Efficient synthesis of polypeptide-α-thioester by the method combining polypeptide expression and chemical activation for the semi-synthesis of interferon-γ having oligosaccharides.pdf

Research Article

Received: 1 July 2014 Revised: 16 September 2014 Accepted: 3 October 2014 Published online in Wiley Online Library: 6 November 2014

(wileyonlinelibrary.com) DOI 10.1002/psc.2709

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Efficient synthesis of polypeptide-α-thioesterby the method combining polypeptideexpression and chemical activation for thesemi-synthesis of interferon-γhaving oligosaccharidesYasuhiro Kajihara,* Yurie Kanemitsu, Mika Nishihara, Ryo Okamotoand Masayuki Izumi

In order to synthesize interferon-γ glycoform having an oligosaccharide at the 97 position by a semi-synthetic method,interferon-γ-polypeptide-(1–94)-α-hydrazide was prepared by the specific Cys-cyanylation of polypeptide-(1–94)-Cys-His6expressed from E. coli and subsequent hydrazinolysis in 22% yield (two steps). This polypeptide-α-hydrazide was then convertedinto corresponding polypeptide-α-thioester under NaNO2/acid conditions followed by thiolysis in 83% yield. Copyright © 2014European Peptide Society and John Wiley & Sons, Ltd.

Keywords: peptide expression; chemical conversion; semi-synthesis; peptide-α-thioester

* Correspondence to: Yasuhiro Kajihara, Department of chemistry, Osaka University,1–1, Machikaneyama, Toyonaka 560–0043 Japan. E-mail: [email protected]

Department of chemistry, Osaka University, 1-1, Machikaneyama, Toyonaka560-0043, Japan

Introduction

Chemical protein synthesis is a robust method to prepare not onlypost-translationally modified protein but also protein including un-natural amino acid derivatives in peptide backbone [1]. Thesechemical syntheses efficiently make use of site-specific peptidecoupling reactions. Of these coupling reactions, a representativeis native chemical ligation (NCL) enabling to couple two peptidesthrough native amide bond by the reaction of a peptide-α-thioesterand a Cys-peptide [2]. Consequently, the preparation method ofpeptide-α-thioester has been extensively studied, and manymethodologies have been reported based on chemical activationconditions [1,3]. Along with this chemical method, expressed pro-tein ligation (EPL) based on inten system has also been developed[4]. Intein system employs unique protein self-splicing system andcatalyzes the formation of polypeptide-α-thioester via a reactionof N-terminal flanking polypeptide with endogenous cysteine-thiolof intein peptide backbone. This thioester bond can be cleaved bythe addition of several exogenous thiols to yield polypeptide-α-thioester that is useful for semisynthesis of protein under the EPLconcept [4–6]. In this method, hydrazine can also be used insteadof exogenous thiol to obtain polypeptide-α-hydrazide that is stableand can be efficiently converted into polypeptide-α-thioestersunder acidic oxidative conditions [7–9]. Recently, a uniquethioesterification method based on N to S acyl migration was alsofound [3,10]. Peptides having a specific sequence of Gly-Cys orHis-Cys were found to undergo the N to S acyl migration by a nucle-ophilic attack of the Cys-thiol to the amide group under the acidicconditions and this specific reaction yields thioester form.Subsequently, this resultant thioester can be converted intopolypeptide-α-thioester and polypeptide-α-hydrazide by the addi-tion of several kinds of thiol and hydrazine, respectively [11].

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Our laboratory has been examining chemical and semichemicalsynthesis of glycoproteins [12]. Human interferon γ (hIFN-γ) consistsof 143 amino acid residues and has N-glycans at the 25 and 97 po-sitions. Oligosaccharides play important roles of glycoprotein bioac-tivity, folding in the endoplasmic reticulum, glycoprotein-lifetime inblood, and interference of protein aggregation. However, oligosac-charides exhibit structural diversity, and therefore it is still difficultto determine what oligosaccharide-structure is essential for proteinactivity [12]. hIFN-γ exhibits considerable heterogeneity in the oli-gosaccharide structure at the 97 position rather than 25 positionand shows at least 12 variants [13,14]. However, it is still unclearwhy the oligosaccharide at the 97 position show more diversitiesthan that at the 25 position. To investigate structure activity relation-ship, it is essential to prepare homogeneous glycoform that is an iso-mer of glycoprotein dependent on oligosaccharide structuresattached and glycosylation positions. Therefore, we set out to syn-thesize hIFN-γ glycoform an oligosaccharide at the only 97 position.To synthesize this type of glycoproteins with NCL conditions, an idealstrategy is the coupling of anN-terminal polypeptide-α-thioester pre-pared from E. coli expression with the chemically synthesized shortglycopeptide having a cysteine at the N-terminus. In this context,we have explored an alternative efficient chemical thioesterificationreaction of a polypeptide prepared from E. coli expression. This EPL

Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

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Figure 1. Synthesis of polypeptide-α-hydrazide. (A) Synthetic strategy ofpolypeptide-α-hydrazide from a non-protected polypeptide. (B) Aminoacid sequence of interferon γ. Glycosylation site is indicated in italic letter.(C) amino acid sequence of interferon γ-polypeptide-(MetGln1-Lys94)-Cys-His6 obtained by E. coli expression.

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SEMI-SYNTHESIS OF POLYPEPTIDE-α-THIOESTER

strategy allows us to expect to prepare hIFN-γ glycoforms varying inoligosaccharide structure at the 97 position.In this paper, we describe a semi-synthesis of interferon-γ-poly-

peptide-(1–94)-α-thioester based on peptide expression and chem-ical activation methods. This chemical activation methodology isapplicable for polypeptides obtained from inclusion body.

Figure 2. E. coli expression of interferon γ-polypeptide-(MetGln1-Lys94)-Cys-His6 1 and its cyanylation. (A) SDS-gel electrophoresis of the expressedpolypeptide 1. Lane 1: maker, Lane 2: crude proteins from E. coliexpression, Lane 3: After Ni/NTA-column purification. (B) ESI-mass andHPLC profile of 1 purified with HPLC. (C) ESI-mass and HPLC profile ofinterferon γ-polypeptide-(MetGln1-Lys94)-Cys(CN)-His6 2 purified withHPLC.

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Materials and Methods

The enzymeswere obtained from commercially available company:NdeI(wako Nippon gene), BamHI( wako Nippon gene ), and ligationbuffer ( TOYOBO, Code No. SMK-101 ).

J. Pept. Sci. 2014; 20: 958–963 Copyright © 2014 European Peptide Society a

Preparation of Vector

The DNA sequence coded interferon-γ-(Gln1-Lys94)-Cys-hexahistidine (His6) was prepared by the custom company, andthe gene was inserted in commercially available pET3a vectorwith Nde I and BamH I enzymes. Ligation between inserted geneand vector gene was performed with commercially availableligation solution (TOYOBO, Code No. SMK-101).

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Table 1. Hydrazinolysis conditions toward cyanylated polypeptides

Entry Condition pH Conversion yield (%)a

1 10% Hydrazine/0.2M phosphate buffer 8.0 0.4



4 10% Hydrazine/6M Gn-HCl, 0.2M phosphate buffer 8.0 0.5



7 10% Hydrazine/H2O 10.8 14.4

8 20% Hydrazine/H2O 11.0 5.2

9 5% Hydrazine/H2O 10.7 21.2

10 1% Hydrazine/H2O 10.5 22.4

11 0.5% Hydrazine/H2O 10.5 29.7

12 0.1% Hydrazine/H2O 10.3 26.7

13 0.01% Hydrazine/H2O 9.2 5.3

14 0.5% Hydrazine/1M Gn-HCl, 33mM phosphate buffer 10.0 53.2

aconversion yields were estimated by the peak area of the corresponding HPLC chromatogram.

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DNA sequence: CATATGCAAGATCCATACGTGAAGGAAGCCGAGAA-CCTGAAGAAGTACTTCAATGCTGGTCACTCTGACGTAGCCGATAACG-GTACTCTGTTTCTGGGTATCCTGAAGAACTGGAAAGAAGAGAGCGA-CCGTAAGATCATGCAGTCTCAGATTGTTAGCTTCTACTTCAAGCTGT-TCAAGAACTTCAAAGACGATCAATCTATCCAGAAGAGCGTTGAAA-CTATCAAAGAAGATATGAATGTTAAGTTCTTCAACTCTAATAAGAA-GAAACGCGATGACTTTGAGAAGTGTCATCACCATCATCACCACTAA-GGATCC

Figure 3. Synthesis of interferon γ -polypeptide-(MetGln1-Lys94)-α-hydrazide 3.

