WO1997035202A1 - Bibliotheque de ligands a base d'hydrates de carbone, dosage et procede correspondants - Google Patents

Bibliotheque de ligands a base d'hydrates de carbone, dosage et procede correspondants Download PDF

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Publication number
WO1997035202A1
WO1997035202A1 PCT/US1997/004639 US9704639W WO9735202A1 WO 1997035202 A1 WO1997035202 A1 WO 1997035202A1 US 9704639 W US9704639 W US 9704639W WO 9735202 A1 WO9735202 A1 WO 9735202A1
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carbohydrate
glycosyl
distinct
mmol
solid support
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PCT/US1997/004639
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English (en)
Inventor
Daniel E. Kahne
Jennifer Loebach
Lin Yan
Rui Liang
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Princeton University
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Priority to JP53375297A priority Critical patent/JP2001504800A/ja
Priority to AU23413/97A priority patent/AU2341397A/en
Priority to EP97916164A priority patent/EP0901629A4/fr
Publication of WO1997035202A1 publication Critical patent/WO1997035202A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives

Definitions

  • the invention relates to a library of carbohydrate-based ligands, which are bound to and presented on a solid support to permit multivalent interactions with a variety of probes having a plurality of carbohydrate binding sites. Methods of the preparation of the library, the library's characteristics and an assay for selecting particular ligand-probe interactions are also described.
  • Carbohydrates play central roles in a wide variety of normal and abnormal biological recognition processes. Among their less benign roles, carbohydrates on cell surfaces have been implicated in chronic inflammation, in viral and bacterial infection, and in tumorigenesis and metastasis. Strategies to block the interactions between cell surface carbohydrates and their protein targets could provide an effective means of preventing, treating, or alleviating the effects of various diseases. Therefore, the identification of ligands, which bind to the protein targets better (i.e., with greater affinity) than the natural cell surface carbohydrates, would be of great interest as potential candidates for mediating biologic or physiologic processes.
  • N-linked glycoside species are described.
  • covalent attachment of the sugar to the peptide is accomplished through an amide bond formed by the condensation of the amino group of an aminosugar with the carboxylic acid group of a resin-bound peptide.
  • the preparation of a multitude of sugar-containing compounds, including 0-linked glycoside species, bound to a solid support, in which new and distinct glycosyl bonds are formed substantially concurrently, has not been described.
  • combinatorial strategies for identifying ligands against particular receptors are evaluated by determining if it is possible with the particular strategy to "pull out" (i.e., to selectively locate and identify) the binding ligand from a library that contains the binding ligand along with a great many other components.
  • the binding ligand which may be a natural or unnatural ligand of the receptor under study, must bind significantly more tightly to the receptor than most of the other compounds in the library; otherwise, the binding ligand cannot be identified easily.
  • Carbohydrates are unusual ligands for proteins because they are relatively hydrophilic and bind with relatively low affinity to their receptors. Accordingly, many naturally occurring carbohydrate ligands bind only weakly to their receptors.
  • Standard in vi tro assays show only a small difference in affinity between the natural ligand and other ligands. Hence, it is usually impossible to select the natural carbohydrate ligand for a carbohydrate- binding protein from a mixture of carbohydrates in solution.
  • the ideal assay must be capable of presenting multiple carbohydrate ligands in the appropriate spatial arrangement to permit multivalent binding.
  • the ideal assay must, therefore, be able to present the multiple ligands with some degree of flexibility, particularly conformational flexibility.
  • Such flexibility permits the individual ligands to orient themselves independently into the optimum position and conformation for binding, yielding ultimately the lowest-energy (and thermodynamically most favored) ligand-receptor geometry.
  • the carbohydrate ligands will be anchored in proximity to one another, greatly reducing the entropy loss that would otherwise weigh against the desired multivalent binding.
  • ⁇ ctins ectins are a group of naturally occurring proteins having the ability to agglutinate erythrocytes and many other types of cells.
  • the term "lectin” may be used to designate any sugar-binding protein or glycoprotein of non-immune origin, which agglutinates cells or precipitates glycoconjugates . They are known to exhibit a variety of unusual biological properties, including specific interactions with human blood groups, induction of lymphocyte proliferation, preferential agglutination of mouse tumor cells over cells from normal tissue, agglutination of virally or chemically transformed cell lines, mediation of nuclear envelope phosphorylation, or facilitation of bone marrow transplants in patients having severe combined immunodeficiency.
  • Lectins are widely distributed in nature and can be found primarily in seeds of plants, although they can also be found in roots, leaves and bark.
  • a lectin of particular interest is that isolated from the seeds of the camel's foot tree, Bauhinia purpurea.
  • This lectin first reported in 1958, is a well-known agglutinin of human red blood cells. The specificity of this lectin for various carbohydrates has been studied extensively, and it is generally accepted that it agglutinates red cells by binding to the O- ⁇ -D-galactopyranosyl- (1 ⁇ 3) -N-acetyl-D- galactosamine group present in the mucin component of the red cell membrane.
  • the lectin consists of four identical subunits, each of which binds to a carbohydrate ligand, and thus this lectin is an example of a multivalent carbohydrate-binding protein (i.e., a potential probe "having a plurality of carbohydrate binding sites") .
  • Such an assay should preferably take advantage of polyvalent binding to allow for the selection and identification of low- affinity binding ligands, which would normally be undifferentiated (and remain undetected) in a solution binding affinity-based assay.
  • the preparation of such a library and the demonstration of such an assay to locate and detect a particular member or members of the library, which bind to a multivalent probe or receptor of interest, would be a great advance in the art .
  • the present invention a method has been developed for synthesizing and screening combinatorial libraries of polyvalent carbohydrate ligands that can be used to investigate key issues involving carbohydrate recognition.
  • the present finding shows that there is not a good correlation between the monovalent affinities and polyvalent avidities of carbohydrate ligands.
  • the presentation of carbohydrates on the polymer surface has a profound influence on the interaction of the ligand with the protein receptor.
  • the present invention exhibits an unanticipated degree of specificity in carbohydrate recognition and suggest that carbohydrates may play a greater function as recognition signals in Nature than has been recognized previously.
  • the present invention is directed to a library of carbohydrate ligands from which specific compounds can be isolated, which compounds exhibit affinity binding characteristics selective for a given receptor.
  • a library of carbohydrate- based ligands bound to a solid support is described in which multiple copies of specific ligands are generated and presented on the surface of the solid support in a way that allows a polyvalent receptor molecule to undergo a multivalent binding interaction with a plurality of carbohydrate-based ligands.
  • Such an approach uniquely exploits the additional binding interactions possible with multivalent receptors and enhances the probability that equally unique binding substances can be isolated from a collection of inherently low affinity binding carbohydrate-based ligands.
  • the preparation of carbohydrate-based ligands bound to a multivalent support is described.
  • the present method gives rise to the preparation of a collection of distinct carbohydrate- based ligands bound to a solid support, which method includes a glycosyl bond-forming step.
  • a plurality of glycosyl acceptors and glycosyl donors can be used, substantially concurrently, each combination giving rise to a newly formed glycosyl bond and, hence, a distinct carbohydrate-based ligand.
  • the resulting library of carbohydrate substances can have a prodigious number of molecularly diverse members.
  • the library of the invention mimics the polydentate binding properties of naturally occurring cell- surface carbohydrates. This mimicry is surprising in that the individual carbohydrate moieties are not in a fixed geometric relationship to one another.
  • the preferred solid support presents a multiplicity of each distinct carbohydrate ligand in spatial proximity, but with relative geometric flexibility, so that the relative orientation of potential binding ligands of the receptor which leads to effective, selective binding is permissible.
  • ligands may, for example, display potential agonist or antagonist activity with respect to a given receptor, or the ligand of interest may inhibit the activity of a particular enzyme.
  • ligands may also form the basis of effective vaccines against disease brought on by infectious agents (e.g., viral or bacterial pathogens) or hyperproliterative conditions (e.g., malignant or non-malignant tumor growth) .
  • the present invention makes possible a method of immunizing an individual comprising administering to an individual in need of immunization an effective amount of a vaccine, in which the vaccine comprises a plurality of one or more distinct carbohydrate-based ligands and, optionally, one or more distinct non-carbohydrate- based ligands, which carbohydrate-based ligands at least are bound to and presented for multivalent interaction on a scaffold or on the surface of a solid support.
  • Carbohydrate ligands having the same identity are preferably assayed using multiple copies of the same ligand presented on a solid support, such as a microtiter plate, a glass slide, or a solid or porous bead.
  • the present invention makes it possible for the first time to prepare rapidly a large number of carbohydrate derivatives by combinatorial synthetic methods, and to screen them rapidly for their binding affinity to proteins or other receptors of interest in a way that permits multivalent binding.
  • the present invention makes it possible to discover novel ligands without resorting to traditional, labor- intensive organic synthesis and which ligands may have gone unrecognized by conventional assays, especially conventional, solution-based, affinity assays .
  • a monosaccharide refers to a pentose, hexose, heptose, or octose sugar, analog, or derivative thereof, including, but not limited to, deoxy sugars, dideoxy sugars, amino sugars and sugar acids. These terms include the protected and unprotected forms thereof (that is, in which selected reactive groups, typically oxygen- or nitrogen-bearing groups, of the carbohydrate monomer or monosaccharide have been either temporarily blocked to prevent their undergoing a reaction under the conditions of a specific transformation or left exposed and available for possible participation in a reaction, respectively) .
  • a protecting group is any chemical moiety that is temporarily attached to a reactive functional group of a given molecule to mask the functional group's reactivity while chemical reactions are permitted to proceed elsewhere on the molecule.
