Protease-catalyzed synthesis of a precursor dipeptide, Z-Asp-Val-NH2 of thymopentin, in organic solvents

The protease-catalyzed, kinetically controlled synthesis of a precursor dipeptide, Z-Asp-Val-NH(2) of thymopentin (TP-5), in organic solvents was studied. Z-Asp-OMe and Val-NH(2) were used as the acyl donor and the nucleophile, respectively. An industrial alkaline protease alcalase was used to catalyze the synthesis of the target dipeptide in water-organic cosolvent systems. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including organic solvents, water content, temperature, pH, and reaction time on the yield of Z-Asp-Val-NH(2). The optimum conditions using alcalase as the catalyst are pH 10.0, 35 degrees C, in acetonitrile/Na(2)CO(3)-NaHCO(3) buffer system (9:1, V/V), reaction time 5 h, with a yield of 63%. The dipeptide product was confirmed by LC- MS.     

Nucleophile specificity of subtilisin in an organic solvent with low water content: investigation via acyl transfer reactions

Nucleophile specificity of subtilisin (subtilopeptidase A) was studied via acyl transfer reactions in acetonitrile containing piperidine and 10 vol% of water. Ac-Tyr-OEt was used as acyl donor and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) were used as nucleophiles. The nucleophilic efficiencies of these peptides were characterized by the values of the apparent partition constants, p(app), determined from the HPLC analysis of the reactions. The order of preference for the P'(1) position was estimated to be: Gly > hydrophilic, positively charged > hydrophobic, aromatic > negatively charged > Leu > Pro side chain. For the P'(2) position the order of preference was: Gly > hydrophilic, charged > hydrophobic, aromatic > Pro side chain. The values of p(app) for Gly-Gly-Xaa tripeptides cover a range of only two orders of magnitude, with lower nucleophile efficiency for those with hydrophobic amino acid residues in the P'(3) position. The dipeptide with Pro in P'(1) did not react at all, but a tripeptide having Pro in P'(3) was a very good nucleophile. The negatively charged amino acid residues in the P'(1) position result in very weak nucleophilic behavior, whereas the peptides with Asp or Glu in P'(2) and P'(3) are well accepted. Generally, peptides of the Gly-Xaa or Gly-Gly-Xaa series were better nucleophiles than peptides of the Xaa-Gly series. The length of the peptide chain or amidation of alpha-carboxyl function had no influence on nucleophilic behavior. No significant difference in nucleophile specificity between subtilopeptidase A and nagarse was observed. (c) 1996 John Wiley & Sons, Inc.     

Synthesis of a precursor dipeptide of thymopentin in organic solvents by an enzymatic method

The protease-catalyzed, kinetically controlled synthesis of a precursor dipeptide of thymopentin(TP-5), Z-Arg-Lys-NH2 in organic solvents was studied. Z-Arg-OMe was used as the acyl donor and Lys-NH2 was used as the nucleophile. An industrial alkaline protease alcalase and trypsin were used to catalyze the synthesis of the target dipeptide in water-organic cosolvent systems. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including organic solvents, water content, temperature, pH, and reaction time on the yield of Z-Arg-Lys-NH2. The optimum conditions using alcalase as the catalyst are pH 10.0, 35 degrees C, in acetonitrile/DMF/Na2CO3-NaHCO3 buffer system (80:10:10, V/V), 6 h, with the dipeptide yield of 71.1%. Compared with alcalase, the optimum conditions for trypsin are pH 8.0, 35 degrees C, in ethanol/Tris-HCl buffer system (80:20, V/V), 4 h, with the dipeptide yield of 76.1%.     

Alcalase-catalyzed, kinetically controlled synthesis of a precursor dipeptide of RGDS in organic solvents

The protease-catalyzed, kinetically controlled synthesis of a precursor dipeptide of RGDS, Z-Asp-Ser-NH2 in organic solvents was studied. Alcalase, an industrial alkaline protease, was used to catalyze the synthesis of the target dipeptide in water-organic cosolvents systems with Z-Asp-OMe as the acyl donor and Ser-NH2 as the nucleophile. Acetonitrile was selected as the organic solvent from acetonitrile, ethanol, methanol, DMF, DMSO, ethyl acetate, 2-methyl-2-propanol, and chloroform tested under the experimental conditions. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including water content, temperature, pH, and reaction time on the Z-Asp-Ser-NH2 yields. The optimum conditions are pH 10.0, 35 degrees C, in acetonitrile/Na2CO3-NaHCO3 buffer system (85:15, v/v), 6 h, with a dipeptide yield of 75.5%.     