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Amino acid sequence: M Q D P Y V K E A E N L K K Y F N A G H S D VA D N G T L F L G I L K NW K E E S D R K I M Q S Q I V S F Y F K L F K NF K D D Q S I Q K S V E T I K E D M N V K F F N S N K K K R D D F E K CH H H H H H

The plasmid was transfected in BL21 (DE5: Wako Nippon gene)and incubated. The cells were treated by microwave trituration inLysis buffer (100mM NaH2PO4, 300mM NaCl, pH8.0). Peptide inlysate was isolated by Ni-column and eluted with imidazolesolution (50mM NaH2PO4, 300mM NaCl, 8M Urea, 20-250mMImidazole, pH 8.0). Fractions containing target peptide 1 werepooled and lyophilized. The residue was purified by HPLC to obtainhomogeneous peptide. HPLC condition: column: Proteonavi C4,5μm, 4.6×250mm, 0.1% TFA: 0.1% TFA in 90% MeCN=70 : 30 to20 : 80 for 20min at the flow rate of 1mL/min. MS analysis foundthat the N-terminus has methionine residue that is an initiationcodon, so this expression yielded polypeptide-(MetGln1-Lys94)-Cys95-His6 1 (ca 20mg/ L amount). ESI-MS: m/z Calcd forC549H834N152O160S4: [M+H]+ 12252.7, found for [M+7H]7+ 1750.9,[M+8H]8+ 1531.9, [M+9H]9+ 1361.9, [M+10H]10+ 1225.9, [M+11H]11+ 1114.6, [M+12H]12+ 1021.8, [M+13H]13+ 943.2, [M+14H]14+ 876.0,[M+15H]15+ 817.6, [M+16H]16+ 766.6, [M+17H]17+ 721.6.(Deconvoluted: 12251.2 [M+H] +)

Cyanylation of Polypeptide-(MetGln1-Lys94)-Cys-His6 2

To a solution of 0.1% trifluoroacetic acid in 30% acetonitrile solution(0.25mL) containing polypeptide 1 (3.0mg, 0.25μmol) was added1-cyano-4-dimethylaminopyridium tetrafluoroborate (CDAP, 0.6mg,2.5μmol) [15–17], and the solution was stirred at room temperaturefor 30min. Cyanylated polypeptide was directly purified with reversephase HPLC, and the fractions containing product were pooled andthen lyophilized. Cyanylated polypeptide 2was obtained in 82% iso-lated yield (2.5mg, 200nmol). HPLC conditions: Proteonavi C4, 5μm,4.6×250mm, 0.1% TFA: 0.1% TFA in 90% MeCN=60 : 40 to 50 : 50for 20min at the flow rate of 1mL/min. ESI-MS: m/z Calcd forC550H833N153O160S4: [M+H]+ 12277.7, found for [M+7H]7+ 1754.5,[M+8H]8+ 1535.2, [M+9H]9+ 1364.8, [M+10H]10+ 1228.4, [M+11H]11+ 1116.9, [M+12H]12+ 1023.9, [M+13H]13+ 945.2, [M+14H]14+ 877.8, [M+15H]15+ 819.3, [M+16H]16+ 768.2, [M+17H]17+ 723.1, [M+18H]18+ 683.1. (Deconvoluted: 12275.6 [M+H] +)

ptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 958–963

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Figure 4. Monitoring of hydrazinolysis. (A) At the beginning ofhydrazinolysis (<5min). (B) At 2 h. (C) interferon γ-polypeptide-(MetGln1-Lys94)-α-hydrazine 3 purified with HPLC. (D) ESI-mass of 3 purified withHPLC.

Figure 5. Synthesis of interferon γ-polypeptide-(MetGln1-Lys94)-α-thioester 7.

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Figure 6. Monitoring of thioesterification (A) interferon γ-polypeptide-(MetGln1-Lys94)-α-hydrazine 3. (B) Starting point of oxidative conversion(<1min). (C) After thiolysis of interferon γ-polypeptide-(MetGln1-Lys94)-α-azide with sodium 2-mercaptoethanesulfonate.