  • Protecting groups preferred for protecting the reactive functional groups of sugars include, but are not limited to, alkyl, benzyl, acyl and silyl protecting groups. Many other s are well known to those of ordinary skill.
  • a carbohydra te monomer is a type of monosaccharide which is capable of influencing the stereoche ical course of a glycosylation reaction such that the resulting glycosylation product bears substantially the stereochemistry desired (e.g., a 1,2 - cis relationship among the substituents on the 1- and 2-positions of the glycosidic ring) .
  • a carbohydrate monomer is a particular type of glycosyl donor, as defined below.
  • a carbohydrate, disaccharide, oligosaccharide , or polysaccharide each refers to a molecule or a portion thereof, which is comprised of two or more monosaccharides that are joined by a glycosidic linkage.
  • a sugar is any carbohydrate, disaccharide, oligosaccharide, polysaccharide, or monosaccharide.
  • carbohydrate -based ligand refers to a substance having an affinity for a given receptor, such as a carbohydrate-binding protein, enzyme, nucleic acid, lipid and the like, and is composed solely or partially of carbohydrate moieties.
  • carbohydrate ligand may be used interchangeably with carbohydrate-based ligand in this disclosure.
  • a low-affini ty ligand is one that, in solution, binds to a receptor with a dissociation constant of from about one hundred micromolar to about one hundred millimolar.
  • a gly cocon jugate refers to any molecule, substance, or substrate, including a solid, that includes a monosaccharide, carbohydrate, disaccharide, oligosaccharide, or polysaccharide covalently attached or adhered to a non-sugar chemical, biochemical, biological, or inorganic moiety.
  • Preferred glycoconjugates include, but are not limited to, small molecules conjugated to the sugar (e.g., heteropolyaromatic-sugar conjugates, nucleosides, nucleoside analogs and the like) , glycopeptide, glycoproteins, glycolipids and the like.
  • a glycosyl donor is a sugar with a leaving group (or potential leaving group) on at least one of its anomeric carbon which, under appropriate conditions, is capable of participating in a glycosylation reaction by which such anomeric carbon becomes covalently attached to a second moiety, typically a glycosyl acceptor, as defined below, or a nucleophile.
  • a glycosyl acceptor is any moiety, including a sugar, having the capacity to participate as the second moiety in the above-mentioned glycosylation reaction by virtue of a nucleophilic (or potentially nucleophilic) group present among the groups or substituents of such moiety, such that a covalent bond is formed between the anomeric carbon of such glycosyl donor and such nucleophilic (or potentially nucleophilic) group.
  • sulfoxide-mediated glycosylation reaction refers to the glycosylation technique first described by Kahne et al . in J. Am. Chem. Soc. (1989) 111:6881.
  • lower alkyl refers to such substituents, as the case may be, having one to five carbon atoms, in either a linear or branched arrangement.
  • aryl refers to aromatic groups, such as phenyl rings, naphthyl rings and the like, optionally bearing one or more substituents on the various ring positions.
  • heterocycle refers to five-membered or six-membered aromatic rings containing one or more nitrogen, oxygen, or sulfur atoms, optionally bearing one or more substituents on the various ring positions.
  • Such optional "substituents” may be any substituent that is chemically compatible with the aryl or heterocyclic ring and with the overall molecule of which such aryl or heterocyclic ring may be a part.
  • a mul tivalent support is any material or acromolecule to which more than one carbohydrate ligand can be attached, and includes, but is not limited to, organic dendrimers and polymers, glasses, metals and metal oxides, in any physical form such as solutions, emulsions, suspensions, beads, fibers, or planar surfaces.
  • the term solid support or solid phase means a solid or porous material that is substantially insoluble in typical aqueous or non ⁇ aqueous solvents. Despite being insoluble, such supports may swell, however. Most solid supports or solid phases generally make adequate multivalent supports.
  • the detection of a substance refers to the qualitative determination that the substance is present and may also refer to the quantitative measurement of the amount of the substance present. Detection may be made by any means, including but not limited to affinity-based, physical, optical, radiometric, photometric, electrochemical, or spectroscopic methods. All such detection methods are intended to be within the scope of the present invention. There are a variety of detection methods known in the art, and it is well within the capacity of the skilled practitioner to choose the method most appropriate or convenient for each situation.
  • the selection of a particular ligand-probe interaction refers to the process of selectively recognizing the interaction of interest from among a potentially large number of possible interactions.
  • the selection step includes, but is not limited to, the detection of the desired interaction. For instance, selection may include the selective resolution of one or a few beads from a vessel filled with different beads or one or two wells from a multi-well plate.
  • a detectable moiety is any particle, molecule, fragment of a molecule, or atom whose presence and concentration can be readily measured, preferably by automated analytical instruments.
  • the detectable moieties include, but are not limited to, radioactive isotopes, fluorescent molecules, chemiluminescent compounds, chromophores, high-affinity ligands or antigens, haptens, colloidal metal, enzymes, or other species or catalysts that can either produce or be manipulated to provide detectable products.
  • An immune system response includes any response by the body to invasion by a pathogen or to a cell affected by a disease caused by a pathogen, such as bacterial or viral infections, or caused by a hyperproliterative condition, such as neoplasticity, tumor growth, cancer, metastasis and the like and further includes any humoral or cell-mediated immune response.
  • a pathogen such as bacterial or viral infections
  • a hyperproliterative condition such as neoplasticity, tumor growth, cancer, metastasis and the like and further includes any humoral or cell-mediated immune response.
  • FIG. 1 illustrates the synthetic scheme for the preparation of compounds 1-20.
  • Fig. 2 illustrates the synthetic scheme for the preparation of compounds 21-D-26.
  • Fig. 3 illustrates the synthetic scheme for the preparation of compounds 27-33.
  • Fig. 4 illustrates the synthetic scheme for the preparation of compounds 34-L-45.
  • Fig. 5 illustrates the synthetic scheme for the preparation of compounds 46-53.
  • Fig. 6 illustrates the synthetic scheme for the preparation of compounds 54-66.
  • Fig. 7 illustrates the synthetic scheme for the preparation of compounds 67-77.
  • Fig. 8 illustrates the synthetic scheme for the preparation of compounds 78-88.
  • Fig. 9 illustrates the synthetic scheme for the preparation of compounds 81-95.
  • Fig. 10 illustrates schematically the synthesis of the disaccharide residue Gal ⁇ (1-3)GalNAc- ⁇ - thiophenyl ether on a solid phase support.
  • Other sugar residues are prepared similarly using different precursor materials, glycosyl donors and/or reagents.
  • Fig. 11 shows the results of an aggregation study of TentaGel beads.
  • the right panel shows that underivatized beads do not aggregate when treated with Arachis Hypogaea lectin at 25 ⁇ g/mL.
  • the left panel shows how derivatized beads aggregate under the same conditions.
  • Fig. 12 illustrates the results of a colorimetric assay of a four-carbohydrate mixture (magnified 100-fold) , along with the structures of the four resin-bound carbohydrates.
  • Fig. 12A tabulates the results of the screening of the four-carbohydrate library.
  • Fig. 13 diagrams the steps of a particular colorimetric version of the assay of the invention.
  • the colorimetric assay is used for the selection of a specific disaccharide-lectin interaction among those possible with the various members of the library. The multivalent nature (and, perhaps, other features) of the interaction is not illustrated in this Figure.
  • Fig. 14 shows the precursors (or monomers) , glycosyl donors and additional reagents used in a particular embodiment of the present library.
  • a library of approximately 1500 distinct carbohydrate- based ligands is prepared by a "split and mix" method described herein.
  • the stereochemical configuration of the putative natural substrate of the lectin used in the subsequent assay is also illustrated.
  • Fig. 15 is a reproduction of a photograph showing the colored bead selected by the assay of the invention among the large population of beads contained in the assay vessel.
  • a number of different carbohydrate-based ligands, particularly unnatural ones, are identified by the methods of the invention.
  • the present invention in general terms, provides a method for discovering or identifying ligands for receptors.
  • the present invention is particularly useful for discovering or identifying ligands for receptors which utilize polyvalent interactions with their ligands. More particularly, the present invention provides a method for identifying or discovering carbohydrate-based ligands for carbohydrate-binding receptors, including peptides or proteins, especially those carbohydrates- based ligands that in solution exhibit a low affinity for the carbohydrate-binding receptor.
  • the methods disclosed are especially suitable for screening libraries of compounds but may also be applied to the study of smaller collections of carbohydrate-based ligands or even single ligands on an individual basis.
  • the invention involves the presentation of multiple copies of a ligand, attached to a solid support, to a receptor, in order to take advantage of any polyvalent interactions that the receptor might be capable of.
  • a receptor-ligand interaction is then detected and/or selected from which information about the ligand can be obtained.
  • the selection/detection methods and identification schemes can be carried out using conventional methods, such as known affinity-binding detection methods, "sequencing" techniques, microanalytical techniques and spectroscopic methods.
  • single or distinct resin-bound carbohydrate ligands and, subsequently, a spatially resolved carbohydrate library are synthesized on polymer beads as a solid support .
  • the individual beads can be encoded using available chemical tagging technologies.
  • chemical tagging techniques facilitate the rapid structural identification of the ligand attached to or derived from a particular bead. See, for example, Barchart, A. and Still, W. C, in J “ . Am. Chem. Soc. (1994) 116:373-374; Nestler, P., et al., in J. Org. Chem . (1994) 59:4723-4724; Ohlmeyer, M. H. J. et al., in Proc. Natl .
  • Still other known methods of identifying library members, which are detected by an appropriate assay include library deconvolution/resynthesis techniques and spatial addressing. Moreover, smaller libraries can also be prepared by parallel synthesis, obviating a decoding step.
  • the preferred method for forming the library of the invention comprises the use of a sulfoxide-mediated glycosylation reaction step, i.e., one that leads to the formation of new glycosyl bonds.
  • the disaccharide ⁇ -Gal- ( (1-3) ) -GalNAc a known ligand for peanut lectin
  • Peanut lectin is representative of any carbohydrate- binding protein that utilizes a multivalent mode of carbohydrate recognition.
  • a similar exposure to peanut lectin of identical beads not bearing the disaccharide failed to result in agglutination. The results of this agglutination study are shown in Fig. 11.
