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Table 1. A summary of synthesis yield and labeling efficiencyAffibodyVariantSyntheticyieldATTO-tecDyeLabelingYieldTheoreticalMw (Da)ExperimentalMw(Da)488 70% 7491.7 7492.6Z HER2 10.6%594 100%* 7707.7 7708.9647N 100%* 7547.7 7548.8488 72% 7331.4 7331.0Z EGFR 21.6%594 100%* 7546.4 7547.8647N 100%* 7387.4 7387.5* No unlabeled product was detectedThe fluorescent-labeled Affibody molecules are currently used in fluorescence microscopyto study the relative expression and spatial distribution of the EGFR and HER2 proteins inhuman breast cancer cell lines with the aim of identifying differences that can be ofdiagnostic or prognostic value in the analysis of patient samples. These labeled binders willalso be used for ultrahigh resolution microscopy (STED = Stimulated emission depletion).ConclusionsTo conclude, a protocol for chemical synthesis of Affibody molecules binding EGFR orHER2 has successfully been established. In addition, a protocol for in solution labeling hasbeen optimized for ATTO-TEC fluorophores enabling high yield conjugation.Affibody molecules have many advantages compared to antibodies in molecular imaging:• Site specific labeling – Affibody molecules provide well defined, homogeneous affinityreagents• High stability – The excellent refolding properties of Affibody molecules enables for highresolution purification using RP-HPLC• Small size – Great value in high resolution optical methods where the reagent size isbecoming the limiting factorAcknowledgmentsThe research leading to these results has received funding from the European Community's SeventhFramework Programme FP7/2007-2011 under grant agreement no. 201837.References1. Friedman, M., et al. J. Mol. Biol. 376, 1388-402 (2008).2. Orlova, A., et al. Cancer Res. 66, 4339-4348 (2006).181
Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Oxyfold: A New Solid Supported Reagent for the Simple andEffective Formation of Disulfide Bond in PeptidesL. Ronga 1 , P. Verdié 2 , M. Cristau 2 , M. Amblard 2 , S. Cantel 2 ,C. Enjalbal 2 , K. Puget 1 , G. Subra 2 , and J. Martinez 21 Genepep, St Clément de Rivière, 34980, France, 2 Institut des Biomolécules MaxMousseron (IBMM), Montpellier, 34093, FranceIntroductionThe importance of disulfide bridges in peptide conformation and activity make theirformation a fundamental step of peptide synthesis. Formation of disulfide bond is probablyone of the most challenging steps to achieve regarding the formation of unwantedby-products and oligomerization. In order to minimize the latter phenomenon, disulfidebridge cyclizations are performed under high diluted conditions which require timeconsuming removal of solvent at the end of the reaction. Dimethyl sulfoxide (DMSO) isone of the oxidizing reactants most commonly used [1]. One of its major drawbacks is itselimination from the reaction medium, which requires evaporation under strong vacuum orrepeated lyophilizations. Furthermore, the dimethyl sulfide generated during the reaction isvolatile and toxic. On the other hand, supported reactants are particularly advantageous forpromoting intramolecular reactions. In fact, they are known to cause a phenomenon of“pseudodilution” which makes it possible to minimize oligomerization and to use muchsmaller amounts of solvents [2]. Here we present the synthesis and the use of novel solidsupported oxidation reactants for disulfide bond formation. This family of supportedreagents consists in a series of oxidized methionines grafted onto a solid support. Wedemonstrate the efficiency and easiness of these supported reagents for the formation ofdisulfide bridges in peptides.Results and DiscussionThree different solid supported methionine sulfoxides were synthesized by anchoring aFmoc-protected Met on different matrices: Amino PEGA, Amino PEG-PS andaminomethyl polystyrene. After Fmoc deprotection and acetylation, methionine oxidationwas performed by treatment of the resin with H 2 O 2 . The ability of these supportedmethionine sulfoxides to promote disulfide bond formation of crustacean cardioactivepeptide CCAP (PFCNAFTGC-NH 2 ) [3] was explored at pH=6.1 (ammonium acetatebuffer) and pH=7.5 (phosphate buffer). Under the same conditions, the CCAP oxidationpromoted by Ellman’s reagent prepared on amino PEGA resin and 10% DMSO solutionwas investigated. Our results (Figure 1) show that Oxyfold is more effective than DMSOand supported Ellman’s reagent. Among the compared resins, amino PEGA resin gives thebest results of CCAP oxidation. Moreover, pH=7.5 seems the ideal pH for Oxyfoldpromotedoxidation.pH 6.1pH 7.5100100% oxydized CCAP80604020% oxydized CCAP8060402000 5 10 15 20 25 30time (h)00 5 10 15 20 25 30time (h)Fig. 1. CCAP oxidation: ■ 10% DMSO,▲ Ac-Met[O]-PEGA,● Ac-Met[O]-PS, ♦ Ellman’sreagent-PEGA,× Ac-Met[O]-PEG-PS.182
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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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Proceedings of the 31 st European P
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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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