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Hydrazinolysis of Polypeptide 2

To a solution of sodium phosphate buffer (30mM, pH10, 1.0mL)containing 1M guanidine-HCl and 0.5% hydrazine was added asolution of polypeptide 2 (11.4mg, 930nmol), and the solution wasincubated at room temperature for 3h. polypeptide-α-hydrazidewas directly purified with reverse phase HPLC and the fractions


containing product were pooled and then lyophilized. Polypeptide-α-hydrazide 3was obtained in 27% isolated yield (2.8mg, 250nmol).HPLC conditions: Proteonavi C4, 5μm, 4.6×250mm, 0.1% TFA: 0.1%TFA in 90% MeCN=70 : 30 to 20 : 80 for 20min at the flow rate of 1 -mL/min. ESI-MS:m/z Calcd for C510H789N135O152S3: [M+H]+ 11340.7,


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found for [M+6H]6+ 1890.4, [M+7H]7+ 1620.6, [M+8H]8+ 1418.1, [M+9H]9+ 1260.8, [M+10H]10+ 1134.9, [M+11H]11+ 1031.7, [M+12H]12+ 945.8, [M+13H]13+ 873.2, [M+14H]14+ 810.9, [M+15H]15+ 756.9,[M+16H]16+ 709.7, [M+17H]17+ 668.0. (Deconvoluted: 11338.0 [M+H] +)

IFN-γ (MetGln1-Lys94)-S(CH2)2SO3H 7

To a solution of 6.0M guanidine-HCl solution (pH4.0, 0.25mL) con-taining polypeptide-α-hydrazide 3 (2.8mg, 250nmol) was addedNaNO2 (70μg, 1.0μmol) at 4 °C and the solution was incubated at4 °C for 5min. To this solution containing azide 6 was added so-dium mercaptoethanesulfonate (6.6mg, 40μmol) and the solutionwas adjusted pH7.0. After 30min, polypeptide-α-thioester 7 wasdirectly purified with HPLC. Polypeptide-α-thioester 7 was obtainedin 83% isolated yield (2.3mg, 200nmol). HPLC conditions: ProteonaviC4, 5μm, 4.6×250mm, 0.1% TFA: 0.1% TFA in 90%MeCN=70 : 30 to20 : 80 for 20min at the flow rate of 1mL/min. ESI-MS: m/z Calcd forC512H791N133O155S5: [M+H]+ 11450.9, found for [M+6H]6+ 1908.9,[M+7H]7+ 1636.4, [M+8H]8+ 1431.8, [M+9H]9+ 1273.0, [M+10H]10+ 1145.7, [M+11H]11+ 1041.6, [M+12H]12+ 955.0, [M+13H]13+

881.5, [M+14H]14+ 818.7, [M+15H]15+ 764.2, [M+16H]16+ 716.5,[M+17H]17+ 674.3. (Deconvoluted: 11447.4 [M+H] +)

Results and Discussion

In order to obtain hIFN-γ varying oligosaccharide structure at the 97position, we considered to prepare a polypeptide-(Gln1-Lys94)-α-hydrazide for the coupling with a glycopeptide having β-mercaptoleucine at the 95 position by NCL followed by a desulfurizationstrategy [12]. For the specific hydrazinolysis, we developed a newstrategy employing an S-cyanylation of Cys-thiol and a basicpeptide cleavagemethodology that has been used for the determi-nation of disulfide bond positions [15–17]. Fortunately, thesequence of hIFN-γ polypeptide-(Gln1-Lys94) does not have anycysteine residues in the sequence (Figure 1b), and therefore wedesigned a polypeptide-(Gln1-Lys94)-Cys95-His6 1 (Figure 1a andc). Toward this peptide, we examined the selective S-cyanylationof Cys at the 95 position and subsequent hydrazinolysis.

