Thermolysin peptide synthesis

Although equihbrium-controUed peptide synthesis has been successfully used on a number of occasions, including thermolysin-catalyzed synthesis of aspartame (126) and semisynthesis of insulin (127), the method has a significant drawback a water-miscible organic cosolvent added to the reaction medium to suppress the ionization of unactivated carboxy components significantly reduces the reaction rate. 

When one is using proteases in a direct reversal of their normal hydrolytic function, the equilibrium position is very important in limiting the attainable yield in equilibrium-controlled enzymatic peptide synthesis. If both reactants and products are largely undissolved in the reaction medium as suspended solids, thermodynamic analysis of such a system shows the reaction will proceed until at least one reactant has dissolved completely, towards either products or reactants ( switchlike behavior). In case of a favorable equilibrium for synthesis, the yield is maximized in the solvent of least solubility for the starting materials (Hailing, 1995). Thermolysin-catalyzed reactions ofX-Phe-OH (X = formyl, Ac, Z) with Leu-NH2 yielded X-Phe-Leu-NH2 with equilibrium yields > 90% over a range of solvents. Some predictions, such as a linear decrease in yield with the reciprocal of the initial reactant concentrations, could be verified (Hailing, 1995). 

M. Erbeldinger, X. Ni, and P. J. Halling, Effect of water and enzyme concentration on thermolysin-catalyzed solid-to-solid peptide synthesis, Biotechnol. Bioeng. 1998, 59, 68-72. 

Peptide synthesis is an extremely important area of chemistry for the pharmaceutical industry, and like any specialized area of chemistry, has its own set of unique problems associated with it. Racemization and purification of final products are two of the most difficult problems in this area. The use of enzymes has been explored as a possible answer to these problems since 1938 [29]. However, proteases needed to catalyze peptide synthesis are subject to rapid autolysis under the conditions needed to affect peptide coupling, so this has generally not been a practical approach until cross-linked enzyme crystals of proteases became available. The synthetic utility of protease-CLCs was demonstrated by the thermolysin CLC (PeptiCLEC -TR)-catalyzed preparation of the aspartame precursor Z-... 

The enzymatic aspartame process of Tosoh can also be regarded as a reactive deracemization of D,L-phenylalanine methyl ester (D.L-Phe-OCHa). In the key step the metalloproteinase thermolysin couples only the L-isomer with Z-aspartic acid in a thermodynamically controlled peptide synthesis the remaining D-Phe-OCH3 is racemized and reused [107]. 

When cross-linked crystals of thermolysin were applied in peptide synthesis in ethyl acetate, they were stable for several hundred hours at amazingly low enzyme consiunption, whereas a soluble enzyme preparation became inactive within a short period of time. Again it is worthwhile to consider the quality of the soluble enzyme preparation. When soluble thermolysin was stored in mixed aqueous-organic solutions, it lost about 50% of its activity within the first day of incubation only to be then quite stable for the next 15 days. It is possible that the initial inactivation was caused by an unstable fraction of thermolysin and that crystals of thermolysin no longer contained this unstable fraction [118]. Productivity comparable to that of crystals was achieved with thermolysin adsorbed on Amberlite XAD-7 resin which was employed in continuous plug flow reactors with tert-amyl alcohol as solvent [119]. 

Fisheries processing. Chapman HaU, London, pp 206-222 Basso A, De Martin L, Ebert C et al. (2000) High isolated yields in thermodynamically controlled peptide synthesis in toluene catalysed by thermolysin adsorbed on Cellite R-640. Chem Com-mun 467- 8... 

Thurst S, Koksche B (2003) Protease-catalysed peptide synthesis for the site specific incorporation of alpha-fluoroalkyl amino acids into peptides. J Org Chem 68 2290-2296 Trusek-Holownia A (2003) Synthesis of Z-Ala-Phe.OMe, the precursor of bitter dipeptide in the two-phase ethyl acetate-water system catalysed by thermolysin. J Biotechnol 102 153-163 Tuchscherer G, Mutter M (1996) Template assisted protein de novo design. Pure Appl Chem 68(11) 2153-2162... 

In another research, an approach by using proteases, enzymes that normally hydrolyze peptide bonds in aqueous medium, to perform the reverse reaction (i.e., peptide synthesis or reversed hydrolysis) to produce amphiphilic peptide hydrog-elators that self-assemble to form nanofibrous stmctures was proposed by Ulijin et al. As shown in Fig. 3.34, Fmoc-amino acids (44) and peptides (45) could reacted to form hydrogelator 46 catalyzed by thermolysin (or chymotrypsin), which was able to form transparent gels in water. The result demonstrates that a protease can be used to selectively trigger the self-assembly of peptide hydrogels via reversed hydrolysis [84]. 

Thermolysin, which is another protease, will also catalyse peptide synthesis, and a new plant will shortly use this enzyme for the manufacture of the artificial sweetener Aspartame, at a scale of2,000 tonnes per year. In this reaction the L-enantiomer of racemic phenylalanine methyl ester reacts specifically with the a-carboxyl group of JV-protected L-aspartate (Scheme 6.26). Thus both the separation of the enantiomers of the phenylalanine and the protection of the y-carboxyl group of the L-aspartate are unnecessary, which simplifies the synthesis. Although the equilibrium favours hydrolysis rather than synthesis, the peptide product, which is the JV-protected precursor ester of Aspartame, forms an insoluble salt with the... 

Thermolysin Fmoc-X + FF Peptide synthesis FHydrogel formation [26]... 