  • the agglutination assay described above demonstrates that a carbohydrate binding protein containing multiple carbohydrate binding sites causes agglutination of the carbohydrate-derivatized polymer beads.
  • the experiment thus shows the availability of the support-bound disaccharide for polyvalent receptor binding.
  • the experiment further demonstrates the usefulness of the invention for detecting qualitatively the binding activity of individually synthesized carbohydrates on a solid support.
  • the concentration of lectin required for agglutination of the beads can be used to quantitate the binding activity of the ligand.
  • the agglutination assay cannot be used to select resin-bound carbohydrates that bind to a receptor from a mixture of resin-bound carbohydrates.
  • the colorimetric assay described in Example 7.35 is used.
  • the disaccharides shown in Fig. 12 are synthesized separately on TentaGel S RAM resin. These disaccharides differ only in the stereochemistry at C4 and at the anomeric linkage of the first sugar. Equal portions of each resin are combined and assayed as described in 7.35.
  • Fig. 12 Approximately 25% of the beads stain dark purple, 25% stain light purple, and 50% of the beads remain white (Fig. 12) .
  • the beads are separated according to color and the products were removed from the beads by hydrolysis with TFA and identified by correlation with authentic standards.
  • the dark purple-staining beads are found to contain Gal ⁇ (l-3)GalNAc- ⁇ -thiophenyl ether (Fig. 12A) .
  • the light purple beads are found to contain Gal ⁇ (l- 3)GlcNAc- ⁇ -thiophenyl ether and Gal ⁇ (1-3)GalNAc- ⁇ - thiophenyl ether.
  • the unstained beads are found to contain Gal ⁇ (1-3)GlcNAc- ⁇ -thiophenyl ether.
  • Example 7.35 This experiment demonstrates that the colorimetric assay described in Example 7.35 can be used to differentiate closely related carbohydrate ligands.
  • carbohydrate ligands have similar affinities for the Bauhunia purpurea lectin in solution as evaluated by an assay which measures inhibition of agglutination of erythrocytes (Laboratory techniques in biochemistry and molecular biology: Glycoprotein and proteoglycan techniques, Beeley, J.G. p. 327-333, Elsevier Science: Amsterdam 1985)
  • the avidities of the carbohydrate-derivatized beads are sufficiently different that the individual carbohydrates can easily be differentiated.
  • a library of 1269 resin-bound carbohydrates is synthesized on polymer beads.
  • the library described herein is synthesized on TentaGel polymer beads, the present invention is not limited to TentaGel or even to a polymer bead.
  • the library members can be arrayed on a planar support, such as a microtiter plate or glass slide.
  • the library members can be attached to a functionalized surface, such as a modified polyethylene substrate.
  • a functionalized surface such as a modified polyethylene substrate.
  • the present invention is intended to include within its scope the use of any modified solid support that allows the bound carbohydrate-based ligands enough geometric flexibility to achieve polyvalent binding to a receptor, i.e., which provides a multivalent support.
  • the presentation of "polydentate" carbohydrate-based ligands permits the practitioner to (1) assay receptor binding to the members of the library, and
  • the active members of the library can be identified by a variety of methods known in the art.
  • the members of the library could be identified by physical location on the array.
  • members of the library could be identified using an associated chemical or physical tag, or by direct structure determination, or by a deconvolution strategy, or by a combination of deconvolution and resynthesis.
  • the method of the present invention can be used to generate and screen carbohydrate-based ligands for potentially any biological receptor. Also, the present method is especially valuable for discovering new, clinically useful compounds that exhibit an affinity for carbohydrate-binding receptors, such as cell adhesion molecules, for example. Such newly discovered compounds have the potential to exhibit useful biological activity, including, but not limited to, agonist, antagonist, inhibitory, augmenting, or simply activity that interferes or disrupts inter or intracellular signal transduction.
  • the carbohydrate library is preferably constructed on a commercially available synthesis resin, a polyether chain-modified polystyrene sold under the trade name TENTAGEL, using a "split and mix" synthesis approach.
  • the resin is composed of a cross-linked polystyrene base to which poly(ethylene glycol) chains are attached.
  • the library members are attached to the poly(ethylene glycol) chains.
  • six different monomers are attached to the resin via a thioether linkage .
  • the resins are mixed and then split into twelve equal batches, each of which is glycosylated with one of twelve different glycosyl donors to produce seventy- two different di- or trisaccharides. Because it is desirable that a library constructed using a split and mix synthesis strategy have a single product on each bead, the glycosylation method used should preferably achieve glycosylation stereospecifically for all the different donor/acceptor pairs in all the reactor vessels. Accordingly, the preferred method of glycosylation is the sulfoxide-mediated glycosylation reaction.
  • the resulting batches are recombined, mixed and split into twenty separate batches. Nineteen of these batches are treated with trimethylphosphine to reduce the azides to amines. Eighteen of these nineteen trimethylphosphine-treated batches are acylated. The twenty batches of resin are recombined. All hydroxyl protecting groups are removed to produce a library containing approximately thirteen hundred carbohydrates.
  • carbohydrate-binding proteins recognize the non-reducing end of their carbohydrate ligand.
  • the carbohydrates in the exemplified embodiment of the invention are deliberately synthesized with the reducing end directed toward the solid surface, with subsequent sugar or other reaction units added with the non-reducing end directed outward. This orientation facilitates library screening while the carbohydrates are still attached to the support.
  • the exemplified library is designed to include the natural ligand for peanut agglutinin, a carbohydrate-binding protein that agglutinates neuraminidase-treated erythrocytes.
  • this protein system is a good model system for many carbohydrate-binding protein-carbohydrate ligand interactions because the affinity of peanut agglutinin for its natural ligand is low. What is more, it utilizes a polyvalent strategy to achieve activity (agglutination) .
  • TentaGel resin is purchased from RAPP Poly ere (TentaGel S NH 2 , 130 ⁇ m, 0.3 mmol g _1 capacity) .
  • N, N-dimethylformamide (DMF) , tetrahydrofuran (THF) , diisopropylethylamine (DIEA) and trifluoromethanesulfonic acid (TFA) , and bovine serum albumin (BSA) are from Aldrich.
  • 1-methyl-2-pyrrolidinone (NMP) , 1- hydroxybenzotriazole (HOBt) and 2- (1H- benzotriazol-1-yl) -1, 1, 3, 3, -tetramethyluronium hexafluorophosphate (HBTU) are from Applied Biosystems .
  • Lyophilized powders of lectins and alkaline phosphatase-conjugated streptavidin, as well as solutions of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) /nitroblue tetrazolium (NBT) liquid substrate system and p-nitrophenyl phosphate (pNPP) are from Sigma.
  • Rabbit blood is purchased from Remel.
  • PBS 150 mM NaCl, 7.3 mM Na 2 HP0 4 , and 2.7 mM NaH 2 P0 4 , adjusted to pH 7.2.
  • PBST 0.05% (v/v)
  • Tris buffered saline (TBS) is 500 mM NaCl and 20 mM Tris, adjusted with dilute HC1 to pH 7.5.
  • TBST is 0.05% (v/v) Tween-20 in TBS.
  • Alkaline phosphatase buffer (AP) is 100 mM NaCl, 5 mM MgCl 2 , and 100 mM Tris, adjusted to pH
  • GalNAc- ⁇ -thiophenyl glycoside and underivatized beads are placed in separate wells of a 96 well microtiter plate and swollen in PBS buffer. The buffer is removed, and 100 ⁇ L of Arachis Hypogaea lectin (10-1000 ⁇ g/mL in PBS buffer) is added to each well. The beads are examined under a microscope after 1-2 hours.
  • a stock solution of the lectin is made by dissolving 5 mg of Bauhinia Purpurea lectin in 2.5 mL of PBS. Serial dilutions of lectin in PBS are prepared, and 50 ⁇ L of each solution is transferred into 12 microtiter plate wells. 50 ⁇ L of a 2% suspension of rabbit erythrocytes in PBS is added to each well and incubated at room temperature on an orbital shaker for 1 hour. Agglutinated cells form a carpet covering the bottom of the well; non-agglutinated cells form a compact button at the center of the well . The titer is defined as the last dilution well before the erythrocyte button begins to form. The HA titer for Bauhinia Purpurea lectin is ca. 1 ⁇ g/mL.
  • HSD Hemagglutination Inhibition Assay
  • each sugar solution is added to single wells of a microtitre plate that contain 25 ⁇ L of a 16 ⁇ g/mL lectin solution in PBS and incubated at room temperature on an orbital shaker for 1 hour.
  • To each well is added 50 ⁇ L of a 2% suspension of erythrocytes. The plate is gently agitated for 10 min and then incubated at room temperature for 1 hour.
  • the final lectin concentration is 4 ⁇ g/mL, which is 4x the dose of the HA titer.
  • the end point is defined as the lowest sugar concentration which inhibits agglutination.
  • Carbohydrate Mixture A portion of resin, which contains equal portions of la-4a (10 mg total) , is washed with PBST buffer (3 x 1 mL, 10 min) . The beads are incubated for 30 min at room temperature in 1 mL of PBST containing 3% BSA and washed with PBST (3 x 1 mL, 5 min) containing 1% BSA. The beads are incubated in 1 L of Bauhinia Purpurea lectin (0.1 ⁇ g/mL in PBST containing 1% BSA) at room temperature for 3 hours and then washed with TBST buffer (3 x 1 mL, 5 min) containing 1% BSA.
  • the resin is incubated for 20 min at room temperature in 1 mL of alkaline phosphatase- conjugated streptavidin (10 ⁇ g/mL in TBST containing 1% BSA) and then washed with alkaline phosphatase buffer (3 x 1 mL, 5 min) .
  • alkaline phosphatase buffer 3 x 1 mL, 5 min
  • a small portion of the resin ( ⁇ l/3) is stained with 200 ⁇ L of BCIP/NBT. Staining is terminated after 30 min by washing the beads twice with 200 ⁇ L of 20 mM sodium ethyleneaminetetracetic acid, pH 7.4.
  • the dark purple, light purple and colorless beads are pulled out for analysis, including decoding if encoded, using 50 ⁇ L micropipettes.
  • Carbohydrate Library Screening of the larger carbohydrate library follows the same procedure as that provided above, except that the lectin concentration is 10 ⁇ g/mL for the larger library.
  • the combined lectin/sugar solutions are incubated at room temperature for 1 hour, and 100 ⁇ L of each solution is added to the resin.
  • the plate is agitated on an orbital shaker at room temperature for 3 hours.
  • the resin is washed with TBST containing 1% BSA (3 x 100 ⁇ L, 5 min) .
  • a solution (100 ⁇ L) of alkaline phosphatase- conjugated streptavidin (10 ⁇ g/mL in TBST containing 1% BSA) is added to each well, and the plate is incubated at room temperature for 20 min.
  • the beads are washed with alkaline phosphatase buffer (3 x 100 ⁇ L, 5 min) and transferred into a
  • Gal ⁇ (l-3)GalNAc- ⁇ -thiophenyl ether (la) is constructed on TentaGel resin.
  • the sulfoxide glycosylation reaction proceeds stereospecifically and in near quantitative yield. Subsequent chemical transformations also worked well on the resin.
  • the synthesis is carried preferably out from the reducing to the non-reducing end of the ligand so that the carbohydrates are presented in a way that mimics cell surface carbohydrates. This orientation also permits direct screening of the derivatized beads for binding.
  • samples of the Gal ⁇ (l-3) GalNAc beads and underivatized beads are treated separately with varying concentrations of Arachis Hypogaea (peanut) lectin, a protein known to recognize Gal ⁇ (1-3) GalNAc.