The DNA sequence [13] of polypeptide-(Gln1-Lys94)-Cys95-His61 was inserted in pET 3a plasmid, and the corresponding polypep-tide was prepared by E. coli expression. The expression amount ofpolypeptide-(Gln1-Lys94)-Cys95-His6 1 was found to be suitableand the polypeptide 1 was purified with nitrilotriacetic acid-Ni(Ni/NTA) column. As shown in Figure 2a, expression and Ni/NTA-column isolation proceeded in moderate yield. During extensivestudies, we found that this expression provides a polypeptide hav-ing an additional methionine residue that is a product of an initia-tion codon at the N-terminal, however. Because methionine atthe N-terminal may not disturb protein bioactivity, we proceededto the next synthetic step. Polypeptide-(MetGln1-Lys94)-Cys95-His6 1 thus obtained was purified on reverse phase HPLC as shownin Figure 2b. Polypeptide-(MetGln1-Lys94)-Cys95-His6 1 wasobtained ca 20mg/L with the expression protocol.

To polypeptide-(MetGln1-Lys94)-Cys95-His6 1, cyanylationwas then examined with 1-cyano-4-dimethylaminopyridiumtetrafluoroborate (CDAP) reagent [15–17] in H2O-MeCN (7 : 3)and this reaction smoothly gave polypeptide-(MetGln1-Lys94)-Cys(CN)95-His6 2 in 82% (isolated yield). As shown inFigure 2c, ESI-mass indicates suitable ion peaks of the desiredpolypeptide 2.

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With polypeptide-(MetGln1-Lys94)-Cys95(CN)-His6 2 in hand, weexamined hydrazinolysis of 2 under the several conditions asshown in Table 1. Hydrazinolysis employing 10% hydrazine/buffersolution (pH8.0-10.8) conditions (entry 1–7) were examined. Thiscondition was found to afford the desired polypeptide-(MetGln1-Lys94)-α-hydrazide 3 (Figure 3). However, these conditions didnot give desired product 3 in good yield. Cyanyl group at the Cysposition was removed by hydrazinolysis and the substrate 1was re-covered (Figure 3). Addition of guanidine-HCl (Gn-HCl) to theseconditions (entry 4–6) in order for peptide to denature did alsonot give the desired product 3 in good yield. On the other hand,the decreasing of hydrazine amount in the H2O increased yieldslightly ranging from 1% to 30% (entry 7–12). The good yield was~30% under the 0.5% hydrazine/H2O condition (entry 11). This con-dition was further optimized with 1M Gn-HCl in a sodium phos-phate buffer (33mM, pH 10.0), and this condition yielded thedesired product polypeptide-(MetGln1-Lys94)-α-hydrazide 3 in53% (HPLC yield, 27% isolated yield). The reaction was monitoredby HPLC and the HPLC profiles are shown in Figure 4a and 4b.Some byproducts such as intramolecular lactam derivative 4, in-termolecular disulfide bond derivative 5, and substrate 1 werealso obtained during the hydrazinolysis reaction (Figure 3). Thepurified polypeptide-(MetGln1-Lys94)-α-hydrazide 3 exhibitedsuitable HPLC profile and ESI-mass as shown in Figure 4c and4d, respectively.This polypeptide-α-hydrazide 3 was then converted into corre-

sponding polypeptide-α-azide 6 in a solution of sodium phosphatebuffer (200mM, pH4.0) containing 10mM NaNO2 and 6M Gn-HCl(Figure 5) [8,9]. This reaction was monitored by reverse phaseHPLC-MS (Figure 6). After confirmation of the formation ofpolypeptide-α-azide 6, sodium 2-mercaptoethanesulfonate wasadded to this solution, and the pH was adjusted 7.0. This conditionsmoothly yielded the desired polypeptide-(MetGln1-Lys94)-α-thioester 7 in 83% isolated yield.In conclusion, we demonstrated the preparation of polypeptide-

α-thioester consists of 94 amino acids by E. coli expression followedby cyanylation, hydrazinolysis, and thiolysis. This reaction can beperformed on over 100mg scale polypeptide after E. coliexpression, and this will enable us to perform a semisynthesis ofpost-translationally modified proteins. This chemical thioesterformation can be applied to a polypeptide isolated from inclusionbody, because insoluble peptides can be activated in the organicsolvent or under the denatured condition such as guanidine solu-tion. Currently, this conversion is applicable only to the polypeptidehaving no cysteine residue in the target sequence. In order to per-form this reaction toward peptide having several cysteine residues,site-specific cyanylation at the C-terminal additional Cys-His6sequence is required. This research is in progress to obtain anypolypeptide-α-thioester.

