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Synthesis of Aspartame by Thermolysin: An X-ray …

19/02/2004 · Synthesis of aspartame precursor with an immobilized thermolysin ..

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Synthesis of Aspartame by Thermolysin: ..

On the basis of the enzyme–substrate complexstructuresreported here and the enzyme kinetics data from the literature summarizedin Table , we propose a chemically reasonablemechanism of action for the TLN-catalyzed synthesis of the aspartameprecursor ZAPM, as shown in Scheme . We hypothesizethat the reaction is initiated by the preferential binding of thecarboxy-donor substrate ZA to the amino-donor (S1′) side ofthe enzyme active site. While still tethered to the enzyme throughits interaction with the catalytic zinc ion, ZA then rotates out ofthe amino-donor site to accommodate PM binding to the enzyme. No stericclashes are evident that would prevent ZA from sampling a productivecarboxy-donor conformation (Figure ) as itreorients within the TLN active site. Conversely, there do not appearto be any compelling interactions in the poorly defined carboxy-donorsite that would appropriately orient ZA absent its initial bindingto zinc. Once both substrates are productively bound to TLN, the enzymecan catalyze the formation of a tetrahedral intermediate, analogousin structure to the inhibitor ZFPLA (Figure ). Collapse of the intermediate would then lead to the formationof the product ZAPM, whose precipitation as the water-insoluble Phe-OMesalt drives the overall reaction in the direction of peptide synthesis.

These amino acids are used as precursors for the organic synthesis of various products; e.g

In spite of their commercial importance, potentially broad applicabilityand technical simplicity, protease-catalyzed peptide synthesis reactionshave not been adequately characterized, optimized, or broadly acceptedin the practice of modern, large-scale, solution enzymology. The crystalstructures described here highlight the degree of complexity thatcan accompany such unnatural reactions in which experimental conditionsare artificially manipulated to achieve an economically viable, albeitenzymatically inefficient, result. These structures may also helpto improve the aspartame precursor synthesis reaction directly, eitherby guiding the rational selection of alternate substrates, or by inferringmodifications of the TLN enzyme (or other closely related enzymes) for increased reaction efficiency. More broadly,these results lead one to consider the extent to which the inefficiency(and unpredictability) associated with this approach to peptide synthesismay be attributed to similarly encrypted substrate inhibition thatmight be correctable, once identified by more powerful methods likeX-ray crystallography. Fortunately, many of the enzymes that havebeen used in the protease-mediated peptide syntheses documented inthe literature have themselves been thesubject of extensive crystallographic study, and these data may help predict how the enzymes will interact withvarious substrates. To prioritize the reactions that would benefitmost from additional analysis by X-ray crystallography, equilibriumdialysis, or other direct ligand binding studies could be used toscreen suboptimal peptide synthesis approaches, in the hope of improvingthe efficiency, and expanding the commercial applicability of protease-mediatedpeptide synthesis reactions.

Synthesis of aspartame precursor: ..

Many aspects of industrial synthesis of aspartame were established by Ajinomoto

Protease mediated peptide synthesis(PMPS) was first describedin the 1930s but remains underexploited today. In most PMPS, the reactionequilibrium is shifted toward synthesis by the aqueous insolubilityof product generated. Substrates and proteases are selected by trialand error, yields are modest, and reaction times are slow. Once implemented,however, PMPS reactions can be simple, environmentally benign, andreadily scalable to a commercial level. We examined the PMPS of aprecursor of the artificial sweetener aspartame, a multiton peptidesynthesis catalyzed by the enzyme thermolysin. X-ray structures ofthermolysin in complex with aspartame substrates separately, and afterPMPS in a crystal, rationalize the reaction’s substrate preferencesand reveal an unexpected form of substrate inhibition that explainsits sluggishness. Structure guided optimization of this and otherPMPS reactions could expand the economic viability of commercial peptidesbeyond current high-potency, low-volume therapeutics, with substantialgreen chemistry advantages.

Aspartame (APM) is a protected dipeptide (-aspartyl--phenylalanine methyl ester) that is widely used as a low-caloriesweetener. More than 19000 metrictons of APM are produced per year, makingit the most highly synthesized peptide in the world. Over 2000 metrictons of the annual output of APM is made enzymatically, using the protease thermolysin (TLN) to catalyzethe condensation of the chiral aspartame-precursor, carbobenzoxy--aspartyl--phenylalanine methyl ester (ZAPM), fromthe protected amino acid substrates carbobenzoxy--asparticacid (ZA) and -phenylalanine methyl ester (PM).,− Deprotection of ZAPM by catalytic hydrogenation yieldsaspartame as the final product., In the ZAPM precursorreaction, TLN is enantioselective for the desired -phenylalaninemethyl ester substrate from a racemic mixture of -phenylalaninemethyl ester. In contrast, although both enantiomers of ZA can bindto TLN, only carbobenzoxy--aspartic acid is used in practicesince carbobenzoxy--aspartic acid inhibits the enzyme.

Enhancement of the aspartame precursor synthetic …

Kinetics and equilibrium of enzymatic synthesis of peptides in aqueous/organic biphasic systems.

Three kinds of acetic esters -ethyl acetate, butyl acetate and amyl acetate -were used as the organic component in an aqueous-organic biphasic mixture for aspartame precursor synthesis, accompanied by solvent extraction.
Firstly, the distribution ratios of the substrates and product between organic and aqueous media were measured at various pH, and it was confirmed that these three acetic esters are suitable as the solvent in this reaction system.
Secondly, the effect of acetic esters on the native enzyme activity was examined by using three solvent systems : water free from any ester, water saturated with the respective ester and aqueous -ester biphasic mixture. Comparison of the latter two with the former showed that the reaction rate was lowered in the acidic range in the case of ethyl acetate, but no remarkable effect was observed in the cases of butyl acetate and amyl acetate.
Thirdly, using butyl acetate as the solvent for substrate feeding and product extraction the batchwise synthesis was performed repeatedly. It was found that free enzyme can be used repeatedly without its deactivation, merely by renewing the organic phase.

Isowa and collaborators discovered thesynthesis of ZAPM by TLNafter a systematic evaluation of the enzyme-mediated coupling of PMto a series of N-protected -aspartic acid analogues. Subsequently, a mechanism of action for the TLN-mediatedsynthesis of ZAPM in aqueous solvent was proposed by Oyama et al.; this accounts for the rate saturation of thereaction observed with increasing concentration of the carboxy-donorsubstrate ZA and the linear rate increase of the reaction within theconcentration range explored for the amino-donor substrate PM. Theseresults were confirmed by Wayne and Fruton and extended to biphasic aqueous–organic solvent mixtures and organic solvents by Nakanishi et al. The enzyme data obtained by these groups, summarizedin Table , illustrate the poor binding ofthe substrates to thermolysin and the sluggishness with which thereaction proceeds. These characteristics make it difficult to studythe reaction using conventional steady-state kinetic methods.

Synthesis of aspartame precursor with an immobilized thermolysin in mixed organic solvents.
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