Thioester Thiolysis

The fourth and last step of the /3-oxidation cycle is catalyzed by acyl-CoA acetyltransferase, more commonly called thiolase, which promotes reaction of /3-ketoacyl-CoA with a molecule of free coenzyme A to split off the carboxyl-terminal two-carbon fragment of the original fatty acid as acetyl-CoA The other product is the coenzyme A thioester of the fatty acid, now shortened by two carbon atoms (Fig. 17-8a). This reaction is called thiolysis, by analogy with the process of hydrolysis, because the /3-ketoacyl-CoA is cleaved by reaction with the thiol group of coenzyme A... 

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. 

The dipeptide L,L-0-(methyl-serinyl)valine was formed without significant loss of label, and at the same time, no label was observed in the AMP released. This result thus excludes a thioester intermediate, which by thiolysis of the adenylate would have led to an even distribution of the 180 between AMP and Val. However, the isomeric dipeptide L,D-0-(methyl-serinyl)valine was recovered with all possible labeling patterns of 180180, 160180, and 160160 [69], To explain the retainment of label in the epimerized dipeptide is not easy. Baldwin and colleagues propose an alternative direct acyl transfer mechanism operating with dipeptidyl adenylates of the type Cys-Val, being epimerized, and transferred to... 

Acetoacetate is formed from acetyl CoA in three steps (Figure 22.19). Two molecules of acetyl CoA condense to form acetoacetyl CoA. This reaction, which is catalyzed hy thiolase, is the reverse of the thiolysis step in the oxidation of fatty acids. Acetoacetyl CoA then reacts with acetyl CoA and water to give 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) and CoA. This condensation resembles the one catalyzed by citrate synthase (Section 17.13). This reaction, which has a favorable equilibrium owing to the hydrolysis of a thioester linkage, compensates for the unfavorable equilibrium in the formation of acetoacetyl CoA. 3-Hydroxy-3-methylglutaryl CoA is then cleaved to acetyl CoA and acetoacetate. The sum of these reactions is... 

The most common source of imidates is nitriles (c/. Volume 6, Chapter 2.7) and, as these can be directly transformed into thioamides (c/. Section 2.4.3.4), this one-step approach is usually preferred. Moreover, depending on the reaction conditions and the substitution pattern, thiolysis of imidates may give thioesters rather than thioamides (c/. Volume 6, Chapter 2.5). However, some high-yield applications of imidates in thioamide synthesis have been reported and are detailed in equations (27) ° and (28). ... 

Harnessing protein splicing, researchers now have the ability to generate recombinant protein a-thioesters through the thiolysis of an appropriately mutated protein-intein fusion. In principle, this means that synthetic and recombinant building blocks can be fused in a semisynthetic version of NCL. Such an approach was first reported in 1998 and has been named expressed protein ligation [8],... 

Fig. 10.1-3 Expressed protein ligation. as a fusion to the N-terminus of an intein. Synthesis of recombinant protein thioesters The CBD allows for purification. The usingthe IMPACT system. Thioesters are thioester resulting from thiolysis can be obtained by expressing a protein of interest ligated under the conditions of NCL...

Scheme 4 Examples of peptide thioester preparation by C-terminal activation and thiolysis...

A similar concept for site-selective thiolysis of peptide bonds via backbone peptide bond activation can be found in Jensen s work (Fig. 18) [115]. The key step involves activation of the carboxy group of a Glu side chain by PyBrOP (19), resulting in the on-resin formation of the pyroglutamyl imide moiety. The activation renders the imide C-N bond susceptible to thiolysis, after which protected peptide thioesters were released from the solid support. 

Interestingly, lipase-catalyzed acyl transfer onto SH-groups (corresponding to ester thiolysis in analogy to aminolysis) does not take place [289]. As a result, the resolution of sec-thiols is not feasible by this method and has to be performed via hydrolysis or alcoholysis of the corresponding thioesters [290-292]. 

Key words. Chiral, crown ether, enzyme model, peptide synthesis, chiral recognition, molecular recognition, thiolysis, aminolysis, thiol, thioester. 

It was already reported that thiol-bearing chiral crown ethers of type 1 showed rate enhancements in the thiolysis of a-amino acid p-nitrophenyl ester salts, due to the intra-complex nature of the reaction, forming the corresponding thioester as shown in Scheme 1 [5]. Evaluating the thioester as the reactive intermediate for nucleophile... 

In an earlier study, we pointed out some important aspects of aminolysis of thioester in our enzyme model. First, the fastest rate for aminolysis of thioester was obtained in the presence of equimolar amounts of acid and base catalysts. Second, the reaction proceeded in aprotic nonpolar solvents such as benzene, ethyl acetate, dichloromethane, and so on [7]. Thus, the peptide syntheses by the enzyme model have been performed in benzene buffered with equimolar amounts of pivalic acid and triethylamine as acid and base catalysts, respectively. Third, the superiority of intramolecular aminolysis over an intermolecular one was clearly demonstrated, despite the large membered cyclic intermediate expected for the intramolecular reaction. The host 10 could achieve the synthesis of the tetrapep-tide derivative (11) by formal turnover of the intra-complex thiolysis and the intramolecular aminolysis, but its efficiency as an enzyme model has remained to be improved [3]. 

Moreover, the same authors employed a closely related organocatalyst and the corresponding urea derivative to promote the enantioselective dynamic kinetic resolution of azalactones with allylic alcohol. In this case of substrates, the urea derivatives proved to be superior to their thiourea analogues and, most usefully, these catalysts were insensitive to the steric bulk of the amino acid residue, allowing alanine-, methionine- and phenylalanine-derived azalactones to undergo dynamic kinetic resolution with unprecedented levels of enantioselectivity, as shown in Scheme 9.4. Furthermore, the compatibility of these catalysts with thiol nucleophiles was exploited in the first enantioselective catalytic dynamic kinetic resolution of azalactones by thiolysis to furnish enantioenriched amino acid thioesters of potential use with moderate enantioselectivities (<64% ee). 

Crown ether systems which display both binding selectivity and chiral recognition have potential in the field of enzyme models, and an important step in this direction is a report of the catalysis of transacylation of amino-acid p-nitro-phenylesters in ethanol by crown compound (60). There is evidence for a thioester intermediate, and the observation that (S)-host is the more efficient catalyst with (L)-guests can be rationalized by consideration of models for the hypothetical tetrahedral intermediate (61) in the thiolysis. ... 

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