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.     

Peptide synthesis using proteases dissolved in organic solvents

Organic solvent-soluble -chymotrypsin (CT) and subtilisin Carlsberg (SC) are effective catalysts for peptide synthesis in homogeneous organic solutions. The soluble enzymes have values of k_cat/K_m for the reaction of N-Bz-L-Tyr-OEt with L-Leu-NH₂ to yield the dipeptide N-Bz-L-Tyr-L-Leu-NH₂ that are over 3 orders of magnitude higher than their suspended counterparts in isooctane (containing 30% (v/v) tetrahydrofuran (THF) to aid in substrate solubility). Both enzymes are substantially more active in hydrophobic organic solvents than hydrophilic solvents. Adding small concentrations of water (<0.2% and 1% (v/v) in isooctane-THF and ethyl acetate, respectively) results in up to a 150-fold activation of -chymotrypsin-catalyzed peptide synthesis. Importantly, added water does not promote hydrolysis in either isooctane-THF or ethyl acetate; thus, -chymotrypsin is highly selective toward peptide synthesis in the nearly anhydrous organic solutions. Unlike CT, the activation of subtilisin Carlsberg upon partial hydration of isooctane-THF or ethyl acetate was not significant and actually resulted in substantial hydrolysis. Using -chymotrypsin, a variety of tripeptides were produced from dipeptide amino acid esters. Reactivity of D-amino acid amides as acyl acceptors and partially unblocked amino acid acyl donors further expands the generality of the use of organic solvent-soluble enzymes as peptide synthesis catalysts.     

Unusual specificity of polyethylene glycol-modified thermolysin in peptide synthesis catalyzed in organic solvents

Summary Polyethylene glycol-modified thermolysin was found to efficiently catalyze peptide synthesis in organic solvents. As in aqueous media, the reaction occurred through a rapid equilibrium random bireactant mechanism. However, the substrate specificity of modified thermolysin was actually changed since hydrophilic as well as acidic amino acids were better carboxyl group donors than hydrophobic residues, contrary to what is observed in both the enzyme-catalyzed synthesis and hydrolysis of peptide bonds in water.     

Peptide synthesis catalyzed by subtilisin-72 in organic solvents

The solubility, stability, and activity of native subtilisin 72 and of its complex with SDS were comparatively studied in a number of polar organic solvents. Subtilisin was found to catalyze peptide bond formation when suspended in acetonitrile or solubilized as a complex with SDS in ethanol and isopropanol. Tripeptide Z-Ala-Ala-Leu-pNA, tetrapeptides A-Ala-Ala-P1-P1'-B, where A = Z or Abz; P1 = Leu, Phe, Met, Trp, Ile, Tyr, Phe(NO2), or Glu(OMe), P1' = Leu, Phe, Glu, Ala, Ile, Val, or Arg; B = NH2, pNA, or 2-(2,4-dinitrophenyl)aminoethylamine residue (Ded); pentapeptides Z-Ala-Ala-Leu-Ala-Ala-pNA and Z-Ala-Ala-Leu-Ala-Phe-pNA; and hexapeptide Abz-Val-Ala-Phe-Phe-Ala-Ala-Ded were synthesized using the SDS-subtilisin complex. The complex also efficiently catalyzed the oligomerization of tripeptide H-Phe-Ala-Leu-OCH3 in ethanol, which resulted in a 63:37 mixture of trioligomer and tetraoligomer. It was demonstrated that SDS-subtilisin is a much more efficient catalyst than the suspension of native enzyme.     

Solid phase synthesis of peptides with polyethylene glycol-modified protease in organic solvents

Summary Trypsin, chymotrypsin, papain, thermolysin and pepsin were modified with a polyethylene glycol derivative (PEG). Each PEG-protease became soluble and active in organic solvents and catalyzed peptide bond formation with their substrate specificity. Using five kinds of PEG-proteases, various kinds of tripeptides were synthesized by solid phase system in methanol or in dimethylformamide.Abbreviations Boc t-butyloxycarbonyl - Z benzyloxycarbonyl - Fmoc 9-fluorenylmethyloxycarbonyl - PAM 4-(oxymethyl)phenylacetoamidomethyl - HMP 4-(oxymethyl)phenoxymethyl.     

