In vitro fenoprofenyl–coenzyme a thioester formation: Interspecies variations

In vitro coenzyme A thioester formation from (?)-(R)-fenoprofen (FPF) and palmitic acid has been studied using liver microsomes from rat, guinea pig, sheep, and dog. In every species with both palmitic acid or (?)-(R)-fenoprofen, the Lineweaver–Burk plot was linear in the substrate concentration range used and as a consequence agrees with the involvement of only one isoenzyme (or different isoenzymes of similar K_m values). The V_max values for the thioesterification of (?)-(R)-fenoprofen present large species variations from 2.1 ± 1.0 with sheep liver microsomes to 60.6 ± 11 nmol/min/mg with dog liver microsomes. These values statistically significantly correlate (r = 0.94) to the V_max values observed when palmitic acid was used as a substrate. Furthermore palmitic acid inhibited (?)-(R)-fenoprofen–CoA formation in the same extent in all animal species. The stereoselectivity of the thioesterification was also species dependent. © 1995 Wiley-Liss, Inc.     

Efficient method of circumventing insolubility problems with fully protected peptide carboxylates via in situ direct thioesterification reactions

A straightforward and convenient protocol is presented for the direct thioesterification of fully protected peptide C‐terminal carboxylates synthesized by Fmoc strategy. This methodology specifically serves to overcome the frequent insolubility problem of these fully protected carboxolate isolates during the thioesterification process by carrying out the reaction as an in situ procedure on the freshly cleaved 1% TFA/DCM solution of carboxylate. The direct thioesterification of a number of insolubility prone peptide systems is explored and compared with some control systems for ease of conversion to the corresponding thioesters. It is shown that although the fully protected carboxylates are indeed insoluble to varying degrees in the thioesterification reactions carried out using the classical approach, full dissolution is maintained and complete conversion is evident using the in situ methodology. This protocol serves to remove a frequent stumbling block in the preparation of peptide thioesters via the direct approach, allowing for facile entry into previously difficult systems traditionally unapproachable through this method. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Structure Elucidation of Gibberellin A32 and Its Acetonide

The structures of two gibberellin-like substances isolated from the immature seeds of Prunus persica, tentatively named PG–I and PG–II, were elucidated. PG–I was an ammonium salt of a novel gibberellin, ent-3α,10,12β,13,15α-pentahydroxy-20-norgibberella-l,16-diene-7,19-dioic-19,10-lactone (1), to which gibberellin number A₃₂ was allocated. PG–II was shown to be gibberellin A₃₂ acetonide (7), and concluded to be an artifact produced from gibberellin A₃₂ in the isolation process.     

Comparison of Acyl-CoA Synthetic Activities and Enantioselectivity toward 2-Arylpropanoic Acids in Firefly Luciferases

Measurement of thioesterification activities for dodecanoic acid (C12) and ketoprofen was done using five firefly luciferases, from Pyrocoelia miyako (PmL), Photinus pyralis (PpL), Luciola cruciata (LcL), Hotaria parvura (HpL), and Luciola mingrelica (LmL). Among these, PmL, PpL, and LcL showed the expected thioesterification activities toward both substrates. All the enzymes exhibited (R)-enantioselectivity toward ketoprofen, which had same tendency as firefly luciferase from Luciola lateralis (LUC-H). HpL and LmL, however, did not accept ketoprofen, although they had thioesterification activity toward C12. These results indicate that the substrate acceptance of luciferases for the thioesterification reaction varies dramatically relying on the origin of firefly. Hence we focused primarily on PmL and investigated the effect of pH on enzymatic activity. In addition, by determining the kinetic parameters at various pH values, we verified that the k _cat parameter contributed to the preferential enantioselectivity of this enzyme.     

A novel post‐ligation thioesterification device enables peptide ligation in the N to C direction: synthetic study of human glycodelin

Human glycodelin consists of 162 amino acid residues and two N‐linked glycans at Asn²⁸ and Asn⁶³. In this study, we synthesized it by a fully convergent strategy using native chemical ligation (NCL) in N to C direction. The four peptide segments corresponding to 1–31, 32–65, 66–105 and 106–162 sequences were synthesized by 9‐fluorenylmethoxycarbonyl based solid‐phase peptide synthesis. At the C‐terminus of the second segment, N‐ethyl‐S‐acetamidomethyl‐cysteine was attached as a post‐ligation thioesterification device. The N‐terminal two segments were condensed by the homocysteine‐mediated NCL at Leu‐Met site, and the product was methylated to convert homocysteine to methionine. After deprotection of acetamidomethyl group on the N‐ethylcysteine residue, the peptide was thioesterified by N‐alkylcysteine‐assisted method. The product was then ligated with the C‐terminal half, which was obtained by the NCL of third and fourth segments, to give the full‐length glycodelin. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.