Metalloproteases and glutamic and aspartic proteases, on the other hand, do not form covalent intermediates, but activate a water molecule that directly attacks the carbonyl carbon of the peptide bond and displaces the amide nitrogen [49]. These enzymes are often the preferred catalysts for thermodynamically controlled coupling (Figure 15.1). An example of a metalloprotease applied for peptide synthesis is thermolysin, which contains a HExxH+E sequence motif that coordinates a zinc ion and a water molecule. Zinc polarizes the carbonyl group and facilitates deprotonation of the... 

The metalloprotease thermolysin has been widely used for peptide synthesis [66,67]. Thermolysin is produced and secreted by the thermophilic bacterium Bacillus thermo-proteolyticus. Catalysis involves water activation by a zinc ion that is coordinated by side chains of two histidines and a glutamate. Thermolysin specificity is defined by its Sr pocket accepting large hydrophobic, polar, and charged residues. In the SI pocket hydrophobic residues are preferred, and Leu > Ala > Phe > Gly is the preference order for the S2 and S2 pockets. This hydrolytic specificity of thermolysin is reflected in its synthetic specificity. 

Many related so-called thermolysin-like proteinases (TLPs) from various Grampositive strains have been described [47], including neutral proteases from Bacillus subtilis, and some of these variants are applied in peptide synthesis. Several metal-loenzymes acting as carboxy- or aminopeptidase have also been characterized, but these variants have not been extensively used in peptide synthesis. A bovine carboxy-peptidase A [39] and orange carboxypeptidase C [68] have been applied for dipeptide synthesis in water-organic solvent mixtures, both under thermodynamic and xmder kinetic control. 

Chemoenzymatic peptide synthesis of aspartame using thermolysin under thermodynamic control. The coupling reaction is stereo- and regioselective. The unreacted isomer o-Phe-OMe forms a precipitate with the product, shifting the equilibrium toward the synthesis. After precursor isolation and hydrogenolysis, the o-isomer is chemically racemized and can be reused [69, 70]. 

Another example of thermolysin-catalyzed peptide synthesis is the production of precursors of enkephalins [57], in particular, coupling of nonnatural amino acids, such as halophenylalanines, is also possible [34]. Similar to a-ch5unotrypsin and papain, thermolysin has been used for peptide polymerization [71]. Using the reversibility of thermolysin-catalyzed peptide bond formation, d5mamic combinatorial libraries of peptides could be established and screened for e formation of nanostructures with special properties [72]. 

Following the demonstration of the exceptional stability and activity of crosslinked crystals (CLECs) of thermolysin in aqueous and organic media, and its application in peptide synthesis [369], this novel technology has been rapidly commercialized and shown to be applicable to a variety of enzymes [370-383]. 

Parallel approaches have been described for the preparation of polyacrylate-protease conjugates [396-400]. Acryloylation of subtilisin and a-chymotrypsin, followed by mixed polymerization with methyl methacrylate, vinyl acetate, styrene, or ethylvinyl ether, provides insoluble, doped polymethyl methacrylate, polyvinyl acetate, polystyrene, and polyethyl vinyl ether polymers [396]. These biocatalytic plastics perform especially well in hydrophilic and hydrophobic solvents, and have been used for peptide synthesis and the regioselective acylation of sugars and nucleosides. Similarly, modification of subtilisin and thermolysin with PEG monomethacrylate, then copolymerization with methyl methacrylate and trimethylolpropane trimethacrylate furnishes protease-polymethyl methacrylate plastics, which show good activities and stabilities in aqueous, mixed, and low-water and anhydrous organic media [397-400]. The protein-acrylate composites are unique in that they enable catalytic densities as high as 50% w/w. 

The potential utility of peptides as therapeutics with clinical applications is limited by its metabolic instability or poor transmembrane mobility. Consequently, the preparation of metabolically stable peptide analogs that can either mimic or block the function of natural peptides or enzymes is an important area of medicinal chemistry research. Synthesis of fluoroolefin amide isosteres, its incorporation in peptidomimetics, and the influence of that isosteric substitution on the inhibition of several enzymes such as peptidyl prolyl isomerases, dipeptidyl peptidase IV, and thermolysin is described. Moreover, protein folding and activity... 

Alternative methods for the synthesis of peptide aldehydes include reduction of acid halides, phenyl esters, thioesters, and anhydrides prepared from corresponding acids, isoxazolidides, and the hydrolysis of thiazolidine peptides 17,54-56 Enzymes such as thermolysin, subtilisin, and pronase E have proven valuable as effective semisynthetic alternatives 40,57 5 62 ... 

U. Eichhorn, A. S. Bommarius, K. Drauz, and H.-D. Jakubke, Synthesis of dipeptides by suspension-to-suspension conversion via thermolysin catalysis - from analytical to preparative scale, J. Peptide Sci. 1997, 3, 245-251. 

RA Persichetti, N St. Clair, JP Griffith, MA Navia, AL Margolin. Cross-linked enzyme crystals (CLECs) of thermolysin in the synthesis of peptides. J Am Chem Soc... 

The enzymatic synthesis approaches are discussed in more detail in Chapter 19. A protease can be used to catalyze the synthesis of a peptide bond (Scheme 31.21). When the stoichiometry of the reactions is such that two moles of phenylalanine methyl ester are used with one mole of Z-Asp, the Z-APM PM product precipitates and shifts the equilibrium to >95% conversion.232 This is the basis of the commercial TOSOH process operated by Holland Sweetener that uses thermolysin.233 One significant variation has been the use of racemic PM instead of the L-isomer. Because the enzyme will only recognize the l-PM isomer to form the peptide bond, the unreacted d-PM isomer forms a salt and then, after acidification, the d-PM can be chemically racemized and recycled. 

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