Peptide and ester synthesis in organic solvents catalyzed by seryl proteases linked to alumina

Trypsin and alpha-chymotrypsin were immobilized to alumina-phosphocolamine complex, activated by glutaraldehyde. The immobilized enzymes show a great stability toward organic solvents miscible or immiscible with water. In the presence of a low concentration of water, the immobilized enzymes catalyzed transesterification reactions as well as peptide synthesis. The synthesized peptides were stable toward the immobilized enzymes.     

Thermodynamic parameters monitoring the equilibrium shift of enzyme-catalyzed hydrolysis/synthesis reactions in favor of synthesis in mixtures of water and organic solvent

The main strategy developed to shift the equilibrium state of a hydrolase-catalyzed hydrolysis/synthesis reaction consists in reducing water activity by addition of organic solvents in the reaction medium. We have used several mixtures of water and 1,4-butanediol, ranging from pure water to pure 1,4-butanediol, to study the hydrolysis/synthesis reaction of the N-Cbz-L-tryptophanyl-glycineamide dipeptide, catalyzed by alpha-chymotrypsin. In the presence of 1,4-butanediol, alpha-chymotrypsin also catalyzed the esterification reaction between this diol and N-Cbz-L-tryptophan; this ester hydrolysis/synthesis reaction has thus also been examined. The dipeptide and ester equilibrium concentrations increase when the water content of the reaction medium is decreased. Using our experimental data, we have determined the equilibrium constants of the hydrolysis/synthesis equilibria involving the nonionized forms of the protected amino acids, the estimated values of which are Ksp = 8 10(5) for the dipeptide and Kse = 78 for the ester respectively. They are true thermodynamic equilibrium constants, each related to a single, well-defined reaction equilibrium and with water activity being taken into account. If an organic solvent is added to the reaction medium these equilibria can be shifted towards synthesis by decreasing the water activity but also by modifying the ionization/neutralization equilibrium constant of the ionizable groups. These two effects depend both on the water content and on the nature of the organic solvent used, and, in particular, on its dielectric constant. Because of the importance of this parameter in our study, we discuss using it as an indicator to select an appropriate organic solvent to perform an enzyme-catalyzed synthesis.     

The role of hydration in enzyme activity and stability: 1. Water adsorption by alcohol dehydrogenase in a continuous gas phase reactor

The enzymatic synthesis of the tripeptide derivative Z-Gly-Trp-Met-OEt is reported. This tripeptide is a fragment of the cholecystokinin C-terminal octapeptide CCK-8. Studies on the alpha-chymotrypsin catalyzed coupling reaction between Z-Gly-Trp-R(1) and Met-R(2) have focused on low water content media, using deposited enzyme on inert supports such as Celite and polyamide. The effect of additives (polar organic solvents), the acyl-donor ester structure, the C-alpha protecting group of the nucleophile, enzyme loading, and substrate concentration were tested. The best reaction medium found was acetonitrile containing buffer (0.5%, v/v) and triethylamine (0.5%, v/v) using the enzyme deposited on Celite as catalyst (8 mg of alpha-chymotrypsin/g of Celite). A reaction yield of 81% was obtained with Z-Gly-Trp-OCam as acyl donor, at an initial concentration of 80 mM. The tripeptide synthesis was scaled up to the production of 2 g of pure tripeptide with an overall yield of 71%, including reaction and purification steps. (c) 1996 John Wiley & Sons, Inc.     

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents.

Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (+/-)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V(0), ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38 degrees C and 56 degrees C, respectively). No relationship between this property and V(0) was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V(0) was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of subtilisin activation by crown ethers, as recently suggested.     

Alteration of the reaction rate in the esterification of (R,S) ibuprofen by addition of crown ether or porphyrin

Summary The reaction yield of the esterification of (R,S) ibuprofen with n-propanol, catalyzed by physically adsorbed Candida antarctica lipase B on anionic resin, is improved by the addition of benzo-[18]-crown-6 or meso-tetraphenylporphyrin but reduced by the presence of metal-porphyrins. The interaction of benzo-[18]-crown-6 or meso-tetraphenyl porphyrin with the lid of pure lipase B on C. antarctica, would produce the activation of the lipase increasing the reaction rate but not the enantioselectivity.     

Synthesis of dermorphin-(1-4) derivatives catalyzed by proteases in organic solvents.