  • the underivatized beads do not aggregate at lectin concentrations ranging from 10-1000 ⁇ g/mL.
  • the carbohydrate-derivatized beads (left panel) aggregate at a lectin concentration of 25 ⁇ g/mL.
  • Arachis Hypogaea lectin also causes erythrocytes to aggregate - or agglutinate - in this concentration range in a process believed to involve polyvalent interactions between the lectin and carbohydrates on the surfaces of different cells.
  • the derivatized beads aggregate due to multivalent interactions between the lectin and carbohydrate ligands on different beads.
  • the aggregation can be prevented by increasing the protein concentration such that the protein coats the entire surface of each bead and makes multivalent interactions involving carbohydrates on different beads impossible.
  • an assay for Detecting Binding.
  • an assay is provided which can discriminate between different carbohydrate- derivatized beads.
  • Gal ⁇ (l-3)GalNAc (la) is a known ligand for Bauhinia purpurea lectin.
  • the structures of the three other carbohydrates differ from la in terms of the stereochemistry at the C4 position and/or at the anomeric position of the sugar directly attached to the resin.
  • the C4 stereochemistry is varied because solution binding studies have shown that the lectin is sensitive to the stereochemistry at this position and binds the C-4 equatorial isomer with a three-fold lower affinity relative to the axial isomer.
  • the configuration of the internal glycosidic linkage is varied to probe the effect of ligand presentation on binding.
  • the beads can be encoded during the synthesis, if desired, using known technology
  • the assay of the present invention clearly distinguishes the best ligand from three other closely related ligands. It is also apparent from the assay that the worst polyvalent ligand is 4a, in which both the C4 and anomeric stereochemistry differ from the known ligand, la.
  • Ligands 2a and 3a have similar avidities, although the ratio of stained to unstained beads suggests that 2b is a better ligand. Hence, the two apparently best ligands contain the ⁇ - stereochemistry at the internal glycosidic linkage.
  • the thiophenyl glycosides lb-4b are each synthesized and evaluated separately for their relative solution affinities for Bauhinia purpurea lectin using a standard hemagglutination inhibition assay.
  • the results from this solution assay show that lb, inhibits agglutination at concentrations four-fold to eight-fold lower than the other three thiophenyl glycosides. There is essentially no difference in the solution affini ties of the other three carbohydra tes .
  • the most avid disaccharide contains an ⁇ glycosidic linkage between the two sugars while the known ligand for this lectin contains a ⁇ linkage.
  • the preferred ligands have an equatorial hydroxyl group at the C4 position of the resin-linked sugar even though previous results suggest that the axial hydroxyl group is preferred.
  • the preferred ligands have an axial anomeric linkage to the resin.
  • the screen of the four compound mixture suggests that the equatorial thiophenyl linkage to the resin is preferred over the axial stereochemistry, at least when the glycosidic linkage between the two sugars is equatorial.
  • carbohydrate- derivatized beads resemble carbohydrate presenting cell surfaces in key respects.
  • the carbohydrate- derivatized beads can be recognized by carbohydrate-binding proteins, and at low protein concentrations the recognition process involves polyvalent interactions. It is, therefore, believed that at least some of the conclusions drawn about the recognition of these carbohydrate- derivatized beads can also be applied to understanding carbohydrate recognition in vivo .
  • One of the conclusions drawn is that the presentation of the carbohydrate ligand on the surface plays a critical role in determining how the carbohydrate ligand interacts with its receptor.
  • there may be critical concentrations of a given carbohydrate ligand on the surface of a cell which determine the activation of inter- or intracellular communications initiated by interaction of the cell surface with a carbohydrate binding partner.
  • a library comprising a collection of distinct carbohydrate-based ligands.
  • a plurality of each ligand is bound to and presented on the surface of a resolvable portion of a solid support to permit: (i) multivalent interactions of the plurality of ligands with one or more probes bearing a plurality of carbohydrate binding sites (that is, the probe has the capacity to bind in a polyvalent fashion) , and (ii) selection of at least one particular ligand-probe interaction.
  • the library is prepared by a method comprising a glycosyl bond- forming step, preferably a sulfoxide-mediated glycosylation reaction and more preferably, conducted in a solid phase.
  • a glycosyl bond- forming step leads to the formation of a C- , N- , O- , S-, or P-linked glycoside.
  • the solid support to which the ligand is bound may comprise any solid or porous material, including but not limited to a planar support, separate wells, a multi-well microtiter plate, or a three- dimensional, spherically shaped substrate.
  • the solid support comprises a plurality of solid or porous beads.
  • the solid support is substantially insoluble in a variety of solvents, including aqueous and non-aqueous solvents.
  • the preferred solid or porous beads are insoluble in tetrahydrofuran, methanol, methylene chloride, N- methyl pyrrolidinone and dimethylformamide.
  • the solid support comprises a synthetic polymer, such as polystyrene or a modified polystyrene. More preferably, the solid support comprises a polyether chain-modified polystyrene, most preferably, commercially available TentaGel resin.
  • the resolvable portion of the solid support may comprise a well of a planar support or a bead of a spherically shaped support.
  • the size of the beads can vary depending on the particular application.
  • the beads may have a wide range of diameters.
  • the bead may have an average diameter ranging from about 50 nm to about 5 ⁇ m, about 50 nm to about 1 ⁇ m, about 50 nm to about 0.5 ⁇ m, about 50 nm to about 250 nm, about 50 nm to about 100 nm, or about 75 nm to about 200 nm.
  • the bead may have an average diameter that is less than or equal to about 0.5 ⁇ m, less than or equal to about 0.3 ⁇ m, less than or equal to about 0.2 ⁇ m, or less than or equal to about 0.1 ⁇ m.
  • the solid support may comprise one or more detachable scaffolds on the surfaces of an insoluble solid or porous bead.
  • the solid support comprises comprise derivatized beads, each having one or more detachable scaffolds on the bead surfaces, a plurality of each ligand being bound to the one or more scaffolds.
  • the scaffold can bear a plurality of one or more distinct carbohydrate-based ligands and, optionally, one or more distinct non-carbohydrate- based ligands. The scaffold can then be detached from the solid support, if desired, prior to conducting an assay or using the derivatized scaffold, e.g., prior to administration to an individual for a therapeutic, diagnostic, or prophylactic application.
  • the present invention contemplates a composition for use as a vaccine comprising a plurality of one or more distinct carbohydrate-based ligands and, optionally, one or more distinct non- carbohydrate-based ligands, which carbohydrate-based ligands at least are bound to and presented on the surface of a solid support to permit the multivalent interaction of the plurality of one or more distinct carbohydrate-based ligands with one or more receptors associated with an immune system response, such that an individual, to whom an effective amount of the composition has been administered, is able to mount an appropriate immune response against a given disease that is caused by a given pathogen or which is characterized by the expression of a given marker on the surface of a cell affected by the disease.
  • composition of the present invention may further comprise a pharmaceutically acceptable carrier and may further include any adjuvants appropriate for amplifying or enhancing the desired immune system response (that is, increase the immunogenicity of the composition) .
  • adjuvants appropriate for amplifying or enhancing the desired immune system response (that is, increase the immunogenicity of the composition) .
  • optional non-carbohydrate-based ligands are also contemplated, which optional ligands may be administered before, after, or along with the carbohydrate-based ligands.
  • optional ligands may also be presented on the surface of the same or separate solid support or scaffold as the carbohydrate-based ligand.
  • optional ligands include, but are not limited to, small molecules, drugs, peptides, glycopeptides, proteins, glycoproteins, nucleic acids (e.g., deoxyribonucleic acids or ribonucleic acids) , lipids, glycolipids, or combinations or complexes thereof.
  • the solid support When meant for administration into and circulation within the vascular system, the solid support preferably comprises an insoluble solid or porous bead having an average diameter that permits the bead to move substantially freely in an individual's circulatory system. Suitable sizes are enumerated above, but preferably sizes are selected which would minimize clogging of an individual ' s blood vessels and/or capillaries.
  • the present invention also contemplates a method of immunizing an individual comprising administering to an individual in need of immunization an effective amount of vaccine comprising a plurality of one or more distinct carbohydrate-based ligands and, optionally, one or more distinct non-carbohydrate-based ligands, which carbohydrate-based ligands at least are bound to and presented for multivalent interaction on a scaffold or on the surface of a solid support .
  • An example of a suitable scaffold is a dendrimer, a glycosaminoglycan, a glycan, or a piece of an extracellular matrix.
  • the ligands may be bound to the solid support or scaffold in any number of ways, directly or indirectly through a linker moiety.
  • the ligands are bound to functional groups on the surface of the solid support or scaffold via linker groups .
  • Any one of a great variety of bifunctional linker groups known to those of ordinary skill in the art can be used as the linker moiety.
  • a probe one may use a substance that comprises one or more receptors.
  • receptors may further comprise a sequence of amino acids (e.g., peptides or proteins, including one or more protein subunits) , a piece of DNA, or a piece of RNA.
  • the receptor may also form part of an enzyme, such as a protease.
  • the preferred receptors bear two or more (i.e., a plurality of, a multiplicity of, or the capacity to interact with more than one carbohydrate ligand) carbohydrate binding sites.
  • the probe may comprise an intact cell or a portion thereof.
  • the cell may further be a prokaryotic cell or a eukaryotic cell, a bacterial, yeast, fungal, mammalian, animal, human, plant, or insect cell.
  • the desired cell probe may be selected from among those involved in a cell-mediated immune response, including B lymphocytes, T lymphocytes, natural killer cells, or neutrophils.
  • the cell may also be selected from among those involved in the production of antibodies.
  • Preferred cells may be phagocytic cells, tumor cells, infected cells, diseased cells, or cells from malignant tissue. Additional cells can be selected from antigen presenting cells or cells involved in a cell adhesion process.
