WO2006079364A1 - Clivage specifique et modere de peptide ou de liaison amide - Google Patents

Clivage specifique et modere de peptide ou de liaison amide Download PDF

Info

Publication number
WO2006079364A1
WO2006079364A1 PCT/EP2005/007233 EP2005007233W WO2006079364A1 WO 2006079364 A1 WO2006079364 A1 WO 2006079364A1 EP 2005007233 W EP2005007233 W EP 2005007233W WO 2006079364 A1 WO2006079364 A1 WO 2006079364A1
Authority
WO
WIPO (PCT)
Prior art keywords
phosphine
azide
peptide
tris
compound
Prior art date
Application number
PCT/EP2005/007233
Other languages
English (en)
Inventor
Jaapwillem Back
Jan Herman Van Maarseveen
Chris Gerdinus De Koster
Original Assignee
Stichting Voor De Technische Wetenschappen
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Stichting Voor De Technische Wetenschappen filed Critical Stichting Voor De Technische Wetenschappen
Publication of WO2006079364A1 publication Critical patent/WO2006079364A1/fr

Links

Classifications

    • 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
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • 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

Definitions

  • the azide functional group is exceptionally well suited for in vivo labeling of biomolecules .
  • Azides combine a high chemical stability under biological conditions with a unique reactivity enabling mild and selective organic transformations under physiological conditions ( Saxon, E . & Bertozzi , C . R. (2000 ) Cell Surface Engineering by a Modified Staudinger Reaction . Science 287 , 2007-2010 ; Prescher, J. A. , Dube , D . H . & Bertozzi , C . R. (2004 ) Chemical remodelling of cell surfaces in living animals . Na ture 430 , 873-7 ; Kohn, M. & Breinbauer, R. (2004 ) The Staudinger Ligation - A Gift to Chemical Biology .
  • the amino acid Azido-homoalanine (Azhal ) has been shown to be effectively incorporated into proteins by the native methionyl tRNA synthase of E coli (Kiick et al . , supra) . Kiick et al . have demonstrated that proteins containing azidohomoalanine can be 5 selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents . It was thus suggested that incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the 10 intracellular environment .
  • proteins containing azide-functionalized amino acids with azide-reducing agents in a protic environment cleavage of the amide bond C-terminal to azide-functionalized amino acid is accomplished to obtain a free amine compound and a secondary residue that varies depending on the azide-functionalized amino acid used .
  • This method may advantageously be used for cleavage of proteins or peptides , but can also be used for the cleavage of other compounds comprising an amide bond C-terminal of the azide-functionalized amino acid .
  • the present invention relates to a method for cleaving of an amide bond in a compound comprising said amide bond and at least one azide-function spaced at 3 or 4 atoms from the carbon of the amide bond to be cleaved, said compound being chosen from the compounds of formulae I-IV :
  • Ri and R 2 may be independently a hydrogen atom, a hydrocarbon group, or a heterocyclic group, which may have a substituent, and the two R ' s may be the same or different and independently a hydrogen atom, a hydrocarbon group, or a heterocyclic group, which may have a substituent
  • said method comprising the steps of : a) providing the compound of any of formulae I-IV, wherein R 1 and R 2 may be independently a hydrogen atom, a hydrocarbon group, or a heterocyclic group, which may have a substituent , and the two R ' s may be the same or different and independently a hydrogen atom, a hydrocarbon group, or a heterocyclic group, which may have a substituent; and b) subj ecting the compound of step a) to an azide-reducing agent in a protic solvent to cleave the amide bond C-terminal to the non-natural amino acid, thereby obtaining RiNH 2 and a secondary residue .
  • Detection of the presence of non-natural amino acids in a peptide chain is part of the routine work for the skilled practitioner ( see e . g . the above citations ) , and can be done by one or more of several techniques , such as mass spectrometry, EcLman degradation, amino acid analysis, or derivatization or reaction with appropriate azido specific reagents that carry a detectable label (e . g . phosphines or alkynes , labeled with fluorophores , biotins , or any other secondary label in conj unction with a suitable assay .
  • a detectable label e . g . phosphines or alkynes , labeled with fluorophores , biotins , or any other secondary label in conj unction with a suitable assay .
  • R 1 is a peptide chain (in case of the compounds of formulae III and IV) or R 1 and R 2 are both peptide chains ( in case of the compounds of formulae I and II ) , such that the non-natural azide-functionalized amino acid is incorporated in a peptide or a protein .