In order to extend the use of proteases to organic synthesis and seek the rules of enzymatic reactions in organic media, we focused on unnatural substrates for proteases to form amide bonds. In this paper, the study of unnatural substrates containing D-amino acid residue, which act as acyl acceptors as well as acyl donors for proteases in organic media, is reported. Dermorphin is a heptapeptide (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) with potent analgesic activity. The N-terminal tetrapeptide is the minimum sequence that retains dermorphin activity, and is selected as the model compound in our study. Two dermorphin-(1-4) derivatives, Boc-Tyr-D-Ala-Phe-Gly-N(2)H(2)Ph and Boc-Tyr-D-Ala-Phe-Gly-NH(2), which contained a d-amino acid residue, were synthesized by proteases in organic media for the first time. The synthesis of these two dermorphin-(1-4) derivatives could be catalyzed by subtilisin with Boc-Tyr-D-Ala-OCH(2)CF(3) as an acyl donor substrate in AcOEt. The synthesis of dermorphin-(1-2) derivative Boc-Tyr-D-Ala-N(2)H(2)Ph was catalyzed by alpha-chymotrypsin in different organic solvents and D-Ala-N(2)H(2)Ph was used as an acyl acceptor substrate. Factors influencing the above enzymatic reactions were systematically studied.     

Enzyme catalyzed biotransformations in aqueous two-phase systems with precipitated substrate and/or product

Biotransformations catalyzed by free and immobilized enzymes have been carried out in aqueous suspensions with up to 25% (w/w) precipitated substrate or product. For the kinetically controlled synthesis of N-Acetyl-Tyr-Arg-NH(2) with up to 0.8 M insoluble activated substrate N-Acetyl-TyrOEt catalyzed by alpha-chymotrypsin (EC3.4.21.1) the dipeptide yield was found to be >90%. This and the space-time yields were higher than those observed for one-phase aqueous systems and much higher than in systems where the insoluble substrate had been solubilized by addition of organic solvents. In the equilibrium controlled hydrolysis of 0.4 M D-phenylglycine-amide catalyzed by immobilized penicillin amidase (EC 3.5.1.11) the product precipitates. The enzyme immobilized in the support with the smallest pores could be reused without reduction in the rate due to precipitation in the pores. This decreases the number of immobilized enzyme molecules that can be used as biocatalysts. The latter was observed for supports with larger pores as the solubility decreases with increasing particle size. These results demonstrate that biotransformations with insoluble substrates or products using free or immobilized enzymes can be easily carried out in aqueous two-phase systems, without organic solvents, provided that the pore sizes of the supports are sufficiently small and that the rate of mass transfer from the precipitated substrate is large. The latter increases with decreasing particle size. (c) 1995 John Wiley & Sons, Inc.     

Synthesis of N-protected peptide alcohols catalyzed by subtilisin or alpha-chymotrypsin in organic solvents

A series of N-protected peptide alcohols were synthesized using amino alcohols with unprotected hydroxy groups as amino components by the catalysis of subtilisin or alpha-chymotrypsin in organic solvents. N-protected aromatic amino acid esters were more suitable as acyl donors for subtilisin. The influences of different N-protecting groups, organic solvents, and content of water on synthesis of N-protected peptide alcohols were systematically studied.     

Why do crown ethers activate enzymes in organic solvents?

One of the major drawbacks of enzymes in nonaqueous solvents is that their activity is often dramatically low compared to that in water. This limitation can be largely overcome by crown ether treatment of enzymes. In this paper, we describe a number of carefully designed new experiments that have improved the insights into the mechanisms that are operative in the crown ether activation of enzymes in organic solvents. The enhancement of enzyme activity upon addition of 18-crown-6 to the organic solvent can be reconciled with a mechanism in which macrocyclic interactions of 18-crown-6 with the enzyme play an important role. Macrocyclic interactions (e.g., complexation with lysine ammonium groups of the enzyme) can lead to a reduced formation of inter- and intramolecular salt bridges and, consequently, to lowering of the kinetic conformational barriers, enabling the enzyme to refold into thermodynamically stable, catalytically (more) active conformations. This assumption is supported by the observation that the crown-ether-enhanced enzyme activity is retained after removal of the crown by washing with a dry organic solvent. A much stronger crown ether activation is observed when 18-crown-6 is added prior to lyophilization, and this can be explained by a combination of two effects: the before-mentioned macrocyclic complexation effect, and a less specific, nonmacrocyclic, lyoprotecting effect. The magnitude of the total crown ether effect depends on the polarity and thermodynamic water activity of the solvent, the activation being highest in dry and apolar media, where kinetic conformational barriers are highest. By determination of the specific activity of crown-ether-lyophilized enzyme as a function of the enzyme concentration, the macrocyclic crown ether (linearly dependent on the enzyme concentration) and the nonmacrocyclic lyoprotection effect (not dependent on the enzyme concentration) could be separated. These measurements reveal that the contribution of the nonmacrocyclic effect is significantly larger than the macrocyclic refolding effect.     

Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18‐crown‐6 on lipase activity,enantioselectivity, and conformation

Lipase from Burkholderia cepacia (lipase BC) and lipase B from Candida antarctica (CALB) show an increase of the transesterification activity in toluene (up to 2.4- and 1.7-fold, respectively), when lyophilized with 18-crown-6. Nevertheless, the increase was observed only for low (less than 100) 18-crown-6/lipase molar ratio, while at higher ratios, the activity decreased for both enzymes to values lower than those obtained in the absence of the additive. In 1,4-dioxane, the activation is lower for lipase BC (1.7-fold) and for CALB (1.5-fold). Concerning enantioselectivity, tested in the kinetic resolution of 6-methyl-5-hepten-2-ol, only in the case of CALB, an effect of the additive (the E value varied from about 120 to 280) was observed. In water, 4% (w/w) of 18-crown-6 caused a loss of activity in the hydrolysis of p-nitrophenyl laurate of about 88 and 99.75%, compared to that observed in the absence of the crown ether for CALB and lipase BC, respectively. These data and the conformational analysis of both lipases, carried out by FT/IR spectroscopy indicate that the enzyme inactivation in water and in organic solvents at 18-crown-6/lipase molar ratios, higher than 100 might be due to conformational changes caused by the additive. Instead, at molar ratios lower than 100, 18-crown-6 might increase the activity - particularly, in toluene - thanks to the fact that in its presence, the enzyme has an hydrogen bonds pattern, more similar to that in water. This suggests that the additive would be able to provide the enzyme with more water.     

Gram-scale enzymatic synthesis of a peptide bond.

The synthesis reaction of the peptide, N-Cbz-L-tryptophanyl-glycineamide, catalyzed by alpha-chymotrypsin was performed in a 20% water/80%, 1,4-butanediol mixture. The synthesis yield reached 90.9% at the end of the reaction and 72.3% after purification. The effects on the yield of both pH and the ratio between total initial concentrations of glycineamide and N-Cbz-L-tryptophan are examined. The high yield, specificity, simplicity and reproducibility of this method make it complementary of the chemical methods.     

Enzymic properties of nitrated alpha-chymotrypsin and delta-chymotrypsin.

Chymotrypsinogen A and alpha-chymotrypsin are both nitrated at tyrosines 146 and 171 by reaction with tetranitromethane. This substitution was essentially without influence on the overall rate constant for hydrolyses of N-acetyl-L-tryptophan methyl ester and N-acetyl-L-tyrosine ethyl ester catalyzed by alpha-chymotrypsin and delta-chymotrypsin, prepared by fast tryptic activation of nitrated chymotrypsinogen. With both ester substrates Km was doubled for nitrated alpha-chymotrypsin. Nitrated alpha-chymotrypsin, nitrated delta-chymotrypsin and delta-chymotrypsin could all bind N-acetyl-L-tryptophan methyl ester at alkaline pH, in contrast to alpha-chymotrypsin. The dissociation constant, Kd, of the complex of alpha-chymotrypsin and basic pancreatic trypsin inhibitor was lowered ten-fold relative to the constant obtained with unmodified alpha-chymotrypsin. The nitrated delta-chymotrypsin and delta-chymotrypsin showed identical Kd values. The nitrated alpha-chymotrypsin is inactivated faster at pH 8.0 and 8.5 than alpha-chymotrypsin and apparently by a different mechanism.     

Alpha-chymotrypsin catalysis in imidazolium-based ionic liquids

The transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol catalyzed by alpha-chymotrypsin was examined in the ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]) and 1-octyl-3-methylimidazolium hexafluorophosphate ([omim][PF(6)]), and in combination with supercritical carbon dioxide (SC-CO(2)). The activity of alpha-chymotrypsin was studied to determine whether trends in solvent polarity, water activity, and enzyme support properties, observed with this enzyme in conventional organic solvents, hold for the novel environment provided by ionic liquids. alpha-Chymotrypsin freeze-dried with K(2)HPO(4), KCl, or poly(ethylene glycol) demonstrated no activity in [bmim][PF(6)] or [omim][PF(6)] at very low water concentrations, but moderate transesterification rates were observed with the ionic liquids containing 0.25% water (v/v) and higher. However, the physical complexation of the enzyme with poly(ethylene glycol) or KCl did not substantially stimulate activity in the ionic liquids, unlike that observed in hexane or isooctane. Activities were considerably higher in [omim][PF(6)] than [bmim][PF(6)]. Added water was not necessary for enzyme activity when ionic liquids were combined with SC-CO(2). These results indicate that [bmim][PF(6)] and [omim][PF(6)] provide a relatively polar environment, which can be modified with nonpolar SC-CO(2) to optimize enzyme activity.     

