trisaccharide diol 17:
[0272] 16 was dried azeotropically with toluene and dissolved in 2 mL of anhydrous methanol under argon. Sodium methoxide (25% by weight in methanol, 100 μL) was added and the reaction mixture was stined for 12 h. Solid ammonium chloride was added and the mixture was stined for 20 min. The reaction mixture was carefully evaporated to solid residues, and the residues were dissolved in ethyl acetate and washed with brine. Evaporation of ethyl acetate layers provided crude products, which was purified by silica gel column chromatography (10% ethyl acetate/dichloromethane) to yield diol 17 in 50% over two steps. [α]o25 +53.1 (c 1.0, CHC13); lB NMR (CDC13, 400 MHz) δ 4.94 (bs, IH), 5.17 (bs, IH), 5.44 (bs, IH). 13C NMR (CDCI3, 125 MHz) δ 138.68, 138.66, 138.4, 138.1, 138.06, 138.03, 134.9, 131.0, 129.3, 128.72, 128.70, 128.67, 128.61, 128.49, 128.47, 128.16, 128.13, 128.10, 128.01, 127.96, 127.88, 127.86, 127.83, 127.82, 127.79, 127.76, 127.71, 127.35, 99.9, 85.3, 80.5, 80.2, 79.6, 75.3, 75.15, 75.07, 74.6, 74.4, 73.8, 73.5, 72.7, 72.3, 72.2, 71.8, 71.7, 71.3, 69.5, 68.94, 68.90, 68.2, 66.4. LRMS (ESI) calcd for C80H84O15SNa+ [M+Na]+ 1339.6, found 1339.5.
Figure imgf000118_0002
[0273] Pentasaccharide 18: To a mixture of 17 (208 mg, 0.158 mmol), 15
(332 mg, 0.631 mmol), molecular sieves, DTBP (0.088 mL, 0.347 mmol) in CH2C12 (13 mL) was added AgOTf (166 mg, 0.646 mmol) at 0 °C. The mixture was stined for 18 h at r.t. and quenched with triethylamine, filtered, diluted with EtOAc, washed with NaHCO3 saturated aqueous solution, brine, dried over anhydrous MgSO4 and filtered. The organic layer was concentrated and residue purified by PTLC using pentane/ether (2/1) as the eluent to afford 18 as a white solid (310 mg, 87%). [α]D 25 443.4 (c 0.49, CHC13). 1H NMR (400 MHz, CDC13) selected signals: δ 2.10 (s, 3 H), 2.11 (s, 3 H), 4.88 (s, 1 H), 5.02 (s, 1 H), 5.04 (s, 1 H), 5.21 (s, 1 H), 5.51 (s, 1 H). 13C NMR (100 MHz, CDC13) δ 14.2, 20.9, 21.0, 21.1, 29.6, 44.6, 60.3,
66.6, 68.7, 68.8, 71.5, 71.7, 71.8, 72.0, 72.1, 73.1, 73.3, 73.4, 74.1, 74.2, 74.4, 74.6,
74.7, 75.0, 75.2, 78.0, 78.1, 79.2, 80.3, 84.8, 89.8, 95.4, 99.0, 99.4, 99.5, 101.2, 116.9, 125.1, 127.1-128.4, 129.1, 130.8, 138.0-138.6, 146.8, 168.3, 170.0. LRMS (ESI) calcd for C138H144O27SNa+ [M+Na]+ 2288.0, found 2287.9.
Figure imgf000119_0001
[0274] Undecasaccharide 10b: The preparation of 10b from 18 and 13 follows the same procedure as the one used for 3. 10b, white solid (529 mg, 63% yield, 85% based on recovered starting material). [α]D 25 214.3 (c 0.23, CHCI3). Η NMR (400 MHz, CDCI3) selected signals: δ 0.07 (s, 3 H), 0.15 (s, 3 H), 0.90 (s, 9 H), 2.01 (s, 3 H), 2.10 (bs, 6 H), 5.05 (bs, 1 H), 5.07 (bs, 1 H), 5.10 (bs, 1 H), 5.12 (bs, 1 H), 5.13 (bs, 1 H), 5.15 (bs, 1 H), 5.23 (bs, 1 H), 5.51 (bs, 1 H), 5.54 (bs, 1 H). 13C NMR (100 MHz, CDC13) δ -5.7, -4.4, 0.0, 1.0, 14.2, 18.0, 21.0, 21.1, 21.2, 25.8, 29.7, 58.0, 58.6, 60.4, 68.6, 68.7, 68.8, 71.8, 72.2, 72.3, 73.0, 73.1, 73.2, 73.3, 74.2, 74.5, 74.8, 75.0, 75.1, 78.2, 78.3, 78.4, 79.4, 92.8, 99.3, 99.5, 100.7, 101.6, 102.3, 127.3-128.4, 138.1-138.7, 140.7, 141.3, 170.0, 170.1, 170.15. LRMS (ESI) calcd for C293H316N2θ61S2SiNa2 [M+2Na]2+ 2488.0, found 2488.0.
Figure imgf000120_0001
[0275] Undecasaccharide triol 24: 24 was prepared using the same procedure as described for 7. 24, white solide (468 mg, 96%). [α]D 25 214.3 (c 0.23, CHC13). 1H NMR (400 MHz, CDC13) selected signals: δ 0.03 (s, 3 H), 0.05 (s, 3 H), 0.90 (s, 9 H), 5.07 (s, 1 H), 5.08 (s, 1 H), 5.13 (s, 1 H), 5.18 (s, 1 H), 5.21 (s, 1 H), 5.30 (s, 1 H). 13C NMR (100 MHz, CDCI3) δ -5.7, -4.5, 0.0, 14.1, 18.0, 21.0, 25.8, 29.6, 57.9, 58.6, 60.3, 67.6, 68.4, 68.6, 68.7, 68.9, 71.5, 71.9, 72.0, 72.3, 73.0, 73.1- 73.5, 74.2, 74.5, 74.7, 74.9, 75.0, 75.2, 79.9, 80.0, 92.7, 99.4, 100.2, 100.7, 101.1, 101.5, 102.3, 126.7-128.7, 138.1-138.8, 140.7, 141.3. LRMS (ESI) calcd for C287H31oN2O58S2SiNa2 [M+2Na]2+ 2425.0, found 2425.2.
Figure imgf000120_0002
[0276] Undecasaccharide tetraol 19: To a solution of 24 (468 mg, 0.097 mmol) in HAc (1.0 M in THF, 2.5 mL) was added TBAF (1.0 M in THF, 2.5 mL) and the reaction mixture was stined for 1 h before additional HAc (1.0 M in THF, 5.0 mL) was added. The mixture was concentrated and residue purified by column chromatography using 2.5% MeOH in CH2C12 as the eluent to afford 19 as a white solid (460 mg, 98%). [α]D 25 121.7 (c 0.32, CHC13). 1H NMR (400 MHz, CDC13) selected signals: δ 4.95 (bs, 2 H), 5.08 (s, 1 H), 5.20 (bs, 2 H), 5.24 (s, 1 H), 5.27 (s, 1 H). LRMS (ESI) calcd for C28ιH296N2θ58S2Na2 [M+2Na]2+ 2367.9, found 2367.6.
Figure imgf000121_0001
[0277] Glycan 20: To a solution of sodium (101 mg, 4.391 mmol) in 15 mL liquid ammonia was added 19 (95 mg, 0.020 mmol) in THF (4 mL) at -78 °C and the reaction mixture was stined for 2 h at -78 °C. The reaction was quenched with solid NH4C1 at -78 °C and then warmed up to r.t. while argon was blowing through the reaction flask to evaporate all liquid. The residue was dried on vaccum for 2 h and dissolved in saturated NaHCO3 aqueous solution (2 mL) and cooled to 0 °C. Ac O (0.1 mL) was then added at 0 °C and the ice bath was then removed and 5 min later additional Ac2O (0.05 mL) was added. 30 min later, low resolution mass spectrum showed reaction is complete. The reaction mixture was loaded on to a Bio-Gel P-2 column (BIO-RAD, catalog number 150-4134, molecular cutoff 2000) using water as the eluent to remove salt and small molecular weight compounds. The fraction containing desired material (illustrated by MassSpectrum) was conbined and lyophilized to afford glycan 20 as a white solid (33 mg, 87% from 19). 1H NMR (400 MHz, CDC13) selected signals: δ 5.07 (bs, 2 H), 5.08 (s, 1 H), 5.13 (s, 1 H), 5.33 (s, 1 H), 5.36 (s, 1 H), 5.40 (s, 1 H). LRMS (ESI) calcd for C7oHπ8N2O56Na+ [M+Na]+ 1905.6, found 1905.6.
Figure imgf000122_0001
[0278] Glycosylamine 21: A solution of 20 (33 mg, 0.018 mmol), NH4C1
(10 g) in 30 mL water was heated to 40 °C for 2 days and Mass spectrum indicated that reaction is complete. So the reaction mixture was frozen and lyophilized. The residue was dissolved in 20 mL water, frozen and lyophilized again. This process was repeated until the weight of the residue is constant (36 mg). LRMS (ESI) calcd for C70Hι19N3O55Na+ [M+Na]+ 1904.7, found 1904.8.
Figure imgf000122_0002
[0279] Gpl20 glycopeptide 23: A solution of peptide acid 32 (21 mg, 0.008 mmol), HATU (6 mg, 0.016 mmol), diehtylpropylamine (DIEPA) (2 μL, 0.011 mmol) in DMSO (150 μL) was stined for 5 min and transfened to the flask containing 21 (5 mg, 0.002 mmol) and the reaction mixture was stined for 2 h. Additional DIEPA was added (0.6 μL at 4 h and 0.6 μL at 6 h). At 7 h, a mixture of hydrazine, piperidine and DMF (volume ratio: 5:15:85, 0.2 mL) was added and the reaction mixture was stined for 5 min and TFA in water (10%, 0.55 mL) was added and stined for 30 min. The crude solution was purified by HPLC using a Varian
C18-DYNAMAX-60 A column. HPLC Conditions: 10%B to 50%B over 50 min,
UV 214 nM (A: 0.05% TFA in water; B: 0.04% TFA in CH3CN). Retention time:
19.8 min. The fraction containing 23 was lyophilized to give 23 as a white solid (1.7 mg, 16% from 20). 1H NMR (400 MHz, CDC13) selected signals: δ 4.99 (s, 1 H),
5.16 (s, 1 H), 5.19 (s, 1 H), 5.25 (s, 1 H), 8.29 (s, 1 H). LRMS (ESI) calcd for
Cι64H275N35O8oS2Na3 [M+3Na]3+ 1360.6, found 1360.7; calcd for
Ci64H275N35θ8oS2Na4 [M+4Na]4+ 1020.7, found 1020.6.
[0280] References:
[0281] (1) Dudkin, V. Y.; Miller, J. S.; Danishefsky, S. J. Tetrahedron
Letters 2003, 44, 1791-1793.
[0282] (2) Zhang, Y.-M.; Mallet, J.-M.; Sinay, P. Carbohydrate Research
1992, 236, 73-88.
[0283] (3) Marra, A.; Mallet, J. M.; Amatore, C; Sinay, P. Synlett 1990,
572-574.
[0284] (4) Matsuo, I.; Wada, M.; Manabe, S.; Yamaguchi, Y; Otake, K.;
Kato, K; Ito, Y. Journal of the American Chemical Society 2003, 125, 3402-3403.
[0285] (5) Calarese, D. A.; Scanlan, C. N.; Zwick, M. B.; Deechongkit, S.;
Mimura, Y; Kunert, R.; Zhu, P.; Wormald, M. R.; Stanfield, R. L.; Roux, K. H.;
Kelly, J. W.; Rudd, P. M.; Dwek, R. A.; Katinger, H.; Burton, D. R.; Wilson, I. A.
Science (Washington, DC, United States) 2003, 300, 2065-2071.
[0286] (6) Likhosherstov, L. M.; Novikova, O. S.; Derevitskaya, V. A.;
Kochetkov, N. K. Carbohydrate Research 1986, 146, C1-C5.