  • a library of carbohydrate ligands can be synthesized on TentaGel beads and screened against neutrophils to select the multivalent carbohydrate ligands that bind the neutrophils best. The beads are then washed to remove unbound neutrophils. Bound neutrophils can be detected by treating the beads with nitroblue tetrazolium, which is converted to an insoluble colored product by oxidative enzymes in the neutrophils. The beads that change color are selected from the library and washed extensively to remove bound neutrophils. The identity of the carbohydrates on the beads can then be determined by decoding (if the library is encoded) or by a deconvolution strategy involving resynthesis, or by some combination of mass spec of the hydrolyzed products and a deconvolution strategy.
  • the plasma membrane proteins of the neutrophils can be solubilized and then passed down a column containing beads derivatized with the appropriate carbohydrate.
  • the protein receptors on the plasma membrane that bind the multivalent carbohydrate ligands adhere to the column.
  • the other membrane components can be washed away.
  • Bound proteins can be eluted with soluble carbohydrate competitor or with urea or like denaturing agent. Partial amino acid sequences from the purified protein can be used to make degenerate oligonucleotide probes to screen a cDNA library which can be sequenced to provide the full protein sequence.
  • the glycosyl bond-forming step includes a condensation reaction between a glycosyl donor (GD) and a solid support-bound glycosyl acceptor (GA-SS) to provide a structural unit (GD-GA-SS) with a newly formed glycosyl bond. More preferably, the glycosyl bond- forming step includes a plurality of condensation reactions taking place substantially concurrently between a glycosyl donor and a plurality of distinct solid support-bound glycosyl acceptors to provide a plurality of distinct structural units with newly formed glycosyl bonds.
  • the glycosyl bond-forming step includes a plurality of condensation reactions taking place substantially concurrently between a plurality of distinct glycosyl donors and a solid support-bound glycosyl acceptor to provide a plurality of distinct structural units with newly formed glycosyl bonds; or, the glycosyl bond-forming step includes a plurality of condensation reactions taking place substantially concurrently between a plurality of distinct glycosyl donors and a plurality of distinct solid support-bound glycosyl acceptors to provide a plurality of structural units with newly formed glycosyl bonds.
  • the plurality of condensation reactions can take place in the same reaction vessel or in separate reactions vessels.
  • the present invention provides an assay (and method) for a carbohydrate-based ligand-receptor interaction comprising the steps of: (a) providing a library comprising a collection of distinct carbohydrate- based ligands, a plurality of each ligand being bound to and presented on the surface of a resolvable portion of a solid support; (b) contacting the library with one or more probes bearing a plurality of carbohydrate binding sites; and (c) selecting at least one particular ligand-probe interaction.
  • the contacting step may be carried out in a vessel containing a plurality of members of the library.
  • the selection step includes selecting those resolvable portions of the solid support to which a probe has bound.
  • the assay is made possible by the library of the invention, which permits multivalent interactions of the plurality of ligands with the one or more probes.
  • a solid support may comprise an insoluble material, such as insoluble polymer.
  • Preferred solid supports comprise a polystyrene resin or a polystyrene resin that is modified by covalently bound polyether chains.
  • the solid support may also comprise a polyamide resin or one that is modified by covalently bound polyether chains.
  • the solid support comprises a polyethylene resin, a poly(ethylene glycol) resin, or a dendrimer polymer.
  • the assay probe may, of course, comprise one or more receptors, including receptors that are labeled with a detectable moiety, su;h as a radioisotope or a fluorescent or chemiluminescent substance.
  • a detectable moiety may comprise an enzyme that generates a detectable product.
  • the detectable moiety may comprise a substance having a selective affinity for a detecting agent.
  • the substance may be biotin and the detecting agent may be avidin or streptavidin.
  • the selection step may be carried out using an anti-probe or anti-ligand-probe antibody.
  • the antibody may be labeled with a detectable moiety, preferably a radioisotope, a fluorescent or chemiluminescent substance, an enzyme that generates a detectable product, a substance having a selective affinity for a detecting agent, or biotin.
  • a detectable moiety preferably a radioisotope, a fluorescent or chemiluminescent substance, an enzyme that generates a detectable product, a substance having a selective affinity for a detecting agent, or biotin.
  • Such ligands can exhibit a variety of characteristics, including but not limited to those consistent with an enzyme inhibitor, a receptor agonist, a receptor antagonist, an antigen, an immunogen, an anti-tumor agent, an anticancer agent, an anti-emetic agent, an anti-inflammatory agent, a neurotransmitter, or a substance that exhibits endocrine-like properties.
  • the present invention contemplates a method of preparing a library comprising a collection of distinct carbohydrate-based ligands each bound to a resolvable portion of a solid support (SS) comprising (a) providing a plurality of distinct solid support-bound glycosyl acceptors (GA ⁇ SS, GA 2 - SS, etc.) , each distinct solid support-bound glycosyl acceptor being bound to a resolvable portion of a solid support, (b) contacting the plurality of distinct solid support-bound glycosyl acceptors with at least one distinct glycosyl donor (GD) such that condensation reactions take place, including glycosyl bond-forming steps, between the at least one distinct glycosyl donor and each of the distinct solid support-bound glycosyl acceptors to provide at least the distinct structural units (GD-GA j -SS, GD-GA 2 -SS, etc.) .
  • the plurality of distinct solid support-bound glycosyl acceptors may be provided in separate reaction
  • At least one distinct glycosyl donor is contacted with each of the distinct solid support-bound glycosyl acceptors.
  • the plurality of distinct solid support-bound glycosyl acceptors is not provided in separate reaction vessels each holding a distinct solid support-bound glycosyl acceptor.
  • a particular method may involve contacting at least one distinct glycosyl donor with the plurality of distinct solid support-bound glycosyl acceptors substantially concurrently in the same reaction vessel .
  • the method can further comprise contacting at least the distinct structural units (GD-GA ⁇ SS, GD-GA 2 -SS, etc.) with one or more additional reagents, including one or more additional glycosyl donors.
  • reaction mixture is diluted with 200 mL of CH 2 C1 2 , washed with H 2 0 (100 mL) , saturated NaCl (100 mL) , dried over Na 2 S0 4 , filtered and concentrated to afford 2.77 g of a clear oil, which is purified by flash chromatography (17% EtOAc/petroleum ether) to give
  • reaction mixture is heated at 100 °C for 12 h, cooled, diluted with 50 mL of CH 2 C1 2 , washed with H 2 0 (100 mL) and saturated NaCl (100 mL) , dried over Na 2 S0 4 , filtered and concentrated to afford 3.25 g of phenyl 6-deoxy-2, 3,4-tri-0- pivaloyl-1-thio- ⁇ -L-galactopyranoside 4 as a brown oil. This oil is purified by flash chromatography
  • reaction mixture (1.53 g, 8.84 mmol) .
  • the reaction mixture is allowed to warm to -15 °C and then quenched with methyl sulfide (5.3 mL, 4.48 g, 17.4 mmol) and allowed to warm to room temperature.
  • the reaction mixture is then diluted with 50 mL CH 2 C1 2 , extracted with H 2 0
  • reaction mixture is diluted with 400 mL of CH 2 Cl 2 , washed with saturated NaHC0 3 (2 x 200 mL) , dried over Na 2 S0 4 , filtered, concentrated and purified by flash chromatography (8% EtOAc/petroleum ether) to give 3.3 g (85%) of phenyl 6-deoxy-3 , 4 -O- isopropylidene-1-thio-L-galactopyranoside 6 as a mixture of anomers.
  • reaction mixture is stirred at room temperature for 5 h and then neutralized with A berlite resin (OH " form) .
  • the solution is filtered, washed several times with MeOH and then concentrated in vacuo.
  • the product is purified by flash chromatography (60% EtOAc/petroleum ether) to give 2.06 g (95%) of isolated anomers of phenyl 6-deoxy-2- (4- methoxybenzyl) -l-thio-L-galactopyranoside 9.
  • Trifluoromethanesulfonic anhydride (2.5 mL, 15 mmol) is added dropwise to a cooled (0 °C) solution of phenyl 6-deoxy-2- (4-methoxybenzyl) -1-thio- ⁇ -L- galactopyranoside ( ⁇ -9) (1.4 g, 3.8 mmol) and pyridine (3.0 mL, 38 mmol) in 50 mL of CH 2 C1 2 .
  • the solution is stirred at 0 °C for 45 min and is allowed to warm to room temperature over 3 h.
  • the reaction mixture is then cooled to 0 °C before quenching with TEA (1 mL, 7.2 mmol) .
  • Trifluoromethanesulfonic anhydride (1.8 mL, 10.8 mmol) is added at 0 °C to a solution of phenyl 6- deoxy-2-O- (4-methoxybenzyl) -1-thio- ⁇ -L-allopyranoside ( ⁇ -14) (1.01 g, 2.69 mmol) and pyridine (2.2 mL, 26.9 mmol) in 30 mL of CH 2 C1 2 .
  • the solution is stirred at 0 °C for 1 h and allowed to warm at room temperature over 3 h.
  • the reaction mixture is cooled to 0 °C before quenching with TEA (1.0 mL, 7.2 mmol) .
  • the reaction mixture is diluted with 10 mL of CH 2 C1 2 , washed with saturated NaHS0 3 (10 mL) , saturated NaHC0 3 (10 mL) , dried over Na 2 S0 4 , filtered and concentrated.
  • the crude product is purified by flash chromatography (60% EtOAc/petroleum ether) to give 231 mg (83%) of 3-azido-4-O-benzoyl-l, 3, 6-tri-deoxy-2-O-pivaloyl-1- (phenylsulfinyl) - ⁇ -L-galactopyranose ( ⁇ -20) as a mixture of diastereomers: R f 0.26 (50% EtOAc/petroleum ether) .
  • the mixed anomers of 1, 3,4-tri-0-acetyl-2- azido-2, 6-dideoxy-L-galactopyranoside (21) are prepared from L-fucose by the method of A. Anisuzzaman and D. Horton, Carjb. Res . , 169, 258-262 (1987) .
  • thiophenol 3.1 mL, 3.3 g, 30.1 mmol
  • BF 3 ⁇ Et 2 0 7.4 mL, 8.5 g, 60.3 mmol
  • reaction mixture is heated at 40 °C for 50 min, then quenched with H 2 0 (20 mL) .
  • the organic layer is washed with brine (100 L) , dried over Na 2 S0 4 , filtered and concentrated to afford a mixture of anomers of phenyl 2-azido-3, 4-di- O-acetyl-2, 6-dideoxy-l-thio-L-galactopyranoside (22) as a clear oil: R f 0.39 (25% EtOAc/petroleum ether) .
  • reaction is quenched with dimethyl sulfide (1 mL) at -40 °C and then poured into a solution of saturated NaHC0 3 (50 mL) and extracted with CH 2 C1 2 (3 x 30 mL) . The organic layers are combined, dried over Na 2 S0 4 , filtered, concentrated, and purified by flash chromatography (EtOAc/petroleum ether) to afford the title compound 26 as a mixture of diastereomers.
  • the ketal 29 is dissolved in 100 mL of MeOH and p-toluenesulfonic acid monohydrate (0.60 g, 3.1 mmol) is added. The reaction is stirred at room temperature for 10 h and then saturated NaHC0 3 (50 mL) is added, followed by water (100 mL) . The product is extracted with CH 2 C1 2 (4 X 100 mL) , and the organic layers are combined, washed with saturated NaCl, (1 X
  • the sulfoxide is purified using flash chromatography
  • reaction mixture is diluted with 100 mL of CH 2 C1 2 and washed with H 2 0 (100 mL) , saturated NaCl (100 mL) , dried over Na 2 S0 4 , filtered, concentrated and purified by flash chromatography (15% EtOAc/hexane) to give 0.59 g
  • Lactose phenyl thioglycoside 49 is dissolved in 80 mL of pyridine, and pivaloyl chloride (20 mL, 170 mmol) and DMAP (0.10 g, 0.82 mmol) are added. The reaction mixture is heated to 110 °C for 24 h.
  • reaction mixture is stirred for 45 min at -25 °C and then diluted with 50 mL of CH 2 C1 2 , washed with H 2 0 (100 mL) , NaHC0 3 (100 mL) , saturated NaCl (100 mL) , dried over MgS0 4 , filtered, and concentrated to afford an orange gel.
