Effects of Acetonitrile-Water Mixtures on α-Chymotrypsin Catalyzed Dipeptide Synthesis

-Chymotrpysin (EC 3.4 21.1) was immobilized by deposition on celite and subsequent cross-linking with glutaraldehyde. The effects of different mixtures of aqueous buffer and acetonitrile on the immobilized preparation were evaluated using a dipeptide synthesis as model reaction. The initial reaction rate at 6-95% of water increased with increasing water content. The maximum yield of peptide had two maxima; the first one at 6% of water (92%) and the second one at 80% of water (39%). The presence of two maxima was due to severe enzyme inactivation at intermediate water contents (50-60%). The immobilisation procedure slowed the inactivation of -chymotrypsin. Cross-linked enzyme was inactivated to a lesser extent than both free enzyme and enzyme that had been deposited on celite. The increased resistance to inactivation was, however, not sufficient to make peptide synthesis attractive at intermediate water contents (50-60%). In order to obtain good peptide yields, low water contents (below 10%) should be used.     

Aspartame precursor synthesis in water miscible cosolvent catalysed by thermolysin

Summary The synthesis of the dipeptideN-benzyloxycarbonyl-L- aspartyl-phenylalanine methyl ester, aspartame precursor, catalysed by thermolysin in aqueous and aqueous methanolic solutions was studied. Thermolysin with concentration as low as 10 M in 25% methanol can catalyse the synthetic reaction. The optimum methanol compositions at 4°C and 37°C were 50% and 25% respectively where an increase in peptide yield of 85% was obtained for both conditions as compared to that in water.Abbreviations N-cbz-L-Asp N-benzyloxycarbonyl-L-aspartic acid - L-Phe-OMe L-phenylalanine methyl ester - N-cbz-L-Asp-Phe-OMe N-benzyloxycarbonyl-L-aspartyl-phenylalanine methyl ester All the % of methanol is a volume % in water unless otherwise specified.     

Solid-phase peptide synthesis by ion-paired alpha-chymotrypsin in nonaqueous media

Solid-phase synthesis of dipeptides in low-water media was achieved using AOT ion-paired alpha-chymotrypsin solubilized in organic solvents. Multiple solvents and systematic variation of water activity, a(w), were used to examine the rate of coupling between N-alpha-benzyloxycarbonyl-L-phenylalanine methyl ester (Z-Phe-OMe) and leucine as a function of the reaction medium for both solid-phase and solution-phase reactions. In solution, the observed maximum reaction rate in a given solvent generally correlated with measures of hydrophobicity such as the log of the 1-octanol/water partitioning coefficient (log P) and the Hildebrand solubility parameter. The maximum rate for solution-phase synthesis (13 mmol/h g-enzyme) was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.30. For the synthesis of dipeptides from solid-phase leucine residues, the highest synthetic rates (0.14-1.3 mmol/h g-enzyme) were confined to solvent environments that fell inside abruptly defined regions of solvent parameter space (e.g., log P > 2.3 and normalized electron acceptance index <0.13). The maximum rate for solid-phase synthesis was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.14. In 90/10 and 70/30 (v/v) isooctane/tetrahydrofuran environments with a(w) set to 0.14, seven different N-protected dipeptides were synthesized on commercially available Tentagel support with yields of 74-98% in 24 h.     

Enzymatic synthesis of silicon-containing dipeptides with 3-trimethylsilylalanine

In this study, the enzymatic synthesis of silicon-containing dipeptides with a silicon-containing amino acid, 3-trimethylsilylalanine (TMS-Ala), was attempted in ethyl acetate, and the effects of TMS-Ala on thermolysin-catalyzed dipeptide synthesis are also discussed. Benzyloxycarbonyl(Z)-TMS-Ala was recognized by thermolysin as a better substrate than Z-Leu, and various silicon-containing dipeptides, Z-(TMS-Ala)-Xaa-OMe (Xaa = Leu, Ile, Phe, etc.), could be obtained. The acceleration of the reaction rate in the synthesis of Z-(TMS-Ala)-Leu-OMe compared with Z-Leu-Leu-OMe synthesis was explained by the higher hydrophobicity of the side-chain of TMS-Ala containing a trimethylsilyl group. On the other hand, TMS-Ala-OMe was not accepted as the amino component because of the bulkiness of the trimethylsilyl group. The enantioselectivity of thermolysin was very high. Z-d-TMS-Ala was not a substrate, while Z-l-TMS-Ala served as a good substrate. Received: 5 October 1998 / Received last revision: 4 December 1998 / Accepted: 26 December 1998     