Figure imgf000123_0001
[0287] Into a 25 mL flask containing donor 15 (0.169 gm, 0.332 mmol) and acceptors 14 (0.037 gm, 0.083 mmol) (azeotropically dried with toluene) in 1.5 dichloromethane was added activated MS 4A and the mixture was stined for 1 hr at room temperature. In a separate flask, AgOTf (0.087 gm, 0.332 mmol) and DTBP (0.078 mL, 0.347 mmol) in 1.5 mL of dichloromethane were stined with MS 4A. After stining for 1 hr, the flask containing the AgOTf/ DTBP was cooled to - 10 °C and the solution containing mixture of donor and acceptor was added over 5 minutes. The solution was stined in dark with gradual warming up to room temperature over 24 hr. The reaction mixture was diluted with ethyl acetate and was added aqueous saturated NaHCO3. After stining for 10 minutes, the reaction mixture was filtered through bed of Celite and the filtrate was washed with water, then with brine, dried over MgSO and evaporated in vacuo. The crude product was purified by silica gel column chromatography (10% ethyl acetate / toluene) to afford semi pure trimer diacetate. This diacetate was dried azeotropically with toluene and dissolved in 2 mL of anhydrous methanol under argon. Sodium methoxide, 25% by weight in methanol (100 μL) was added and the reaction mixture was stined for 12h. Solid ammonium chloride was added and the resulting solution was stined for 20 min. The reaction mixture was carefully evaporated to solid residues, and the solid residues were washed with ethyl acetate. Evaporation of ethyl acetate layer provided crude product, which was purified by silica gel column chromatography (10% ethyl acetate / dichloromethane) to yield diol 17 in 65% yield (over two steps, [α] + 53.1 (c 1, CHC13); 1H - NMR (CDC13, 400 MHz) δ 7.33-7.03 (45H, m, aromatic), 5.44 (IH, br-s), 5.17 (IH, br-s), 4.94 (IH, br-s), ; 13C-NMR (CDC13, 125 MHz) δ 138.68, 138.66, 138.4, 138.1, 138.06, 138.03, 134.9, 131.0, 129.3, 128.72, 128.70, 128.67, 128.61, 128.49, 128.47, 128.16, 128.13, 128.10, 128.01, 127.96, 127.88, 127.86, 127.83, 127.82, 127.79, 127.76, 127.71, 127.35, 99.9, 85.3, 80.5, 80.2, 79.6, 75.3, 75.15, 75.07, 74.6, 74.4, 73.8, 73.5, 72.7, 72.3, 72.2, 71.8, 71.7, 71.3, 69.5, 68.94, 68.90, 68.2, 66.4. ESI-MS calcd for C80H84O15S Na [M+Na]1+ m/z = 1339.5: found 1339.5
Figure imgf000125_0001
22
[0288] Into a 25 mL flask containing 17 (0.099 gm, 0.0689 mmol, azeotropically dried with toluene) in 0.4 mL mL of dry dichloromethane under argon and cooled to 0 °C. Pyridine (55 μL, mmol, 6.8 mmol) and chloroacetic anhydride (0.047 gm, .0275 mmol), were added successively and resulting reaction mixture was stined for 2 h at 0 °C, and then diluted with ethyl acetate, washed two times with 0.5N HCl, water, sat NaHCO3, brine, and dried with MgSO4. Evaporation of ethyl acetate layer followed by silica gel column chromatography (20 ethyl acetate in hexanes) provided 0.166 gm (79% yield) of 22. R/ 0.33 (20% ethyl acetate in hexanes). [α] + 58.2 (c 1, CHC13); 1H - NMR (CDC13, 400 MHz) δ 7.30-7.03 (45H, m, aromatic), 5.47 (2H, m), 5.41 (IH, br-s), 5.15 (IH, s), 4.88 (IH, s), 4.75 (2H, t, J = 10.5 Hz); 13C-NMR (CDCI3, 125 MHz) δ 166.88, 166.78, 138.57, 138.51, 138.3, 137.79, 137.76, 137.74, 134.7, 130.9, 129.3, 128.7, 128.6, 128.59, 128.51, 128.48, 128.43, 128.30, 128.10, 128.0, 127.97, 128.86, 127.78, 127.75, 127.74, 127.7, 127.4, 99.5, 97.9, 84.9, 79.0, 78.1, 77.7, 75.4, 75.2, 75.1, 74.3, 74.1, 73.7, 73.5, 72.43, 72.37, 72.29, 71.78, 71.69, 70.7, 70.4, 69.0, 68.7, 66.8, 41.2, 41.0
Ph°2SHN Ph°2SHN 0TBS
Figure imgf000125_0002
23 [0289] Into a 25 mL flask containing donor 2 (125 mg, 0.0696 mmol, azeotropically dried with toluene) and 4A molecular sieves in dry acetonitrile were stined for 1 hr under argon. Tris (4-bromophenyl) aminium hexachloroantimonate [(BrC6H )3NSbCl6] (140 mg, promoter) and then a solution of acceptor 1 (100 mg, 0.0696 mmol) were added slowly while cooling the flask at 15 °C. After stirring for 15 min, another portion of tris (4-bromophenyl) aminium hexachloroantimonate [(BrC6H )3NSbCl6] (46 mg) was added and the reaction mixture was warmed to room temperature and stined for 3 hr. Freshly distilled triethyl amine (1.5 mL) was added to neutralize the reaction. The reaction mixture was filtered through a bed of Celite and concentrated. The crude product was purified by silica gel column chromatography to afford tetrasaccharide (0.110 gm). R/ 0.65 (20% ethyl acetate in toluene). Under argon this material was dissolved in mixture of dry methanol (2 mL) and dichloromethane (1.5 mL). Sodium methoxide, 25% by weight in methanol (0.038 mL) was added and stined for 12 hr. Solid ammonium chloride was added and the mixture was evaporated to dryness. The solid residue was washed several times with ethyl acetate and concentrated. Purification by silica gel column chromatography afforded the 0.092 gm (89% yield) of 23. R 0.42 (40% ethyl acetate in hexanes). [α] - 8.8 (c 1, CHCh^H - NMR (CDC13, 400 MHz) (selected signals) δ 7.75 (2H, d, J = 7.6 Hz), 7.72 (2H, ά, J = 6.8 Hz), 5.41 (IH, br-s), 5.26 (IH, d, J = 2.0 Hz), 5.11 (IH, d, J = 2.4 Hz), 3.14 (IH, m), 3.0 (2H, m), 1.57 (IH, br-s), 0.908 (9H, s), 0.09 (3H, s), 0.03 (3H, s); 13C-NMR (CDC13, 100 MHz) δ 141.7, 140.9, 138.7, 138.65, 138.61, 138.4, 138.0, 137.9, 137.8, 137.5, 132.6, 132.4, 129.5, 129.1, 129.0, (128.9-127.6), 127.4, 127.3, 127.2, 126.2, 101.5, 101.3, 101.1, 100.4, 93.0, 80.3, 80.0, 76.2, 75.8, 75.6, 75.5, 75.2, 74.8, 74.4, 73.96, 73.87, 73.6, 72.2, 72.0, 69.9, 69.3, 68.8, 68.5, 68.2, 67.8, 67.2, 37.5, 33.8, 33.6, 32.1, 30.3, 30.2, 29.9, 29.5, 29.1, 27.3, 26.9, 26.0, 23.4, 22.9, 19.9, 18.2, 14.4, 14.3, 7.6, - 4.2, - 5.4;
Figure imgf000127_0001
[0290] Into a 25 mL round-bottomed flask containing acceptor 23 (0.100 gm, 0.0535 mmol, azeotropically dried with toluene) in 1 mL dry acetonitrile was added 4A activated molecular sieves and was stined at room temperature under argon for 1 hr. Similarly the donor 24 (0.1498 mmol, 0.158 gm) and 4A molecular sieves in 1 mL dry acetronitrile were stined at room temperature for 1 hr. The flask containing donor was cooled to 15 °C and 0.091 gm of tris (4-bromophenyl) aminium hexachloroantimonate [(BrC6H4)3NSbCl6] (promoter) was added followed by the solution of acceptor. The reaction mixture was stined at 15 °C for 20 min and then additional 0.031 gm of promoter was added. The cooling bath was removed and the reaction mixture was stined for 3 hr. The reaction mixture was cooled to 0 °C and triethyl amine (1 mL) was added. After stining 10 min at 0 °C, the reaction mixture was warmed to room temperature and stined for additional 10 min. Filtering through a pad of Celite and evaporation of the filtrate afforded the crude product, which was purified by preparative TLC (first using 5% ethyl acetate in dichloromethane, then 30% ethyl acetate in hexanes) to afford .091 gm of 25 (60% yield), [α] - 16.6 (c 1, CHC13); !H - NMR (CDC13, 400 MHz) (selected signals) δ 7.72 (2H, d, J = 7.2 Hz), 7.67 (2H, d, J = 7.2 Hz), 5.21 (IH, br-s), 5.07 (IH, br-s), 5.01 (IH, br-s), 4.98 (IH, d, J = 8Hz), 2.99 (IH, m), 2.89 (IH, t, J = 8.4 Hz), 2.80 (IH, m), 2.64 (2H, m), 0.875 (9H, s), 0.07 (3H, s), 0.05 (3H, s); 13C-NMR (CDC13, 100 MHz) δ 140.5, 139.7, 138.04, 138.01, 137.7, 137.69, 137.63, 137.59, 137.56, 137.47, 137.41, 137.3, 137.0, 136.8, 136.5, 136.4, 132.4, 131.3, 131.1, 130.9, 128.4, 128.1, 127.8, (127.5-126.1), 125.8, 125.3, 122.4, 122.0, 102.3, 100.3, 99.9, 99.4,
96.5, 94.7, 91.8, 81.5, 78.9, 77.6, 77.36, 77.31, 75.1, 74.4, 74.2, 74.1, 73.9, 73.8,
73.6, 73.5, 73.1, 72.8, 72.7, 72.4, 72.2, 71.9, 71.8, 71.6, 71.5, 71.2, 57.8, 56.9, 51.3, 28.7, 24.8, 16.9, 7.6, - 5.4, - 6.6
Figure imgf000128_0001
[0291] To the azeotropically dried 25 (0.307 gm, 0.107 mmol) in 25 mL round bottomed flask was added 8 mL of BH3.THF (1 molar) and stined for 5 minutes at room temperature. The reaction mixture was cooled to 0 °C and 0.35 mL of «Bu2OTf (1 molar in CH2C12) was added. The resulting reaction mixture was stined at 0 °C for 9 hr. Freshly distilled triethyl amine (0.492 mL) was added and followed by careful addition of methanol until the evolution of H2 had ceased. The reaction was evaporated to dryness, twice codistilled from methanol to afford the crude product as clear oil. Purification by silica gel column chromatography (30% ethyl acetate in hexanes) provided the 26 in 75% yield (0.231 gm). [0292] [α] - 7.0 (c 1, CHC13); 1H - NMR (CDC13, 400 MHz) (selected signals) δ 7.74 (2H, d, J= 7.2 Hz), 7.69 (2H, d, J = 6.8 Hz), 5.07 (2H, m), 2.96 (IH, m), 2.75 (2H, m), 0.90 (9H, s), 0.07 (3H, s), 0.02 (3H, s); 13C-NMR (CDCI3, 100 MHz) δ 168.5, 167.7, 141.4, 140.9, 139.3, 139.2, 138.97, 138.91, 138.73, 138.66, 138.64, 138.58, 138.50, 138.45, 138.27, 138.1, 137.8, (129.2-126.9), 123.5, 123.4, 103.3, 101.0, 100.9, 99.2, 96.5, 92.9, 82.6, 80.1, 79.9, 78.7, 78.4, 76.5, 76.0, 75.9, 75.4, 75.1, 74.9, 74.8, 74.68, 74.63, 74.49, 74.34, 74.29, 74.24, 73.84, 73.77, 73.6, 73.45, 73.3, 73.2, 73.0, 72.8, 72.7, 61.4, 60.6, 58.6, 59.2, 55.8, 26.0, 21.3, 19.3, 18.2, 14.4, 14.1, - 4.2, - 5.4;
Figure imgf000129_0001
[0293] Into a 5 mL v vial were taken azeotropically dried donor 22 and acceptor in 26 mL anhydrous acetonitrile and activated 4A MS was added. The resulting reaction mixture was stined under argon for 1 hour at room temperature and then was cooled to 15 °C. At this point Tris (4-bromophenyl) aminium hexachloroantimonate [(BrC6H4)3NSbCl6] was added to the reaction mixture. The cooling bath was removed and the reaction mixture was stined at room temperature for 12 h or TLC indicated the disappearance of the acceptor. The reaction mixture was cooled to 0 °C and 2 mL triethyl amine was added and stined for 30 minutes with gradual warming up to room temperature. The reaction mixture was filtered through a pad of Celite and concentrated to provide crude material, which was purified by preparative TLC (20x20cm x 1 mm thickness PK6F plates) using 40% ethyl acetate in hexanes to yield 27. [α] + 9.4 (c 1, CHC13); Η - NMR (CDCI3, 400 MHz) (selected protons) δ 5.41 (IH, br-s), 5.32 (IH, br-s), 5.09 (IH, br-s), 4.97 (2H, m), 0.83 (9H, s), 0.05 (s, 3H), 0.03 (s, 3H); 13C-NMR (CDCI3, 100 MHz)D 168.6,
167.6, 166.70, 166.67, 141.5, 140.9, 139.30, 139.23, 139.0, 138.9, (138.8-138.1), 137.89, 137.86, 137.6, 133.6, 132.5, 132.3, 132.0, (129.0-126.9), 126.7, 103.3,
101.7, 100.9, 99.3, 98.0, 97.8, 96.3, 92.9, 82.6, 81.3, (78.9-65.1), 58.6, 58.1, 55.8, 39.9, 39.8, 28.7, 24.8, 17.0, - 5.4, - 6.7.
Figure imgf000130_0001
[0294] To azeotropically dried 27 ( 100 mg, 0.023 mmol) in a v 5 mL v vial equipped with spin bar were added fresh toluene (2 mL) and «-butanol (4 mL). Ethylenediamine was added and the reaction mixture was heated at 90 o C for 18 hr. After cooling to room temperature, the reaction mixture was concentrated under vacuo. The crude product was dissolved in 5 mL of toluene and evaporated to dryness. Pyridine and acetic anhydride were added and the reaction mixture was stined for 16 hr at room temperature. The reaction mixture was evaporated to dryness, twice from toluene, yielding foam with some solid. This material was dissolved in 5 mL of methanol and 2 mL of THF under argon and 0.35 mL of 25% sodium methoxide in methanol was added and the reaction mixture was stined for 12 hr. Solid ammonium chloride was added and stined for 30 min. Careful evaporation of this biphasic reaction mixture provided white solid residue, which was washed three times by ethyl acetate. Concentration of ethyl acetate layer yielded the crude product, which was purified by preparative TLC (10% ethanol in toluene) to afford 28 in 69% yield (0.064 gm). R = 0.67 (10 ethanol in toluene), [α] + 14.6 (c 1, CHC13); 1H - NMR (CDCI3, 500 MHz) (selected protons) 7.65 (2H, d, J = 9 Hz), 7.60 (2H, d, J= 7.5 Hz), 5.22 (IH, d, J= 8 Hz), 5.10 (IH, br-s), 5.07 (2H, br-s), 3.06 (IH, m), 2.96 (IH, m), 2.24 (2H, d, J = 14.5 Hz), 1.68 (3H, s), 0.90 (9H, s), 0.07 (3H, s), 0.027 (3H, s). 13C-NMR (CDC13, 125 MHz)δ 169.6, 141.5, 140.9, 139.6, 139.3, 139.1, 138.97, 138.95, 138.89, 138.7, 138.6, 138.5, 138.39, 138.36, 138.3, 138.2, 138.0, 137.7, 132.6, 132.4, 129.0, 128.9, (128.7-127.3), 127.2, 126.7, 103.0, 102.0, 100.9, 100.0, 99.9, 98.3, 97.793.0, 82.5, 81.4, 80.2, 80.1, 79.7, 79.4, 78.8, 78.6, 78.1, 77.9, 77.8, 76.6, 76.0, 75.3, 75.2, 74.9, 74.8, 74.7, 74.4, 74.38, 74.35, 74.2, 73.9, 73.7, 73.6, 73.5, 73.49, 73.45, 73.37, 73.2, 73.1, 72.0, 71.9, 71.8, 71.4, 71.4, 71.3, 71.1, 70.0, 69.7, 69.1, 68.9, 68.7, 68.4, 67.9, 67.8, 66.7, 65.7, 58.8, 58.2, 57.2, 26.0, 23.6, 18.2, 1.2, -4.2, 5.4.
Figure imgf000131_0001
[0295] To the azeotropically dried 28 in a 25 mL round bottomed flask equipped with stir bar was added 0.5 mL IM acetic acid in THF and the reaction mixture was cooled to 0 °C. To this ice cooled reaction mixture was added 0.5 mL TBAF (IM in THF). The cooling bath was removed and the reaction mixture was stined for 3 hr. Additional 2 mL IM acetic acid in THF was added and the reaction mixture was stined for 15 min. The reaction mixture was evaporated to dryness and the crude product was purified by preparative TLC (10% ethanol in toluene) to afford 0.055 gm (89% yield)of 29. [α] + 9.40 (c 1, CHC13); Η - NMR (CDC13, 500 MHz) (selected protons) δ 7.66 (2H, d, J= 8 Hz), 7.60 (2H, d, J= 7.6 Hz), 5.17 (IH, d, J = 7.2 Hz), 5.07 (IH, br-s), 5.01 (2H, m), 2.34 (IH, br), 2.18 (IH, br), 1.61 (s, 3H),
Figure imgf000132_0001
[0296] Into a three necked round bottomed flask, equipped with dry ice acetone condenser was condensed 15 mL ammonia under argon. Sodium metal (0.095 gm, 153 equiv.) was added in three portions. The resulting blue solution was stined for 30 min at - 78 °C. The compound 29 (0.104 gm, 0.027 mmol) in 1.5 mL (3 x 0.5 mL) was added to the solution and the reaction mixture was stined for 2 hr. Solid ammonium chloride (0.263 gm, 4.97 mmol) was added to quench the reaction and the reaction mixture was warmed to room temperature slowly. Evaporation of the residual liquid provided solid residue, which was dissolved in 5 mL pyridine. To this mixture was added acetic anhydride (3 mL) and DMAP (5 mg) and the resulting mixture was stined with slowly warming to room temperature over 12 hr. The reaction mixture was evaporated to dryness and purified carefully by silica gel column chromatography to afford peracetate. The peracetate in 5 mL methanol was added solution of NaOMe, 25% by weight in methanol (0.4 mL) and was stined for 24 hr. The resulting cloudy solution was treated with water at 0 °C and stined for another 24 hr. The reaction mixture was neutralized using Amberlyst - 15 acidic regin and evaporated to afford crude product, which was purified by size exclusion chromatography using Bio-Gel P2 regin yielding 30 mg of free glycan.
Figure imgf000133_0001
[0297] Free glycan (10 mg) in 15 mL of saturated ammonium bicarbonate was heated at 40 °C. Additional ammoniumhydrogen carbonate was added time to time to keep the solution saturated. After two days of stirring the content of the flask was shell frozen, lyophilized, dissolved in water (10 mL), lyophilized; this process was repeated until the white solid residue reached constant mass of 10 mg, which was used directly in the next step.
Figure imgf000133_0002
[0298] Glycopeptide 34: [0299] A solution of acid 33 (6 mg, 0.007 mmol), HATU (5 mg, 0.013 mmol), DIEPA (1.7 μL, 0.012 mmol) in DMSO (0.1 mL) was stined for 10 min and transfened to a falcon tube (25 mL) containing 4.2 mg of 21. The solution was stined for 2 h and additional DIEPA (1.2 μL) was added. The reaction mixture was purified by semiprep HPLC column (30 to 50%B over 20 min) to afford Fmoc- protected glycopeptide (3.6 mg, 60%). LRMS (ESI) calcd for C1nH,77Ni2O64S2Na++ [M+H+Na]"1-1" 1394.5, found 1394.5. This Fmoc-protected glycopeptide was dissolved in 0.4 mL pipyridine/DMF (1 :4) solution and stined for 15 min and quenched by THF/H2O (10%) until the pH = 2-3. The crude mixture was purified on semiprep HPLC column (5 to 25%B over 20 min) to afford 34 (2 mg, 40%). LRMS (ESI) calcd for C96H167N,2O62S2Na++ [M+H+Na]^ 1283.5, found 1283.6. 1H NMR (400 MHz, CDCI3) selected signals: δ 4.99 (s, 1 H), 5.02 (s, 1 H), 5.16 (s, 1 H), 5.18 (s, 1 H), 5.25 (s, 1 H).