Protease-Catalyzed Synthesis of a Precursor Dipeptide,Z-Asp-Val-NH2 of Thymopentin,in Organic Solvents

Abstract

The protease-catalyzed, kinetically controlled synthesis of a precursor dipeptide, Z-Asp-Val-NH₂ of thymopentin (TP-5), in organic solvents was studied. Z-Asp-OMe and Val-NH₂ were used as the acyl donor and the nucleophile, respectively. An industrial alkaline protease alcalase was used to catalyze the synthesis of the target dipeptide in water-organic cosolvent systems. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including organic solvents, water content, temperature, pH, and reaction time on the yield of Z-Asp-Val-NH₂. The optimum conditions using alcalase as the catalyst are pH 10.0, 35°C, in acetonitrile/Na₂CO₃-NaHCO₃ buffer system (9:1, V/V), reaction time 5 h, with a yield of 63%. The dipeptde product was confirmed by LC- MS.     

Effect of mass-transfer limitations on the selectivity of immobilized alpha-chymotrypsin biocatalysts prepared for use in organic medium

The selectivity of preparations of alpha-chymotrypsin immobilized on Celite or polyamide and carrying out syntheses of di- and tripeptides in acetonitrile medium were studied. The study concerns the effect of mass-transfer limitations on three different kinds of selectivity: acyl donor, stereo- and nucleophile selectivities, defined respectively as the ratio of initial rates with different acyl donors; the enantioselectivity factor (E); and the ratio of initial rates of peptide synthesis and hydrolysis of the acyl donor. Strong mass-transfer limitations caused by increased enzyme loading had a very strong effect on acyl donor selectivity, with reductions of up to 79%, and on stereoselectivity, with reductions of up to 77% in relation to optimum values, both on Celite. Nucleophile selectivity was not affected as strongly by mass-transfer limitations. Using a small molecule (AlaNH(2)) as nucleophile, the onset of these limitations caused only minor reductions in selectivity, while when using a larger nucleophilic species (AlaPheNH(2)) it was reduced by up to 60% when increasing enzyme loading on Celite from 2 to 100 mg/g. The different way these kinds of selectivity are affected by the onset of mass-transfer limitations can be explained by a combination of different aspects: the kinetic behavior of the enzyme toward nucleophile and acyl donor concentrations, the relative concentrations of reagents used in the reaction media, and their relative diffusion coefficients. In short, higher concentrations of nucleophile than acyl donor are generally used, and the nucleophile most often used in the experiments hereby described (AlaNH(2)) diffuses faster than the acyl donors employed. These factors combined are expected to give rise to concentration gradients inside porous biocatalyst particles higher for acyl donor than for nucleophile under conditions of mass-transfer limitations. This explains why acyl donor selectivity and stereoselectivity are much more influenced by mass transfer limitations than nucleophile selectivity.     

Trypsin-catalyzed kinetically controlled synthesis of a precursor dipeptide of thymopentin in organic solvents, using a free amino acid as nucleophile

Trypsin-catalyzed, kinetically controlled synthesis of a precursor, dipeptide of thymopentin (TP-5), Bz-Arg-Lys-OH (or-OEt) in organic solvents was studied. Bz-Arg-OEt was used as the acyl donor and Lys-OH and Lys-OEt were used as the nucleophiles. Ethanol was selected as the organic solvent from ethanol, methanol, acetonitrile, and ethyl acetate tested under the experimental conditions. As expected, Lys-OEt is not a suitable nucleophile in trypsin-catalyzed reaction, due to its competition with the protective Arg-OEt as acyl donor for the active site of trypsin, while Lys-OH does not have this problem. The optimal reaction condition for the synthesis of Bz-Arg-Lys-OH was set up as 20% Tris-HCl buffer, pH 8.0, 35 degrees C for 6 h with the yield of 52.5%, or for 18-24 h with the yield of about 60%.     

Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A

A proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac-Phe-OEt and Bz-Ala-OMe. Tur-NH(2), Trp-NH(2), Leu-pNA, and Val-pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz-ala-Tyr-NH(2) was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz-ala-OMe, 25 mM Tyr-NH(2), 70 degrees C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz-Ala-OMe and 615 mM Tyr-NH(2) at 40 degrees C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; -betaNA and -pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield. (c) 1994 John Wiley & Sons, Inc.     