Conformation aspects of peptide interaction with proteolytic enzymes. Alpha-chymotrypsin catalyzed hydrolysis of the cyclopeptides containing leucyltyrosyl fragments]

The studies were made on the interaction of alpha-chymotrypsin with a series of cyclopeptides cyclo(-L-leucyl-L-tyrosyl-glycyln-), n=4, 6 and 8 (I, II and III respectively), and cyclo(-L-leucyl-L-tryosyl-beta-aminovalero-yl2-) (IV). Compounds I and IV are resistant to enzyme action whereas cyclopeptides II and III proved to be the substrates, their kinetic constants being Km=15.4 and 13.2 mM and kcat=0.54 and 9.53 sec-1 respectively. The binding capacity of cyclopeptides I-IV is evaluated by their competitive inhibition of alpha-chymotrypsin catalyzed hydrolysis of N-acetyl-L-tyrosine methyl ester.     

Molecular simulation of Tyr105 phosphorylated pyruvate kinase M2 to understand its structure and dynamics

Microstructure of dibenzo-18-crown-6 (DB18C6) and DB18C6/Li⁺ complex in different solvents (water, methanol, chloroform, and nitrobenzene) have been analyzed using radial distribution function (RDF), coordination number (CN), and orientation profiles, in order to identify the role of solvents on complexation of DB18C6 with Li⁺, using molecular dynamics (MD) simulations. In contrast to aqueous solution of LiCl, no clear solvation pattern is found around Li⁺ in the presence of DB18C6. The effect of DB18C6 has been visualized in terms of reduction in peak height and shift in peak positions of g_Li-Ow. The appearance of damped oscillations in velocity autocorrelation function (VACF) of complexed Li⁺ described the high frequency motion to a “rattling” of the ion in the cage of DB18C6. The solvent-complex interaction is found to be higher for water and methanol due to hydrogen bond (HB) interactions with DB18C6. However, the stability of DB18C6/Li⁺ complex is found to be almost similar for each solvent due to weak complex-solvent interactions. Further, Li⁺ complex of DB18C6 at the liquid/liquid interface of two immiscible solvents confirm the high interfacial activity of DB18C6 and DB18C6/Li⁺ complex. The complexed Li⁺ shows higher affinity for water than organic solvents; still they remain at the interface rather than migrating toward water due to higher surface tension of water as compared to organic solvents. These simulation results shed light on the role of counter-ions and spatial orientation of species in pure and hybrid solvents in the complexation of DB18C6 with Li⁺. Graphical Abstract

DB18C6/Li⁺ complex in pure solvents (water, methanol, chloroform, and nitrobenzene) and water/nitrobenzene interface     

Sol-gel immobilization of serine proteases for application in organic solvents

The serine proteases alpha-chymotrypsin, trypsin, and subtilisin Carlsberg were immobilized in a sol-gel matrix and the effects on the enzyme activity in organic media are evaluated. The percentage of immobilized enzyme is 90% in the case of alpha-chymotrypsin and the resulting specific enzyme activity in the transesterification of N-acetyl-L-phenylalanine ethyl ester with 1-propanol in cyclohexane is 43 times higher than that of a nonimmobilized lyophilized alpha-chymotrypsin. The activities of trypsin and subtilisin Carlsberg are enhanced with 437 and 31 times, respectively. The effect of immobilization on the enzyme activity is highest in hydrophobic solvents.     

A comparison of lipase-catalysed ester and lactone synthesis in low-water systems: analysis of optimum water activity.

We investigated the effects of the lyophilisation medium (enzyme plus buffer salt and additives) and of water activity (a(w)) on the catalytic properties of lipase from Chromobacterium viscosum (lipase CV) in organic solvents; catalysis of ester and lactone synthesis were compared and, despite the similarities of the reactive groups involved in these reactions, some interesting differences were observed. Including 2-[N-morpholino]ethanesulfonic acid (MES) buffer in the lyophilisation medium of lipase CV increased its catalytic activity in transesterification and lactonisation, although the buffer salt requirement for maximal activity differed between the two reactions. Sorbitol, glucose, lactose, 18-crown-6 (crown ether 18-C-6), beta-cyclodextrin and bovine serum albumin were employed as alternative additives in the transesterification reaction, but were not as effective as MES buffer. Salt hydrates were used to investigate the effect of a(w) on esterification and lactonisation reactions catalysed by lipase CV. The maximum rate of hexadecanolide synthesis in toluene occurred at a(w) = 0.48. The optimum a(w) for the transesterification reaction in heptane/alcohol mixtures depended on the alcohol substrate employed (1-heptanol, 2-heptanol, or 3-methyl-3-hexanol) but not on the acyl donor (p-NP acetate or caprylate). The optimum a(w) values for both reactions were unchanged when a common solvent system (toluene/1-heptanol) was employed, indicating that the dependence of enzyme activity on a(w) is an intrinsic property of the enzyme-catalysed reaction and not a function of the solvent or other additives.     