[0300] Glycopeptide 35:
[0301] To a solution of 34 (2 mg, 0.0008 mmol) in phosphorous buffer
(NaH2PO4 and Na2HPO4, pH=7.4, 0.5 mL) was added HSCH2CH2SO3Na (10 mg, 0.061 mmol) and stined for 2 days. TCEP (30 mg, 0.104 mmol) was then added and the resulting solution was stined for 1 h. The residue was purified on semiprep HPLC column (5 to 25%B over 20 min) to afford 35 (1.7 mg, 60%). LRMS (ESI) calcd for
Figure imgf000134_0001
[M+2HT 1228.5, found 1228.5. 1H NMR (400 MHz, CDCI3) selected signals: δ 4.90 (s, 1 H), 4.99 (s, 1 H), 5.15 (s, 1 H), 5.18 (s, 1 H), 5.25 (s, 1 H).
Figure imgf000135_0001
Glycopeptide, when dissolved in H2O and exposed to air, formed dimmer 36. HPLC (Varian Microsorb 100-5-C18) retention time, 12.75 min (0% to 40% acetonitrile in water). LRMS: (ESI) C184H317N24Oi24S2 : [M-i-3H]"H+: calculated: 1636.95, found, 1636.99.
Figure imgf000135_0002
[0302] To a 15 mL polypropylene conical tube equipped with stir bar in 0.2 mL DMSO was added peptide 33 (11 mg, 3 equiv.) and HATU (15 mg, 5.9 equiv.). The solution was stined for 1 min and was added diisopropyl ethyl amine (3.58 μL, 3 equiv.) and was stined for another minute. This orange-yellow solution was transfened via 0.5 mL syringe to the flask containing glycosylamine 31 (11 mg). The conical tube was rinsed with additional 0.1 mL of DMSO and transfened to the flask containing glycosyl amine using the same 0.5 mL syringe. Monitoring by LCMS showed that no additional product formation after 6 hr. Purification of the reaction mixture by size exclusion chromatography provided the 37. To this Fmoc protected 38 was added a 1 : 3 : 16 mixture of hydrazine : piperidine : DMF (200 μL). The resulting yellowish solution was stined for 30 min before addition of a solution of TFA to bring the pH to 3. The reaction mixture was purified by semiprep HPLC column (5 to 25%B over 25 min) to afford the Fmoc deprotected 38 in 30% yield. 1H - NMR (CDC13, 500 MHz) (selected protons) δ 4.78 (2H, d, J= 12.4 Hz), 4.70 (IH, d, J = 9.6 Hz), 4.57 (2H, d, J = 13.2 Hz), 4.26 (4H, m), 4.14 (IH, d, J = 7.2 Hz), 1.01 (9H, s), 0.575 (6H, m).
Figure imgf000136_0001
39
[0303] Compound 39 was prepared similar to 34.
ABBREVIATIONS AND GLOSSARY
[0304] A: alanine
[0305] Ac: acetyl
[0306] ACT: αl-antichymotrypsin
[0307] Ala: alanine
[0308] Arg: arginine
[0309] Asn: asparagine
[0310] Asp: aspartic acid
[0311] Bn: benzyl [0312] Boc: tert-butyloxycarbonyl
[0313] BPH: benign prostatic hyperplasia
[0314] BSP: benzenesulfinyl piperidine
[0315] Bu: butyl
[0316] Bz: benzoyl
[0317] CAN: eerie ammonium nitrate
[0318] coll: -f /w-collidine
[0319] C-terminus: peptide carbonyl terminus
[0320] Cys: cysteine
[0321] D: aspartic acid
[0322] DIEA: NN-diisopropylethylamine
[0323] DMF: dimethyl formamide
[0324] DMSO: dimethyl sulfoxide
[0325] DTBMP: di-tert-butylmethylpyridine
[0326] DTBP: di-tert-butylpyridine
[0327] Et: ethyl
[0328] Fmoc: 9-fluorenylmethyloxycarbonyl
[0329] G: glycine
[0330] Gal: galactose
[0331] Glc: glucose
[0332] Gin: glutamine
[0333] Glu: glutamic acid
[0334] Gly: glycine
[0335] H: histidine
[0336] HATU: 7-azahydroxybenzotriazolyl tetramethyluronium hexafluorophosphate
[0337] His: histidine
[0338] He: isoleucine
[0339] K: lysine
[0340] kDa: kilodaltons
[0341] KLH: keyhole limpet hemocyanin
[0342] L: leucine [0343] Leu: leucine
[0344] Lys: lysine
[0345] Man: mannose
[0346] MES-Na: 2-mercaptoethanesulfonic acid, sodium salt
[0347] MHC: major histocompatibility complex
[0348] N: asparagine
[0349] NAc: N-acetyl
[0350] ΝCL: native chemical ligation
[0351] Ν-terminus: peptide amine terminus
[0352] O-linked: linked through an ethereal oxygen
[0353] Pam3Cys: tripalmitoyl-S-glycerylcysteinylserine
[0354] PBS: phosphate-buffered saline
[0355] Ph: phenyl
[0356] Phth: phthalimido-
[0357] PMB: j3-methoxybenzyl
[0358] Pro: proline
[0359] Gpl20: prostate specific antigen
[0360] Py: pyridine
[0361] QS21 : a glycosteroidal immunoaduvant
[0362] R: arginine
[0363] S: serine
[0364] sat. aq.: saturated aqueous
[0365] Ser: serine
[0366] T: threonine
[0367] TBAF: : tetra-H-butylammonium fluoride
[0368] TBS: tert-butyldimethylsilyl
[0369] tBu: tert-butyl
[0370] Tf: trifluoromethanesulfonate
[0371] THF: tetrahydrofuran
[0372] Thr: threonine
[0373] t-Gpl20: total prostate specific antigen
[0374] Tip: tryptophan [0375] V: valine
[0376] Val: valine
[0377] W: tryptophan
- APPENDIX A -
Pierce Chemical Technical Library
cross-linking
Applications for Use of Cross-linkers
Cell Surface Cross-linking
To ensure cell-surface specific cross-linking for identification of surface receptors or their ligands, it is best to use membrane-impermeable cross- linkers. In the past, researchers used water-insoluble cross-linkers and carefully controlled the amount of cross-linker and the cross-linking duration. This prevented penetration of the membrane by the cross-linker and subsequent reaction with membrane proteins. Many references cite the use of membrane-permeable cross-linkers for cell surface cross-linking. Staros developed water-soluble sulfo-NHS analogs as alternatives to membrane permeable, homobifuπctional NHS-ester and imi- doester cross-linkers.'8 The sulfo-NHS-ester, ho- mobifuπctioπa! cross-linker BS3 (Product #21579) is very useful for cell surface cross-linking of ligands to receptors through primary amines on each. The sulfonyl groups attached to the succin- imidyl rings of sulfo-NHS cross-linkers make them membrane-impermeable and non-reactive with inner membrane proteins. Therefore, cross-linking time and quantity of cross-linker are less critical when using sulfo-NHS-esters. Pierce offers a variety of sulfo-NHS-ester cross-linkers, both homobi- functional and heterobifunctional. Homobifunc- tional sulfo-NHS-esters, heterobifunctional sulfo-NHS-esters and photoreactive phenyl azides are good choices for cross-linking on the surface of a cell. See Tables 3, 5 and 9 for specific characteristics and selection of cross-linkers for cell surface applications.
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cross-linking
Table 3: Homobifunctional NHS-Ester Cross-linkers (Continued)
DSP 22585 40442 12 A One of the most • Examining spatial relationships of the capsid polypeptides
Dιthιobιs(succιnιπιιdyl widely used cross- ofthe mengo virion" propionate) linkers, also known • Studying renal Na+ and K+-ATPase" as Lomants • Nearest neighbor relationships of bovine mitochondnal
Reagent " Water- H+-ATP" insoluble, thiol- • Producing interactions between protein components cleavable— can be of the chemotaxis mechanism in E coli" cleaved with 10-50 • Chemical cross-linking of a-CPI" mM DTT at 370C • Identifying cross-linked cytochrome P-450 in rat liver for 30 minutes or microsomes*
Figure imgf000142_0001
with 5% β-mercap- • Studying the influence of metal ions on prothrombin toethanol in SDS- self-association"
PAGE sample • Studying glycoprotein topology on intact human red blood buffer (2% SDS, cells"
625 mM Tris base, • Molecular identification of receptors for vasoactive
10% glycerol) at intestinal peptide in rat intestinal epithelium"
100°C for 5 minutes ■ Characterization of a cell surface receptor for colony stim ulating factor (CSF-2a)100 • Determining membrane antigens by covalent cross-linking to monoclonal antibodies'"
DTSSP [3,3'- 21577 60851 12 A Water-soluble analog Cross-linking the extracytoplasmic domain of the
Dithiobis (sulfosuccinim idyl of DSP" anion exchange channel in intact human erythrocytes" propionate) Cross-linking studies on Novikoff ascites hepatoma cytokeratin filaments"
Characterization of the B lymphocyte Fc receptor for IgE" Cross-linking platelet glycoprotein lb,H Characterization of a membrane-ribosome complex m B subti s""
Figure imgf000142_0002
DTSSP
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cross-linking
Table 3: Homobifunctional NHS-Ester Cross-linkers (Continued)
Figure imgf000143_0001
Sulfo-EGS 21566 66047 161 A Water-soluble analog of EGS 104
Ethylene glycobislsulfo- Reactions similar to EGS "" succimmidylsuccinate)
Figure imgf000143_0002
Sulfo-EGS
DST 20590 34424 64A Water-insoluble sample Cross-linking of ubiquinone cytochrome c reductase
Disuccimmidyl tartarate cross linked with DST (complex III)"0 in first-dimensional Characterization of the cell surface receptor for colony- gel Cleavable by stimulating factor (CSF-2a)'" soaking in 0015 M Cross-linking study of the Ca2', Mg!' activated adenosine sodium penodate, Iπphosphateof E coir
01% SDS, 002 M Human promyelocytic ceil line cross-linking of cell lysate
Figure imgf000143_0003
sodium phosphate, with DSP" pH 70 for 2 hours
DST (with several changes) at room temperature "0
Sulfo-DST 20591 54834 64 A Water-soluble analog of Disulfosucciπimidyl tartarate DST 100,110-112
Figure imgf000143_0004
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cross-linking
Table 3: Homobifunctional NHS-Ester Cross-linkers (Continued)
Figure imgf000144_0001
Sulfo-EGS 21566 660 7 161 A Water-soluble analog of EGS 104
Ethylene glycobιs(sulfo- Reactions similar to EGS1" succinimidylsucαnate)
Figure imgf000144_0002
Sulfo-EGS
DST 20590 34424 64A Water-insoluble sample • Cross-linking of ubiquinone cytochrome c reductase
Disuccinimidyl tartarate cross-linked with DST (complex III)"0 in first-dimensional • Characterization of the cell surface receptor for colony- gel Cleavable by stimulatiπg factor (CSF-2a)"' soaking in 0015 M • Cross-linking study of the Ca", Mg1' activated adenosine sodium penodate, tnphosphateof E coir 01%SDS, 002 M • Human promyelocytic cell line cross linking of cell lysate
Figure imgf000144_0003
sodium phosphate, with DSP" pH 70 for 2 hours
DST (with several changes) at room temperature '"
Sulfo-DST 20591 54834 64A Water-soluble analog of
Disulfosuccini idyl tartarate DST 100,110-112
Figure imgf000144_0004
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cross-linking
Table 5: NHS-Ester-Maleimide Heterobifunctional Cross-linkers
Figure imgf000145_0001
SMCC 22320 33433 11 6 A Water-insoluble, • Conjugation of glucose oxidase from Aspergillus
Succimmidyl noncleavable, very niger to rabbit antibodies"
4-(N-maleιmιdo- stable. Maleimide- • Conjugating Fab' to horseradish peroxidase'" '" "s ethyl) cyclo- reactive group • Conjugating affinity-purified antιdιgoxιn F(ab)2 hexane-1- fragments to B-galactosidase'M carboxylate • Enzyme labeling of antibodies and antibody fragments"
• Conjugating alkaline phosphatase and human IgG F(ab )2 fragments for phase change immunoassays'"
• Preparing immunogens """
Figure imgf000145_0002
SMCC
Sulfo SMCC mil 43637 11 6 A Water-soluble analog A comparison of maleimide containing heterobifunctional
Sulfo succimmidyl 4-(N- of SMCC, very stable cross-linkers in the conjugation of Fab' fragments maleimidomethyl) cyclo- maleimide-reactive to horseradish peroxidase'" hexane-1-carboxylate group This noncleavable Preparation of enzyme-antibody conjugates'" cross-linker is membrane impermeable
Figure imgf000145_0003
Sulfo-SMCC
MBS 22310 314 2 99 A Water-insoluble, • Preparing an insulin β-galactosιdase conjugate"' m Maleimidobenzoyl-N- noncleavable cross- • Conjugating hen egg ovalbumin with thiolated synthetic hydroxysucciπimide ester linker copolymers of D-glutamic acid and D-lysιne"'
• Preparing antibody β galactosidase conjugates'™
• Producing nciπ immunotoxins'" "!
• Preparing hapten-carπer protein conjugates from peptides'" """ "0-"'
• Coupling blastiαdin S to bovine serum albumin"'
• Preparing Fab'-β galactosidase conjugates'"
• Preparing synthetic peptide antigens for making antibodies to detect oncogene-related proteins'"
Figure imgf000145_0004
• Investigating the mechanism of cytotoxicity of diphtheria toxin coupled to antι-CD3 MAb"f
MBS • Preparing enzyme labeled vιomycιπ'B
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cross-linking
Table 5: NHS-Ester-Maleimide Heterobifunctional Cross-linkers (Continued)
Figure imgf000146_0001
Sulfo-MBS 22312 416.24 99 A Water-soluble analog An alternative method utilizing small quantities of ligand m-Maleimidobenzoyl-N- of MBS, noncleavable, for affinity purification of monospecific antibodies'" hydroxysulfosuccinimide ester membrane impermeable Coupling of antibody to β-D-galactosιdase'"
Figure imgf000146_0002
Sulfo-MBS
SMPB 22315 35632 14 5A Water-insoluble, Conjugation of preformed vesicles and Fab'
Succimmidyl 4-(p maleimido- extended spacer arm fragments in a study of liposomes as phenyl)-butyrate to limit steπc a carrier system'4' hindrance, noncleavable Attaching insulin molecules to reconstituted
Sendai virus envelopes'"
Targeting of loaded Sendai virus envelopes by covalently attached insulin molecules to virus receptor-depleted cells'"
Figure imgf000146_0003
Forming alkaline phosphatase-Fab' fragment conjugates for an enzyme immunoassay system'"
Preparing peptide-protein immunogens'"
Sulfo-SMPB 22319 45836 14 5A Water-soluble analog Studying the transport of the vaπent surface glycoprotein Sulfosuccinimidyl of SMPB. Extended of Trypanosome brucia'" 4-(p-maleιmιdophenyl)- spacer arm to limit Using aromatic cross-linkers such as Sulfo-SMPB to butyrate steπc hindrance, improve the yield of immunotoxin conjugates'" non-cleavable, membrane impermeable
Figure imgf000146_0004
Sulfo-SMPB
BMH 22319 27629 16.1A Water-insoluble homobifunctional Structural and functional studies of cross-linked Bismaleimidohexane cross-linker employing two G01 eg protein subunits'™ maleimide functional Studies of lymphocyte function-associated antιgen-3 groups, noncleavable (LFA-3)'"
Producing multimeπc forms of CD4'M
Figure imgf000146_0005
BMH
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cross-linking
Table 5: NHS-Ester-Maleimide Heterobifunctional Cross-linkers (Continued)
Figure imgf000147_0001
GMBS 22314 280.24 10.2 A Water-insoluble, Acylation of antibody to introduce maleimide groups"
N-(g-maleimidobutyryloxy) noncleavable. succini ide ester
Figure imgf000147_0002
GMBS
Sulfo-GMBS 22324 382.28 10.2 A Water-soluble analog 154-155
N-(g-maleimidobutyryloxy) of GMBS; noncleavable, sulfosuccinimide ester membrane impermeable.