  • R 1 may be different from R 2 , but R 1 and R 2 may also be the same .
  • the non-natural amino acid is preferably chosen from the group, consisting of azidohomoalanine (the azide-functionalized amino acid moiety displayed in formula I wherein both R ' s are H) , azidonorvaline (the azide-functionalized amino acid moiety displayed in formula I I wherein both R' s are H) and derivates thereof (the azide- functionalized amino acid moiety displayed either in formula I or II wherein the two R ' s may be the same or different and independently a hydrogen atom, a hydrocarbon group, or a heterocyclic group, which may have a substituent; R may e . g .
  • Azidohomoalanine , azidoalanine, azidonorvaline and azidonorleucine are shown to be methionine analogs .
  • the azide group can survive cellular metabolism.
  • the incorporation of the above methionine analogs into proteins is controlled most stringently by the methionyl-tRNA synthetase of the host .
  • a methionine analog can be incorporated into the peptide chain, e .
  • Ri and R 2 may be independently a hydrogen atom, a hydrocarbon group, or a heterocyclic group, which may have a substituent, and the two R ' s may be the same or different and independently a hydrogen atom, a hydrocarbon group, or a heterocyclic group, which may have a substituent , as indicated above .
  • step b) the compound of any of formulae I-IV is subj ected to an azide-reducing agent in a protic solvent to cleave the amide bond C-terminal to the non-natural amino acid, thereby obtaining R 1 NH 2 and a secondary residue .
  • the azide-reducing agent may be any azide-reducing agent known in the art, such as LiAlH 4 , H 2 /catalyst, Cr (II ) /H + , ⁇ 3 P/NH 4 OH,
  • H 2 S/pyridine/water, H 2 /Lindlar catalyst, trivalent phosphines , and thiol-containing agents Boyley H . , Standring, D . N . and Knowles , J . R. ( 1978 ) Propane-1 , 3-dithiol : a selective reagent for the efficient reduction of alkyl and aryl azides to amines . Tetrahydr. Lett . 39, 3633-3634 ) .
  • a “protic solvent” as herein used refers to a solvent that is capable of donating a hydrogen atom for hydrogen bonding . This usually requires an NH or OH bond .
  • Non-limiting examples thereof are aqueous solutions , such as water and several types of buffer solutions , and alcohols such as ethanol , nitriles such as acetonitrile , organic acids such as acetic acid, furanes such as tetrahydrofurane , formamides such as dimethylformamide and any mixture of these solvents .
  • the protic solvent should allow for solubilisation of the peptide chain as well as the azide-reducing agent .
  • the "subj ecting the peptide chain to an azide- reducing agent in a protic solvent” refers to a reaction that occurs between the peptide chain and an azide-reducing agent, e . g . phosphine, 2-mercaptoethanol or dithiothreitol , when these are simultaneously present in the protic solvent .
  • the peptide chain will thus be subj ected to the azide-reducing agent when a sample of peptide chain in protic solvent is mixed with the azide-reducing agent in the same protic solvent or a protic solvent that is miscible with the protic solvent used to solubilise the peptide chain .
  • the amide bond C-terminal to the non-natural amino acid participates in the reaction with the triazene that results from the reaction of the azido moiety of this amino acid with the azide-reducing agent to accomplish cleavage of this amide bond, as is further exemplified using a phosphine as azide-reducing agent in Fig . 1. Due to the reaction with the azide- reducing agent, the resultant peptide C-terminal to the scissile bond becomes a peptide with an N-terminal free amine, whereas the resultant peptide N-terminal to the scissile bond becomes a so-called secondary residue ( Fig . 1 ) .
  • the secondary residue is dependent on the non-natural amino acid that is used in step a) , and is e . g . a homoserine lactone residue in the case of the non-natural amino acid being azidohomoalanine .
  • homoserine and homoserine lactone are in equilibrium, and the equilibrium is dependent on pH, as the skilled practitioner is well aware of .
  • a nucleophile e . g . a primary amine
  • Proteins may be produced in the host of choice (see e . g . Wang & Schulz, supra) if so desired as a fusion protein as to solubilise them and may accordingly undergo regular post- translational processing, and may then be cleaved from the fusion moiety to yield the desired protein in fully processed form.
  • the present invention finds further application in organic synthesis .
  • the non-natural amino acid is azidohomoalanine and the secondary residue is a homoserine lactone residue .
  • azidohomoalanine is most efficiently incorporated into a protein by means of the cell ' s native translational apparatus (Kiick et al . , supra and Link et al . , supra) .
