Cyclic disulfide analogues of the complement component C3a Synthesis and conformational investigations

Abstract

The flexible C‐terminal region of the anaphylatoxic peptide C3a was reported to contain the receptor binding site. To elucidate the receptor binding conformation of the C‐terminus, as well as to examine a synthetic approach to potential C3a‐antagonists, 26 cyclic disulfide bridged C3a analogues were synthesized. Solid phase peptide synthesis was performed on different polymeric supports by individual peptide synthesis, with Fmoc strategy, and simultaneous multiple peptide synthesis, using Boc and Fmoc strategies. Both strategies gave open‐chain peptides in comparable yields. Syntheses using the Boc strategy employed the HF‐labile 4(methoxy)benzyl group (Mob) for β‐thiol protection of cysteine; in contrast, the TFA‐stable protecting groups, acetamidomethyl (Acm) and trityl (Trt), were chosen for syntheses employing Fmoc strategy. Ring closure reactions by iodine oxidation were carried out starting from protected (Acm/Acm, Trt/Acm) or unprotected dithiols. The resulting cyclic C3a analogues were characterized by HPLC, amino acid analysis, and FAB‐MS. Conformational investigations using CD spectroscopy and theoretical structural investigations by means of molecular dynamics calculations revealed that slight variations in sequence result in pronounced conformational consequences. The potential of cyclic C3a analogues to activate or to desensitize guinea pig platelets, a standard test system for biological activities of anaphylatoxic peptides like C3a, revealed relatively low activities for cyclic peptides (<0.1% C3a activity). N‐terminal acylation with cationic, arginine‐rich sequences like YRRGR‐ led to amplified biological effects. Three of the synthesized peptides, namely CAALCLAR (P1), YRRGR°CGGLCLAR (P5) and YRRGRAhx°CGGLCLAR (P8), point in the direction of C3a antagonists.

Large Scale Solid Phase Synthesis of Peptide Drugs: Use of Commercial Anion Exchange Resin as Quenching Agent for Removal of Iodine during Disulphide Bond Formation

“…To help limit these, excess iodine should be quenched or adsorbed as quickly as possible after completion of the disulfide bond formation by addition of sodium bisulfite [9], sodium thiosulfate [10], ascorbic acid [11], powdered zinc dust, activated charcoal [12], or by dilution with water followed by extraction with carbon tetrachloride [13]. However, the quenching reagents themselves can sometimes cause additional side reactions including formation of thiosulfate adduct of the peptide [11].…”

“…However, the quenching reagents themselves can sometimes cause additional side reactions including formation of thiosulfate adduct of the peptide [11]. The large volumes of highly diluted aqueous peptide solutions, post-Acm removal, can also encumber the subsequent RP-HPLC purification.…”

Stepwise Construction of Disulfides in Peptides

“…In the synthesis of the single‐disulfide‐containing domain of protein HIV‐2 gp41, the oxidation was completed within 20 min in a solution of 3 % H 2 O 2 . I 2 and N ‐halosuccinimides have both been employed for converting free thiols to disulfides; however, such conditions have mostly been used for the direct oxidation of two protected thiols . Recently, NCS has been re‐established for the oxidation of two free thiols on resin or in solution .…”

“…Two S−Trt groups can be readily converted to a disulfide by treatment with I 2 or thallium(III) trifluoroacetate (Tl(CF 3 CO 2 ) 3 ) . Similarly, a twin S−Acm pair can also be oxidized by these two reagents.…”

Πεπτίδια Με Πολλαπλούς Δισουλφιδικούς Δεσμούς

“…(1-30)-OH Cys(Trt) 3,8,15,17,22,29 3,8,15,17,22,29 -S-R Cys(Trt) 3,8,15,17,22,29 -OH Cys(Trt) 3,8,15,17,22,29 -OH Cys(Acm) 8,22 Cys(Trt) 3,17 Cys(Mmt) 15,29 ..................…”