Figure imgf000147_0003
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cross-linking
Table 9: Photoreactive Cross-linkers
Figure imgf000148_0001
ABH 21510 177 17 Hydrazide Glycoprotein receptor studies'6
Azidobenzoyl 21509 Phenylazide
Hydrazide
Figure imgf000148_0002
ABH
ANB-NOS 21551 30521 77 A NHS-ester Cross-linking cobra venom
N-5-Azιdo-2- Phenylazide phospholipase A2 aggregation state"' nitrobenzoyloxysuccinimide Photo-cross-linking of the signal sequence of nascent preprolactin to a polypeptide of the signal recognition particle"0
Figure imgf000148_0003
ANB-NOS
APDP 27720 446.55 Pyπdyl Cross-linking of protein subunits and ligand
N-[4-(p-azιdosalιcylamιdo) disulfide by introduction of disulfide bonds'" butyl]-3'(2'-pyπdyldιthιo) Phenylazide propionamide
Figure imgf000148_0004
APDP
APG 20107 19316 93 A Phenylazide Inhibiting bovine heart lactic dehydrogenase, p-Azidophenyl Phenyf glyoxal eggwhite lysozyme, horse liver alcohol glyoxal monohydrate dehydrogenase, and yeast alcohol dehydro genase'"
Cross-linking πbonucleic acid-protein in
-r@i E coli πbosomes"1
CHO* H,0 Identifying regions of brome mosaic virus coat protein chemically cross-linked in situ
APG to viral RNA'"
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cross-linking
Table 9: Photoreactive Cross-linkers (Continued)
Figure imgf000149_0001
24927 163A Carbonyl reactive Phenylazide
Figure imgf000149_0002
ASBA
ASIB 21511 41721 188 A lodoacetyl
1-(p-Azιdosalιcyl- Phenylazide amιdo)-4-(ιodo- acetamidojbutane
Figure imgf000149_0003
ASIB
BASED 21564 47454 Phenylazide Receptor location Bιs-[β 4- (homobifunctional) Near neighbor analysis azιdosalιcylamιdo)ethyl] Protein structural studies disulfide Appropriate in the absence of primary amines and thiols
Figure imgf000149_0004
BASED
HSAB 21560 26021 80A NHS-ester Photoaffinity labeling of peptide hormone
N-Hydroxysuccinimidyl- Phenylazide binding sites'"
4-azιdobenzoate Photoaffinity labeling of insulin receptor with an insulin analog1"
Identifying nerve growth factor receptor proteins in sympathetic ganglia membranes'"
Photoaffinity labeling the hormone receptor of both α and β subunits of
Figure imgf000149_0005
human choriogonadotropin'"
Isolating in situ cross-linked ligand- receptor complexes'"
Cross-linking vasoactive intestinal polypeptide to its receptors on intact human lymphocytes"
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cross-linking
Table 9: Photoreactive Cross-linkers (Continued)
Figure imgf000150_0001
Sulfo-HSAB 21561 36225 90A NHS-ester Photoaffinity labeling of peptide hormone
N-Hydroxysulfo-succinimidyl 4- Phenylazide binding sites'" azidobenzoate See applications for HSAB
Figure imgf000150_0002
Sulfo-HSAB
NHS-ASA 27715 27621 80A NHS-ester Photoaffinity labeling of "sl-AS-Con A to
N-Hydroxysucαnimidyl- Phenylazide erythrocyte ghosts'" 4-azιdosalιcylιc acid Deπvatizing human choπogonadotropin wιth'"l-NHS-ASA and photo-cross-linkmg the αB dimer'"
Radiolabeling D-glucose and cross-linking the sugar to the human erythrocyte mono saccharide transporter"'
Photoaffinity labeling of a bacterial
Figure imgf000150_0003
sialidase"1
NHS-ASA
Sulfo NHS-ASA 27725 37825 80A NHS-ester See applications/references for NHS-ASA N-Hydroxysulfo- Phenylazide succιnιmιdyl-4- azidosalicylic acid
Figure imgf000150_0004
Sulfo-NHS-ASA
Sulfo-NHS-LC-ASA 27735 491 41 18A NHS-ester See applications/references for NHS-ASA Sulfosuccmimidyl- Phenylazide (4-azιdosal icylamido) - hexanoate
Figure imgf000150_0005
Sulfo-NHS-LC-ASA
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cross-linking
Table 9: Photoreactive Cross-linkers (Continued)
PNP-DTP 20669 27615 • Diazo • Photoaffinity labeling of thyroid hormone nup-Nitrophenyl- clear receptors in intact cells'" '"
2-dιazo-3,3,3-tπfluoropropιonate
Figure imgf000151_0001
PNP-DTP
DTP 20670 • Sulfhydryls • Pierce offers this product for researchers
2-Dιazo-3,3,3,-trιfluoro- • Amines who require the acid chloride precursor of propionylchlonde PNP-DTP
N, 0
II II
F3C-C-C-CI
DTP
SADP 21552 352 38 139 A • NHS ester • Cross-linking concanavahn A to receptors
• Phenylazide on the human erythrocyte membrane"1 ■ Preparing photoactivatable glycopeptide reagents for site-specific labeling of lectins'"
• Attaching a Sendai viπon envelope and a mouse surface membrane polypeptide on newly infected cells'"
• Cross-linking platelet glycoprotein 1b'ra
Figure imgf000151_0002
Sulfo-SADP 21553 45445 139 A • NHS-ester • See applications/references for SADPimidyl-
Sulfosuccin • Phenylazide
(4-azιdophenyldιthιo) propionate
Figure imgf000151_0003
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cross-linking
Table 9: Photoreactive Cross-linkers (Continued)
Figure imgf000152_0001
SAED 33030 621 60 236A NHS-ester Functionally directed region specific
Sulfosuccinimidyl Phenylazide fluorescent labeling of proteins"'
2-(7-azιdo-4- Fluorescent Assessing conformational changes in the foot methylcoumaπn-3- protein of the sarcoplasmic reticulum by acetamide) ethyl- site directed fluorescent labeling'"
1,3'-dιthιopropιonate
Figure imgf000152_0002
Sulfo-SAMCA 33025 45834 128A NHS-ester Specific fluorescent labeling
Sulfosuccinimidyl 7-azιdo-4- Phenylazide methylcoumarιn-3 acetate Fluorescent
Figure imgf000152_0003
Sulfo-SAMCA
SAND 21549 57052 185 A NHS-ester Demonstration of the aggregation state
Sulfosuccinimidyl Phenylazide of Phosphohpase A2'H 2-(m-azιdo-o- nitrobenzamido) ethyl- 1,3'-dιthιopropιonate
Figure imgf000152_0004
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cross-linking
Table 9: Photoreactive Cross-linkers (Continued)
SANPAH 22588 39095 182 A • NHS-ester • Cross-linking ligand-receptor complexes in situ '"
Λ-succιnιmιdyl-6-(4'- • Phenylazide • Preparing photoactivatable glycopeptide azιdo-2'-nιtrophenyl- reagents for site-specific labeling of lectms'* amιno)hexanoate • Photoaffinity labeling of the N-formyl peptide receptor binding site of intact human polymorphonuclear leukocytes'"
• Cross-linking vasoactive intestinal peptide to receptors on intact human lymphoblasts"
Figure imgf000153_0001
Sulfo-SANPAH 22589 49239 182 A • NHS-ester • See applications/references for SANPAH
Sulfosuccinimidyl 6-(4'-azιdo-2'- • Phenylazide nitrophenylammojhexanoate
Figure imgf000153_0002
SASD 27716 541 51 189 A • NHS ester • Deπvatization of bacteπal lipopolysacchaπde'"
Sulfosuccinimidyl • Phenylazide • Identification of the muπne interleukin receptor
2-( azιdosalιcyl- and N for yl peptide receptor"1 amιdo)ethyl-1,3'- • Compaπson of SASD radiolabeling techniques"1 dithiopropionate • Cross-linking of factor V and Va to lodinated peptides'"
Figure imgf000153_0003
Sulfo-SAPB 21562 40432 128A NHS-ester
Sulfosuccinimidyl 4-(p-azιdophenyl)- Phenylazide butyrate
Figure imgf000153_0004
Sulfo-SAPB
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cross-linking
References
26 Staros, J V, Morgan, D G and App ng, D R (1981) A membrane-imper-meant, cleavable cross-linker J Biol Chem 256(11), 5890-5893
71 Pilch, PF and Czech, M P (1979) Interaction of crosslinking agents with the insulin effector system of isolated fat cells J Biol Chem 254, 3375-3381
72 Rebois, R V , Omedeo-Sale, F and Fishman, PH (1981) Covalent crosslinking of human choπonic gonadotropin to its receptors in rat testes Proc Natl Acad Sci USA 78, 2086 2089
73 Caamano, C A , Fernandez, H N and Paladani, A C (1983) Specificity of covalently stabilized complexes of 'Bl-labeled human so atotropin and components of the lactogenic binding sites of rat liver Biochem Biophys Res Comm 115, 29-37
74 Morgan, C J and Stanley, E R (1984) Chemical crosslinking of the mononuclear phagocyte specific growth factor CSF-1 to its receptor at the cell surface Biochem Biophys Res Comm 119, 35-41
75 Sen, I , Bull, H G and Sutler, R L (1984) Isolation of an angioteπsm II binding protein from liver Proc Natl Acad So USA 81, 1679 1683
76 Wood, C L and 0'Dorιsιo, M S (1985) Covalent crosslinking of vasoactive intestinal polypeptide to its receptors on intact human lymphoblasts J Biol Chem 260, 1243-1247
77 Petruzel , , Herrer, R , Garcia-Arenas, R and Rosen, R M (1985) Acquisition of insulin-dependent protein tyrosine kinase activity during Drosophiha embryogenesis J Biol Chem 226, 16072-16075
78 Cox, G W , Mattieson, B J , Giardina, S L and Varesio, L (1990) Characterization of IL-2 receptor expression and function on muπne macrophages J Immunol 145, 1719-1726
79 Vandlen, R L , Arcuπ, K E and Napier, M A (1985) Identification of a receptor for atπal natπuretic factor in rabbit aorta membranes by affinity crosslinking J Biol Chem 260, 10889 10892
80 Rashidbaigi, A , Langer, J A , Jung , Jones, C , Morse, R G , Tischfield, J A , Trill, J J , Kung, H -F and Pestka, S (1986) The gene for the human immune interferon receptor is located on chromosome 6 Proc Natl Acad Sci USA 83, 384 388
81 Tsudo, M , Kozak R W , Goldman, C K and Waldmann, T A (1986) Demonstration of a non-Tac peptide that binds interleukin 2 a potential participant in a multichain interleukin 2 receptor complex Proc Natl Acad Sci USAM, 9694-9698
82 Kehrl, J H , Taylor, A S , Delsing, G A , Roberts, A B , Sporn, M B and Fauci, A S (1989) Further studies in the role of transforming growth factor-β in human B cell function J Immunol 143, 1868-1874
83 Helmesta, D M , Hammonds, Jr , R G and Li, C H (1986) Preparation of ["Sl-Tyr", Leus] Bh-endorphin and its use for crosslinking of opioid binding sites in human stπatum and NG108-15 neuroblastoma-glioma cells Proc Natl Acad Sci USA 83, 4622-4625
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Figure imgf000155_0001
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Subunit Cross-linking and Protein cumstances, the cross-linking pattern or success Structural Studies may be affected by the cross-linker's solubility. Hydrophobic cross-linkers tend to cross-link more ef¬
Cross-linkers can be used to study the structure fectively in hydrophobic regions of molecules. and composition of proteins in biological samples.
If the three-dimensional structure of a protein is to Some proteins are difficult to study because they be determined or confirmed, cleavable cross-linkexist in different conformations under varying pH ers with increasing spacer arm lengths can be used or salt conditions. One way to avoid conformato determine the distance between two subunits. tional changes is to cross-link the subunits together. Amine-, carboxyl- or sulfhydryl-reactive Experiments using cross-linkers with different rereagents are employed for identification of particuactive groups may indicate the locations of specific amino acids. Once conjugated, the proteins are lar amino acids or for the determination of the subjected to two-dimensional electrophoresis. In number, location and size of subunits in a protein. the first dimension, the proteins are run under Short-to-medium spacer arm cross-linkers are senon-reducing conditions. The molecular weight of lected when intramolecular cross-linking is perthe non-reducing sample is recorded. It should be formed. If the spacer arm is too long, intermolecu- lar cross-linking can occur. Carbodii ides that noted that some of the subunits may not be cross- linked and will run according to their individual result in no spacer arm, along with short length molecular weights. Other subunits will be comconjugating reagents, such as amine-reactive bined and, under nonreducing conditions, will run DFDNB (Product #21524, Table 10) or the phoaccording to the combined molecular weight. The toactivatable amine-reactive cross-linker NHS-ASA second dimension of the gel is then run using con(Product #27715), can cross-link between sub- units without cross-linking to extraneous moleditions to cleave the cross-linked subunits. The individual molecular weights of the cross-linked sub- cules if used in optimal concentrations and condiunits can be determined. If the cross-linked tions. Slightly longer cross-linkers such as DMP subunits were not reduced, the pattern of the sec(Product #20666), DMS (Product #20668), DTBP ond dimension would be a diagonal. However, with (Product #20665), DSS (Product #21555) or DSP the cleavable cross-linker, the cross-linked sub- (Product #22585) can also cross-link between units will be released under reducing conditions, subunits, but they may result in intermolecular coupling. Intermolecular cross-linking can be conand the individual molecular weights of the sub- units will be approximated. The cleaved subunits trolled by adjusting the amount of cross-linker and will be off the diagonal. The molecular weights of the concentration of the material to be cross- the individual subunits should be compared with linked. Dilute protein solutions and high concenpre-determined molecular weights of the protein trations of cross-linker favor intramolecular cross- subunits under reducing SDS-polyacrylamide gel linking when homobifunctional cross-linkers are electrophoresis. employed. BMH (Product #22319) and other noncleavable, homobifunctional, sulfhydryl-reactive linkers can be used to link subunits of proteins that were joined by disulfide bonds. After reduction of the disulfides, and by cross-linking through the generated sulfhydryls, the protein will run as its full molecular mass using polyacrylamide gel elec- trophoresis and reducing conditions. In some cir¬
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cross-linking
Table 10: Bifunctional Aryl Halide
Figure imgf000160_0001
DFDNB 21524 204.1 3A Aryl halide-amine and Cross-linking phospholipids
1,5-Difluoro- sulfhydryl-reactive in human erythrocyte membranes'*
2,4-dinitrobenzene Coupling peptides to albumin'" Studies of near neighbor relationships of proteins in the myelin membrane"* Cross-linking cytochrome oxidase subunits'"
Figure imgf000160_0002
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cross-linking
Intermolecular Cross-linking for gation reaction of a particular cell membrane preparation to a known protein or radioactive label the Study of Protein Interactions in the presence of water-soluble or water-insoluble and Associations cross-linkers. Upon conjugation the cells may be
Cross-linkers are widely used for identification of washed, solubilized and characterized by SDS- near-neighbor protein relationships, ligand-recepPAGE. The gel electrophoresis results can be used tor identification and interactions, and enzyme- to determine whether the protein of interest was substrate orientations. The cross-linkers chosen conjugated. Any integral membrane protein will for these applications are usually longer than those conjugate in the presence of a water-insoluble used for subunit cross-linking. Homobifunctional, cross-linker, but not in the presence of water-soluamine-reactive NHS-esters or imidates and heterble cross-linkers. Surface membrane proteins obifunctional, amine-reactive, photoactivatable should conjugate in the presence of both water- phenyl azides are the most commonly-used cross- soluble and water-insoluble cross-linkers. linkers for these procedures. Occasionally, a BASED (Product #21564), a homobifunctional sulfhydryl- and amine-reactive cross-linker such photoactivatable phenyl azide, is one of the more as Sulfo-SMCC (Product #'s 22522, 22322) may versatile cross-linkers for the study of protein inbe employed if one of the two proteins or moleteractions and associations. It is cleavable and can cules is known to contain sulfhydryls. Cleavable or be radiolabeled with "5I using IODO-BEADS® lodi- noncleavable cross-linkers are typically used. Benation Reagent (Product #28665). After cleavage, cause the distances between two molecules are both of the dissociated molecules will still be iodi- not always known, the optimum length of the nated. Because both reactive groups on this cross- spacer arm of the cross-linker may be determined linker are nonspecific, the cross-linking is not deby the use of a panel of similar cross-linkers with pendent on amino acid composition for successful different lengths. DSS (Product #21555) or its conjugation. cleavable analog DSP (Product #22585) are among the shorter cross-linkers used for protein- SDBP (Product #22340) is a cross-linker that is protein associations. NHS-ester, phenyl azides are amine-reactive at both ends, but contains two difvery useful for this type of cross-linking because ferent reactive groups with varying reactivity. Please see Table 11 for more information on SDBP. they usually result in some successful, if not efficient, cross-linking. SASD (Product #27716) is a The reaction is controlled by temperature. SDBP is an NHS-ester with amine reactivity that is only unique sulfo-NHS-ester, photoactivatable phenylazide that is iodinatable and cleavable. Its characslightly affected by temperature; however, its second amine-reactive functional group is a dibro- teristics allow for detection and analysis of small quantities of protein. moacetyl group that is slow to react with amines at physiological pH at 4°C This cross-linker can be
Cross-linkers can be used to determine whether a useful for studying conformational changes in proparticular protein is located on the surface or the teins. integral part of the membrane. These studies are possible because water-soluble cross-linkers are membrane-impermeable, while water-insoluble cross-linkers are membrane-permeable. The experiment can be carried out by performing a conju¬
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cross-linking
Table 11: Heterobifunctional Amine-Reactive Cross-linker
Figure imgf000162_0001
SDBP 22340 328.96 5.0 A • NHS-ester • Preparation of immunotoxins"0
N-Hydroxysuccin• Alkyl dibromide imidyl 2,3-Dibromopropionate
Figure imgf000162_0002
SDBP
M W. 328.96 Spacer Arm 5.0 A
References
200. McKenzie, J.A., Raison, R.L. and Rivett, E.E. (1988). Development of a bifunctional crosslinking agent with potential for the preparation of immunotoxins. l Profe/π C/?em.7(5), 581-592.