  • the method according to the invention can be very efficiently carried out as is shown below in the examples and in figure 1.
  • the protic solvent is an aqueous solution .
  • protons are available for the reaction that is depicted in fig . 1.
  • the phosphine of formula V may be any phosphine of formula V
  • R 4 , R 5 and R 6 are independently optionally substituted alkyl or aryl chains and may be the same or different .
  • R 4 , R 5 and R 6 groups that render the phosphine soluble in a protic solvent, in particular in water .
  • Non- limiting examples of R 4 , R 5 and R 6 include carboxylic acids (e . g . propionic acid, acetic acid) , alkylamines (e . g . propylamine , ethylamine ) , alkylhydroxyls (e . g .
  • the thiol-containing agent may be any thiol-containing agent known in the art, particular one of formulae VII or VIII
  • R 7 is an independently optionally substituted alkyl or aryl chain .
  • Non-limiting examples thereof include hydroxylalkylthiols , e . g . (2- ) mercaptoethanol , mercaptopropanol , mercaptobutanol , dithiothreitol , dithioerytol ; aminoalkylthiols , e . g . cysteine , cystamine ; alkylthiols , e . g . ethanedithiol , propanedithiol , butanedithiol ; and carboxyalkylthiols , e . g . thioglycolic acid, 2 , 3- dimercaptosuccinic acid.
  • Such thiol-containing agents are readily soluble in protic solvents in the applicable pH ranges and are therefore the preferred thiol-containing agents to be used .
  • the azide-reducing agent is water-soluble such that reaction between the azide-reducing agent and the peptide chain according to the method of the invention is facilitated and most efficient .
  • R 4 , R 5 and R 6 are independently optionally substituted alkyl or aryl chains and may be the same or different . It was found that such phosphines are particularly suitable for use in the method of the invention .
  • the phosphine is chosen from the group, consisting of tris (carboxyethyl ) phosphine , tris (carboxypropyl ) phosphine , tris (hydroxyethyl ) phosphine , tris (hydroxypropyl ) phosphine , tris (ethylamine ) phosphine , tris (propylamine) phosphine .
  • These phosphines are readily soluble in protic solvents in the applicable pH values and are therefore the preferred phosphines to be used .
  • the phosphine is tris (carboxyethyl ) - phosphine, as this compound is readily available at a relatively low cost price . It is expected that the trisalkylaminephosphines and the trisalkylhydroxylphosphines will be particularly effective at low pH .
  • the thiol-containing agent is a dithiol-containing agent, as it was found that dithiol-containing agents are 300-1 , 000 times more efficient in the reduction of the azide than monothiol-containing agents . It was found that dithiol- containing agents are about equally efficient in comparison to trivalent phosphines .
  • dithiothreitol butanedithiol or propanedithiol is used, as these compounds are readily available on the market, the latter two at a relatively low cost price .
  • the compound of step a) is peptide chain, which peptide chain is obtained by in vivo incorporation of at least one non-natural azide-functionalized amino acid, since this in particular allows for post-translational processing as indicated above .
  • the azide-functionalized amino acid may be any amino acid as disclosed above , such as azidohomoalanine and azidonorvaline and derivatives thereof .
  • step b) of the method according to the present invention is carried out at a pH in the range of 3-10.
  • the preferred pH is dependent on the protic solvent used and the solubility of the azide-reducing agent used.
  • One skilled in the art will readily be capable of determining a suitable system for carrying out the invention .
  • One example of such system is the system set forth below in example 2.
  • the present invention relates to a peptide obtained by a method according to the present invention .
  • This peptide may originate from a larger peptide from which it was liberated by the method according to the present invention .
  • the peptide C-terminal to the scissile bond may closely or fully resemble a native peptide or protein .
  • Figure 1 shows the mechanism proposed for the reaction :
  • Phosphines add to the electron deficient centre of the azide 1 initially forming intermediate 2 , that may either hydrolyze through postulated intermediate 3 into the triazene 4 or fragment into aza- ylide 5.
  • Triazenes have previously been shown react through an S N 2 reaction with suitable nucleophiles of either intra- or intermolecular origin . It is worthy to mention that at elevated pH conversion of 1 to homoserine without peptide cleavage occurs , indicative of S N 2 attack by OH " .