  • reaction mixture is diluted with 25 mL of CH 2 C1 2 , washed with H 2 0 (10 mL) , saturated NaCl (10 mL) , dried over Na 2 S0 4 , filtered, and concentrated to afford 0.232 g of crude 2-azido-l, 2-dideoxy-l- (4- hydroxyphenylthio) -3,4, 6-tri-0-acetyl- ⁇ , ⁇ -D- glucopyranose 58 as a yellow oil, a mixture of anomers which is then taken on to the next step without further purfication.
  • the reaction mixture is heated at 45 °C for 12 h, cooled, diluted with 50 mL of CH 2 C1 2 , washed with saturated NaHC0 3 (75 mL) and then extracted with CH 2 C1 2 (2 x 50 mL) , washed with H 2 0 (75 mL) , saturated NaCl (75 mL) , dried over Na 2 S0 4 , filtered and concentrated to afford 5.40 g of the anomers 61 and 62 of 2- (trimethylsilyl) ethyl 2- ⁇ 4- [ (2-azido-4, 6-0- benzylidene-1, 2-dideoxy-D-glucopyranosyl) thio] - phenoxy ⁇ acetate.
  • tetra-n-butylammonium fluoride solution 1.0 M in THF, 0.36 mL, 0.36 mmol.
  • the reaction mixture is stirred for 20 min at room temperature and diluted with 3 mL of CH 2 C1 2 , washed with 5% HCl (5 mL) , extracted with CH 2 Cl 2 (2 x 2 mL) , washed with saturated NaCl (5 mL) , dried over Na 2 S0 4 , filtered, and concentrated to afford 0.067 g of a clear oil.
  • reaction is stirred at room temperature for 20 min and then diluted with 3 mL of CH 2 C1 2 , washed with NaHC0 3 (10 mL) , extracted with CH 2 C1 2 (2 x 3 mL) , washed with saturated NaCl (5 mL) , dried over Na 2 S0 4 , filtered and concentrated to afford 0.612 g of a yellow oil.
  • tetra- ⁇ -butylammonium fluoride solution 1.0 M in THF, 3.5 mL, 3.53 mmol.
  • the reaction mixture is stirred for 5 min at room temperature and diluted with 10 mL of CH 2 C1 2 , washed with 5% HCl (10 mL) , extracted with CH 2 C1 2 (2 x 5 L) , washed with saturated NaCl (10 mL) , dried over Na 2 S0 4 , filtered, and concentrated to afford 0.65 g of a clear oil.
  • reaction suspension is stirred vigorously at -15 to -20 °C for 24 h and then filtered through Celite.
  • nitrate ester 68 (17.5 g, 46.5 mmol) in 500 mL of glacial acetic acid is added sodium acetate (7.63 g, 93.0 mmol) .
  • the solution is stirred at 100 °C for 3 h and then allowed to cool to room temperature.
  • the reaction mixture is diluted with 1000 mL of ice water and extracted with CH 2 C1 2 (2 x 200 mL) .
  • the organic layers are combined and washed with ice water (2 x 400 mL) , saturated NaHC0 3
  • reaction mixture is diluted with 200 mL of EtOAc, washed with saturated NaCl (3 x 200 mL) , dried over Na 2 S0 4 , filtered, concentrated and purified by flash chromatography (35% EtOAc/hexane) to give 1.30 g (89%) of 2-azido-4, 6-O-benzylidene- 1, 2-dideoxy-l- (4-hydroxyphenylthio) - ⁇ , ⁇ -D- galactopyranose 72.
  • the mixed anomers are used directly for the next step, although the anomers are separable by flash chromatography (35% EtOAc/hexane) :
  • reaction mixture is stirred at 50-60 °C for 3 h and then allowed to cool to room temperature.
  • the reaction mixture is diluted with EtOAc (150 mL) , washed with H 2 0 (3 x 80 mL) and saturated NaCl (80 mL) , dried over Na 2 S0 4 , concentrated and purified by flash chromatography (45% EtOAc/hexane) to give 1.60 g (68%) of 2- (trimethylsilyl)ethyl 2- ⁇ 4- [ (2-azido- 4, 6-O-benzylidene-l, 2-dideoxy- ⁇ , ⁇ -D- galactopyranosyl)thio]phenoxy ⁇ acetate 73 as a mixture of anomers.
  • the mixed anomers are used directly for the next step, but could be separated by flash chromatography (25% EtOAc/hexane to elute the ⁇ - anomer, 45% EtOAc/hexane to elute the ⁇ -anomer) : R f
  • the reaction is diluted with 100 mL of CH 2 C1 2 and washed with saturated NaHC0 3 (80 mL) , dried over Na 2 S0 4 , filtered, concentrated and purified by flash chromatography to give a combined yield of 1.31 g (76.4%) of the anomers of 2- (trimethylsilyl) ethyl 2- ⁇ 4- [ (3-0-acetyl-2-azido-4, 6- O-benzylidene-1, 2-dideoxy- ⁇ , ⁇ -D-galactopyranosyl) - thio] phenoxy ⁇ acetate (15% EtOAc/hexane to elute the ⁇ -anomer 75, 35% EtOAc/hexane to elute the ⁇ -anomer 74) .
  • the title compound is prepared from 75 by treatment with tetrabutylammonium fluoride, and purified by flash chromatography, as described above:
  • thioglycoside 78 (1.40 g, 3.10 mmol) in 15 mL of methylene chloride is added N,N- diisopropylethylamine (0.64 mL, 3.70 mmol) , followed by 2- (trimethylsilyl)ethoxymethyl chloride (0.60 mL, 3.40 mmol) .
  • the mixture is stirred at room temperature for 2 h, poured into saturated NaCl (50 mL) and extracted with CH 2 C1 2 (2 x 50 mL) , dried over
  • N,N- diisopropylethylamine (0.090 mL, 0.51 mmol)
  • acetic anhydride (0.021 mL, 0.23 mmol)
  • DMAP (4 mg, 0.03 mmol)
  • the solution is stired at -78 °C for 30 min, then diluted with 25 mL of EtOAc and washed with saturated NH 4 C1 (25 mL) and saturated NaHC0 3 (25 mL) .
  • tetrabutylammonium fluoride solution (IM in THF, 2.46 mL, 2.46 mmol) .
  • IM in THF tetrabutylammonium fluoride solution
  • EtOAc 25 mL
  • reaction mixture is stirred at room temperature for 45 min and then is filtered though celite and washed with 10 mL of aqueous NaHC0 3 .
  • the aqueous solution is extracted with CH 2 C1 2 (2 x 10 mL) and the organic layers are combined and dried over Na 2 S0 4 , filtered and concentrated.
  • TentaGel S NH 2 resin (0.500 g) is suspended in N-methylpyrrolidinone (NMP, 15 mL) , and to this mixture is added acid 64 (0.115 g, 0.230 mmol) , diisopropylethylamine (0.22 g, 1.3 mmol) , and
  • the reaction mixture is then shaken for 2-5 h until the resin gives a negative Kaiser test.
  • the resin is washed with CH 2 C1 2 (3 x 15 mL, 5 min) , NMP (3 x 15 mL, 5 min) , and DMF (3 x 15 mL, 5 min) .
  • glycosyl acceptors (glycosylated acids 64, 66, 76, 77, 88 and 95) are attached to six separate batches of resin (e.g., TentaGel) and deprotected using the procedure described above. Afterwards, 0.450-gram portions of each resin are combined, suspended in 15 mL of CH 2 C1 2 , shaken for 15 min and dried on the lyophilizer for 12 h. 7.32. Procedure For Solid Phase Glycosylation
  • Predetermined portions (0.225 g) of the mixed resin are placed in twelve glycosylation reaction vessels equipped with glass-fritted bottoms, suspended in 5 mL of CH 2 C1 2 and agitated by slowly bubbling argon through the glass frit for 10 min. Each of the twelve resin samples is then glycosylated as follows: The 1-sulfinyl hexose derivative 5 (0.137 g, 0.270 mmol) and 2, 6-di- ert-butyl-4-methylpyridine
  • the dissolution and drying step is repeated at least once more.
  • the residue is then dried under vacuum for 1 h.
  • the sulfoxide and base are dissolved in CH 2 C1 2 (10 mL) and added to the first resin sample.
  • the suspension is then cooled to -65 °C, and a solution of trifluoromethanesulfonic anhydride (23 ⁇ L, 0.13 mmol) in 1 mL of CH 2 C1 2 is added dropwise over 10 min.
  • the reaction mixture is allowed to warm to 0 °C over 1-2 h, quenched using saturated aqueous NaHC0 3 (10 mL) and agitated for 10 min.
  • the resin is then washed sequentially with 10 mL portions (3 x 10 mL, 5 min for each portion) of the following solvents: NaHC0 3 , H 2 0, methanol, diethyl ether, CH 2 C1 2 and toluene.
  • the resin is dried on the lyophilizer (in vacuo) for 12 h and resubjected to the glycosylation reaction conditions for a second time to ensure complete reaction.
  • the twelve resin samples are glycosylated with the 1- sulfinyl hexose derivatives 5, 11, 13, D-25, L-25, 33, 37, 41, D-45, L-45, 51 and 53. All 12 portions of the resin are then combined, suspended in 15 mL of CH 2 C1 2 , shaken for 15 min and dried on the lyophilizer for 12 h. 7.33. Azide Reduction And Amine Acylation
  • Predetermined portions (0.139 g) of the resin are placed in 19 reaction vessels. Eighteen of the resin portions are subjected to the following reduction conditions to convert the azide groups to amino groups:
  • the resin is suspended in anhydrous THF (8 mL) , treated with trimethylphosphine (1.0 M solution in THF, 0.5 mL, 0.5 mmol) and shaken at room temperature for 4 h.
  • To the suspension is added 1 mL of H 2 0, and the reaction vessel is shaken for an additional 37 h at 48 °C.
  • the resin is washed with THF (3 x 15 mL, 5 min) to remove the trimethylphosphine.
  • the resin is then resuspended in
  • Typical conditions for the coupling of pyridine-4-carboxylic acid, pyridine-4-carboxylic acid N-oxide, N-acetyl-D- alanine and N-acetyl-L-alanine are as follows: The resin is suspended in a solution of 1:1 DMF/CH 2 C1 2 (6 mL) . Then one of the above-mentioned acids (0.42 mmol) and diisopropyl carbodiimide ( 66 ⁇ L, 0.052 g, 0.42 mmol) are added.
  • reaction mixture is shaken at room temperature for 12-24 h or until the resin gives a negative Kaiser test.
  • the resin is washed sequentially (3 x 8 mL portions, 5 min for each portion) with the following solvents: DMF, isopropanol and CH 2 C1 2 .
  • Typical conditions for the coupling of methanesulfonyl chloride, methyl isocyanate, methyl isothiocyanate, 3-methylbutyryl chloride, pentanoyl chloride, methyl chloroformate, benzoyl chloride, 4- nitrobenzoyl chloride, 2-thiophenecarbonyl chloride, 2-iodobenzoyl chloride and glutaric anhydride are as follows: The resin is suspended in CH 2 C1 2 (6 mL) , and to the suspension is added triethylamine (70 ⁇ L, 0.50 mmol) or diisopropylethylamine (88 ⁇ L, 0.50 mmol) .
  • DMAP (0.010 g, 0.080 mmol) is added in the cases of acetic anhydride and diketene.
  • One of the acylating reagent listed above (0.42 mmol) is then added, and the reaction mixture is shaken at 4 °C for 12 h.
  • the acylation reactions for reagents methyl isocyanate and methyl isothiocyanate are conducted at 50 °C for 12 h. All resin portions give a negative Kaiser test, thus indicating a complete reaction.
  • the resin is next washed with CH 2 C1 2 (3 x 8 mL, 5 min for each portion) .
  • the resin is suspended in a solvent mixture of THF:MeOH (1:4 v/v, 20 mL) for 10 min, and ground LiOH'H 2 0 (0.20 g, 4.8 mmol) is added.
  • the reaction mixture is shaken at room temperature for 12 h.
  • the resin is then washed with H 2 0 until the pH of the filtrate is determined to be neutral.
  • the neutral resin is then dried in vacuo for 12 h.
  • the resin sample is then incubated for 30 min at room temperature in 1 mL of PBST containing 3% bovine serum albumin (BSA) to block any nonspecific protein binding sites and then washed three times for 5 min each with 1 mL of PBST containing 1% BSA.
  • the resin sample is incubated in 1 mL of a lectin solution (10 ⁇ g/mL in PBST containing 1% BSA) at room temperature for 3 h.
  • the resulting resin sample is then washed three times for 5 min each with 1 mL of TBST buffer (20 mM TrisHCl, pH 7.5/500 mM NaCl/0.05% Tween-20) containing 1% BSA.
  • the resin sample is incubated for 20 min at room temperature in 1 mL of alkaline phosphatase-coupled streptavidin (10 ⁇ g/mL in TBST containing 1% BSA) .
  • Each of the thirty-eight purple beads, identified above by their color as having bound to the lectin probe, is treated to release the saccharide-containing moiety from the solid support.
  • the released moiety is subsequently analyzed by a variety of methods, including but not limited to, GC- mass spectroscopy, GC-Fourier Transform Spectrophotometry, nuclear magnetic resonance spectroscopy and the like. Knowing the starting reagents used in building the combinatorial library and the sequence in which each is used, the identity of each binding moiety is determined from the additional analytical data (such as mass spectral data) obtained above.
  • any compound or substance including but not limited to small molecules, purines, pyrimidines, nucleosides, nucleic acids, other sugars, amino acids, peptides, proteins, other natural polymers, unnatural polymers, synthetic polymers and the like, having a nucleophilic group capable of forming a covalent bond with a glycosyl donor can be utilized in the present invention.
  • purines pyrimidines
  • nucleosides nucleic acids
  • other sugars amino acids
  • peptides, proteins other natural polymers
  • unnatural polymers, synthetic polymers and the like having a nucleophilic group capable of forming a covalent bond with a glycosyl donor