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cross-linking doester cross-linkers (imidates) are water-soluble,
Cell Membrane Structural Studies but they are still able to penetrate membranes
Cell membrane structural studies require reagents DTBP (Product #20665) is an amine-reactive imi- of varying hydrophobicity to determine the locadoester that is cleavable by sulfhydryls Sulfhydryl- tion and the environment within a cell's lipid bi- reactive cross-linkers may be useful for targeting layer Fluorescent tags are used to locate proteins, molecules with cysteines to other molecules within lipids or other molecules inside and outside the the membrane membrane Various cross-linkers with differing
EDC (Product #'s 22980, 22981), water insoluble spacer arm lengths can be used to cross link prodicyclohexylcarbodiimide, or DCC (Product teins to associated molecules within the mem#20320), and other water-soluble and water-insolbrane to determine the distance between moleuble coupling reagents are used to study memcules Successful cross-linking with shorter branes and cellular structure,"" protein subunit cross-linkers is a strong indication that two molestructure and arrangement,"" enzyme-substrate cules are interacting in some manner Failure to interactions,'"' and cell surface59 and membrane obtain cross-linking with a panel of shorter cross- receptors *" The hydrophihc character of EDC can linkers, while obtaining conjugation with the use of result in much different cross-linking patterns in longer reagents, generally indicates that the molemembrane and subunit studies than with hycules are located in the same part of the membrane drophobic carbodiimides such as DCC S3SS Often it but are not interacting Homobifunctional NHS-esis best to attempt cross-linking with a water-soluters, imidates or heterobifunctional NHS-ester, ble and water-insoluble carbodiimide to obtain photoactivatable, phenyl azides are commonly a complete picture of the spacial arrangements or used for these procedures Because they are memprotein-protein interactions involved brane impermeant, sulfo-NHS-esters are not useful for cross-linking within the membrane Imi-
References
52 Buisson, M and Reboud, A M (1982) Carbodnmide-induced protein- RNA crosslinking in mammalian πbosomal subunits FEBS Lett 148(2), 247-250
53 Zurrer, H , Snozzi, M and Bachofen, R (1983) Specific binding of DCCD to reaction centers of the photosynthetic bacterium Rhodospirillυm rubrum and its effect of certain photosynthetic reactions FEBS Lett 153(1), 151-155
54 Lotscher, H -R , deJong, C and Capaldi, R (1984) Inhibition of the adenosinetπ phosphatase activity of Eschenchia coll F1 by the water-soluble carbodiimide 1 -ethyl-3-[3-(dιmethylamιno)propyl]carbodιιmιde is due to modification of several carboxyls in the β subunit Biochem 23(18), 4134-4140
55 Lotscher, H -R and Capaldi, R A (1984) Structural asymmetry of the F1 of Eschenchia colias indicated by reaction with dicyclohexylcarbodi-imide Biochem Biophys Res Comm 121(1), 331-339
56 Yamada, H , Imoto, T, Fujita, K , Okazaki, K and Motomura, M (1981) Selective modification of aspartic acιd-101 in lysozyme by carbodiimide reaction Biochem 20, 4836-4842
57 Davidson, V L , Jones, L H and Kumar, M A (1990) pH-dependent semiquinone formation by methylamme dehydrogenase from Parcoccus denitnicans Evidence for intermolecular electron transfer between quinone cofactors Biochem 29, 10786-10791
58 Gutweniger, H E , Grassi, C and Bisson, R (1983) Interaction between Cytochrome C and ubiquinoπe-Cytochrome C oxidoreductase a study with water- soluble carbodiimides Biochem Biophys Res Comm 116(1), 272-283
59 Grob, PM , Berlot, C and Bothwell, M A (1983) Affinity labeling and partial purification of nerve growth factor receptors from rat pheochromocytoma and human melanoma cells Proc Natl Acad Sci USA 80, 6819-6823
60 Taniuchi, M , Schweitzer, J B and Johnson Jr , E M (1986) Nerve growth factor receptor molecules in rat brain Proc Natl Acad Sci USA 83, 1950-1954 61 Taniuchi, M , Clark, H B and Johnson, Jr , E M (1986) Induction of nerve growth factor receptor in Schwann cells after axotomy Proc Natl Acad Sci USA tt, 4094-4098
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cross-linking
Immunotoxins Other water-soluble SPDP analogs, such as Sulfo- LC-SPDP (Product #'s 21650, 21649), are avail¬
Specific antibodies can be covalently linked to able for immunotoxin production, allowing for ease toxic molecules and then used to target antigens of use or avoidance of organic solvents. In addion cells. Often these antibodies are specific for tion, Sulfo-LC-SPDP and LC-SPDP (Product #'s tumor associated antigens. Immunotoxins are 21651, 21652) have longer spacer arms and can brought into the cell by surface antigens and, once offer better conjugation efficiency. internalized, they proceed to kill the cell by ribo- SMPT (Product #21558) is a reversible, NHS-ester, some inactivation or other means. The type of pyridyl disulfide cross-linker developed to provide cross-linker used to make an immunotoxin can afincreased stability of immunotoxins in vivo. The fect its ability to locate and kill the appropriate disulfide bond in SMPT is protected, making it less cells. For immunotoxins to be effective, the conjulikely to be cleaved in vivo prior to reaching the gate must be stable in vivo. In addition, once the antigenic target. In addition, the NHS-ester of immunotoxin reaches its target, it is important that SMPT is much more stable in aqueous solution the antibody be separable from the toxin to allow than typical NHS-ester compounds, showing little the toxin to kill the cell. Thiol-cleavable, disulfide- degradation even after several hours in aqueous containing conjugates have been shown to be solution. A water-soluble long chain version of more cytotoxic to tumor cells than noncleavable SMPT is also offered— Sulfo-LC-SMPT (Product conjugates of ricin A immunotoxins. Cells are able #'s 21569, 21568). to break the disulfide bond in the cross-linker, allowing the release of the toxin within the targeted cell.
SPDP (Product #'s 21757, 21657, 21557) is a reversible NHS-ester, pyridyl disulfide cross-linker used to conjugate amine-containing molecules to sulfhydryls. For several years, this has been the "workhorse" cross-linker for production of immunotoxins. The amine-reactive NHS-ester is usually reacted first with the antibody. In general, toxins do not contain surface sulfhydryls; therefore, sulfhydryls must be introduced onto them by reduction of disulfides, which is common for procedures involving ricin A chain and abrin A chain, or through chemical modification reagents. A second SPDP molecule can be used for this purpose. It is reacted with amines on the immunotoxin, then reduced to yield sulfhydryls. Another chemical modification reagent that is commonly used for production of immunotoxins is 2-imiπothiolane, also known as Traut's Reagent (Product #26101). Traut's Reagent reacts with amines and yields a sulfhydryl when its ring structure opens during the reaction.
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cross-linking
Carrier Protein Hapten/ very efficient and yield an immunogen that is capable of eliciting a good response upon injection. A Peptide/Polypeptide good choice of cross-linker with these characteristics is Pierce's Sulfo-SMCC (Product #22322). This Conjugates for Use as cross-linker is an amine-reactive NHS-ester that Immunogens contains a cyclohexyl group in its spacer and a very stable maleimide group at the other end of the mol¬
Pierce offers many products in this area of imecule. The maleimide of Sulfo-SMCC is more stable munological research. Easy-to-use kits are availthan the maleimide on other NHS-ester maleimide able for coupling ligands using several different cross-linkers because of the stability imparted by chemistries. These kits and the use of immunogens the cyclohexyl ring. Pierce uses Sulfo-SMCC to proare discussed in the Antibody Production Technical duce its entire selection of Maleimide Activated CarSection of this catalog. There are many cross-linkrier Proteins and Kits. Please see the Antibody Proers used for the production of these conjugates, duction Technical Section for additional and the best choice is dependent on the reactive information. Other cross-linkers that can be used to groups present on the hapten and the ability of the make immunogens are MBS (Product #'s 22510, hapten-carrier conjugate to function successfully 22310), SMPB (Product #'s 22316, 22315) and as an immunogen after its injection. Carbodiimides GMBS (Product #22314). Water-soluble analogs are good choices for producing peptide carrier conare also available, including Sulfo-MBS (Product #'s jugates because both proteins and peptides usually 22313, 22312), Sulfo-SMPB (Product #'s 22318, contain several carboxyls and primary amines. Car22317) and Sulfo-GMBS (Product #22324). bodiimides such as EDC (Product #'s 22980, SDBP (Product #22340) is a cross-linker that is 22981) react with carboxyls first to yield highly reamine-reactive at both ends, but contains two difactive unstable intermediates. The intermediates ferent reactive groups with varying reactivity. The can then couple to primary amines. Many different reaction is controlled by temperature. SDBP is an carboxyl- or amine-containing small molecules can NHS-ester with amine reactivity that is only slightly be attached to carrier proteins using this easy-to- affected by temperature; however, its second use chemistry. amine-reactive functional group is a dibromoacetyl
Other heterobifunctional cross-linkers can also be group, which is slow to react with amines at physiused to make immunogen conjugates. Often pepological pH at 4°C A possible application for this tides are synthesized with terminal cysteines to cross-linker is to allow the NHS group to react with allow for their attachment to supports or to carrier amines on the carrier protein. After quick removal proteins through a part of the molecule that is not of the excess cross-linker from the carrier protein, important for activity or recognition. Sulfhydryl-rean amine-containing hapten can be added to the soactive, heterobifunctional cross-linkers can be coulution, and the reaction can be allowed to warm to pled to carrier proteins through their other funcroom temperature, then proceed for several hours. tional group and then can be linked to peptides through terminal cysteines. This method can be
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Solid-Phase to be bound. Steric effects are usually most pronounced when the ligand is a small molecule. ReImmobilization action times are generally in the range of 1-3 hours for EDC coupling of molecules to solid supports.
Proteins, peptides and other molecules can be imThe amide bond formed by EDC coupling is relamobilized on solid-phase matrices for use as affintively stable, especially at neutral pH. ity supports or for sample analysis. The matrices may be agarose, beaded polymers, polystyrene Heterobifunctional cross-linkers that can be replates or balls, porous glass or glass slides, and acted in two-steps are often more useful and effinitrocellulose or other membrane materials. Some cient for producing solid-phase supports than hosupports can be activated for direct coupling to a mobifunctional cross-linkers. Amine-activated ligand. Other supports are made with nucleophiles supports can be converted to sulfhydryl-reactive or other functional groups that can be linked to supports using NHS-ester maleimide cross-linkers proteins or other ligands using cross-linkers. Carsuch as Sulfo-SMCC (Product #'s 22522, 22322). bodiimides such as DCC (Product #20320) and For some compounds that are difficult to immobiEDC (Product #'s 22980, 22981) are very useful lize, it may be possible to use NHS-ester, photoacfor coupling proteins to carboxy- and amine-acti- tivatable, phenyl azides to attach them to amine- vated glass, plastic and agarose supports. Caractivated supports. The photoactivatable, phenyl bodiimide procedures are usually one-step methazide is unreactive in the dark but, once exposed to ods; however, two-step methods are possible if the appropriate wavelength range of light, it bereactions are performed in organic solvents, or if comes extremely reactive and able to nonselec- NHS (Product #24500) or Sulfo-NHS (Product tively couple to almost any ligand. #24510) are used to enhance the reaction. The cross-linker DMP (Product #20666) has been
EDC is useful for coupling ligands to solid supemployed in the production of immobilized antiports.62* It can also be used to attach leashes onto bodies on protein A or protein G columns for use affinity supports and for subsequent coupling of as antigen purification supports.39 After antibody ligands. Useful spacers are diaminodipropylamine binds to the Fc-binding proteins, most or all of the (DADPA)," ethylenediamine, hexanediamine,63-61 6- antibody can be oriented so that the Fab region is amino-caproic acid,™* and any of several amino available for antigen recognition. DMP is applied to acids or peptides." Useful solid supports for imthe bound antibody column to link the two proteins mobilization are agarose,6"5 plastic," or cellulose through primary amines. matrices.66 Leashes become necessary to overcome steric effects when the ligand is immobilized too near the matrix to allow access by the molecule
References
39. Schneider, C, Newman, R.A., Sutherland, D.R., Asser, U. and Greaves, M.F. (1982). A one-step purification of membrane proteins using a high efficiency immunomatrix. J. Biol. Chem.257(18), 10766-10769.
62. Hermanson, G.T., Mallia, A.K. and Smith, P.K. (1992). Immobilized Affinity Ligand Techniques. California: Academic Press.
63. Stevens, D.A., Schreurs, J., Ihle, J.N. and May, W.S. (1991). Characterization of three related murine interleukin-3 surface receptor proteins. J. Biol. Chem. 266(7), 4151-4158.
64. Martzen, M.R., McMullen, B.A., Smith, N.E., Fujikawa, K. and Peanasky, RJ. (1990). Primary structure of the major pepsin inhibitor from the intestinal parasitic nematode Ascarissuum. Biochem.29, 7366-7372.
65. Burton, S.C, Haggarty, N.W. and Harding, D.R.K. (1991). Efficient substitution of 1,1'-carbonyldiimidazole activated cellulose and sepharose matrices with amino acyl spacer arms. J. Chrom.587, 271-275.
66. Mazid, M.A. and Kaplan, M. (1991 ). Immunoadsorbents with synthetic oligosaccharide hapten representing blood group A substances. Bioconjugate Chem. 2, 32-37.
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cross-linking
Protein-Protein Heterobifunctional cross-linkers are perhaps the best choices for antibody-enzyme or other protein- Conjugates to-protein cross-linking. Unwanted self-conjugation inherent when using homobifunctional NHS-
One of the most widely used applications for ester reagents or glutaraldehyde can be avoided cross-linkers is the production of protein-protein when using a reagent such as SMCC (Product #'s conjugates. Biological assays require methods for 22321 , 22320) or Sulfo-SMCC (Product #'s 22522, detection, and one of the most common methods 22322). Sulfo-SMCC is conjugated to one protein, for quantitation of results is to conjugate an enand the second is thiolated with SATA (Product zyme, fluorophore or other molecule to a protein #26102) or Traut's Reagent (Product #26101). Althat has affinity for one of the components in the ternatively, disulfides in the protein are reduced, biological system being studied. Antibody-enzyme and the two activated proteins are incubated toconjugates (primary or secondary antibodies) are gether to form conjugates that are free of dimers of among the most common protein-protein conjueither protein. Any of the other NHS-ester gates used. Secondary antibodies are relatively inmaleimide or pyridyl disulfide cross-linkers can be expensive and are available from Pierce (see the substituted for Sulfo-SMCC in this reaction Antibody Ordering Section of this catalog). Howscheme. Heterobifunctional photoactivatable ever, enzyme labeled primary antibodies are usuphenylazide cross-linkers are seldom used for ally expensive and can be difficult to obtain. Many making protein-protein conjugates because conjuresearchers find it necessary to label their primary gation efficiencies antibodies.
There are many reagents used for the production of antibody-enzyme conjugates. These have been produced by glutaraldehyde cross-linking in one- and two-step procedures. These conjugates are easy to make but often yield conjugates that give high background in immunoassays. Carbohydrate moieties of antibodies can be oxidized and then coupled to primary amines on enzymes, such as horseradish peroxidase, in a procedure called reductive alkylation or amination. These conjugates give less background in enzyme immunoassays and are relatively easy to prepare. Some self-conjugation of antibody may occur in the protocol. Homobifunctional NHS-ester or imidoester cross- linkers can be substituted for glutaraldehyde in a one-step protocol; however, polymerization and self-conjugation are still likely to occur. Homobifunctional sulfhydryl-reactive cross-linkers such as BMH (Product #22319) and DPDPB (Product #21701) may be useful if both proteins to be conjugated contain sulfhydryls.