Ester synthesis catalyzed by polyethylene glycol-modified lipase in benzene

Lipoprotein lipase, which catalyzes hydrolysis of emulsified triglycerides or water-insoluble esters, was modified with 2,4-bis(o-methoxy-polyethylene glycol)-6-chloro-s-triazine(activated PEG2). The modified lipase, in which 55% of the total amino groups in the lipase molecule, was soluble in organic solvents such as benzene, toluene, chloroform and dioxane. The modified lipase could catalyze ester synthesis reaction in benzene. When very hydrophobic substrates of lauryl alcohol and stearic acid were used, the ester synthesis reaction proceeded efficiently in the transparent benzene solution with the maximum activity of approximate 5.0 mumoles/min/mg of protein. Ester exchange and aminolysis reactions were also conducted with the modified lipase in benzene.     

Glycosidases in organic solvents: II. Transgalactosylation catalysed by polyethylene glycol-modified β-galactosidase

β- -Galactoside galactohydrolase (E.C. 3.2.1.23) was chemically modified with 1,1′-carbonyldiimidazole-activated polyethylene glycols (MW 2,000, 8,000, and 20,000). The modified β-galactosidases had over 50% of amino groups coupled to polyethylene glycol but retained over 50% of the original activity. The hydrophobically modified enzymes were soluble in chlorinated organic solvents in which transferase activity has been demonstrated. The transferase activity, its dependency on water content, and the thermostability of all three modified enzymes were compared.     

Effect of immiscible organic solvents on activity/stability of native chymotrypsin and immobilized-stabilized derivatives

We have developed different activity/stability tests to evaluate the possibilities of fully dispersed chymotrypsin derivatives as industrial catalysts in biphasic systems. We have tested different immiscible organic solvents (log P ranged from 0.65 to 2.8) and used different enzyme derivatives (soluble chymotrypsin and one-point and multipoint covalent attached derivatives). Special emphasis has been given to the role of the "exact composition of the aqueous phase."High phosphate concentrations largely protect every hymotrypsin derivative from the distorting effects of dissolved solvent molecules. The effects on the activity and stability of soluble chymotrypsin due to saturating solvent concentrations in an aqueous solution, and the much more severe effects of contact with the phase interface in a stirred biphasic system, all show the opposite trend for the influence of solvent polarity to that generally observed for biocatalysts. For example, deleterious effects decline in the order chloroform, dichloromethane, ethyl acetate. On the contrary, with or without stirring, our stabilized chymotrypsin-agarose derivatives are much more stable against these water-immiscible solvents, and their relative effects follow the normal trend. From these integrated activity and stability tests we can conclude that fully dispersed immobilized-stabilized derivatives seem to be an interesting alternative to develop industrial biphasic processes catalyzed by chymotrypsin.     

Lipase catalyzed esterification of glycidol in nonaqueous solvents: solvent effects on enzymatic activity

We studied the effect of organic solvents on the kinetics of porcine pancreatic lipase (pp) for the resolution of racemic glycidol through esterification with butyric acid. We quantified ppl hydration by measuring water sorption isotherms for the enzyme in the solvents/mixtures tested. The determination of initial rates as a function of enzyme hydration revealed that the enzyme exhibits maximum apparent activity in the solvents/mixtures at the same water content (9% to 11% w/w) within the associated experimental error. The maximum initial rates are different in all the media and correlate well with the logarithm of the molar solubility of water in the media, higher initial rates being observed in the solvents/mixtures with lower water solubilities. The data for the mixtures indicate that ppl apparent activity responds to bulk property of the solvent. Measurements of enzyme particle sizes in five of the solvents, as function of enzyme hydration, revealed that mean particle sizes increased with enzyme hydration in all the solvents, differences between solvents being more pronounced at enzyme hydration levels close to 10%. At this hydration level, solvents having a higher water content lead to lower reaction rates; these are the solvents where the mean enzyme particle sizes are greater. Calculation of the observable modulus indicates there are no internal diffusion limitations. The observed correlation between changes in initial rates and changes in external surface area of the enzyme particles suggests that interfacial activation of ppl is only effective at the external surface of the particles. Data obtained for the mixtures indicate that ppl enantioselectivity depends on specific solvent-enzyme interactions. We make reference to ppl hydration and activity in supercritical carbon dioxide. (c) 1994 John Wiley & Sons, Inc.     

Biosynthesis of cholesterol linoleate by polyethylene glycol-modified cholesterol esterase in organic solvents.

