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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Identification of Sulphated Peptides Binding FGF1 Using aMicro Particle Matrix (MPM) Encoded LibraryManat Renil and Morten MeldalCenter for Solid-Phase Organic Combinatorial Chemistry (SPOCC),Department ofChemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500, Valby, DenmarkIntroductionStructure elucidation of highly active hits from a ‘one bead one compound’ combinatoriallibrary has been a challenge ever since the introduction of ‘on-bead’ screening techniques.A relatively high concentration of ‘on bead’ ligands are required for the structuralelucidation of active compounds by e.g. Edman’s micro sequencing or mass spectrometricanalysis frequently leading to false positives in ‘on bead’ screening analysis targetinginteractions were nano-molar or even lower concentration of the bead bound ligand isdesirable. Micro-Particle-Matrix (MPM) encoding of PEGA based beads has been recentlyintroduced from our laboratory to circumvent this draw back [2]. Heparin mimeticsulphated peptide libraries [1] were used to identify sulphated peptides that binds to FGF1.Results and DiscussionIt has previously been shown that short O-sulphated peptides can inhibit heparin-FGF1/FGFR interaction using a combinatorial library approach [1]. In the previous libraryhigh concentration of on-bead peptide libraries were used in the screening to obtain severalmM to M inhibitors. In thepresent work we employed anMPM-encoded library [2,3] of20.000 encoded beads where thepeptide library structure wasbiased, based on a previouslyidentified sulphated peptideligand to FGF1. The library had apeptide concentration of only 1mol/mL to screen for activityand selectivity between ROXlabelledanti-thrombin and FGF1binding. The ten amino acidresidue library was synthesizedby cycles of split/mix with code(image) recording during thesplitting immediately prior tonext coupling reaction. Hits werealso recorded and codes wereFig. 1. Ac-ETET*S*S*ES*ES*K-NH 2 - H 1 compared. Four amino acidsNMR of (Table1) were coupled at eachsulphated (above) and non-sulphated peptide (below). step i.e. with 5000 encoded beadsper well.Progress of coupling reactions were monitored (Dhbt-OH). O-Sulphonation wasperformed at the end of synthesis on dry resin using sulphur trioxide pyridine (SO 3 -Pyr)complex in DMF at 60 o C. The library was incubated in BSA/PBS buffer at pH 7.5 withROX labeled anti-thrombin. Beads with high fluorescence intensity were isolated anddecoded. Of 20 beads 15 structures were determined and 5 determined partially.Table1. The amino acids used for library synthesis (*Post assembly sulphatation)R9 R8 R7 R6 R5 R4 R3 R2 R1 R0Glu Ser Glu Ser* Ser* Ser* Glu Ser* Glu Ser*Asp Thr Asp Thr* Thr* Thr* Asp Thr* Asp Thr*Gln Thr* Gln Hyp* Hyp* Hyp* Gln Hyp* Gln Hyp*Asn Ser* Asn Tyr* Tyr* Tyr* Asn Tyr* Asn Tyr*148