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cross-linking
DNA/RNA Cross-linking to Proteins
Cross-linking of DNA or RNA to proteins is more limited because the reactivities of most cross-linkers favor protein-protein cross-linking over pro- tein-DNA cross-linking. To assist in these cross- linking methods, DNA probes are often synthesized with primary amines or thiols attached to specific bases. After insertion of the bases into DNA, amine- or sulfhydryl-reactive cross-linkers can be used for their conjugation to proteins. EDC (Product #'s 22980, 22981) has been reportedly used to cross-link RNA to ribosomal protein sub- units. Other specialized chemistries are reviewed in Wong's book, Chemistry of Protein Conjugation and Cross-linking (Product #15010) .
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cross-linking
Other Applications
There are many additional applications for cross- linkers that are either antiquated methods, new technologies or for more specialized needs. Older methods for peptide synthesis involve use of carbodiimide cross-linkers such as DCC (Product #20320) and EDC (Product #'s 22980, 22981) for the step-wise addition of individual amino acids to support bound peptides. Cross-linkers such as glutaraldehyde and dimethylpimelimidate have been used for tissue fixation. Newer cross-linkers are being developed that have more than two functional groups. Some trifuπctionals are already reported in the literature.
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- APPENDIX B -
TOTAL SYNTHESIS OF /V-LINKED GLYCOPOLYPEPTIDES
DESIGN AND PREPARATION OF ARTIFICIAL HIV ANTIGENS BASED ON THE 2G12 EPITOPE
COMPLICATING FACTORS IN CREATING EFFICIENT HIV VACCINE
□ Low antigenicity of the envelope spike surface (very little of surface is available for antibody binding)
■high degree of glycosylation ■oligomerization of constituent proteins
□ Low immunogenicity of the envelope proteins ( the mature oligomer typically stimulates only weak antibody response)
-4
© ■most of the epitopes on unprocessed gplβO are unaccessible on mature envelope
■strong initial response to gplβO may supress response to lower concentrations of mature oligomer
■"glycan shield" problem
■conformational masking of receptor binding sites
□ High degree of viral variation
Burton Proc. Natl. Acad. Sci. USA 1997(94) 10018-10023
Kwong et al. Nature 2002(420) 678-682
Wei et al. Nature 2003^422) 307-312
MAJOR STRATEGIES FOR CANDIDATE VACCINE DESIGN
1. Vaccination with attenuated virus
+ typically mature oligomer is displayed - safety issues
- low immunogenicity of viral oligomer
2. Vaccination with virus in an inactivated form
+ mature oligomer may be displayed - inactivation difficult without conformation disruption + less safety concerns - low immunogenicity
DNA immunization
+ mature oligomer may be displayed - needs a suitable carrier
- needs to ensure efficient gp160 processing
4. Immunization with a recombinant oligomeric molecule
+ immunization with mature oligomer - hard to fully reproduce correct oligomer conformation
- low immunogenicity
5. Preparation and immunization with epitope mimics of potent neutralizing antibodies
+ highly focused response could difficult to produce appropriate epitope mimics potentially be elicited (especially for discontinuous epitopes)
ADVANTAGES OF TARGETING ENVELOPE GLYCANS
The target carbohydrate epitope is
located on the "silent face" of gp120 easily accessible on mature trime c gp41 -gp120 complex does not overlap with receptor binding sites "entropically disfavored binding" problem is minimized
-4 onserved carbohydrate epitopes can be utilized
bility to use HIV "Glycan Shield" defense mechanism against the virus
MOLECULES BINDING GP120 CARBOHYDRATES
DC-SIGN Cyanovirin-N
Figure imgf000175_0001
Figure imgf000175_0002
high-mannose high-mannose/
Figure imgf000175_0003
hybrid
Femberg et al Science 2001 (294) 2163-2166 Botos et al J Biol Chem 2002 (227) 34336-34342 Sanders et al J Virology 2002 (76) 7293-7305 Scanlan et al J Virology 2002 (76) 7306-7321
MAPPING OF THE 2G12 EPITOPE ON GP120: AA SEQUENCE
Figure imgf000176_0001
256 295 335 355 386 410 445
Scanlan et al. J. Virology 2002 (76) 7306-7321
MAPPING OF THE 2G12 EPITOPE ON GP120: AA SEQUENCE
Figure imgf000177_0001
Summary:
• Binding is sensitive to mutations at N295, N332, N339, N386, N392
• N295, N332, N392 are highly conserved across a broad range of primary isolates
• 1-2 α-mannose linkages are important for 2g12 binding
Conclusion:
> Epitope is formed from high mannose type glycans at N295 and N332
Scanlan et al J Virology 2002 (76) 7306-7321
MAPPING OF THE 2G12 EPITOPE ON GP120: GLYCAN STRUCTURE
Figure imgf000178_0001
Bi-antcnnary Tri-anteimar Tetma-anteiinary ϊte disruption of 2G12 binding; Complex ect on b12 Complex Complex
endo F2: no effect
Figure imgf000178_0002
Conclusion: Hybrid High Mannose
Glycans involved in the epitope are more likely to be hybrid t an high-mannose type
Sanders et al., J Virology 76(14) 2002 p. 7293
FIRST GENERATION TARGET GLYCOPEPTIDES
HIV gp120316 335 Glycopeptide Fragments
Manαi — 2Manαi ^ 'R6 1 - "High-Mannose" type glycopeptide
-»-3 M ιvιaan,,ααi |vχ Manαi →- 2Manα1 6 Manβ1 _^ 4GlcNAcβ1 → 4GlcNAcβ1-NH
Manαi →- 2Manα1 →~2 Manαl^
H2N-Ala-Phe-Val-Thr-lle-Gly-Lys-lle-Gly-Asn-Met-Arg-Gln-Ala-His-Cys-Asn-lle-Ser-Arg-NH2
-4 -4
Manαi -*- 2Manα1 2. "Hybrid" type glycopeptide
Λ ^ Q MMaannαai Manαi →- 2Manα1 -^° x § Manβl →- 4GlcNAcβ1 — 4GlcNAcβ1-NH
Galccl -* 4GlcNAcβ1 →2 Manα1^"
H2N-Ala-Phe-Val-Thr-lle-Gly-Lys-lle-Gly-Asn-Met-Arg-Gln-Ala-His-Cys-Asn-lle-Ser-Arg-NH2
ADVANTAGES OF THE TOTAL SYNTHESIS STRATEGY
Total synthesis vs. Isolation form natural sources
1. Homogeneous, defined glycans are produced
> the molecule is made step by step, the structure is controlled throughout the synthesis
> microheterogeneiety problem is eliminated
> better defined therapeutic formulations
2. Larger quantities available
> hundreds of milligrams can be obtained compared with submilligram quantities typical for glycoprotein purification
-4
90
Lower cost
.. Structural modifications can easily be introduced
5. Glycomimetics and unnatural glycans can only be prepared by synthesis, and possibly used to
> improve stability
> overcome the "degeneracy in carbohydrate recognition" problem
> help induce cellular immune response
"N-LINKED" PROJECT IN DANISHEFSKY LABORATORIES
□ Glycal Assembly technology
□ Solid phase oligosaccharide synthesis
□ Synthesis of "Symmetrically" branched complex type glycans
□ Preparation 15-mer glycan carrying H-type blood group determinants:
Figure imgf000181_0001
GENERAL APPROACH TO N-LINKED GLYCOPEPTIDES
Figure imgf000182_0001
Native Chemical Ligation
Figure imgf000182_0002
Figure imgf000182_0004
Figure imgf000182_0003
Miller J., Dudkin V., Lyon G., Muir T., and Danishefsky J. Toward fully synthetic Λ/-linked glycoproteins. Angew Chem Int. Ed. 2003; 42(4), 431-434
PSA GLYCOPEPTIDES
normal and transformed PSA27-47 glycoforms
Figure imgf000183_0001
Figure imgf000183_0002
Galβl -^4GlcNAcβ1 ^fi r IRI AΓ\ MΔ Ri -*-2'V'anα^
Galβl *4GlcNAcβ1 ^4 §Manβ1 -^4GlcNAcβ1 -^4GlcNAcβ1 -NH
Galβl -4GlcNAcβ1 -2Manα1
H2N-Gly-Gly-Val-Leu-Val-His-Pro-Gln-Trp-Val-Leu-Thr-Ala-Ala-His-Cys-lle-Arg-Asn-Lys-Ser-NH2
Okada et al Biochim. Biophys. Acta-Gen. Subj. 2001, 1525, 149-160 Prakash, S.; Robbins, P. W. Glycobiology 2000, 10, 173-176.
A POTENTIAL PROSTATE CANCER DIAGNOSTIC ASSAY
Glycan Synthesis
Animal Immunization
Figure imgf000184_0001
Antibodies
Figure imgf000184_0002
Peptide Identify, Synthesis screen, isolate
Prostate Cancer Screen against normal
PSA and "transformed" PSA
Figure imgf000184_0003
Binding Assay
Antibodies selective for particular PSA glycoforms
CORE SUBSTITUTION IN GP120 AND PSA CARBOHYDRATES
Figure imgf000185_0001
90
Figure imgf000185_0002
CORE SUBSTITUTION IN GP120 AND PSA CARBOHYDRATES
Figure imgf000186_0001
NOVEL SYNTHETIC STRATEGY: BUILDING ASYMMETRICALLY BRANCHED GLYCANS
Figure imgf000187_0001
COMPLEX-TYPE HIGH-MANNOSE GLYCANS AND HYBRID-TYPE GLYCANS
SYNTHESIS OF PSA27'47 FRAGMENTS
Preparation of selectively deprotected pentasaccharides
Figure imgf000188_0001
Dudkin, Miller, and Danishefsky. submitted to J Amer Chem Soc
SYNTHESIS OF PSA27'47 FRAGMENTS
Preparation of "normal" PSA fragment
Figure imgf000189_0001
Figure imgf000189_0002
Dudkin, Miller, and Danishefsky. submitted io JAmer Chem Soc
SYNTHESIS OF PSA27'47 FRAGMENTS
Preparation of tribranched "transformed"
Figure imgf000190_0001
Figure imgf000190_0002
Dudkin, Miller, and Danishefsky. submitted to J Amer Chem Soc
SYNTHESIS OF PSA27'47 FRAGMENTS
Figure imgf000191_0001
Dudkin, Miller, and Danishefsky. submitted to JAmer Chem Soc
GENERAL STRATEGY FOR GP120 FRAGMENTS PREPARATION
"mannose layers" or
Figure imgf000192_0001
"blocks"
Figure imgf000192_0002
NCL or direct coupling
Figure imgf000192_0003
2G12/MAN-9 CRYSTAL STRUCTURE
Domain exchange in 2g12 antibody
Figure imgf000193_0001
Calarese et al Science 2003 (300) 2065-2071
2G12/MAN-9 CRYSTAL STRUCTURE
Figure imgf000194_0001
Calarese et al. Science 2003 (300) 2065-2071
GLYCAN CLUSTERING: POTENTIAL SOLUTIONS
Second-generation targets: multiple glycans in a single construct
Figure imgf000195_0001
GLYCAN CLUSTERING: POTENTIAL SOLUTIONS
Figure imgf000196_0001
Mimicking a clustered epitope:
Φ Synthesis of a real glycopeptide fragment Φ Placing glycans on an "artificial" linker system Φ Multiple conjugation to a carrier protein
CURRENTLY AVAILABLE FRAGMENTS
type: high mannose hybrid
Manαi -»2Manα1 --..£ free glycans
Manαi Manαi -«-2Manα1 Manαi -»2Manα1 -""2 , i Manαi
§ Manβl —4GlcNAcβ1 -»4GlcNAcβ1 -OH Manαi -«-2Manα1 • ,
§ Manβl →4GlcNAcβ1 — 4GlcNAcβ1 -OH Manαi -*-2Manα1 -*-2 Manαi•""' Ga!(t1 - ► 4GIcKAc(!1 — 2 Manαi'"'
Manαi -«-2Manα1 glycosylamines
Manαi Manαi -*2Manα1 Manαi -*-2Manα1 , '§ Manαi
§ Manβl — 4GlcNAcβ1 — 4GlcNAcβ1 -NH2 Manαi -»2Manα1 ,
§ Manβl — 4GlcNAcβ1 — 4GlcNAcβ1 -NH2
Manul -*- 2Manα1 -*-2 Manαl^ i* -<■ 4GlcNAc|)1 — 2Manα1^
gpl∑ό33'1335 fragments
Manαi -*-2Manα1
[Manαi Manαi -«-2Manα1 V,
§ Manβl — 4GlcNAcβ1 Manβl -► 4GlcNAcβ1 — 4GlcNAcβ1 -NH
Manαi -— 2Manα1 — -2ManαA
Figure imgf000197_0001
H2N-Cys-Asn-lle-Ser-Arg-NH2
Figure imgf000197_0002
SECOND GENERATION TARGETS
1. Multiple monoglycosylated fragments/ carrier protein conjugates
available immediately
2. Gp120 fragments with multiple glycosylation/ carrier protein
potentially requires significant synthetic effort
Multiple glycans on a linker system (polypeptide or non-peptide)/ carrier protein
may be easily accessible potential solution to building a clustered epitope

Claims

What is claimed is:
An isolated compound having the structure:
Figure imgf000199_0001
(I) wherein each occurrence of R1 is independently hydrogen or an oxygen protecting group; eeaacchh ooccccurrence of R2A and R2B is independently hydrogen or a nitrogen protecting group; each occurrence of R is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000199_0002
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R5, R6 and R7 is independently hydrogen, OH, OR', NRnR , NHCOR, F, CH2OH, CH2OR', or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R1, R" and R"1 is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COORlv, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R'", taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occurrence of R,v is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group;
W1, W2 and W3 are independently optionally substituted mannose, galactose or lactosamine moieties; and wherein R4 is -OR4Λ or -NHR4Λ; wherein R4A is hydrogen, aliphatic, heteroaliphatic, aryl, heteroaryl, an amino acyl moiety, an amino acyl residue of a peptide, an amino acyl residue of a protein, or R4A comprises a protein, peptide or lipid moiety covalently linked to the rest of the construct, or to the N or O atom to which it is attached, either directly or through a crosslinker; with the proviso that the compound is not a naturally occurring gpl20 glycoprotein or a compound having the structure:
Figure imgf000200_0001
or
Figure imgf000201_0001
2. The compound of claim 1, wherein W3 is R1, R3, as defined above, or a moiety having the structure:
Figure imgf000201_0002
wherein X is -OR1 or -NR2ΛR2B; and each occurrence of R8 is independently R1 or a sialic acid moiety.
3. The compound of claim 1, wherein W1 and W2 are independently R1, R3 or a moiety having the structure:
Figure imgf000201_0003
wherein each occurrence of R8 is independently R1 or a sialic acid moiety.
The compound of claim 1 having the structure:
Figure imgf000202_0001
The compound of claim 1 having the structure:
Figure imgf000202_0002
wherein X is OR1 or NR^ 2AARπ2B
6. The compound of claim 1, 4 or 5, wherein each occurrence of R1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, -Si(RIA)3, -C(=O)R1A - C(=S)R1A, -C(=NR1A)R1B, -SO2R1A, wherein R1A and R1B are each independently hydrogen, alkyl, alkenyl, alkynyl cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, -C(=O)Rlc or - ZR1C, wherein Z is -O-, -S-, -NRID, wherein each occurrence of R1C and R1D is independently hydrogen, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety.
7. The compound of claim 6, wherein each occurrence of R1 is independently hydrogen, alkylaryl, -Si(RIA)3 or -C(=O)R1A
8. The compound of claim 7, wherein each occurrence of R1 is independently hydrogen, Bn or Bz.
9. The compound of claim 1 , 4 or 5, wherein for each occurrence of -NR2AR2B, at least one occurrence of R Aor R B is independently a nitrogen protecting group.
10. The compound of claim 1, 4 or 5, wherein each occurrence of NR AR2B, R2A and R2B is independently hydrogen, alkyl, alkenyl, -C(=O)R2C, -C(=O)OR2C, -SR2C, SO R , or R and R , taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; wherein each occurrence of R2C is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, -C(=O)R2D or -ZR2D, wherein Z is -O-, -S-, -NR2E, wherein each occurrence of R D and R is independently hydrogen, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety.
