Enzymes amide bond formation

The notion that RNA existed prior to enzymes and that RNA molecules can be catalytically active is by now well accepted. Indeed, RNA molecules have been observed to catalyze phophodiester bond breaking and synthesis (as occurs during replication and splicing) but also reactions more distant to its stmcture, such as amide bond formation (Wiegand, 1997). RNA thus provided the means to assemble peptides which may have led to the protein world of today (Zhang and Cech, 1998). Reactions catalyzed by RNA molecules have thus far not been employed in biocatalysis and it is unlikely that... 

Enzymes such as proteases (122), subtilisin (123), acylases, peptidases, amidases, and lipases (124) are reported to catalyze amide bond formation with, in some cases, enantiospeciflcity of over 99%. Despite limited enzyme-substrate compatibility, specific conditions have been developed to reverse their natural reactivity, which is in favor of the hydrolysis. For example, Kyotorphin (Tyr-Arg) (125), a potent analgesic, was produced on an industrial scale using a-chymotrypsin, a peptidase isolated from bovine pancreas. 

Enzymes can be utilized to affect a number of transformations the broad spectrum of reactions, including amide bond formation, hydrolysis, esterification, reduction, oxidation, and carbon-carbon bond formation, has been reviewed elsewhere (114). 

In addition to the widely reported techniques of amide bond formation, transesterification, and hydrolysis, enzymic enantioselective oxidation is also used in the synthesis of single isomer drugs. Patel described the elficient oxidation of benzopyran (75), an intermediate in thesynthesisof potassium channel openers (123). The transformationwas ef-fected w i t h a cell suspension of MortiereUa raman-niana with glucose over a 48-h period, the isolated product (77) was obtained in a 76%yield with an optical purity of 97%and a chemical purity of 98%, as shown in Pig. 18.32. 

Many natural peptides are synthesized by a sequence of enzyme-controlled processes carried out by a multifunctional enzyme of modular arrangement, similar to some polyketide synthases. These nonribosomal peptide synthetases (NRPSs) typically consist of an adenylation domain, a peptidyl carrier protein domain, and a condensation or elongation domain in order to carry out amide bond formation and some derivations of amino acid residues. 

After the attachment of the initiation unit, formylmethionyl fRNA, the next aminoacyl fRNA is inserted into the ribosomal acceptor site with the aid of a transferase enzyme. The a-amino group of this aminoacyl fRNA rapidly reacts with the activated carboxyl terminal residue of the initiator, or peptidyl fRNA, component. The new peptide bond is thus formed. The fRNAs must have their anticodons properly aligned opposite their respective triplets (i.e., codon) on the mRNA. The amino acids attached to them are then adjacent to each other on the 50S ribosomal subunit. At this point the peptide synthetase enzyme can catalyze the amide bond formation. 

A conceptually different approach to assemble fully unprotected peptides is to use an enzyme to attain both specificity and catalysis of the amide bond formation. This strategy has been developed using proteases, enzymes that cleave peptide backbone amide bonds. Following the principle of microscopic reversibility, any enzyme can be coerced to catalyze a reaction not only in the forward direction but also in the reverse direction. Such reverse proteolysis methods typically use substrates containing activated C-termini,... 

In the absence of acids or bases peptide bonds are quite resistant to hydrolysis, but their hydrolytic cleavage is extremely accelerated in the presence of proteolytic enzymes. The remarkable catalytic effect of these enzymes tempted many investigators, through a long period of time (Fruton 1982), to adopt them for synthesis, rather than hydrolysis of peptide bonds. Since enzymes are catalysts and merely accelerate the establishment of equilibria, it is possible to use proteolytic enzymes for amide bond formation if the equilibrium of the reaction can be modified. Thus anilides of blocked amino acids could be prepared with the help of papain ... 

E,-SH = ubiquitin activating enzyme. E2-SH = a transferase which transfers ubiquitin to the conjugation site. Ej = a ligase which catalyses amide bond formation, All three enzymes have been purified [A. Hershko J. Biol. Chem. 258 (1983) 8206-8214], U-Gly-COOH represents ubiquitin with Its C-terminal glycine. Deubiquitinating enzymes have also been characterized [C.M.Pickart I.A.Rose J. Biol. Chem. 261 (1986) 10210-10217 S.-l Matsul etal. Proc. Natl. Acad. Sci. USA 79(1982) 1535-15391. 

Another example where aggregation of a library member drives its synthesis was recently reported by Ulijn ct al. [24, 25]. They used reversible amide bond formation, mediated by thermolysin, which is an enzyme that can catalyze both amide bond hydrolysis and formation, and is only moderately peptide-sequence-dependent. The authors reported that starting from dipeptides and fluorenyl-protected amino acids, the action of thermolysin gives rise to a dynamic mixture of peptides of different lengths (containing typically one to five amino acid residues). When using phenylalanine or leucine as the starting amino acids the... 

In contrast to the widespread examples for artificial hydrolases which cleave ester or amide bonds only rare examples for transferase enzyme mimics—catalyzing the reverse reaction—are known. This could be due to the fact that amide bond formation is, in general, done under water-free conditions by condensation of an amine and a carboxylic acid. Transferases belong to the second main group of enzymes and transfer atomgroups from a donor (coenzyme A) to an acceptor molecule. The transacylases play an important role in the citric acid cycle or fatty acid biosynthesis (Figure 32) and degradation. The coenzyme A plays... 

Fermentation of Streptomyces niveus in the presence of Me-[ C]-L-methionine yielded novobiocin with an incorporation of 10 per cent of the added isotope °°. The radioactivity was distributed between the 0-methyl and gem-dimethyl groups of the sugar noviose (66 per cent) and the C-methyl group of the coumarin fragment (123,35 per cent) and thus indicated that the coumarin methyl group was formed by a process of C-methylation. The timing of this step is, however, not known. Enzyme systems have been isolated from Streptomyces niveus which are capable of rapid amide bond formation between the isoprenyl 4-hydroxybenzoic acid (125) and the coumarin (123) to give novobiocic acid. The reaction requires ATP and presumably involves some form of activation of the p-hydroxybenzoyl carboxyl function. 

Although selenolsubtilisin is essentially inactive in cleaving amides, it could still be useful as a ligase for preparing amides from esters and amines. In such a scheme the modified enzyme would be acylated with an activated ester and subsequently transfer its acyl group to a suitable nucleophile. However, efficient amide bond formation will require high selectivity for aminolysis with respect to hydrolysis of the... 

Many of the early examples of maerocyeles in drug discovery relied on these classical reaction types for the formation of the ring and they remain in regular use. This was due, in part, to the peptidomimetic nature of many of these structures, which often were targeted at protease enzyme inhibition, and thus lent themselves readily to macrolactamization for amide bond formation or macrolactonization for cyclic depsipeptide-like compounds. Representative examples of these two general transformations are shown in Scheme 11.1 (BOP (benzotriazol-l-ylo5ytris(dimethylamino)-phosphonium hexafluorophosphate, Castro s reagent), EDC (l-ethyl-3-(3-dimethylamino-propyl)-carbodiimide), DMAP (4-dimethylamino-pyridine)) for the matrix metalloproteinase (MMP) inhibitor template 2 and the renin inhibitor scaffold 4. °... 

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