Thermolysin as a catalyst in enzymatic synthesis of asparagine-containing peptides

The following di- and tripeptides were synthesized to study the potential utility of thermolysin as a catalyst in reactions of incorporation of N_α-acyl-l-asparagine into esters of amino acids and peptides: Boc-Asn-Ile-OBzl, Z-Asn-Ile-OBzl, Moz-Asn-Ile-OBzl, Boc-Asn-Leu-OBzl, Z-Asn-Leu-OBzl, Moz-Asn-Leu-OBzl, Boc-Asn-Phe-OBzl, Z-Asn-Phe-OBzl, Moz-Asn-Phe-OBzl, Z-Asn-Val-OBzl, Moz-Asn-Val-OBzl, Moz-Asn-Ile-Gly-OBzl, Moz-Asn-Ile-Ala-OBzl, Moz-Asn-Ile-Leu-OBzl, and Moz-Asn-Ile-Phe-OBzl. All of these peptides were obtained in pure form in good yield and characterized by thin-layer chromatography, melting point, elemental analysis, amino acid analysis, and proton magnetic resonance. The use of benzyloxycarbonyl (Z) and p-methoxybenzyloxycarbonyl (Moz) as protecting groups for asparagine gave excellent yields of the dipeptides. Relative to the dipeptides, the synthesis of the tripeptides was found to require lower enzyme concentrations and reaction times. Since the yields of the tripeptides failed to exhibit significant differences, it was not possible to establish the existence of a secondary specificity of thermolysin for the residue P′₂. A methodological study was also performed to determine the optimum conditions for synthesis of Boc-Asn-Ile-OBzl. This study consisted of an analysis of the influence of pH, enzyme concentration, volume and concentration of the solution of sodium acetate, relative proportions of carboxyl and amine components, temperature, and addition of organic solvent to the reaction medium.     

Biocatalytic properties of thermolysin immobilized on polyvinyl alcohol cryogel

Preparations with different contents of thermolysin were obtained by the immobilization of the enzyme on granulated polyvinyl alcohol cryogel. Their activity and stability in an aqueous medium and in mixtures of polar organic solvents of different composition were investigated. The catalytic properties of the preparations in reactions of peptide bond formation were studied, and the optimal amount of the biocatalyst, the concentrations of initial reagents, and the ratios of organic solvents and water necessary for effective enzymatic peptide synthesis catalyzed by immobilized thermolysin were determined. A series of peptides of the general formula Z-Ala-Ala-Xaa-pNA, where Xaa = Leu, Ile, Phe, Val, or Ala, were synthesized, and the immobilized enzyme was shown to retain substrate specificity in an organic medium.     

Kinetics of enzymatic solid-to-solid peptide synthesis: synthesis of Z-aspartame and control of acid-base conditions by using inorganic salts

Enzymatic peptide synthesis can be carried out efficiently in solid-to-solid reaction mixtures with 10% (w/w) water added to a mixture of substrates. The final reaction mass contains >/=80% (by weight) of product. This article deals with acid-base effects in such reaction mixtures and the consequences for the enzyme. In the Thermoase-catalyzed synthesis of Z-Asp-Phe-OMe, the reaction rate is strongly dependent on the amount of basic salts added to the system. The rate increases 20 times, as the KHCO(3) or K(2)CO(3) added is raised 2.25-fold from an amount equimolar to the Phe-OMe. HCL starting material. With further increases in KHCO(3) addition, the initial rate remains at the maximum, but with K(2)CO(3) it drops sharply. Addition of NaHCO(3) is less effective, but rates are faster if more water is used. With >1.5 equivalents of basic salt, the final yield of the reaction decreases. Similar effects are observed when thermolysin catalyzes the same reaction, or Z-Gln-Leu-NH(2) synthesis. These effects can be rationalized using a model estimating the pH of these systems, taking into account the possible formation of up to ten different solid phases.     

Enzymatic synthesis of the precursor of Leu-enkephalin in water-immiscible organic solvent systems.

