Thiol-thioester Exchange Research Articles

Dynamic Cyclic Thiodepsipeptide Libraries From Thiol-Thioester Exchange

Thiol-thioester exchange was found to readily generate libraries of cyclic thiodepsipeptides under thermodynamic control, which will enable their use in a variety of dynamic combinatorial chemistry assays. The kinetic determinants of macrocycle formation and the role of amino acid structure on the reaction dynamics are discussed.

In situ monitoring of backbone thioester exchange by 19F NMR.

backbone thioester exchange; coiled coil; fluorine; NMR spectroscopy; protein foldingBackbone thioester exchange (BTE) is a new technique for assessing the stability of foldingpatterns adopted by polypeptides.[1–5] Implementation of this method requires thereplacement of a single backbone amide group in the polypeptide of interest with a thioester;this replacement is an approximately isostructural substitution.[ 6,7] Combining the backbone-modified polypeptide analogue (a thiodepsipeptide) with a small thiol in pH 7 aqueous bufferinitiates a thiol–thioester exchange reaction.[8] The equilibrium constant for this exchange isstrongly influenced by the stability of the folded conformation adopted by the full-lengththiodepsipeptide. We have shown that BTE enables examination of sequence–stabilityrelationships for a variety of small folding motifs under native conditions including β-hairpins,coiled coils and a small protein, the chicken villin headpiece subdomain.[1–5] BTE analysisis complementary to traditional methods of assessing protein conformational stability, in whichthe folding pattern is disrupted with heat or chemical additives.[ 9] The traditional approachesrequire extrapolation to obtain folding parameters that apply under native conditions, whileBTE enables direct measurement under native conditions. BTE has provided new insights oncommon protein folding motifs[ 1,3–5] and on contributions of unnatural side chains to tertiarystructural stability,[2] but continued development is necessary to maximize the utility of thisnew tool.Here we report that quantitative BTE analysis can be conducted in situ by using

The use of a cysteinyl prolyl ester (CPE) autoactivating unit in peptide ligation reactions

A peptide containing a cysteinyl prolyl ester (CPE) moiety at the C-terminus (CPE peptide) is spontaneously transformed into a diketopiperazine thioester via an intramolecular N– S acyl shift reaction, followed by diketopiperazine formation. The CPE peptide can be ligated with a Cys-peptide in a one-pot procedure. The peptide diketopiperazine thioester can also be transformed into a peptide thioester by intermolecular thiol–thioester exchange with external thiol compounds such as sodium mercaptoethanesulfonate. Since CPE peptides can be prepared by standard Fmoc solid-phase synthesis, it is a versatile alternative to the peptide thioester, providing a flexible ligation strategy that promises to be useful in polypeptide synthesis.

Thermodynamic Analysis of β‐Sheet Secondary Structure by Backbone Thioester Exchange