11. The compound of claim 1, 4 or 5, wherein for each occurrence of-NR2AR2B, at least one occurrence of R2A or R2B is independently -C(=O)R2A or SO2R2A; or R2A and R2B, taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety.
12. The compound of claim 11, wherein for each occurrence of -NR2AR2B, at least one occurrence of R2A or R2B is independently acyl, -SO2Ph or R2A and R2B, taken together with the nitrogen atom to which they are attached, form an azide or a substituted or unsubstituted phthalimide moiety.
13. The compound of claim 5, wherein X is -OR1 :
14. The compound of claim 1 , wherein each occurrence of R3 is independently hydrogen, alkylaryl, -Si(R3A)3 or -C(=O)R3A, wherein R3A is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, -C(=O)R or -ZR , wherein Z is - O-, -S-, -NR , wherein each occurrence of R and R is independently hydrogen, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety.
15. The compound of claim 1, 4 or 5, wherein each occurrence R1 and R3 is independently hydrogen, alkylaryl, -Si(R3A)3 or -C(=O)R3A, wherein R3A is hydrogen, alkylaryl, -Si(R3A)3 or -C(=O)R3A, wherein R3A is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, -C(=O)R3B or -ZR3B, wherein Z is
-O-, -S-,
Figure imgf000204_0001
is independently hydrogen, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety.
16. The compound of claim 1, 4 or 5, wherein R is -OR 4A and the saccharide unit bearing R4 has the structure:
Figure imgf000204_0002
wherein R1, R2A and R2B are as defined generally above and in classes and subclasses herein; R4A is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, an amino acyl moiety, an amino acyl residue of a peptide, an amino acyl residue of a protein, - Si(R4B)3, -C(=O)R4B, -C(=S)R4B, -C(=NR4B)R4C, -SO2R4B, wherein R4B and R4C are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl, heteroaryl, -C(=O)R4D or -ZR4D, wherein Z is -O-, -S-, -NR4E, wherein each occurrence of R4D and R4E is independently hydrogen, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety; or R4A comprises a protein, peptide or lipid moiety covalently linked to the O atom to which it is attached, either directly or through a crosslinker.
17. The compound of claim 1 , 4 or 5, wherein R4 is -NHR4A and the saccharide unit bearing R >4 has the structure:
Figure imgf000205_0001
wherein R1, R Λ and R U are as defined generally above and in classes and subclasses herein; and R is hydrogen, aliphatic, heteroaliphatic, aryl, heteroaryl, an amino acyl moiety, an amino acyl residue of a peptide, an amino acyl residue of a protein, or R4A comprises a protein, peptide or lipid moiety covalently linked to the rest of the construct, or to the N atom to which it is attached, either directly or through a crosslinker.
18. The compound of claim 17, wherein R4A is hydrogen.
19. The compound of claim 17, wherein R4A comprises an amino acyl residue of a peptide whose structure is either identical or closely related to that of gpl20 near an N-glycosylation site.
20. The compound of claim 19, wherein the amino acyl residue is Asn.
21. The compound of claim 1, 4 or 5, wherein R4 is -NHR4A wherein R4A comprises an Asparagine residue (Asn) of a peptide whose structure is either identical or closely related to that of gpl20 near an N-glycosylation site and the saccharide unit bearing R4 has the structure:
R2AR2BN
Cys-Asn-lle-Ser-Arg; or
R2AR2BN
Lys-lle-Gly-Asn-Met-Arg-Gln-Ala-His-Cys— Asn — lle-Ser-Arg Gly-lle-Thr-Val-Phe-Ala wherein any of the amino acid residues may bear one or more protecting groups.
22. The compound of claim 21, wherein the saccharide unit bearing R4 has the structure:
Figure imgf000206_0001
23. The compound of claim 21, wherein the saccharide unit bearing R has the structure:
Figure imgf000207_0001
24. The compound of claim 1 having the structure:
Figure imgf000207_0002
25. The compound of claim 1 having the structure:
Figure imgf000207_0003
26. The compound of claim 1 having the structure:
Figure imgf000208_0001
27. The compound of claim 1 having the structure:
Figure imgf000208_0002
28. A multi-antigenic construct comprising one or more carbohydrate domains having the structure:
Figure imgf000208_0003
wherein each occurrence of R1 is independently hydrogen or an oxygen protecting group; each occurrence of R2A and R2B is independently hydrogen or a nitrogen protecting group; each occurrence of R3 is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000209_0001
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R5, R6 and R7 is independently hydrogen, OH, OR1, NR11^11, NHCOR1, F, CH2OH, CH2OR', or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R', R" and Rm is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COORlv, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R1", taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occurrence of R1V is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group;
W , W and W are independently optionally substituted mannose, galactose or lactosamine moieties; wherein each carbohydrate domain is independently covalently bound to a linker system, said linker system being a peptide or non-peptide nature, and wherein the linker system may be cyclic or acyclic.
29. The construct of claim 28, wherein W is R , R , as defined in claim 28, or a moiety having the structure:
Figure imgf000210_0001
wherein X is -OR or -NR > A RD2B ; and each occurrence of R is independently
R1 or a sialic acid moiety.
30. The construct of claim 28, wherein W and W are independently R , R or a moiety having the structure:
Figure imgf000210_0002
8 1 wherein each occurrence of R is independently R or a sialic acid moiety.
31. The construct of claim 28, wherein one or more carbohydrate domains have the structure:
Figure imgf000210_0003
32. The construct of claim 28, wherein one or more carbohydrate domains have the structure:
Figure imgf000211_0001
33. The construct of claim 28, wherein the linker system is a peptide.
34. The construct of claim 28, wherein the linker system is designed to approximate the spatial position(s) of carbohydrate(s) in gpl20.
35. The construct of claim 28, wherein the linker system is further attached to a carrier immunostimulant.
36. The multi-antigenic construct of claim 28, wherein the construct comprises a backbone made up of two or more amino acids or other structural units, wherein one or more of said amino acids or structural units is/are independently substituted with a glycosidic moiety having the structure:
-LH wherein each occurrence of L is independently a substituted or unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydiate domain of formula:
Figure imgf000212_0001
σdet)
Figure imgf000212_0002
(IIdet) or
Figure imgf000212_0003
(IIIdet)
wherein each occurrence of R1 is independently hydrogen or an oxygen protecting group; each occurrence of R2A and R2B is independently hydrogen or a nitrogen protecting group; each occurrence of R3 is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000213_0001
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R5, R6 and R7 is independently hydrogen, OH, OR1, NR'R111, NHCOR', F, CH2OH, CH2OR', or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R", R" and R"1 is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COOR'v, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R'", taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occurrence of R'v is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group;
W , W and W are independently optionally substituted mannose, galactose or lactosamine moieties.
37. The construct of claim 36 having the structure:
Figure imgf000214_0001
wherein q is 0 or 1; each occurrence of s is independently an integer from 2-20; t is an integer from 1 -6;
RX1 is hydrogen, alkyl, acyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl) or a nitrogen protecting group; or RX1 is covalently bound to a substituent on the last occurrence of the spacer, thereby forming a cyclic backbone;
R is hydrogen or an immunogenic carrier; each occurrence of the structural unit SU is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; each occurrence of the spacer is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; the linker is either a free carboxylic acid, -O-, (carboxamido)alkyl carboxamide, MBS, primary carboxamide, mono- or dialkyl carboxamide, mono- or diarylcarboxamide, linear or branched chain (carboxy)alkyl carboxamide, linear or branched chain (alkoxycarbonyl)alkyl-carboxamide, linear or branched chain (carboxy)arylalkylcarboxamide, linear or branched chain
(alkoxycarbonyl)alkylcarboxamide, an oligoester fragment comprising from 2 to about 20 hydroxy acyl residues, a peptidic fragment comprising from 2 to about 20 amino acyl residues, or a linear or branched chain alkyl or aryl carboxylic ester; each occurrence of L1 is independently a substituted or unsubstituted aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydrate domain of formula (Idet), (IIdet) or (IIIdet).
38. The construct of claim 37, wherein each occurrence of L is independently - O(CHRaa)n- or -NHC(=O)(CHRaa)n- wherein each occurrence of n is independently an integer from 1-10; and each occurrence of R33 is hydrogen, lower alkyl, aryl, heteroaryl, -alkyl(aryl) or -alkyl(heteroaryl).
39. The construct of claim 37, the structural unit SU, for each occurrence, is independently an amino acid residue, a peptidyl moiety, a bivalent aryl or heteroaryl moiety or a substituted or unsubstituted d-όalkylidene or C2.6alkenylidene chain wherein up to two non-adjacent methylene units are independently optionally replaced by CO, CO2, COCO, CONRzl, OCONRzl, NRZ1NRZ2, NRzlNRZ2CO, NRzlCO, NRzlCO2, NRzlCONRZ2, SO, SO2, NRzlSO2, SO2NRzl, NRzlSO2NRZ2,
71 71 79
O, S, or NR ; wherein each occurrence of R and R is independently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl.
40. The construct of claim 39, wherein each occurrence of the structural unit SU is an amino acid residue, and the clustered multi-antigenic construct has the structure:
Figure imgf000215_0001
wherein q is 0 or 1 ; each occurrence of s is independently an integer from 2-20; t is an integer from 1-6;
RX1 is hydrogen, alkyl, acyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl) or a nitrogen protecting group;
R is hydrogen or an immunogenic carrier; each occurrence of the spacer is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; the linker is either a free carboxylic acid, -O-, (carboxamido)alkyl carboxamide, MBS, primary carboxamide, mono- or dialkyl carboxamide, mono- or diarylcarboxamide, linear or branched chain (carboxy)alkyl carboxamide, linear or branched chain (alkoxycarbonyl)alkyl-carboxamide, linear or branched chain (carboxy)arylalkylcarboxamide, linear or branched chain
(alkoxycarbonyl)alkylcarboxamide, an oligoester fragment comprising from 2 to about 20 hydroxy acyl residues, a peptidic fragment comprising from 2 to about 20 amino acyl residues, or a linear or branched chain alkyl or aryl carboxylic ester; each occurrence of L1 is independently a substituted or unsubstituted aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydrate domain of formula (Ide,), (IIdet) or (IIIdet).
41. The construct of claim 37 or 40, wherein the spacer, for each occurrence, is independently -(CHRsp)n-, where n is 1-8 and each occurrence of Rsp is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, -alkyl(aryl), - alkyl(heteroaryl), -ORspl, -SRspl or-NRsplRsp2 where Rspl andRspl are independently hydrogen or lower alkyl; a peptidyl moiety comprising one or more α-amino acid residues, or a bivalent aryl moiety having the structure:
Figure imgf000216_0001
42. The construct of claim 40 having the structure:
Figure imgf000216_0002
/ \
X = |— LIN <ER - CROSSLINKER J) — R
\ q wherein each occurrence of L1 is independently -O CHR33),,- or - NHC(=O)(CHRaa)n- wherein each occurrence of n is independently an integer from 1-10; and each occurrence of Raa is hydrogen, lower alkyl, aryl, heteroaryl, - alkyl(aryl) or -alkyl(heteroaryl); each occurrence of Rsp is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl), -ORspl, -SRspl or -NRsplRsp2 where Rspl and Rspl are independently hydrogen or lower alkyl; or a peptidyl moiety comprising one or more α-amino acid residues; sl, s2 and s3 are independently integers from 2-5;
Aj-A3 are independently a carbohydrate domain of formula (Id t), (IIdet) or (IIIdet), and are different from each other; and
R is hydrogen, alkyl, acyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl) or a nitrogen protecting group.
43. The construct of claim 42 having the structure:
Figure imgf000217_0001
wherein R, R*2, Rsp, sl, s2 and s3 and A A3 are as defined in claim 30; each occurrence of n is independently an integer from 1-10; and each occurrence of Raa is hydrogen, lower alkyl, aryl, heteroaryl, -alkyl(aryl) or -alkyl(heteroaryl).
44. The construct of claim 37 or 40, wherein each occurrence of A is independently a carbohydrate domain having one of the following structures:
Figure imgf000218_0001
45. The construct of claim 40 having the structure:
Figure imgf000218_0002
wherein the linker is -O-, -NRG-, -NRG(CRHRJ)ICNRI -, NRG(CRHRj) NRK(C=O)(CRHRj)kS-, -(CRHRj)kNRK-, -O(CRHRj)kNRK-, an oligoester fragment comprising from 2 to about 20 hydroxy acyl residues, a peptidic fragment comprising from 2 to about 20 amino acyl residues, or a linear or branched chain alkyl or aryl carboxylic ester, wherein each occurrence of k is independently 1-5; wherein each occurrence of RG, RH, RJ or RK is independently hydrogen, a linear or branched, substituted or unsubstituted, cyclic or acyclic alkyl moiety, or a substituted or unsubstituted aryl moiety; wherein the crosslinker is a moiety derived from a crosslinking reagent capable of conjugating the carrier with the linker; wherein the carrier is a peptide, protein or lipid; wherein n is 1, 2, 3 or 4; wherein q is 0 or 1 ; wherein each occurrence of RA, RB and Re is independently hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl; and wherein each occurrence of RD, RE and RF are each independently a carbohydrate domain of formula (Idet), (IIdet) or (IIIdet).
46. The construct of claim 37, 40 or 45 wherein the linker is a moiety having the structure -NH(CH2)tNHC(=O)(CH2)vS- wherein t and v are each integers from 1-6.
47. The construct of claim 46 wherein , t is 3 and v is 1.
48. The construct of claim 40 having the structure:
Figure imgf000219_0001
wherein m', n' and p' are integers between about 8 and 20; j is an integer between 1 and about 8;
Rv, RA> RB and Re are independently hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl; and
RD, RE and Rp are each independently a carbohydrate domain of formula fldet), (πdet) or (mdet).
49. The construct of claim 40 having the structure:
Figure imgf000220_0001
wherein n and p are each independently an integer from 1-6; m', n' and p' are independently integers between about 8 and 20; j is an integer between 1 and about 8;
R is a nitrogen protecting group;
Rv, and RA, RB, Re, RE and RF, for each occurrence, are independently hydrogen, substituted or unsubstituted linear or branched lower alkyl or substituted or unsubstituted phenyl; each occurrence of RD is independently a carbohydrate domain of formula (Idet), (IIdet) or (IIIdet).
50. The construct of claim 48 or 49 wherein each occurrence of RD, RE and RF is independently a carbohydrate domain having one of the following structures:
Figure imgf000220_0002
or
Figure imgf000221_0001
51. A dimeric glycopeptide having the structure:
Figure imgf000221_0002
wherein each peptide may be the same or different; and each occurrence of A is independently a carbohydrate domain having the structure:
Figure imgf000221_0003
de
(iαeι)
Figure imgf000222_0001
(IIdet) or
Figure imgf000222_0002
(IIIdet)
wherein each occurrence of R is independently hydrogen or an oxygen protecting group; each occurrence of R2A and R2B is independently hydrogen or a nitrogen protecting group; each occurrence of R is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000222_0003
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R5, R6 and R7 is independently hydrogen, OH, OR1, N 'R"1, NHCOR1, F, CH2OH, CH2OR', or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R', R" and R1" is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COOR'v, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R'", taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occurrence of R'v is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group;
W1, W2 and W3 are independently optionally substituted mannose, galactose or lactosamine moieties.
52. The glycopeptide of claim 51 , wherein each occurrence of L1 is independently -©(CHR33^- or -NHC(=O)(CHRaa)n- wherein each occurrence of n is independently an integer from 1-10; and each occurrence of Raa is hydrogen, lower alkyl, aryl, heteroaryl, -alkyl(aryl) or -alkyl(heteroaryl).
53. The glycopeptide of claim 52, wherein each occurrence of L1 is an aspartyl side chain.
54. The glycopeptide of claim 52, wherein the peptide has a structure that is either identical or closely related to that of gpl20 near an N-glycosylation site.
55. The glycopeptide of claim 54, wherein the peptide comprises the amino acid sequence: Cys- Asn-lle-Ser-Arg, wherein any one or more of the amino acid residues may bear one or more protecting groups.
56. The glycopeptide of claim 54, wherein the peptide comprises the amino acid sequence: Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His-Cys- Asn-Ile-Ser-Arg, wherein any one or more of the amino acid residues may bear one or more protecting groups.