  • the protonated triazene 6 enables an energetically favorable five-membered membered ring closure resulting in cyclic imido ester 7 , a pathway analogous to the cyanogen bromide induced cleavage of the methionine peptide bond.
  • hydrolysis of the imido ester 7 produces the homoserine lactone 8 and amine 9. This mechanism is supported by the 18 O labeling experimental results described below which allow the carbonyl oxygen to be conserved in the lactone .
  • the phosphine activated azide , aza-ylide 5 can be reduced to amine or may become protonated generating intermediate 11 which via intramolecular S N 2 displacement involving the amide oxygen atom yields the common intermediate imido-ester 7 , that will then again hydrolyze into 8 and 9.
  • A Coomassie Blue stained gel of equal amounts (23 ⁇ g) of Azhal-PYP either mock incubated or incubated with 2ME, DTT, or TCEP in the absence and presence of urea as indicated .
  • B MALDI-FTMS spectrum of TCEP cleaved Azhal-PYP .
  • C sequence of His- tagged PYP from E. halophila , in which X denotes azidohomoalanine . Intact molecule mass and tryptic peptide mapping confirmed >95% incorporation of azidohomoalanine at methionine coded residues .
  • a coverage map obtained with TCEP (above the sequence ) and DTT (below the sequence) is presented . Solid lines indicate peptides resulting from cleavage that were detected with MALDI-TOF; dashed lines indicate peptides that were detected with ESI-FTMS .
  • Figure 3 shows a comparison of the cleavage of Azhal-PYP (all methionines replaced with Azhal ) with TCEP and methionine containing PYP with cyanogen bromide .
  • Digests were loaded onto an LCMSMS system.
  • B Deconvoluted MSMS fragmentation spectra of the doubly charged ion indicated in panel A with annotation of fragment ions and parent ion (par) . Identical fragment spectra prove the identicity of both peptides .
  • C sequence of the peptide and annotation of retrieved fragment ions .
  • U denotes homoserine lactone .
  • HIS- tagged recombinant Photoactive Yellow Protein ( PYP) from Ectothiorhodospira halophila was produced in methionine auxotrophic E. coli grown on L-Azhal containing media as follows .
  • L-Azhal was synthesized from L-BOC-2 , 4-diaminobutyric acid (Chem-Impex, Wood Dale, USA) by diazo transfer using Triflic-azide (TfN 3 ) (Lundquist, J. T . & Pelletier, J. C . (2001 ) Improved solid- phase peptide synthesis method utilizing alpha-azide-protected amino acids . Org. Lett . 3, 781-783 ) , followed by BOC deprotection with dioxane/HCl in dichloromethane .
  • Optimal cleavage of peptides and protein was achieved with a peptide concentration of about 0.2 mg/ml in 50 mM Na-acetate buffer pH 4.4 and 1-100 mM of Tris-carboxy-ethyl-phosphine (TCEP) .
  • TCEP Tris-carboxy-ethyl-phosphine
  • For hydrolysis of homoserine lactone an excess of unbuffered IM tris (hydroxymethyl ) amine was added and left for 48 h before sample cleanup with ZipTip Ci 8 (Millipore, Bedford, USA) .
  • TCEP-induced reduction and cleavage of purified Azhal-labelled PYP was analysed by SDS-PAGE ( figure 2a) and mass spectrometry ( figure 2b) .
  • MALDI-TOF MS or ESI-FTMS we were able to detect fragments N- or C- terminal to all Azhal residues (figure 2b) of TCEP treated protein . All fragments carried a free amine at the N-terminus and all , except the C-terminal peptide that ended in valine, having a homoserine lactone residue at the C-terminus . This was confirmed by tandem mass spectrometry using collision induced dissociation on ESI-FTMS .
  • the cleavage process disclosed in example 2 was performed in a broad range of pH values (tested from 3 to 10 ) .
  • the rate of reaction is slow at pH below 4 , due to decreased solubility of TCEP .
  • the highest ratio of cleavage : reduction occurs at pH 4.5 - 5. Under these conditions approximately only 5% of PYP is still intact and fully or partially reduced .
  • Peptide FFRXGFRF wherein X denotes a Azhal residue
  • TCEP TCEP at pH 5.
  • the products of this cleavage were FFRO, wherein 0 denotes a homoserine lactone residue , and GFRF as a free amine .
  • Example 5 Mass spectrometry
  • Reflectron MALDI-TOF mass spectra were recorded on a Micromass TofSpec 2EC (Micromass, Whyttenshawe, UK) .
  • ESI-FTMS spectra were acquired on an APEX-Q FTMS (Bruker Daltonics, Billerica, USA) , for low energy CID the ions were activated in the external collision cell or produced by SORI-CID in the FTMS cell.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention a trait à un procédé pour le clivage d'une liaison amide dans une chaîne peptidique, permettant d'obtenir un peptide N-terminal sous la forme d'une amine libre et un résidu secondaire, ainsi qu'à un peptide obtenu par un tel procédé.
PCT/EP2005/007233 2005-01-31 2005-07-01 Clivage specifique et modere de peptide ou de liaison amide WO2006079364A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP05075233.6 2005-01-31
EP05075233 2005-01-31