Effects of subzero temperatures on the kinetics of protease catalyzed dipeptide synthesis in organic media

A depeptide synthesis was drastically influenced by the reaction temperature, in the range from -30 degrees to 25 degrees C. This article shows the kinetic reasons of this effect. alpha-Chymotrypsin was immobilized on celite and used in four different water-miscible solvents containing small amounts of water-miscible solvents containing small amounts of water. The reaction studied was the aminolysis of N-acetyl-L-phenylalanine ethyl ester (Ac-PheOEt) with L-alaninamide (Ala-NH(2)) and water for the acylenzyme complex, the nucleophile was favoured by low reaction temperatures. This effect (quantified as p-values) was observed in all four solvents, and it was greatest in acetonitrile and tetrahydrofuran. The esterase and amidase activities of the enzyme were studies using AcPheOEt and N-acetyl-L-phenylalanyl-L-ananinamide (AcPheAla-NH(2)) as substrates. The Michaelis-Menten parameters, K(m,app) and V(max), were determined for ester hydrolysis and dipeptide hydrolysis. Both K(m,app) and V(max) tended to increase with increasing temperature. Secondary hydrolysis was reduced at subzero temperatures because ester hydrolysis was favoured in relation to depeptide hydrolysis. Depeptide synthesis was thus favored by low temperatures in two ways: first, in the competition between the nucleophile and water for the acyl enzyme; and, second, in the competition between the ester substrate and the peptide substrate for the free enzyme. As a result, in acetonitrile containing 10% water, the maximal yield was 99% at -20%C compared with 84% at 25 degrees C. (c) 1995 John Wiley & Sons, Inc.     

Synthesis of a Precursor Dipeptide of Thymopentin in Organic Solvents by an Enzymatic Method

Abstract

The protease‐catalyzed, kinetically controlled synthesis of a precursor dipeptide of thymopentin(TP‐5), Z‐Arg‐Lys‐NH₂ in organic solvents was studied. Z‐Arg‐OMe was used as the acyl donor and Lys‐NH₂ was used as the nucleophile. An industrial alkaline protease alcalase and trypsin were used to catalyze the synthesis of the target dipeptide in water‐organic cosolvent systems. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including organic solvents, water content, temperature, pH, and reaction time on the yield of Z‐Arg‐Lys‐NH₂. The optimum conditions using alcalase as the catalyst are pH 10.0, 35°C, in acetonitrile/DMF/Na₂CO₃‐NaHCO₃ buffer system (80∶10∶10, V/V), 6 h, with the dipeptide yield of 71.1%. Compared with alcalase, the optimum conditions for trypsin are pH 8.0, 35°C, in ethanol/Tris‐HCl buffer system (80∶20, V/V), 4 h, with the dipeptide yield of 76.1%.     

A novel and efficient enzymatic method for the production of peptides from unprotected starting materials

We report herein the development of a novel and efficient enzymatic method for the production of oligopeptides. This newly discovered method is a simple, cost-effective process, using unprotected amino acids as substrates in an aqueous solution and producing peptides in high yield. The target of our initial screen was l-alanyl-L-glutamine, a dipeptide of significant industrial interest by virtue of its widespread use in infusion therapy. By means of the screening of microorganisms that can catalyze the peptide-forming reaction producing l-alanyl-L-glutamine from L-alanine methylester (acyl donor) and L-glutamine (nucleophile), we discovered that Empedobacter brevis ATCC 14234 produced l-alanyl-L-glutamine most efficiently. The newly found enzyme purified from E. brevis ATCC 14234 facilitates significantly high production yields of l-alanyl-L-glutamine from L-alanine methylester and L-glutamine in an aqueous solution--more than 80% yield based on L-alanine methylester. In addition, this enzyme has wide substrate specificity--both for acyl donors and nucleophiles--and can catalyze peptide-forming reactions not only to produce various dipeptides from the corresponding amino acid esters and amino acids but also to produce various oligopeptides from the corresponding amino acid esters and peptides.     

Optimization of Protease-Catalyzed Acyl Transfer Reactions. Determination of the Partition Constant Characterizing Nucleophile Efficiency

The partitioning of the acyl-enzyme between aminolysis by an added nucleophile and hydrolysis plays a key-role in protease-catalyzed acyl transfer reactions. It can be characterized by the partition constant, which is equal to the nucleophile concentration for which aminolysis and hydrolysis proceed at the same velocity. We describe a method for calculation of the partition constant from the product ratio which is based on the integrated rate equation. Therefore, it can be applied to reactions performed under synthesis-like conditions, i.e. a high degree of nucleophile consumption during the reaction. In principle, the dependence of the partition constant on nucleophile concentration can be determined from a single reaction. V8-protease-catalyzed acyl transfer reactions using Z-Glu-OMe as acyl donor and amino acid amides as nucleophiles were investigated as an application of the method. The central role of the partition constant in optimization of preparative protease-catalyzed acyl transfer reactions is discussed.     