The precursor of Leu-enkephalin, Z-L-TyrGlyGly-L-Phe-L-LeuOEt, was synthesized from amino acid derivatives with three proteinases without the protection of the side chain of L-Tyr. First, Z-GlyGlyOBut and Z-L-TyrGlyGlyOBut were synthesized in quite a high yield, 83% and 99%, in an aqueous/organic biphasic system by papain and alpha-chymotrypsin, respectively. Then, Z-L-Phe-L-LeuOEt was synthesized by thermolysin from Z-L-Phe and L-LeuOEt either in buffer or in a biphasic system; the yields were 95% and 100%, respectively. The synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-L-TyrGlyGly and L-Phe-L-LeuOEt was performed effectively by thermolysin immobilized on Amberlite XAD-7 in a buffer and in an aqueous/organic biphasic system, as well as in saturated ethyl acetate, while the yield was low in reactions by free thermolysin. In the reaction with the immobilized enzyme (IME) in saturated ethyl acetate, the maximum yield of the precursor of Leu-enkephalin was 68%. The reasons for effective synthesis with IME are: (1) higher concentration of L-Phe-L-LeuOEt inside support, which resulted in rising the rate of the synthesis reaction and protecting the competitive hydrolysis of Z-L-TyrGlyGly by thermolysin, (2) entrapment of the product inside the support where thermolysin could not act in the case of reaction in buffer, and (3) extraction of the product with the organic solvent in the case of reaction in a biphasic system or in saturated organic solvent.     

Synthesis of Aspartame Precursor Using Protease Suspended in Microaqueous Molten Amino Acids Mixture

Enzymatic synthesis of the aspartame precursor, N -(benzyloxycarbonyl)- l -aspartyl- l -phenylalanine methyl ester (Z-AspPheOMe) was performed with highly concentrated molten substrates. A mixture composed of molten N -(benzyloxycarbonyl)- l -aspartic acid (Z-Asp) and l -phenylalanine methyl ester (PheOMe) mixtures of 20 M could be prepared at 50°C. This Z-Asp/PheOMe mixture was applied to the enzymatic synthesis of Z-AspPheOMe using free thermolysin. Synthesis of Z-AspPheOMe was observed in the range of 100-150 &#119 l of NaOH solution (12.5 M) addition to a reaction mixture consisting of 1.0 mmol Z-Asp and 1.0 mmol PheOMe at 50°C. The enzymatic activity increased with increasing water addition, and reached a maximum at 100 &#119 l in addition to the reaction mixture of 1.0 mmol Z-Asp, 1.0 mmol PheOMe and 125 &#119 l of the NaOH solution. In this reaction system, the conversion at the reaction equilibrium was about 60%, the initial reaction rate calculated on the basis of the enzyme weight was 2.2 &#119 mol/g s, and the productivity calculated on the basis of the reaction mixture volume was 300 mol/m 3 h.     

Effect of electron withdrawing substituents on substrate hydrolysis by and inhibition of rat neutral endopeptidase 24.11 (enkephalinase) and thermolysin

A series of N-acylphenylalanylglycine dipeptides were synthesized and examined as substrates for neutral endopeptidase 24.11 (NEP) and thermolysin. Those N-acyl dipeptides containing an N-acyl group derived from an acid whose pKa is below 3.5 were considerably more reactive with both enzymes than those peptides containing an N-acyl group derived from an acid whose pKa is above 4. The data are interpreted to suggest that electron withdrawal at the scissile bond increases kappa cat for both NEP and thermolysin. The pH dependence for inhibition by the dipeptides Phe-Ala, Phe-Gly, and Leu-Ala showed binding dependent upon the basic form of an enzyme residue with a pKa of 7 for NEP and a pKa of 6 for thermolysin. In the case of thermolysin this pKa was decreased to 5.3 in the enzyme-inhibitor complex. When examined as alternate substrate inhibitors of NEP, N-acyl dipeptides showed three distinct profiles for the dependence of Ki on pH. With N-trifluoroacetyl-Phe-Gly as inhibitor, binding is dependent upon the basic form of an enzyme residue with a pKa value of 6.2. N-methoxyacetyl-Phe-Gly inhibition appears pH independent, while N-acetyl-Phe-Gly inhibition is dependent upon the acidic form of an enzyme residue with a pKa of approximately 7. All inhibitions of thermolysin by N-acyl dipeptides exhibit a dependence on the acidic form of an enzyme residue with a pKa of 5.3 to 5.8. These results suggest that with NEP, binding interactions at the active site involve one or more histidine residues while with thermolysin binding involves an active site glutamic acid residue.     

Enzymatic peptide synthesis in organic media: a comparative study of water-miscible and water-immiscible solvent systems.