Understanding the factors responsible for the stability of common protein secondary structures has been a longstanding goal. Analysis of b sheets has lagged behind the study of a helices because the development of suitable model systems has been more challenging for the former than for the latter. Over the past decade, however, rules for the design of b sheets that fold autonomously in water have been elucidated, with a particular emphasis on two-stranded antiparallel b sheets (“b hairpins”), which represent a minimum increment of this secondary structure. These model systems have proved useful for evaluating the contributions of factors such as side-chain–side-chain interactions, interstrand linker composition, strand length, and strand number to the stability of b sheets. As an outgrowth of this work, b hairpins have emerged as excellent platforms for the evaluation of noncovalent interactions that do not necessarily occur naturally in b sheets. bHairpins and hairpin-like molecules stabilized by cyclization are also attractive systems for biomedical applications and fundamental studies. The use of designed peptides to probe the origins of bsheet stability, or to study noncovalent attractions between moieties that become spatially juxtaposed upon folding, requires the ability to determine the extent of b-sheet folding in solution. If only two conformational states are populated, unfolded and b sheet, then determining the population of these two states gives the folding equilibrium constant (Kfold), which provides insight into the stability of the folded state (DGfold= RT lnKfold). For proteins that adopt defined tertiary structures, conformational stability is often assessed by using heat or a chemical denaturant to disrupt the folded state while monitoring the extent of folding by a conformationally sensitive spectroscopic probe (for example, circular dichroism). This approach is convenient because globular proteins are typically completely folded near room temperature and in the absence of denaturant (“native conditions”), which establishes the spectroscopic signature of the folded state, and because it is often straightforward to identify the spectroscopic signature of a fully unfolded state generated at high temperature or high denaturant concentration. In contrast, most of the autonomously folding b-sheet model systems described to date cannot be driven by changing the conditions to the limiting states. Therefore, identifying the spectroscopic signatures for the fully unfolded and fully folded states of b-sheet model systems has frequently required the preparation and characterization of distinct reference peptides or the implementation of elaborate data analysis techniques. We have recently developed a new approach for studying the conformational stability of small proteins, and here we describe the extension of this approach to b hairpins. This method involves polypeptide analogues in which one backbone amide group has been replaced by a thioester (namely, thiodepsipeptides). The conformational stability is assessed by monitoring the equilibrium constant for a thiol–thioester exchange reaction that causes the full-length thiodepsipeptide to be reversibly cleaved, which precludes adoption of a native-like fold. The “backbone thioester exchange” (BTE) measurements can be conducted under native conditions, and it is not necessary for the full-length molecule to be completely folded under these conditions. Our previous BTE studies have focused on polypeptides that adopt a discrete tertiary structure. Here we show that the BTE method can be extended to a secondary structure model system (Figure 1). Our implementation of BTE began with a designed b hairpin designated HP5W4 (Figure 2) that was developed by Andersen and co-workers. 12] The sequence of HP5W4 incorporates key design features introduced originally by Cochran et al. in their development of the “tryptophan zipper” peptides. The turn segment of a b hairpin appears to be an optimal region for the amide-to-thioester modification that is required for implementation of BTE. Thioesters are poorer hydrogenbonding partners than are secondary amides, and our previous efforts have therefore involved replacement of the backbone amide groups that do not form intramolecular hydrogen bonds in the folded state so as to minimize the conformational destabilization that might result from the modification. The optimized b hairpin turn segment of Andersen and co-workers, Asn-Pro-Ala-Thr-Gly-Arg, seemed to offer a particularly attractive site for thioester insertion at the Thr Gly bond. Our first thiodepsipeptide design (NT-C’) contains a thioester linkage at this position as well as three conservative side-chain modifications compared to HP5W4: all three Lys residues were mutated to Arg. These mutations were made to avoid the possibility of acylation of [*] E. B. Hadley, A. M. Witek, Dr. F. Freire, A. J. Peoples, Prof. S. H. Gellman Department of Chemistry University of Wisconsin–Madison 1101 University Avenue, Madison, WI 53726 (USA) Fax: (+1)608-265-4534 E-mail: gellman@chem.wisc.edu

Peptide Thioester Synthesis via an Auxiliary-Mediated N–S Acyl Shift Reaction in Solution

The 4,5-dimethoxy-2-mercaptobenzyl (Dmmb) group attached to a main chain amide in a peptide is easily transformed into an S-peptide via an intramolecular N–S acyl shift reaction under acidic conditions, and the S-peptide produces a peptide thioester through an intermolecular thiol–thioester exchange reaction. In order to develop a method for efficiently preparing peptide thioesters based on the N–S acyl shift reaction, the factors involved in this process were analyzed in detail. The general features of the transformation at the Dmmb group attached amide bond in a trifluoroacetic acid (TFA) solution and the generation of a peptide thioester were examined by 13C-NMR spectral measurements, reversed-phase (RP) HPLC analyses, mass measurements, and amino acid analyses. The methoxy group of the Dmmb group was not essential for the N–S acyl shift reaction, but played a role in stabilizing the thioester form. The addition of water to the TFA solution accelerated the N–S acyl shift reaction mediated by the Dmmb group and also suppressed the acid-catalyzed cleavage of the Dmmb group. A peptide thioester was produced from the S-peptide via an intermolecular thiol–thioester exchange reaction with minimal epimerization of the amino acid residue that constituted the thioester bond. Undesirable side reactions, such as the hydrolysis of the thioester bond and an S–N acyl shift reaction occurred during the synthetic process, which is a subject of further investigation.

Peptide synthesis using unprotected peptides through orthogonal coupling methods.

We describe an approach to the synthesis of peptides from segments bearing no protecting groups through an orthogonal coupling method to capture the acyl segment as a thioester that then undergoes an intramolecular acyl transfer to the amine component with formation of a peptide bond. Two orthogonal coupling methods to give the covalent ester intermediate were achieved by either a thiol-thioester exchange mediated by a trialkylphosphine and an alkylthiol or a thioesterification by C alpha-thiocarboxylic acid reacting with a beta-bromo amino acid. With this approach, unprotected segments ranging from 4 to 37 residues were coupled to aqueous solution to give free peptides up to 54 residues long with high efficiency.

Does butyryl-CoA dehydrogenase catalyse thiol-thioester exchange?

Does butyryl-CoA dehydrogenase catalyse thiol-thioester exchange?