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OD ratio33031530011060105432102/1 4/3 6/5 8/7 10/9 2/1 4/3 6/5 8/7 10/9peptidesCCP+ serahealthy seraFig. 3. Indirect ELISA with biotinylatedpeptides, pre-coated with NeutrAvidin.specific secondary antibodies (Figure 2).Comparing the short sequences offilaggrin and vimentin, we have found thatthe 5-mer filaggrin-peptide was recognizedby the RA sera with sensitivity andspecificity comparable with the currentlyused tests.We detected lower sensitivity andspecificity than in our first test. However,OD ratios of all peptides have shown asignificant correlation with the MCV(mutated citrullinated vimentin) titer, whichis associated with activity of disease. Onthe other hand, OD ratios did not showcorrelation with the CCP3 titer. These datasuggest that the short citrulline containingfilaggrin peptide (Filaggrin 311-315 XX)might be a useful biomarker for RA.Selected peptides (Table 1) weresynthesised manually by SPPS, accordingto Fmoc/ t Bu strategy using Rink-Amide MBHA or MBHA resin. For the coupling Fmocaminoacid : HOBt : DIC = 2 : 2 : 2 activation was used. C- or N-terminally biotinylatedforms were made using biotin, biotinyl-6-aminohexanoic acid and 4,7,10-trioxa-1,13-tridecanediamino succinic acid linker (Ttds) [3]. The ratio of the coupling reagents wasbiotin : HOBt : PyBOP : DIEA = 3 : 3 : 3 : 6. We coupled the Fmoc-Ttds as a protectedamino acid. The crude products were purified by reversed-phase chromatography. Thestructure of the peptides was proved by electron spray ionization (ESI) mass spectrometry.We have used these peptides in an indirect ELISA, on NeutrAvidin pre-coated plates, thereaction was detected by anti IgG-HRP (Figure 3).The CCP3+ serum samples specifically recognized the C terminally biotinylated5-mer filaggrin peptide, while showed no reaction with the N-terminally biotinylated ones.Table 1. Structures of studied peptides1 biotin-6-amino-hexanoyl- Filaggrin 311-315 RR -NH 22 biotin-6-amino-hexanoyl- Filaggrin 311-315 XR -NH 23 biotinyl- Filaggrin 311-315 RR -NH 24 biotinyl- Filaggrin 311-315 XR -NH 25 Ac- Filaggrin 311-315 RR -K(biotin-6-amino-hexanoyl)-NH 26 Ac- Filaggrin 311-315 XR -K(biotin-6-amino-hexanoyl)-NH 27 Ac- Filaggrin 311-315 RR -K(Ttds-biotin)-NH 28 Ac- Filaggrin 311-315 XR -K(Ttds-biotin)-NH 29 Ac- Filaggrin 311-315 RR -Ttds-K(biotin-6-amino-hexanoyl)-NH 210 Ac- Filaggrin 311-315 XR -Ttds-K(biotin-6-amino-hexanoyl)-NH 2As a conclusionwe can say that theshort Filaggrin 311-315XX peptide andMultipin ELISA maybe a useful addition tothe array of diagnostictools of early RA.Our results werevalidated by conventionalindirect ELISA,also showing thatonly the C-terminalbiotinylation can beapplied for the 5-merfilaggrin-peptide.AcknowledgmentsWe thank to EPS for travel grant. This work was supported by National Office for Research andTechnology (RET-06/2006, GVOP-3.1.1.-2004-05-0183/3.0, OTKA-NKTH CK_80689), Hungary.References1. Vossenaar, E.R., Despres, N., Lapointe, E., van der Heijden, A., Lora, M., Senshu, T., VanVenrooij, W.J., Menard, H.A. Arthritis Res. Ther. 6(2), 86-89 (2004).2. Schellekens, G.A., de Jong, B.A.W., van den Hoogen, F.H.J., van de Putte, L.B.A., Van Venrooij,W.J. J. Clin. Invest. 101, 273-281 (1998).3. Bartos, Á., Uray, K., Hudecz, F. Peptide Science 92, 110-115 (2009).463
Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 20104-Methylpseudoproline Analogues of Cyclolinopeptide A:Synthesis, Conformation and BiologyJ. Katarzyńska 1 , S. Jankowski 1 , K. Huben 1 , M. Zimecki 2 , andJ. Zabrocki 1,31 Institute of Organic Chemistry, Technical University of Lodz, Łódź, 90-924, Poland;2 Institute of Immunology and Experimental Therapy, Polish Academy of Sciences,R. Weigla 12, Wrocław, 53-114, Poland; 3 Peptaderm Inc., KrakowskiePrzedmieście 13, Warszawa, 00-071, PolandIntroductionPseudoprolines ( pro), 1,3-oxazolidines and 1,3-thiazolidines derived from Ser, Thr orCys, have been introduced by Mutter and coworkers [1]. They have been applied as aversatile tool in structure-activity relationship studies due to enhanced rate of cis/transisomerisation, depending on stereochemistry and degree of substitution at the C-2 atomposition of the proline ring.4-Alkylpseudoprolines derived from -alkyl- -hydroxymethyl amino acids can betransformed into suitable pseudoproline (oxazolidine) unit [2]. The presence of alkylsubstituent at the C-4 atom of the ring changes their chemical and conformationalproperties in comparison to pseudoprolines derived from serine or threonine.Results and DiscussionSynthesis and conformational analysis of the short peptides containing (R)- and (S)-4-metylpsudoproline were reported previously [3]. Now we present synthesis, conformationand biological activity of new analogues of cyclolinopeptide A (CLA), containing4-methylpseudoproline instead of proline residues in position 6 or 7. It is known thatimmunosuppressive activity of CLA, comparable with cyclosporine A, is connected to thepresence of the tetrapeptide Pro-Pro-Phe-Phe fragment containing Pro-Pro cis amide bond [3].Leu 1 - Val 2 - (R)-MeSer( pro) 3 - Pro 4 - Phe 5 - Phe 6 - Leu 7 - Ile 8 - Ile 9Leu 1 - Val 2 - Pro 3 - (R)-MeSer( pro) 4 - Phe 5 - Phe 6 - Leu 7 - Ile 8 - Ile 9cyclo - (Leu 1 - Val 2 - (R)-MeSer( pro) 3 - Pro 4 - Phe 5 - Phe 6 - Leu 7 - Ile 8 - Ile 9 )cyclo - (Leu 1 - Val 2 - Pro 3 - (R)-MeSer( pro) 4 - Phe 5 - Phe 6 - Leu 7 - Ile 8 - Ile 9 )Leu 1 - Val 2 - (S)-MeSer( pro) 3 - Pro 4 - Phe 5 - Phe 6 - Leu 7 - Ile 8 - Ile 9Leu 1 - Val 2 - Pro 3 - (S)-MeSer( pro) 4 - Phe 5 - Phe 6 - Leu 7 - Ile 8 - Ile 9cyclo - (Leu 1 - Val 2 - (S)-MeSer( pro) 3 - Pro 4 - Phe 5 - Phe 6 - Leu 7 - Ile 8 - Ile 9 )cyclo - (Leu 1 - Val 2 - Pro 3 - (S)-MeSer( pro) 4 - Phe 5 - Phe 6 - Leu 7 - Ile 8 - Ile 9 )7R6R7S6SThe linear precursors of modified CLA analogues were prepared manually by standardsolid-phase procedure “step by step” on Wang resin, using Fmoc group for N -aminoprotection and TBTU as a coupling reagent. N-9-Fluorenylmethyloxycarbonyl derivativesof 4-methylpseudoproline were obtained in good yield (~70%) by one potcyclocondensation of the corresponding -methylserine with formaldehyde andN-(9-fluorenylmethoxycarbonyloxy)succinimide in alkaline solution. The NH-group in4-methylpseudoproline residue was acylated ("difficult coupling") by using BTC(bis-(trichlomethyl)carbonate) in the presence of collidine. The cyclization of linearprecursors has been achieved in solution using EDC/HOBt as coupling reagents. Structuresof all compounds were characterized by MS and NMR spectroscopy.NMR-studiesThe replacement of one or both Pro residues by 4-methylpseudoproline changedsignificantly the 1 H NMR spectra of 6R and 7R in comparison to that recorded for theunmodified CLA in CDCl 3 at 298 K and 700 MHz. All signals except signals of methylgroups are broad and unresolved. The presence of MeSer( pro) residue decreased the rateof the chemical exchange among conformers of similar energy, characteristic for CLA.464
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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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