57. The glycopeptide of claim 52 having the structure:
Figure imgf000224_0001
wherein each occurrence of A is independently a carbohydrate domain having one of the structures:
Figure imgf000224_0002
or
Figure imgf000225_0001
58. The glycopeptide of claim 52 having the structure:
Figure imgf000225_0002
wherein each occurrence of A is independently a carbohydrate domain having one of the structures:
Figure imgf000225_0003
or
Figure imgf000226_0001
59. A method for preparing an isolated compound having the structure:
Figure imgf000226_0002
wherein each occurrence of R1 is independently hydrogen or an oxygen protecting group; each occurrence of R A and R2B is independently hydrogen or a nitrogen protecting group; each occurrence of R is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000226_0003
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R5, R6 and R7 is independently hydrogen, OH, OR', NR^R1'1, NHCOR1, F, CH2OH, CH2OR, or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R', R" and R'" is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COOR'v, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R"1, taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occurrence of R'v is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group;
1 9 ^
W , W and W are independently optionally substituted mannose, galactose or lactosamine moieties; said method comprising steps of:
(a) providing an α-O-protected carbohydrate construct having the structure:
Figure imgf000227_0001
wherein R is hydrogen or a suitable oxygen protecting group; (b) reacting the construct of step (a) under suitable conditions to form a β- amino carbohydrate construct having the structure:
Figure imgf000227_0002
(c) reacting said β-amino carbohydrate construct under suitable conditions with a peptide whose structure is either identical or closely related to that of gpl20 near an N-glycosylation site and which comprises a -CH2CO H moiety, to form a glycopeptide having the structure:
Figure imgf000228_0001
60. The method of claim 59, wherein in the step of reacting the carbohydrate construct of step (a) under suitable conditions to form the β-amino carbohydrate construct, Kochetkov amination conditions are used.
61. The method of claim 60, wherein NH4HCO3/H2O is used.
62. The method of claim 59, wherein, in the β-amino carbohydrate construct of
1 " step (b), each occurrence of R and R is hydrogen and each occurrence of - NR2AR2B is -NHAc.
63. The method of claim 59, wherein, in the step of reacting the β-amino carbohydrate construct under suitable conditions with a peptide whose structure is either identical or closely related to that of gpl20 near an N-glycosylation site, the reaction conditions comprise HATU and Hunig's base is a suitable solvent.
64. The method of claim 63, wherein the peptide has the following structure:
Figure imgf000229_0001
wherein R is hydrogen or -StBu.
65. The method of claim 59, wherein the glycopeptide of step (c) has the structure:
Figure imgf000229_0002
66. The method of claim 59, wherein, in the β-amino carbohydrate construct formed in step (b), each occurrence of R1 is hydrogen, each occurrence of-NR2AR2B is -NHAc.
67. The method of claim 59, wherein the α-O-protected carbohydrate construct of step (a) has the structure:
Figure imgf000230_0001
and the glycopeptide formed in step (c) has the structure:
Figure imgf000230_0002
68. The method of claim 59, wherein the α-O-protected carbohydrate construct of step (a) has the structure:
and the glycopeptide formed in step (c) has the structure:
Figure imgf000231_0001
69. The method of claim 59, wherein the α-O-protected carbohydrate construct of step (a) has the structure:
Figure imgf000231_0002
and the glycopeptide formed in step (c) has the structure:
Figure imgf000231_0003
70. The method of claim 59, wherein the α-O-protected carbohydrate construct of step (a) has the structure:
Figure imgf000232_0001
wherein MCA represent monochloroacetate and the glycopeptide formed in step (c) has the structure:
Figure imgf000232_0002
71. The method of claim 59, further comprising a step of subjecting the glycopeptide formed in step (c) to Native Chemical Ligation conditions in the presence of a suitable polypeptide to form a glycopolypeptide having the structure:
Figure imgf000233_0001
72. The method of claim 71, wherein the polypeptide comprises the amino acid sequence: Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His-Cys- Asn-Ile-Ser-Arg wherein any one or more of the amino acid residues may bear one or more protecting groups or a moiety suitable for Native Chemical Ligation.
73. The method of claim 71 , wherein the polypeptide has the structure: Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His -SR; where R is a functional group suitable for effecting chemical ligation; and the resulting glycopeptide has the structure:
Figure imgf000233_0002
74. The method of claim 71, wherein the polypeptide has the structure: Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His -SR; where R is a functional group suitable for effecting chemical ligation; and the resulting glycopeptide has the structure:
Figure imgf000234_0001
75. A method for preparing an α-O-protected carbohydrate construct having the structure:
Figure imgf000234_0002
wherein each occurrence of R9 is independently Bz or Ac; said method comprising steps of:
(a) coupling a trisaccharide having the structure:
Figure imgf000234_0003
with a monosaccharide having the structure:
Figure imgf000235_0001
wherein R , 10 is lower alkyl or aryl; in the presence of an activating agent under suitable conditions to form a protected tetrasaccharide having the structure:
Figure imgf000235_0002
(b) partially deprotecting the protected tetrasaccharide formed in step (a) under suitable conditions to form a partially deprotected tetrasaccharide having the structure:
Figure imgf000235_0003
(c) coupling the partially deprotected tetrasaccharide formed in step (b) with a monosaccharide having the structure:
Figure imgf000235_0004
wherein R10 is lower alkyl or aryl; in the presence of an activating agent under suitable conditions to form a protected pentasaccharide having the structure:
Figure imgf000236_0001
(d) partially deprotecting the pentasaccharide formed in step (c) under suitable conditions to form a partially deprotected pentasaccharide having the structure:
Figure imgf000236_0002
(e) coupling the partially deprotected pentasaccharide formed in step (d) with a monosaccharide having the structure:
Figure imgf000236_0003
wherein R10 is lower alkyl or aryl; in the presence of an activating agent under suitable conditions to form an octasaccharide having the structure:
Figure imgf000236_0004
(f) partially deprotecting the octasaccharide formed in step (e) under suitable conditions to form a partially deprotected octasaccharide having the structure:
Figure imgf000237_0001
(g) coupling the partially deprotected octasaccharide formed in step (f) with a monosaccharide having the structure:
Figure imgf000237_0002
in the presence of an activating agent under suitable conditions to the α-O- protected carbohydrate construct.
76. A method for preparing an α-O-protected carbohydrate construct having the structure:
Figure imgf000237_0003
said method comprising steps of:
(a) coupling a trisaccharide having the structure:
Figure imgf000238_0001
with a monosaccharide having the structure:
Figure imgf000238_0002
wherein R1 is lower alkyl or aryl; in the presence of an activating agent under suitable conditions to form a protected tetrasaccharide having the structure:
Figure imgf000238_0003
(b) partially deprotecting the protected tetrasaccharide formed in step (a) under suitable conditions to form a partially deprotected tetrasaccharide having the structure:
Figure imgf000238_0004
(c) coupling the partially deprotected tetrasaccharide formed in step (b) with an ethylthioglycoside having the structure:
Figure imgf000238_0005
under suitable conditions to form a protected hexasaccharide having the structure:
Figure imgf000239_0001
(d) partially deprotecting the hexasaccharide formed in step (c) under suitable conditions to form a partially deprotected hexasaccharide having the structure:
Figure imgf000239_0002
(e) coupling the partially deprotected hexasaccharide formed in step (d) with a monosaccharide having the structure:
Figure imgf000239_0003
wherein R10 is lower alkyl or aryl; in the presence of an activating agent under suitable conditions to form an heptasaccharide having the structure:
Figure imgf000239_0004
(f) partially deprotecting the heptasaccharide formed in step (e) under suitable conditions to form a partially deprotected heptasaccharide having the structure:
Figure imgf000240_0001
(g) coupling the partially deprotected heptasaccharide formed in step (f) with a monosaccharide having the structure:
Figure imgf000240_0002
in the presence of an activating agent under suitable conditions to the α-O- protected carbohydrate construct.
77. An antibody or antibody fragment which is specific to any one or more of the carbohydrate antigens present on a multi-antigenic glycoconjugate comprising one or more carbohydrate domains having the structure:
Figure imgf000240_0003
(I) wherein each occurrence of R is independently hydrogen or an oxygen protecting group; each occurrence of R and R is independently hydrogen or a nitrogen protecting group; each occurrence of R3 is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000241_0001
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R5, R6 and R7 is independently hydrogen, OH, OR1, NR'R111, NHCOR1, F, CH2OH, CH2OR', or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R', R" and R'" is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COOR'v, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R'", taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occurrence of R'v is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group;
W1, W2 and W3 are independently optionally substituted mannose, galactose or lactosamine moieties; wherein each carbohydrate domain is independently covalently bound to a linker system, said linker system being a peptide or non-peptide nature; and wherein the linker system may be cyclic or acyclic; and wherein said antibody is a purified polyclonal antibody or a monoclonal antibody.
78. The antibody or antibody fragment of claim 77, wherein, in the antigen, a carbohydrate domain has the structure:
Figure imgf000242_0001
79. The antibody or antibody fragment of claim 77, wherein a antigen has the structure:
Figure imgf000242_0002
wherein X is OR1 1 o „r_ NτRr>2ΛARr Z2B
80. The antibody or antibody fragment of claim 77, wherein the antigen is a multi-antigenic construct comprising a cyclic or acyclic peptidic or non-peptidic backbone made up of two or more structural units, wherein one or more of said structural units is/are independently substituted with a glycosidic moiety having the structure:
■H wherein each occurrence of L1 is independently a substituted or unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydrate domain of formula:
Figure imgf000243_0001
ιd"> or
Figure imgf000243_0002
(in dαeeι)
wherein each occurrence of R1 is independently hydrogen or an oxygen protecting group; each occurrence of R2A and R2B is independently hydrogen or a nitrogen protecting group; each occurrence of R is independently hydrogen, a protecting group or a carbohydrate domain comprising a saccharide moiety having the structure:
Figure imgf000244_0001
wherein Y is NH or O; wherein a, b and c are each independently 0, 1 or 2; d is an integer from 1-3; with the proviso that the d bracketed structure represents a furanose or pyranose moiety and the sum of b and c is 1 or 2; wherein R° is hydrogen, a linear or branched chain alkyl, acyl, arylalkyl or aryl group; wherein each occurrence of R5, R6 and R7 is independently hydrogen, OH, OR1, N 'R111, NHCOR', F, CH2OH, CH2OR', or a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein each occurrence of R', R" and R1" is independently hydrogen, a protecting group, a sialic acid moiety, CHO, COOR'v, or a substituted or unsubstituted linear or branched chain alkyl, acyl, arylalkyl or aryl group, or R" and R"1, taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted heterocyclic or heteroaryl moiety; and wherein each occurrence of R'v is independently H, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group; and
1 9 "
W , W and W are independently optionally substituted mannose, galactose or lactosamine moieties.
81. The antibody or antibody fragment of claim 80, wherein the antigen has the structure:
Figure imgf000245_0001
wherein q is 0 or 1 ; each occurrence of s is independently an integer from 2-20; t is an integer from 1-6;
R is hydrogen, alkyl, acyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl) or a nitrogen protecting group; or RX1 is covalently bound to a substituent on the last occurrence of the spacer, thereby forming a cyclic backbone;
R is hydrogen or an immunogenic carrier; each occurrence of the structural unit SU is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; each occurrence of the spacer is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; the linker is either a free carboxylic acid, -O-, (carboxamido)alkyl carboxamide, MBS, primary carboxamide, mono- or dialkyl carboxamide, mono- or diarylcarboxamide, linear or branched chain (carboxy)alkyl carboxamide, linear or branched chain (alkoxycarbonyl)alkyl-carboxamide, linear or branched chain (carboxy)arylalkylcarboxamide, linear or branched chain
(alkoxycarbonyl)alkylcarboxamide, an oligoester fragment comprising from 2 to about 20 hydroxy acyl residues, a peptidic fragment comprising from 2 to about 20 amino acyl residues, or a linear or branched chain alkyl or aryl carboxylic ester; each occurrence of L1 is independently a substituted or unsubstituted aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydrate domain of formula
(idet), (iιdet) or (iπdet).
82. The antibody or antibody fragment of claim 80, wherein the antigen has the structure:
Figure imgf000246_0001
wherein q is 0 or 1 ; each occurrence of s is independently an integer from 2-20; t is an integer from 1-6;
R is hydrogen, alkyl, acyl, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl) or a nitrogen protecting group;
R is hydrogen or an immunogenic carrier; each occurrence of the spacer is independently a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, heteroaryl or peptidic moiety; the linker is either a free carboxylic acid, -O-, (carboxamido)alkyl carboxamide, MBS, primary carboxamide, mono- or dialkyl carboxamide, mono- or diarylcarboxamide, linear or branched chain (carboxy)alkyl carboxamide, linear or branched chain (alkoxycarbonyl)alkyl-carboxamide, linear or branched chain (carboxy)arylalkylcarboxamide, linear or branched chain
(alkoxycarbonyl)alkylcarboxamide, an oligoester fragment comprising from 2 to about 20 hydroxy acyl residues, a peptidic fragment comprising from 2 to about 20 amino acyl residues, or a linear or branched chain alkyl or aryl carboxylic ester; each occurrence of L1 is independently a substituted or unsubstituted aliphatic or heteroaliphatic moiety; and each occurrence of A is independently a carbohydrate domain of formula (Idet), ide ) or (IIIdet).
83. The antibody or antibody fragment of claim 80, wherein the antigen has the structure:
Figure imgf000247_0001
wherein the linker is -O-, -NRG-, -NRG(CRHRj)kNRK-, NRG(CRHRj)kNRκ(C=O)(CRHRj)kS-, -(CRHRj)kNRK-, -O(CRHRj)kNRK-, an oligoester fragment comprising from 2 to about 20 hydroxy acyl residues, a peptidic fragment comprising from 2 to about 20 amino acyl residues, or a linear or branched chain alkyl or aryl carboxylic ester, wherein each occurrence of k is independently 1-5; wherein each occurrence of RG, RH, RJ or RK is independently hydrogen, a linear or branched, substituted or unsubstituted, cyclic or acyclic alkyl moiety, or a substituted or unsubstituted aryl moiety; wherein the crosslinker is a moiety derived from a crosslinking reagent capable of conjugating the carrier with the linker; wherein the carrier is a peptide, protein or lipid; wherein n is 1 , 2, 3 or 4; wherein q is 0 or 1 ; wherein each occurrence of RA, RB and Re is independently hydrogen, substituted or unsubstituted linear or branched chain lower alkyl or substituted or unsubstituted phenyl; and wherein each occurrence of RD, RE and RF are each independently a carbohydrate domain of formula (Idet), (IIdet) or (IIIdet).
84. The antibody or antibody fragment of claim 80, wherein the antigen has the structure:
Figure imgf000248_0001
wherein n and p are each independently an integer from 1-6; m', n' and p' are independently integers between about 8 and 20; j is an integer between 1 and about 8;
R is a nitrogen protecting group;
Rv, and RA, RB, RC, RE and Rp, for each occurrence, are independently hydrogen, substituted or unsubstituted linear or branched lower alkyl or substituted or unsubstituted phenyl; each occurrence of RD is independently a carbohydrate domain of formula (Idet),
(iιdet) or (iπdet).
85. The antibody or antibody fragment of claim 80, wherein the antigen has the structure:
Figure imgf000248_0002
wherein each peptide may be the same or different; and each occurrence of A is independently a carbohydrate domain of formula (Idet), (IIdet) or (IIIdet).
86. The antibody or antibody fragment of claim 85, wherein the antigen has the structure:
Figure imgf000249_0001
wherein each occurrence of A is independently a carbohydrate domain having one of the structures:
Figure imgf000249_0002
87. The antibody or antibody fragment of claim 85, wherein the antigen has the structure:
Figure imgf000250_0001
wherein each occurrence of A is independently a carbohydrate domain having one of the structures:
Figure imgf000250_0002
88. The antibody or antibody fragment of claim 77, wherein the antibody is a monoclonal antibody.
89. A pharmaceutical composition comprising an effective amount of a construct of claim 28 or 29; in admixture with a pharmaceutically suitable diluent or carrier.
90. A composition for eliciting an immune response in a subject comprising an effective amount of a construct of claim 28 or 29, said amount being effective to induce antibodies in a subject; in admixture with a suitable immunogenic stimulant.
91. The composition of claim 90 wherein the immunogenic stimulant comprises Salmonella minnesota cells, bacille Calmette-Guerin or QS21.
92. A method of eliciting antibodies in a subject comprising administering to the subject a composition of claim 90.
93. A method of preventing infection with HIV comprising administering to a subject an effective amount of a composition of claim 90.
94. A method of treating an HIV infection comprising administering to a subject in need thereof an effective amount of a composition of claim 90.
95. A method of eliciting an immune response wherein the response is directed against an antigen comprising a carbohydrate epitope expressed on the surface of gpl20, said antigen being a construct of claim 28 or 29.
96. The method of claim 94 further comprising administering an adjuvant.
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