Peptide thioesters

L Xiangqun, T Kawakami, S Aimoto. Direct preparation of peptide thioesters using an Fmoc solid-phase method. Tetrahedron Lett 39, 8669, 1998. 

A second approach that can be adopted to overcome the intrinsic requirement for cysteine at the N-terminus of C-terminal fragment utilizes the enzyme subtiligase, a double mutant of subtilisin, which is able to join two unprotected peptides. Thioester-modified proteins were shown to present good substrates of subtiligase [65]. However, although this approach could be potentially useful for general isotope labeling, the efficiency of this process remains to be proven. 

Since the thioester linkage is susceptible to nucleophilic attack but stable to TEA treatment during solid-phase peptide synthesis (using standard Boc protection), Weigel and colleagues " have envisioned that hydroxylamine derivatives could directly cleave resin-bound peptide thioesters 201 or 202 to form the corresponding peptide hydroxamates 203 (Scheme 88). 

Synthesis of the Peptide-Thioester 8 Incorporating the SCAL Linker ... 

Scheme 18 Synthesis of 2,3-Bis(phytanoxy)propanamine and Conjugation to a Peptide Thioester Synthesized...

The linear peptide thioester was dissolved in 0.2 M phosphate buffer (pH 7.2, 0.1-0.6 mM soln) containing 50% DMF and TCEP (6 equiv). After completion of the reaction (3-24 h, monitored by HPLC) the cyclic peptide was purified by preparative HPLC (small amounts of byproducts result from hydrolysis of the peptide thioester). 

The crude peptide thioester H-Cys-Ser-Cys-Lys-Ser-Lys-Val-Cys(Acm)-Tyr-Lys-Gln-Ser-Ile-Pro-Cys-Gly-Glu-Ser-Cys-Val-Phe-Ile-Pro-Cys(Acm)-Thr-Val-Thr-Ala-Leu-Leu-Gly-COS(CH2)2CONH2 (50-80mg), obtained after cleavage from the resin support by HF (HF/PhOMe 9 1), was extracted into a 8M urea soln (150 mL) at pH 7.6 in a highly reductive environment containing 5- to 10-fold excess of TCEP and then dialyzed against 8 M urea (2000 mL) to allow cyclization in a descending concentration... 

Formation of the internal thioester bond can be also accomplished by mixed anhydride assisted coupling of the N-protected peptide with a mercaptoacetylated C-terminal peptide fragment. 6 Sulfanylacids used for the preparation of peptide thioesters can be obtained from the common amino acids by the method of Pfister et al. 41 further described by Yan-keelov et al. 2 ... 

A more versatile route to synthesize peptide thioesters is by the preparation of a peptide thioacid via the solid-phase method and then reaction with a halo derivative to form the thioester. This route can be achieved via the preparation of thioester linkers that can be applied in stepwise solid-phase peptide synthesis. The general structure of the N-protected amino thioacid attached to the linker is shown in Scheme 14. t65-80 ... 

Powers, J.C. and Kam, C.-H. 1995. Peptide thioester substrates for serine peptidases and metalloen-dopeptidases. Methods Enzymol. 248 3-18. 

Peptide thioesters (Section 15.1.10) are generally prepared by coupling protected amino acids or peptides with thiols and are used for enzymatic hydrolysis. Peptide dithioesters, used to study the structures of endothiopeptides (Section 15.1.11), may be prepared by the reaction of peptide nitriles with thiols followed by thiolysis (Pinner reaction). Peptide vinyl sulfones (Section 15.1.12), inhibitors of various cysteine proteases, are prepared from N-protected C-terminal aldehydes with sulfonylphosphonates. Peptide nitriles (Section 15.1.13) prepared by dehydration of peptide amides, acylation of a-amino nitriles, or the reaction of Mannich adducts with alkali cyanides, are relatively weak inhibitors of serine proteases. 

Scheme 22 Synthesis of Peptide Thioester as a Precursor for Cysteine-Thioester Cyclization on a Thioester...

Peptide thioester 84 was prepared from 81 (0.3 g of resin). All amino acids were protected with N-terminal Boc groups. The side-chain protections were as follows Arg(Tos), Asp(OCy), Cys[Bzl(4-Me)], Glu(OCy), His(Tos), Lys[Z(2-Cl)], Ser(Bzl), and Thr(Bzl). Each synthesis cycle consisted of (1) a 25-min deprotection with 55% TFA/CH2C12 and (2) coupling with Boc amino acid (4 equiv) and BOP or HBTU (4 equiv) in the presence of DIPEA (6 equiv) in DMF for 1 h. All couplings were monitored by the... 

Scheme 1 Peptide Thioesters by Coupling of Peptides with Thiols...

Partially protected peptide thioesters that are prepared from SPPS and purified by RP-HPLC can condense with other peptide segments to form highly homologous peptides which can comprise as many as 100 amino acid residues. 9 Syntheses of peptide thioesters using Boc-SPPS have been quite successful.161214 However, the preparation of thioesters by Fmoc chemistry is difficult because the piperidine used to remove the Fmoc group attacks the carbonyl moiety of the resin-bound thioester to release the peptide from solid support. However, peptide thioesters have been prepared by SPPS using Fmoc chemistry. 9 ... 

Scheme 2 Peptide Thioesters by Boc Solid-Phase Peptide Synthesis18-141...

Scheme 4 Peptide Thioesters by Fmoc Solid-Phase Peptide Synthesis1 1...

Peptide thioester H-Val-Lys-Lys-Thr-Ser-Trp-Thr-Glu-Glu-Asp-Arg-Ile-Ile-Tyr-Gln-Ala-His-Lys-Arg-Leu-Gly-SCH2CH2CONH2 (25 mg, 9.0 pmol), prepared by Boc-SPPS and cleaved from resin, was dissolved in DMSO (0.4 mL) containing TEA (9.6 pL). Boc-OSu (15 mg, 72 pmol) was added to the soln and stirred at rt for 2.5 h, after which EtOAc and Et20 were added. The precipitate was washed with EtOAc and lyophilized from a dioxane suspension to give the peptide yield 25.8mg (90%) MS mlz. calcd for [M + l], 3174.8 found, 3174.8. 

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