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The canonic M2e epitope entailed considerable synthetic difficulties already as a simplemonomer, which were substantially overcome by the use of ChemMatrix resin, systematicdouble couplings and, especially, pseudoproline dipeptide units at the two Ser-Ser pairs [4].All these improvements, plus flexibilizing Ahx units at every branching point, wereincorporated to the all-SPPS synthesis of tetravalent construct C (Figure 2), resulting in aclean, easily purifiable and satisfactorily characterized end product (C 517 H 838 N 146 O 196 S 5 ,MW = 12387.86 Da; [M+H + ] = 12387.79; 4.8% yield).To evaluate the convergent (chemical ligation) approach, constructs D and E (seeFigure 3) were prepared [4]. For thioether ligations, a tetravalent core functionalized withchloroacetyl groups (ClAc 4 -Lys core), andeither the sM2e or the M2e monomers (for Dand E, respectively; each C-terminallyelongated with Cys) were prepared in highlypure form. Ligations were conducted inTris·HCl, pH 7.6, 2 M guanidinium chloride,at 50 ºC, using 10-12-fold molar excess ofpeptide. Reaction progress was monitored byanalytical HPLC and revealed in both casesrather complex processes (Figure 3) [4].MALDI-TOF MS analysis showed MAPproducts containing different numbers ofepitope copies, along with substantial amountsof the disulfide-bound dimers of the linearpeptides. HPLC resolution of the reactionmixtures was challenging and did not allowefficient separation of the target tetravalentconstructs from less substituted species.Additionally, in the ligation for E, an M2etetramer, coeluting with the disulfide dimerand identified as a non-covalent double dimer,further complicated the situation [4].In view of these difficulties with theligation approach, the all-SPPS route appearsFig. 3. Progress of thioether ligationmonitored by HPLC (triangles indicatethioether linkage).advantageous, particularly for epitopes such asM2e, where factors such as size (sterichindrance preventing access of the Cys thiol tounreacted ClAc groups), solubility andaggregation (both difficult to predict) can haveseverely limiting effects. Ligation is also more expensive and time-consuming than the all-SPPS approach, and in thiol-based chemistries tends to generate sizable amounts ofdisulfide dimer whose recycling back to the thiol form is costly in time and effort.Regarding the all-SPPS approach, on the basis of the above results we would proposetwo recommendations: (i) incorporating flexibility-enhancing units (e.g., Ahx) at branchingpoints can be synthetically beneficial; (ii) an exploratory synthesis of the linear epitope canidentify trouble spots to be smoothed before the synthesis of the considerably morecomplicated MAP structures is undertaken.AcknowledgmentsWork supported by MICINN, the Spanish Ministry of Science and Innovation (grants BIO2005-07592-CO2-02 and BIO2008-04487-CO3-02 to D.A.), by the regional government of Catalonia (SGR2005-00494). W.K. is a fellow in the Juan de la Cierva Program of MICINN.References1. Tam, J.P. Proc. Natl. Acad. Sci. U.S.A. 85, 5409-5413 (1988).2. Tam, J.P., In Goodman M (Ed.) Peptide Dendrimers and Protein Mimetics, Thieme, Stuttgart, 2000,p. 129.3. Cady, S.D., Luo, W., Hu, F., Hong, M. Biochemistry 48, 7356-7364 (2009).4. Kowalczyk, W., De la Torre, B.G., Andreu, D. Bioconj. Chem. 21, 102-110 (2010).161
Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Synthesis of the Thiolactone Derivative of Enterococcus faecalisGelatinase Biosynthesis-Activating Pheromone Using theGyrA Mini-InteinKoji Nagata 1 , Yosuke Yamanaka 1 , Shoichiro Horita 1 ,Hidekazu Katayama 2 , Kou Hayakawa 1 , Akihiro Yamamura 1 ,Mami Sato 3 , Kenzo Nishiguchi 3 , Kenji Sonomoto 3,4 , Jiro Nakayama 3 ,and Masaru Tanokura 11 Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi,Bunkyo-ku, Tokyo, 113-8657, Japan; 2 Institute of Glycoscience, Tokai University, 1117Kitakaname, Hiratsuka, Kanagawa, 259-1292, Japan; 3 Faculty of Agriculture, GraduateSchool, 4 Bio-Architecture Center, Kyushu University, Fukuoka, 812-8581, JapanIntroductionThe expression of pathogenicity-related extracellular proteases in Enterococcus faecalis ispositively regulated by the quorum sensing system mediated by an autoinducing peptidetermed gelatinase biosynthesis-activating pheromone (GBAP) [1]. GBAP is an 11-residuecyclic peptide containing a lactone linkage formed by the side-chain hydroxyl group of Ser 3and the main-chain carboxyl group of Met 11 in the following amino-acid sequence,Gln-Asn-Ser-Pro-Asn-Ile-Phe-Gly-Gln-Trp-Met. Secreted GBAP binds to its membraneboundreceptor FsrC and triggers a two-component signal transduction cascade of FsrC andFsrA. We previously reported the chemical synthesis [2] and the solution structuredetermination of GBAP [3]. In order to analyze the intermolecular interaction of GBAP andFsrC by heteronuclear NMR, we need to establish a preparation method of isotopicallylabeled GBAP. Here, we describe a new method to synthesize the thiolactone derivative ofGBAP, a GBAP agonist, from the fusion protein of [Cys 3 ]GBAP and Mycobacteriumxenopi GyrA mini-intein [4]. With this method, we obtained 0.25 mg of 15 N-labeled GBAPthiolactone from 1 L of Escherichia coli culture, and measured its 1 H- 15 N 2D HSQC.Fig. 1. Mechanism of GBAP thiolactone formation using GyrA mini-intein and MESNA.162