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention se rapporte à une bibliothèque de ligands à base d'hydrates de carbone, qui comprend une pluralité de ligands distincts contenant du sucre, chacun lié à une partie résoluble d'un support solide. La bibliothèque est construite selon un procédé qui comprend une étape de formation d'une liaison glycosyle. Des bibliothèque de tailles différentes peuvent être constituées selon le procédé décrit dans cette invention, dans lesquelles un grand nombre d'espèces distinctes sont réalisées sensiblement concurremment par formation de liaisons glycosyles entre de nombreux types de participants. En outre, l'invention porte sur un dosage, qui permet le criblage sensiblement simultané de pratiquement tous les membres de la bibliothèque. L'isolement de ligands nouveaux de faible affinité est ainsi facilité.
PCT/US1997/004639 1996-03-21 1997-03-21 Bibliotheque de ligands a base d'hydrates de carbone, dosage et procede correspondants WO1997035202A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP53375297A JP2001504800A (ja) 1997-03-21 1997-03-21 糖質を骨格とするリガンドライブラリー、アッセイおよび方法
AU23413/97A AU2341397A (en) 1996-03-21 1997-03-21 Carbohydrate-based ligand library, assay and method
EP97916164A EP0901629A4 (fr) 1996-03-21 1997-03-21 Bibliotheque de ligands a base d'hydrates de carbone, dosage et procede correspondants