Acknowledgements

This work was supported by Financial support from the Japan Soci-ety for the Promotion of Science (No. A 23245037).

References1 Hackenberger CP Schwarzer D Chemoselective ligation and

modification strategies for peptides and proteins. Angew. Chem. Int. Ed.2008; 47: 10030–10074.

2 Dawson PE, Muir TW, Clark-Lewis I Kent SBH Synthesis of Proteins byNative Chemical Ligation. Science 1994; 266: 776–779.

ptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 958–963


3 Kang J Macmillan D Peptide and protein thioester synthesis via N→ Sacyl transfer. Org. Biomol. Chem. 2010; 8: 1993–2002.

4 Shah NH Muir TW Inteins: nature’s gift to protein chemists. Chem. Sci.2014; 5: 446–461.

5 Muir TW, Sondhi D Cole PA Expressed protein ligation: A generalmethod for protein engineering. Proc. Natl. Acad. Sci. U. S. A. 1998;95: 6705–6710.

6 Noren CJ, Wang J Perler FB Dissecting the chemistry of protein splicingand its applications. Angew. Chem. Int. Ed. 2000; 39: 450–466.

7 Li Y-M, Yang M-Y, Huang Y-C, Li Y-T, Chen PR, Liu L. Ligation of expressedprotein alpha-hydrazides via genetic incorporation of an alpha-hydroxyacid. ACS Chem. Biol. 2012; 7: 1015–1022.

8 Wang P, Shaw KT, Whigham B Ramage R Synthesis of peptide C-terminalderivatives using the transfer active ester condensation technique.Tetrahedron Lett. 1998; 39: 8719–8720.

9 Fang G-M, Li Y-M, Shen F, Huang Y-C, Li J-B, Lin Y, Cui H-K, Liu L. Proteinchemical synthesis by ligation of peptide hydrazides. Angew. Chem. Int.Ed. 2011; 50: 7645–7649.

10 Kang J, Reynolds NL, Tyrrell C, Dorin JR Macmillan D Peptide thioestersynthesis through N→ S acyl-transfer: application to the synthesis of abeta-defensin. Org. Biomol. Chem. 2009; 7: 4918–4923.


11 Adams AL, Cowper B, Morgan RE, Premdjee B, Caddick S, Macmillan D.Cysteine promoted C-terminal hydrazinolysis of native peptides andproteins. Angew. Chem. Int. Ed. 2013; 52: 13062–13066.

12 Unverzagt C Kajihara Y Chemical assembly of N-glycoproteins: a refinedtoolbox to address a ubiquitous posttranslational modification. Chem.Soc. Rev. 2013; 42: 4408–4420.

13 Curling EMA, Hayter PM, Baines AJ, Bull AT, Gull K, Strange PG, Jenkins N.Recombinant human interferon-y. Biochem. J. 1990; 272: 333–337.

14 Sareneva T, Mortz E, Tolo H, Roepstorff P Julkunen I Biosynthesis and N-glycosylation of human interferon-γ Asn25 and Asn97 differ markedly inhow efficiently they are glycosylated and in their oligosaccharidecomposition. Eur. J. Biochem. 1996; 242: 191–200.

15 Degani Y Patchornik A Cyanylation of Sulfhydryl Groups by 2-Nitro-5-thiocyanobenzoic Acid. High-Yield Modification and Cleavage ofPeptides at Cysteine Residuest. Biochemistry 1974; 13: 1–11.

16 Wu J Watson JT Optimization of the cleavage reaction for cyanylatedcysteinyl proteins for efficient and simplified mass mapping. Anal.Biochem. 1998; 258: 268–276.

17 WuW,HuangW,Qi J, Chou Y-T, Torng EWatson JT ‘Signature Sets’, minimalfragment sets for identifying protein disulfide structures with cyanylation-based mass mapping methodology. J. Proteome Res. 2004; 3: 770–777.


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Efficient synthesis of polypeptide-α-thioester by the method combining polypeptide expression and chemical activation for the semi-synthesis of interferon-γ having oligosaccharides.pdf