Acyl group transfer by proteases forming an acylenzyme intermediate: kinetic model analysis (including hydrolysis of acylenzyme- nucleophile complex)

The quantitative analysis of peptide synthesis via transfer of the acyl moiety from the activated donor (S) to the nucleophile (N), catalysed by proteases forming an acylenzyme intermediate, has been continued. The new kinetic model takes into account the hydrolysis of an acylenzyme-nucleophile complex (EAN). The intensity of the hydrolysis is characterized by parameter gamma equal to the ratio of the rate constant of EAN hydrolysis and the rate constant of peptide formation. The ability of the EAN complex to hydrolyse leads to a decrease in the apparent nucleophile reactivity (beta) of the aminocomponent. As a result, the maximal fractional conversions of S and N to the peptide decrease, and the apparent nucleophile reactivity becomes dependent on the nucleophile concentration. The pattern of parameter gamma influence on maximal fractional conversions depends on which component is in an excess. It is with the donor excess that hydrolysis of the EAN complex affects the peptide yield dramatically. Analytical expressions for the estimation of maximal product concentration were obtained and their accuracy evaluated.     

Optimization of Protease-Catalyzed Acyl Transfer Reactions. Determination of the Partition Constant Characterizing Nucleophile Efficiency

The partitioning of the acyl-enzyme between aminolysis by an added nucleophile and hydrolysis plays a key-role in protease-catalyzed acyl transfer reactions. It can be characterized by the partition constant, which is equal to the nucleophile concentration for which aminolysis and hydrolysis proceed at the same velocity. We describe a method for calculation of the partition constant from the product ratio which is based on the integrated rate equation. Therefore, it can be applied to reactions performed under synthesis-like conditions, i.e. a high degree of nucleophile consumption during the reaction. In principle, the dependence of the partition constant on nucleophile concentration can be determined from a single reaction. V8-protease-catalyzed acyl transfer reactions using Z-Glu-OMe as acyl donor and amino acid amides as nucleophiles were investigated as an application of the method. The central role of the partition constant in optimization of preparative protease-catalyzed acyl transfer reactions is discussed.     

Enzymatic condensation of cholecystokinin CCK-8 (4-6) and CCK-8 (7-8) peptide fragments in organic media

The kinetically controlled condensation reaction of Z-Gly-Trp-Met-OR(1) (R(1): Et, Al, Cam) and H-Asp-(OR(2))-Phe-NH(2) (R(2): H, Bu(t)) catalyzed by alpha-chymotrypsin deposited onto polyamide in organic media was studied. The effect of the drying process of the enzyme-support preparation, substrate concentrations, reaction medium, acyl donor, and nucleophile structure on both enzymatic activity and pentapeptide yield was investigated. The immobilized preparation directly equilibrated at a(w) = 0.113, gave higher enzymatic activities than dried with vacuum first, and then equilibrated at a(w) = 0.113. The addition of triethylamine to the reaction medium increased dramatically the enzymatic activity. However, the pentapeptide yield was affected neither by the drying procedure nor by the addition of triethylamine. The donor ester Z-Gly-Trp-Met-OAl gave initial reaction rates 2.6 times higher than the conventional ethyl ester derivative but rendered similar yields. The best results were obtained using Z-Gly-Trp-Met-OCam as acyl-donor ester; 80% yield and initial reaction rates 4 times higher than the ethyl ester derivative. In all cases, acetonitrile containing Tris-HCl 50 mM pH 9 buffer (0.5% v/v) and triethylamine (0.5% v/v) was found to be the best reaction system. Under these conditions, it was possible to use the nucleophile H-Asp-Phe-NH(2) with beta-unprotected aspartic acid residue. In this case, 50% yield was obtained, but economic considerations could lead to select it as nucleophile. Finally, the fragment condensation reaction was carried out at gram scale, obtaining a 39% yield which included the reaction, removal of protecting groups and purification steps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 456-463, 1997.     

Lipase catalysed acylation of hydroxylamine and hydrazine derivatives

The lipase catalysed acylation of hydroxylamine-and hydrazine as well as their derivatives by octanoic acid is very efficient. Cross-linked crystals of Candida rugosa lipase (ChiroCLEC-CR) mediated the conversion of racemic ibuprofen into (S)-ibuproxam. A number of lipases also catalysed the condensation of hydrazine with an excess of octanoic acid giving N,N′-dioctanoylhydrazine. The hydrazide of 2-(4-isobutylphenyl)propanoic acid (ibuprofen), prepared by non-enzymatic reaction of ibuprofen methyl ester with hydrazine, acted as nucleophile towards several lipases that do not accept ibuprofen derivatives as acyl donor.     