Investigation of the effect of freezing on protease-catalyzed peptide synthesis using cryoprotectants and frozen organic solvent

In order to investigate the effect of freezing on aqueous protease-catalyzed peptide synthesis systems, the influence of polyethylene glycols as cryoprotecting substances on alpha-chymotrypsin-catalyzed coupling of a N-protected acyl donor ester and various nucleophilic amino components was studied. Changes in S'-specificity of alpha-chymotrypsin in frozen aqueous systems were suppressed by polyethylene glycols even at concentrations below 1% (w/v). Furthermore, the influence of freeze-concentration in organic solvents on protease-catalyzed peptide synthesis was investigated for the first time. In frozen tert-butanol, alpha-chymotrypsin-catalyzed peptide synthesis took advantage from freeze-concentration, but in contrast to frozen aqueous systems, no changes in S'-specificity of the biocatalyst were observed. The results suggest that freeze-concentration is not the only cause of freezing-induced yield improvement in aqueous peptide synthesis systems, but interactions between enzyme and ice structures strongly contribute to the observed effects.     

Circular dichroism of host-guest complexes of achiral pyridino- and phenazino-18-crown-6 ligands with the enantiomers of chiral aralkyl ammonium salts

Circular dichroism (CD) spectroscopy was used for distinguishing different types of chiral interactions in host-guest complexes of achiral pyridino- and phenazino-18-crown-6 ligands with chiral aralkyl ammonium salts. The general feature of the CD spectra of many homochiral (e.g., (R,R)-host and (R)-guest) and heterochiral (e.g., (R,R)-host and (S)-guest) alpha-(1-naphthyl)ethylamine hydrogenperchlorate salt (NEA) complexes with chiral pyridino- and phenazino-18-crown-6 hosts is exciton interaction. The most interesting example is the coupling of the transitions of the chiral guest NEA with the energetically close transitions of the achiral phenazino-18-crown-6 host 6. The CD spectrum of the complex is predominated by exciton coupling between the (1)B(b) transition of the chiral guest and the (1)B(b) transition of the achiral host. The redshifted intense spectra of the complexes of (R)- or (S)-1-phenylethylamine hydrogenperchlorate salt (PEA) with the achiral diester-pyridino-18-crown-6 host 4 are indicative of merging the pi electron systems into one joint charge transfer chromophore. The appearance of weak bands with alternating sign in the spectrum of PEA complexes of the achiral "parent" pyridino-18-crown-6 host (1) indicates the presence of two or more conformers. The CD spectra of the complexes of achiral phenazino-18-crown-6 host 6 with PEA are also determined by pi-pi interaction. In addition to charge transfer bands, CD bands are also induced in the long-wavelength spectral region of the achiral host. The weak pi-pi interaction between the achiral phenazino-18-crown-6 host and methyl phenylglycinate hydrogenperchlorate (PGMA) or methyl phenylalaninate hydrogenperchlorate (PAMA) does not result in a definite spectral effect in the (1)L(a) region of the spectrum of the chiral guest, but its existence is proven by the weak CD bands induced in the long-wavelength spectral region of the achiral host.     

Protease-catalyzed synthetic reactions and immobilization-activation of the enzymes in hydrophilic organic solvents.

The catalytic feature of serine proteases for synthetic reactions in hydrophilic organic solvents and effects of immobilization by complexation with polysaccharides are described. Free alpha-chymotrypsin and subtilisin Carlsberg catalyze esterification, transesterification, and peptide synthesis in hydro-organic cosolvents with less than 10% water. Subtilisin BPN' is catalytically less active. The medium effects on the reaction kinetics and product yield were investigated in terms of the nature of solvent and water content in the reaction systems. The substrate- and stereo-specificities of the enzymes suggest that the enzymes maintain their native conformations in these low-water organic solvents. The catalytic activities of the proteases markedly increase by immobilization or complexation with polysaccharides, such as chitin or chitosan. The results of the rate measurements suggest that the primary role of the support materials is the activation of the enzymes and the increase in substrate concentration at reaction sites.