Applications Claiming Priority (2)

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US1537996P 1996-03-21 1996-03-21
US60/015,379 1996-03-21

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011436A1 (fr) * 1996-09-13 1998-03-19 Whitehead Institute For Biomedical Research Augmentation de l'affinite non specifique pour identifier des membres d'une bibliotheque combinatoire
EP0910568A1 (fr) * 1996-03-21 1999-04-28 Princeton University Derives de sulfinyl-hexose servant a la glycosylation
WO1999061583A2 (fr) * 1998-05-28 1999-12-02 Incara Pharmaceuticals Corp. Composes d'echafaudage a base d'hydrate de carbone, banques combinees, et leurs procedes de construction
WO2000042067A1 (fr) * 1999-01-12 2000-07-20 Princeton University Saccharides portes par des composes se liant a des proteines ou des peptides cellulaires de surface
US6335155B1 (en) 1998-06-26 2002-01-01 Sunesis Pharmaceuticals, Inc. Methods for rapidly identifying small organic molecule ligands for binding to biological target molecules
WO2002056016A2 (fr) * 2001-01-11 2002-07-18 Theravance, Inc. Procede d'application d'un ligand pour un substrat biologique
WO2002063299A1 (fr) * 2001-02-05 2002-08-15 Graffinity Pharmaceuticals Ag Procede de criblage des faibles affinites
WO2002063303A1 (fr) * 2001-02-07 2002-08-15 Graffinity Pharmaceuticals Ag Methode de criblage utilisant des supports solides modifies par des monocouches autoassemblees
US6579725B1 (en) 1999-03-05 2003-06-17 Massachusetts Institute Of Technology Linkers for synthesis of oligosaccharides on solid supports
US6919178B2 (en) 2000-11-21 2005-07-19 Sunesis Pharmaceuticals, Inc. Extended tethering approach for rapid identification of ligands
US6972172B2 (en) 1999-02-17 2005-12-06 Glycominds Ltd. Combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof
US6994966B2 (en) * 2000-02-17 2006-02-07 Glycominds Ltd. Combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof
US6998233B2 (en) 1998-06-26 2006-02-14 Sunesis Pharmaceuticals, Inc. Methods for ligand discovery

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WO1995003315A2 (fr) * 1993-07-21 1995-02-02 Oxford Glycosystems Ltd Saccharides leurs syntheses et utilisation
WO1995018971A1 (fr) * 1994-01-11 1995-07-13 Affymax Technologies N.V. Procede de synthese chimique en phase gazeuse de glycoconjugues
US5510240A (en) * 1990-07-02 1996-04-23 The Arizona Board Of Regents Method of screening a peptide library
US5575324A (en) * 1995-04-07 1996-11-19 Hwang; Charles Sunshield and method of manufacture of sunshield

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EP0601417A3 (fr) * 1992-12-11 1998-07-01 Hoechst Aktiengesellschaft Physiologiquement compatible et dégradable bloques de récepteur d'hydrate de carbone à base de polymère, procédé de leur préparation et leur utilisation
JPH08511234A (ja) * 1993-02-23 1996-11-26 ザ トラスティーズ オブ プリンストン ユニヴァーシティー グリコシド結合の溶液及び固相形成
EP0828729A1 (fr) * 1995-05-19 1998-03-18 Glycomed Incorporated Collection de composes de glycosides actives et leur usage biologique

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US5510240A (en) * 1990-07-02 1996-04-23 The Arizona Board Of Regents Method of screening a peptide library
WO1995003315A2 (fr) * 1993-07-21 1995-02-02 Oxford Glycosystems Ltd Saccharides leurs syntheses et utilisation
WO1995018971A1 (fr) * 1994-01-11 1995-07-13 Affymax Technologies N.V. Procede de synthese chimique en phase gazeuse de glycoconjugues
US5575324A (en) * 1995-04-07 1996-11-19 Hwang; Charles Sunshield and method of manufacture of sunshield

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Title
PROC. NATL. ACAD. SCI. U.S.A., December 1993, Vol. 90, OHLMEYER M. et al., "Complex Synthetic Chemical Libraries Indexed with Molecular Tags", pages 10922-10926. *
SCIENCE, November 1996, Vol. 274, LIANG R. et al., "Parallel Synthesis and Screening of a Solid Phase Carbohydrate Library", pages 1520-1522. *
See also references of EP0901629A4 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0910568A1 (fr) * 1996-03-21 1999-04-28 Princeton University Derives de sulfinyl-hexose servant a la glycosylation
EP0910568A4 (fr) * 1996-03-21 2002-04-24 Univ Princeton Derives de sulfinyl-hexose servant a la glycosylation
WO1998011436A1 (fr) * 1996-09-13 1998-03-19 Whitehead Institute For Biomedical Research Augmentation de l'affinite non specifique pour identifier des membres d'une bibliotheque combinatoire
WO1999061583A2 (fr) * 1998-05-28 1999-12-02 Incara Pharmaceuticals Corp. Composes d'echafaudage a base d'hydrate de carbone, banques combinees, et leurs procedes de construction
WO1999061583A3 (fr) * 1998-05-28 2000-04-06 Incara Pharmaceuticals Corp Composes d'echafaudage a base d'hydrate de carbone, banques combinees, et leurs procedes de construction
US6335155B1 (en) 1998-06-26 2002-01-01 Sunesis Pharmaceuticals, Inc. Methods for rapidly identifying small organic molecule ligands for binding to biological target molecules
US6998233B2 (en) 1998-06-26 2006-02-14 Sunesis Pharmaceuticals, Inc. Methods for ligand discovery
WO2000042067A1 (fr) * 1999-01-12 2000-07-20 Princeton University Saccharides portes par des composes se liant a des proteines ou des peptides cellulaires de surface
US6972172B2 (en) 1999-02-17 2005-12-06 Glycominds Ltd. Combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof
US6579725B1 (en) 1999-03-05 2003-06-17 Massachusetts Institute Of Technology Linkers for synthesis of oligosaccharides on solid supports
US6994966B2 (en) * 2000-02-17 2006-02-07 Glycominds Ltd. Combinatorial complex carbohydrate libraries and methods for the manufacture and uses thereof
US6919178B2 (en) 2000-11-21 2005-07-19 Sunesis Pharmaceuticals, Inc. Extended tethering approach for rapid identification of ligands
US6656694B2 (en) 2001-01-11 2003-12-02 Theravance, Inc. Method for identifying a ligand for a biological substrate
WO2002056016A3 (fr) * 2001-01-11 2003-02-06 Theravance Inc Procede d'application d'un ligand pour un substrat biologique
WO2002056016A2 (fr) * 2001-01-11 2002-07-18 Theravance, Inc. Procede d'application d'un ligand pour un substrat biologique
WO2002063299A1 (fr) * 2001-02-05 2002-08-15 Graffinity Pharmaceuticals Ag Procede de criblage des faibles affinites
WO2002063303A1 (fr) * 2001-02-07 2002-08-15 Graffinity Pharmaceuticals Ag Methode de criblage utilisant des supports solides modifies par des monocouches autoassemblees

Also Published As

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EP0901629A1 (fr) 1999-03-17
EP0901629A4 (fr) 2000-02-02
CA2249290A1 (fr) 1997-09-25
AU2341397A (en) 1997-10-10

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