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Peptides 2010Tales of PeptidesProce
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Peptides 2010Tales of PeptidesProce
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IntroductionWe had the distinct hon
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Scientific programIn planning the 3
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31 st EUROPEAN PEPTIDE SYMPOSIUMSep
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published by John Wiley & Sons as a
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The Josef Rudinger AwardThis award
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Young Investigators' SymposiumThe y
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xxiv
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Design of Peptidyl-Inhibitors for G
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Synthetic Antifreeze Glycopeptide A
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Synthesis and Oxidative Folding of
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Convergent Syntheses of Huprp106-12
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Bactericidal Activity of Small Beta
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Bradykinin Analogues Acylated on Th
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Antimicrobial Activity of Small 3-(
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Interaction of Curcumin with A-Synu
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Fluorescent and Luminescent Fusion
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Cellular Expression of the Human An
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2D IR Spectroscopy of Oligopeptides
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large, non-redundant subset of prot
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The aberrantly glucosylated peptide
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Table 1. Proline cis/trans ratios o
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Table 1. Yields, UV-vis absorption
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Table 1. Purity of the targeted tri
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processes are blocked. Interestingl
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(tb4 n ,1 m )MTII(tb1 n ,4 m )MTIIF
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Fig. 2. a) Part of the 400 MHz HR-M
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Once printed, the amino acid partic
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A) PSA, few seconds73B) PSA, 45 min
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short β strands. In contrast, nume
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neg. control Cs9-Rhd MM-218Fig. 2.
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Table 1. The general structure of t
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% ID in the liver 1 h p.i.100908070
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Peptidyl phosphoranes were first re
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obtained from the same sardines and
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MccJ25 variants were produced by si
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Fig. 2. Proposed signaling mechanis
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Table 1. mCD4-HS12 antiviral activi
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Subject index(S)-2-(1-adamantyl)gly
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chip 18chiral triazine condensing r
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heat stress 348helical conformation
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O-acyl isopeptide method 112obesity
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SH2-ligands 210short collagen-relat
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Author indexAboye, Teshome Leta 142
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Chi, Hongfang 480Chiche, Laurent 6C
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Geronikaki, Athina 444Gessmann, Ren
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Kostova, Kalina 528Kotzia, Georgia
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Nagata, Koji 162Nagayev, Igor Yu. 5
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Salvarese, Nicola 518Sammet, Benedi
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Vauquelin, Georges 138Veiga, Ana Sa
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