Publications (1)

Publication Number Publication Date
WO2006079364A1 true WO2006079364A1 (fr) 2006-08-03

Family

ID=34938027

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2005/007233 WO2006079364A1 (fr) 2005-01-31 2005-07-01 Clivage specifique et modere de peptide ou de liaison amide

Country Status (1)

Country Link
WO (1) WO2006079364A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030199084A1 (en) * 2000-03-16 2003-10-23 Eliana Saxon Chemoselective ligation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030199084A1 (en) * 2000-03-16 2003-10-23 Eliana Saxon Chemoselective ligation

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
BALBAA MAHMOUD ET AL: "Secondary substrate binding in aspartic proteinases: Contributions of subsites S-3 and S'2 to k-ca!1t", ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, vol. 306, no. 2, 1 November 1993 (1993-11-01), pages 297 - 303, XP002347354, ISSN: 0003-9861 *
BAYLEY, H; STANDRING, DN; KNOWLES, JR: "Propane-1,3-dithiol: A selective reagent for the efficient reduction of alkyl and aryl azides to amines", TETRAHEDRON LETTERS, vol. 19, no. 39, 1978, Great Britain, pages 3633 - 3634, XP002347106 *
BIBART, RICHARD T. ET AL: "Development of a Second Generation Coenzyme A Analog Synthon", JOURNAL OF ORGANIC CHEMISTRY, CODEN: JOCEAH; ISSN: 0022-3263, vol. 64, no. 8, 16 April 1999 (1999-04-16), pages 2903 - 2909, XP002329926 *
BLOM P ET AL: "Design, synthesis, and evaluation of N-aroyloxy-2-thiopyridones as DNA photocleaving reagents.", BIOORGANIC & MEDICINAL CHEMISTRY. MAY 1999, vol. 7, no. 5, May 1999 (1999-05-01), pages 727 - 736, XP002347292, ISSN: 0968-0896 *
EKSTROM B ET AL: "SEMISYNTHETIC PENICILLINS III. AMINOPENICILLINS VIA AZIDOPENICILLINS", ACTA CHEMICA SCANDINAVICA, MUNKSGAARD, COPENHAGEN, DK, vol. 19, no. 2, 1965, pages 281 - 299, XP009048328, ISSN: 0904-213X *
GOMEZ-VIDAL, JOSE A. ET AL: "Potent and Selective Conformationally Restricted Neuronal Nitric Oxide Synthase Inhibitors", JOURNAL OF MEDICINAL CHEMISTRY, CODEN: JMCMAR; ISSN: 0022-2623, vol. 47, no. 3, 29 January 2004 (2004-01-29), pages 703 - 710, XP002329918 *
KAMAL A ET AL: "Selective reduction of aromatic azides with hexamethyldisilathiane: synthesis of new 2-azidopyrrolo[2,1-c][1,4]benzodiazepines", TETRAHEDRON LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 45, no. 17, 19 April 2004 (2004-04-19), pages 3499 - 3501, XP004500108, ISSN: 0040-4039 *
KIICK K L ET AL: "Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 99, no. 1, 8 January 2002 (2002-01-08), pages 19 - 24, XP002904363, ISSN: 0027-8424 *
LINK A JAMES ET AL: "Cell surface labeling of Escherichia coli via copper(I)-catalyzed (3+2) cycloaddition.", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 125, no. 37, 17 September 2003 (2003-09-17), pages 11164 - 11165, XP002329889, ISSN: 0002-7863 *
LUNDQUIST JOSEPH T IV ET AL: "A new tri-orthogonal strategy for peptide cyclization", ORGANIC LETTERS, vol. 4, no. 19, 19 September 2002 (2002-09-19), pages 3219 - 3221, XP002329919, ISSN: 1523-7060 *
NISHINO, NORIKAZU ET AL: "New synthetic substrates and inhibitors of serratial 56K protease", PEPTIDE CHEMISTRY , VOLUME DATE 1986, 24TH, CODEN: PECHDP; ISSN: 0388-3698, 1987, pages 233 - 236, XP001206436 *
PEI Y ET AL: "Regioselective syntheses of 3-aminomethyl-5-substituted isoxazoles: A facile and chemoselective reduction of azide to amine by sodium borohydride using 1,3-propanedithiol as a catalyst", TETRAHEDRON LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 34, no. 47, 19 November 1993 (1993-11-19), pages 7509 - 7512, XP002952884, ISSN: 0040-4039 *
POPELOVA A ET AL: "A concise synthesis of 4-nitrophenyl 2-azido-2-deoxy- and 2-acetamido-2-deoxy-d-mannopyranosides", CARBOHYDRATE RESEARCH, ELSEVIER SCIENTIFIC PUBLISHING COMPANY. AMSTERDAM, NL, vol. 340, no. 1, 17 January 2005 (2005-01-17), pages 161 - 166, XP004693143, ISSN: 0008-6215 *
REDDY G V ET AL: "A Novel, Simple, Chemoselective and Practical Protocol for the Reduction of Azides Using In / NH4Cl", TETRAHEDRON LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 40, no. 20, 14 May 1999 (1999-05-14), pages 3937 - 3938, XP004163786, ISSN: 0040-4039 *
VANDERHAEGHE H ET AL: "PREPARATION OF PENICILLIN N AND ISOPENICILLIN N", JOURNAL OF ANTIBIOTICS, JAPAN ANTIBIOTICS RESEARCH ASSOCIATION, TOKYO, JP, vol. 27, no. 3, March 1974 (1974-03-01), pages 169 - 177, XP009048331, ISSN: 0021-8820 *