Solid-phase acyl donor as a substrate pool in kinetically controlled protease-catalysed peptide synthesis

Recently we have demonstrated the advantage of solid- phase substrate pools mainly in equilibrium controlled protease-catalysed peptide syntheses. The extension of this approach to protease-catalysed acyl transfer reactions will be presented. The model reaction was systematically investigated according to both the influence of solid phases present in the system on enzyme activity as well as nucleophile concentration on peptide yield. The key parameter for obtaining high peptide yield via acyl transfer is the ratio between aminolysis and hydrolysis. We combined high nucleophile concentrations with solid-phase acyl donor pools. This approach enabled us to supply ester substrate and nucleophile in equimolar amounts in a high-density media without the addition of any organic solvent. Several multi-functional di- to tetrapeptides were obtained in moderate to high yields. ©1997 European Peptide Society and John Wiley & Sons, Ltd.     

Kinetic mechanism of direct transfer of Escherichia coli SSB tetramers between single-stranded DNA molecules

The kinetic mechanism of transfer of the homotetrameric Escherichia coli SSB protein between ssDNA molecules was studied using stopped-flow experiments. Dissociation of SSB from the donor ssDNA was monitored after addition of a large excess of unlabeled acceptor ssDNA by using either SSB tryptophan fluorescence or the fluorescence of a ssDNA labeled with an extrinsic fluorophore [fluorescein (F) or Cy3]. The dominant pathway for SSB dissociation occurs by a "direct transfer" mechanism in which an intermediate composed of two DNA molecules bound to one SSB tetramer forms transiently prior to the release of the acceptor DNA. When an initial 1:1 SSB-ssDNA complex is formed with (dT)(70) in the fully wrapped (SSB)(65) mode so that all four SSB subunits are bound to (dT)(70), the formation of the ternary intermediate complex occurs slowly with an apparent bimolecular rate constant, k(2,app), ranging from 1.2 x 10(3) M(-1) s(-1) (0.2 M NaCl) to approximately 5.1 x 10(3) M(-1) s(-1) (0.4 M NaBr), and this rate limits the overall rate of the transfer reaction (pH 8.1, 25 degrees C). These rate constants are approximately 7 x 10(5)- and approximately 7 x 10(4)-fold lower, respectively, than those measured for binding of the same ssDNA to an unligated SSB tetramer to form a singly ligated complex. However, when an initial SSB-ssDNA complex is formed with (dT)(35) so that only two SSB subunits interact with the DNA in an (SSB)(35) complex, the formation of the ternary intermediate occurs much faster with a k(2,app) ranging from >6.3 x 10(7) M(-1) s(-1) (0.2 M NaCl) to 2.6 x 10(7) M(-1) s(-1) (0.4 M NaBr). For these experiments, the rate of dissociation of the donor ssDNA determines the overall rate of the transfer reaction. Hence, an SSB tetramer can be transferred from one ssDNA molecule to another without proceeding through a free protein intermediate, and the rate of transfer is determined by the availability of free DNA binding sites within the initial SSB-ssDNA donor complex. Such a mechanism may be used to recycle SSB tetramers between old and newly formed ssDNA regions during lagging strand DNA replication.     

Effect of acyl donor chain length and sugar/acyl donor molar ratio on enzymatic synthesis of fatty acid fructose esters

Lipase-catalyzed synthesis of fatty acid sugar esters through direct esterification was performed in 2-methyl 2-butanol as solvent. Fructose and saturated fatty acids were used as substrates and the reaction was catalyzed by immobilized Candida antarctica lipase. The effect of the initial fructose/acyl donor molar ratio and the carbon-chain length of the acyl donor as well as their reciprocal interactions on the reaction performance were investigated. For this purpose, an experimental design taking into account variations of the molar ratio (from 1:1 to 1:5) and the carbon-chain length of the fatty acid (from C8 to C18) was employed. Statistical analysis of the data indicated that the two factors as well as their interactions had significant effects on the sugar esters synthesis. The obtained results showed that whatever the molar ratio used, the highest concentration (73 g l⁻¹), fructose and fatty acid conversion yields (100% and 80%, respectively) and initial reaction rate (40 g l⁻¹ h⁻¹) were reached when using the C18 fatty acid as acyl donor. Low molar ratios gave the best fatty acid conversion yields and initial reaction rates, whereas the best total sugar ester concentrations and fructose conversion yields were obtained for high molar ratios.