Peptide synthesis was carried out in a variety of organic solvents with low contents of water. The enzyme was deposited on the support material, celite, from an aqueous buffer solution. After evaporation of the water the biocatalyst was suspended in the reaction mixtures. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH2 was used as a model reaction. Under the conditions used ([Z-Phe-OMe]0 less than or equal to 40 mM, [Leu-NH2]0/([Z-Phe-OMe]0 = 1.5) the reaction was first order with respect to Z-Phe-OMe. Tris buffer, pH 7.8, was the best buffer to use in the preparation of the biocatalyst. In water-miscible solvents the reaction rate increased with increasing water content, but the final yield of peptide decreased due to the competing hydrolysis of Z-Phe-OMe. Among the water-miscible solvents, acetonitrile was the most suitable, giving 91% yield with 4% (by vol.) water. In water-immiscible solvents the reaction rate and the product distribution were little affected by water additions in the range between 0% and 2% (vol. %) in excess of water saturation. The reaction rates correlated well with the log P values of the solvent. The highest yield (93%) was obtained in ethyl acetate; in this solvent the reaction was also fast. Under most reaction conditions used the reaction product was stable; secondary hydrolysis of the peptide formed was normally negligible. The method presented is a combination of kinetically controlled peptide synthesis (giving high reaction rates) and thermodynamically controlled peptide synthesis (giving stable reaction products).     

Reversal of enzymatic hydrolysis: Rate and extent of ester synthesis as catalyzed by chymotrypsin and subtilisin Carlsberg at low water concentrations

Immobilized chymotrypsin catalyzes esterification of N-acetyltyrosine in a medium containing high concentrations of alcohols. The hydrophilic support and inclusion of glycerol protect the enzyme activity and allow catalysis to proceed in the presence of only 10% (v/v) water. The same equilibrium concentration of ester is obtained whether reaction proceeds from ester or from free acid. Hates of ester synthesis and hydrolysis are similar when measured under the same conditions, but are at least one order of magnitude slower than optimal rates of hydrolysis. Subtilisin Carlsberg in the free, unmodified form catalyzes ester synthesis at even lower water concentrations; optimal rates are obtained at 5–15% H₂O. Hydrolytic enzymes can thus be utilized as catalysts of synthesis reactions in nonaqueous solvents where synthesis is thermodynamically favored over hydrolysis; in some cases this may provide economic and/or energetic advantages over conventional techniques.     

Enzymatic Peptide Synthesis in Organic Media. Synthesis of CCK-8 Dipeptide Fragments

The enzymatic synthesis of the seven consecutive dipeptide fragments of the cholecysto kinin C-terminal octapeptide (CCK-8) in organic media is reported. The influence of the reaction medium composition, the protease, and the structure of N-α and C-α protecting groups of both carboxyl and amino components was evaluated. α-Chymotrypsin, papain and thermolysin adsorbed on Celite were used as catalysts, under thermodynamic and kinetic control. The carboxamidomethyl, methyl and allyl ester derivatives of acetyl, benzyloxycarbonyl, tert-butyloxycarbonyl and fluoren-9-ylmethoxycarbonyl amino acids, were assayed as carboxy components. Amino acid amide and ester derivatives were employed as nucleophiles with a preference for the latter, since the dipeptide product obtained could be used directly, without any further chemical modification, as acyl-donor in subsequent coupling steps. All dipeptides selected were successfully synthesized, using the optimal combination of protecting groups, reaction media and enzyme different for each coupling reaction. The information gained with this study should be instrumental in designing an optimal strategy for the total enzymatic synthesis of cholecystokinin C-terminal octapeptide (CCK-8).     

Trichoderma reesei acetyl esterase catalyzes transesterification in water

Partially purified Trichoderma reesei RUT-C30 acetyl esterase preparation was found to catalyze acyl transfer reactions in organic solvents, mixtures of organic solvents with water and even in water. Using different acyl donors, the best results for acetyl transfer in water were obtained using vinyl acetate. As acetyl acceptors, a variety of hydroxyl bearing compounds in aqueous solutions were used. Degree of conversion and the number of newly formed acetates varied according to the acceptor used. Conversions over 50% were observed for the majority of several common monosaccharides, their methyl and deoxy derivatives and oligosaccharides. In several cases, the transesterification reaction exhibited strict regioselectivity, leading to only one acetyl derivative. Preparative potential of the transesterification in water was demonstrated by acetylation of methyl β- -glucopyranoside, 4-nitrophenyl β- -glucopyranoside and kojic acid, yielding 56.4% of methyl 3-O-acetyl β- -glucopyranoside, 70.2% of 4-nitrophenyl 3-O-acetyl β- -glucopyranoside and 30.9% of 7-O-acetyl-kojic acid as the only reaction products.