Similar Documents

Publication Publication Date Title
Yang et al. Synthesis of K48-linked diubiquitin using dual native chemical ligation at lysine
EP3424941A1 (fr) Procédé de production d'un complexe peptide-acides nucléiques non cyclique présentant, sur une extrémité n terminale, un acide aminé doté d'un groupe thiol à proximité du groupe amino, bibliothèque correspondante, et bibliothèque du complexe peptide-acides nucléiques cyclique ainsi dérivée
Ollivier et al. A simple and traceless solid phase method simplifies the assembly of large peptides and the access to challenging proteins
Wang et al. Chemoselective modifications for the traceless ligation of thioamide-containing peptides and proteins
Back et al. Mild and Chemoselective Peptide‐Bond Cleavage of Peptides and Proteins at Azido Homoalanine
Zeng et al. Genetic introduction of a diketone-containing amino acid into proteins
EP1651664B1 (fr) Procede de ligature
Guan et al. Synthetic thiol and selenol derived amino acids for expanding the scope of chemical protein synthesis
Guidotti et al. Controlling the supramolecular assembly of nucleosomes asymmetrically modified on H4
EP2493909B1 (fr) Procede de ligation native de polypeptides
WO2006079364A1 (fr) Clivage specifique et modere de peptide ou de liaison amide
US9206224B2 (en) Method for synthesizing proteins
US7351797B1 (en) Labeled peptides, and processes and intermediates useful for their preparation
Söveges et al. A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags
Jiang et al. Synthesis of Thiol‐Containing Oligopeptides via Tandem Activation of γ‐Thiolactones by Silver‐DABCO Pair
Sánchez-Campillo et al. A versatile o-aminoanilide linker for native chemical ligation
EP1511760A2 (fr) Strategies de protection de carboxyle pour des acides amines a terminal c acides dans la ligature chimique d'oligopeptides
KR101676515B1 (ko) 펩티드 티오에스테르체의 제조방법
KR20180026350A (ko) 새로운 탄소-탄소 결합 기반 위치 특이적으로 변형된 단백질의 제조방법
Shelton Chemical Approaches to Investigate the Biochemical Crosstalk Between Histone Sumoylation, Methylation and Acetylation
Qiao SITE-SPECIFIC PROTEIN FUNCTIONALIZATION USING ACTIVATED CYSTEINE BASED CHEMICAL BIOLOGY METHODS
Oualid et al. Synthesis of isopeptide-linked diubiquitin chains
Warner Multifunctionalization of proteins: Strategies for combining semi-synthetic and bioorthogonal techniques
Weller Development of auxiliary-mediated protein semisynthesis methods toward the study of histone SUMOylation
KR20210029156A (ko) 유입에서 천연 화학적 라이게이션을 이용한 신규 합성 방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05756573

Country of ref document: EP

Kind code of ref document: A1

WWW Wipo information: withdrawn in national office

Ref document number: 5756573

Country of ref document: EP