Efficient chemical synthesis of human complement protein C3a.

We report the total chemical synthesis of human C3a by one-pot native chemical ligation of three unprotected peptide segments, followed by efficient in vitro folding that yielded the anaphylatoxin C3a in high yield and excellent purity. Synthetic C3a was fully active and its crystal structure at 2.1 Å resolution showed 3 helices and a C-terminal turn motif.

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

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 beta-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), YRRGRCGGLCLAR (P5) and YRRGRAhxCGGLCLAR (P8), point in the direction of C3a antagonists.