This enzymatically catalyzed transacetylation in water, which is applied to transformation of saccharides for the first time, opens a new area in chemoenzymatic synthesis. Its major advantages are simplicity, highly regioselective esterification of polar compounds, high yields, low enzyme consumption and elimination of the need to use toxic organic solvents.     

Assembly of proteolytically cleaved tubulin

Conditions have been found for limited proteolysis of purified tubulin, in which 70-90% of the molecules are cleaved at one or two sites. Thermolysin and chymotrypsin cleave the alpha and beta subunits, respectively, at single sites. Trypsin cleaves the alpha subunit at two sites. The chymotrypsin site and one of the trypsin sites are apparently inaccessible on assembled microtubules. The different samples of proteolyzed tubulin were all fully competent to assemble in a buffer containing 1 M sodium glutamate. In another buffer (50 mM morpholinoethanesulfonic acid, 3.4 M glycerol) tubulin digested by thermolysin assembled as well as native tubulin, but samples digested by chymotrypsin or trypsin would not assemble even at high protein concentrations.     

Enzymatic dipeptide synthesis by surfactant-coated alpha-chymotrypsin complexes in supercritical carbon dioxide

Enzymatic dipeptide synthesis by surfactant-coated alpha-chymotrypsin complexes was performed in supercritical CO(2) and liquid CO(2) at 308.2 and 333.2 K at pressures of 6.1 and 10.1 MPa. The enzymatic activity of coated alpha-chymotrypsin complexes for dipeptides synthesis at 10.1 MPa in supercritical CO(2) (SC-CO(2)) was higher than that in a liquid CO(2) and ethyl acetate solution at 6.1 MPa. The behavior of alpha-chymotrypsin in SC-CO(2) was similar to that in liquid ethyl acetate. And increasing the pressure and temperature increased the maximum conversion and the enzymatic reaction rate in SC-CO(2). Furthermore, the control of the water content in the reaction media had a dominant effect on the enzymatic activity. The maximum conversion for the dipeptide synthesis by the surfactant-coated alpha-chymotrypsin was obtained at 4% water content. The alpha-chymotrypsin complexes exhibited a higher enzymatic activity than native alpha-chymotrypsin in SC-CO(2). The nonionic surfactants l-glutamic acid dialkyl ester ribitol amide and sorbitan monostearate were more favored than the anionic surfactant sodium bis(2-ethylhexyl)sulfosuccinate.     

Biocatalytic properties of native and immobilized subtilisin 72 in aqueous-organic and low water media

Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8 M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200 mM.     

Native and modified subtilisin 72 as a catalyst of peptide synthesis in media with low water content

The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content > 80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N-acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p-nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70-98% in DMF-MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids.     

Comparison of methods for thermolysin-catalyzed peptide synthesis including a novel more active catalyst

This is a comparative study of the performance of thermolysin for enzymatic peptide synthesis by reversed hydrolysis in several different reaction systems. Z-Gln-Leu-NH(2) was synthesized in acetonitrile containing 5% water (with various catalyst preparation methods) as well as by the "solid-to-solid" and frozen aqueous methods. Reaction rates (values in nanomoles per minute per milligram) in acetonitrile depended significantly on the method of addition of enzyme: (a) direct suspension in the reaction mixture as freeze-dried powders gave 60 to 95; (b) addition as an aqueous solution, so that enzyme precipitates on mixing with acetonitrile, gave 230; (c) addition as an aqueous suspension gave a remarkable increase in reaction rates (up to 780); (d) immobilized enzymes (adsorbed at saturating loading on celite, silica, Amberlite XAD-7, or polypropylene, then dried by propanol rinsing) all gave <230. It is postulated that, starting with the enzyme already in the form of solid particles in aqueous buffer, there is a minimum chance of alteration of its optimal conformation during transfer to the organic medium. For solid-to-solid synthesis with 10% water content we found initial rates of 670 under optimized conditions. In frozen aqueous synthesis, rates were <10. Equilibrium yields were always around 60% in low water organic solvent, whereas they were found to >80% in the aqueous systems studied.     

Peptide synthesis catalyzed by polyethylene glycol-modified chymotrypsin in organic solvents

Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene-soluble modified enzyme readily catalyzed both aminolysis of N-benzoyl-L-tyrosine p-nitroanilide and synthesis of N-benzoyl-L-tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L- as well as D-amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivatives. The acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl-enzyme with subsequent synthesis of a peptide bond.