Cynthia G. Fields

Sequence dependence of aspartimide formation during 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis

We have examined the sequence dependence of aspartimide formation during Fmoc-based solid-phase synthesis of the peptide Val-Lys-Asp-X-Tyr-Ile. The extent of aspartimide formation and subsequent conversion to the α- or β-piperidide was characterized and quantitated by analytical reversed-phase high-performance liquid chromatography and fast atom bombardment mass spectrometry. Aspartimide formation occurred for X=Arg(Pmc), Asn(Trt), Asp(OtBu), Cys(Acm), Gly, Ser, Thr and Thr(tBu). No single approach was found that could inhibit this side reaction for all sequences. The most effective combinations, in general, for minimization of aspartimide formation were (i) tert-butyl side-chain protection of aspartate, piperidine for removal of the Fmoc group, and either 1-hydroxybenzotriazole or 2,4-dinitrophenol as an additive to the piperidine solution; or (ii) 1-adamantyl side-chain protection of aspartate and 1,8-diazabicyclo[5.4.0]undec-7-ene for removal of the Fmoc group.

Minimization of tryptophan alkylation following 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis

Abstract We have examined Pmc and Pbf side-chain protection of Arg and Boc side-chain protection of Trp in an attempt to minimize side-chain protecting group “scavenger” use following Fmoc-based solid-phase synthesis. The extent of Trp alkylation was characterized and quantified by analytical RP-HPLC. Edman degradation sequence analysis, and ESMS. The Pbf group offered lower TFA-induced Trp alkylation than the Pmc group. The combination of Trp(Boc) and Arg(Pbf) resulted in extremely low levels of Trp alkylation during TFA treatment of the peptide-resin in the absence of scavengers.

Solid-phase synthesis of triple-helical collagen-model peptides*

The triple-helical conformation of collagen has been proposed to be important for mediation of cellular activities, such as adhesion and activation, extracellular matrix assembly, and enzyme function. We have developed synthetic protocols that allow for the study of biological activities of specific collagen sequences in triple-helical conformation. These methods primarily involve solid-phase assembly and covalent linkage of three peptide chains. The resultant triple-helical peptides have sufficient thermal stabilities to permit structural and biological characterization under physiological conditions. The present article critically reviews the various approaches for constructing synthetic triple-helices.

The development of high-performance liquid chromatographic analysis of allyl and allyloxycarbonyl side-chain-protected phenylthiohydantoin amino acids

Ten phenylthiohydantoin (PTH) amino acids possessing allyl (Al) or allyloxycarbonyl (Aloc) side-chain-protecting groups have been characterized by high-performance liquid chromatography for use in Edman degradation sequence analysis. Optimized separation of side-chain-protected and -unprotected PTH amino acids was achieved on a C-18 reversed-phase column with a two-step gradient spanning 32 min. Five of the side-chain-protected amino acids [Cys(Al), Cys(Aloc), Lys(Aloc), Thr(Aloc), Tyr(Al)] were completely stable to the conditions of PTH derivatization, four [Asp(OAl), Arg(Aloc)2, Glu(OAl), Ser(Aloc)] were partially deprotected during PTH derivatization, and one [His(Aloc)] was completely deprotected during PTH derivatization. All allyl-based derivatives were well resolved from their side-chain-unprotected counterparts. Studies on the stability to piperidine treatment showed Asp(OAl), Cys(Al), Glu(OAl), Lys(Aloc), Thr(Aloc), and Tyr(Al), and possibly Arg(Aloc)2 and Ser(Aloc), to be suitable for peptide synthesis by 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry. Edman degradation of Al and Aloc side-chain-protected Conus geographus Lys9-alpha-conotoxin GI synthesized on 4-methylbenzhydrylamine-copoly(styrene-1%-DVB)-resin demonstrated the usefulness of these derivatives for solid-phase preview sequence analysis.

Solid-phase synthesis of O-palmitoylated peptides

Solid-phase synthesis of an O-palmitoylated peptide has been achieved by either palmitoylation of the resin-bound peptide or synthesis of palmitoylated Fmoc-Thr and subsequent solid-phase incorporation. The desired peptides containing palmitoylated Thr were isolated by reversed-phase C18 HPLC. An O → N acyl shift was encountered when palmitoylated Fmoc-Thr was utilized.