Cholesterol esterase modified with polyethylene glycol was able to dissolve in some highly hydrophobic solvents such as benzene and toluene, and catalyze the synthesis of cholesterol linoleate with time dependency in the reverse of the usual reaction in aqueous solvents. Enzymatic cholesterol linoleate synthesis followed Michaelis-Menten kinetics which depended on the concentration of cholesterol and linoleic acid. When more than 20 mM of both substrates was used, the specific activity in this esterification was 200-250 nmol/min/mg protein. The apparent Km value for cholesterol and linoleic acid was 3.7 and 7.6 mM, respectively. The possibility of using such a modified enzyme for the synthesis of less stable cholesterol esters is discussed.     

Enhancement effect of polyethylene glycol on enzymatic synthesis of cephalexin

In an enzymatic synthesis of cephalexin (CEX) using an acylase from Xanthomonas citri, the effect of polyethylene glycol (PEG) on the synthetic reaction of 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D-alpha-phenyl-glycine methyl ester (PGM) to CEX was investigated. The addition of PEG (MW 300-20,000) increased the yield significantly. This yield enhancement effect tended to increase with the increasing molecular weight of PEG. Addition of PEG to the reaction system did not affect both the CEX and PGM hydrolytic reactions. The PEG added to the reaction medium used in these experiments did not depress the water activity significantly, and the product yield improvement could not be explained by the activity alone. The PEG stabilized the enzyme activity to some extent, but this stabilizing effect was only partially attributable to the yield enhancement of CEX. The enhancing effect of PEG on the synthetic yield increased with the increasing PEG molecular weight or the length of the poly(oxy-1,2-ethanediyl) chain, which increases the hydrophobicity of PEG. This finding consequently has led to the conclusion that the PEG structure renders the affinity between enzyme and 7-ADCA, which is a hydrophobic substrate. The microenvironmental hydrophobicity of PEG and its interaction with the hydrophobic substrate was found to be the main reason for the improvement of the CEX yield. In fact, the Michaelis-Menten kinetic constant for 7-ADCA, K(7-ADCA) in the presence of PEG was smaller than that in the control system (without PEG addition). (c) 1993 John Wiley & Sons, Inc.     

Lipase catalyzed esterification of glycidol in organic solvents

We studied the resolution of racemic glycidol through esterification with butyric acid catalyzed by porcine pancreatic lipase in organic media. A screening of seven solvents (log P values between 0.49 and 3.0, P being the n-octanol-water partition coefficient of the solvent) showed that neither log P nor the logarithm of the molar solubility of water in the solvent provides good correlations between enantioselectivity and the properties of the organic media. Chloroform was one of the best solvents as regards the enantiomeric purity (e. p.) of the ester produced. In this solvent, the optimum temperature for the reaction was determined to be 35 degrees C. The enzyme exhibited maximum activity at a water content of 13 +/- 2% (w/w). The enantiomeric purity obtained was 83 +/- 2% of (S)-glycidyl butyrate and did not depend on the alcohol concentration or the enzyme water content for values of these parameters up to 200 mM and 25% (w/w), respectively. The reaction was found to follow a BiBi mechanism. (c) 1993 John Wiley & Sons, Inc.     

Enzymatic properties of polyethylene glycol-modified cholesterol esterase in organic solvents.

The solvent dependency and substrate specificity of polyethylene glycol (PEG)-modified cholesterol esterase (CEH) catalyzing cholesterol ester synthesis in organic solvents were studied. When cholesterol and linoleic acid were used as the substrates, PEG-modified CEH synthesized cholesterol linoleate only in water-immiscible organic solvents. Among some solvents capable of solubilizing all of the reaction components (PEG-modified CEH, cholesterol, and linoleic acid), chloroform was most suitable for enzymatic cholesterol linoleate synthesis, and the synthetic activity for cholesterol linoleate decreased in the order chloroform, benzene, toluene, and cyclohexane. PEG-modified CEH synthesized various cholesterol esters with significant substrate specificity. The substrate specificity for cholesterol ester synthesis in benzene was analogous to that for cholesterol ester hydrolysis in aqueous solution.     

The equilibrium and kinetics of N-acetyl-tryptophan phenylethyl ester synthesis by agarose-chymotrypsin in organic media

The synthesis of N-acetyl tryptophan phenylethyl ester in organic media is catalyzed by suspended agarose beads with multipoint covalently attached chymotrypsin. A dilute aqueous phase is trapped within the gel beads and may be manipulated separately from the organic phase. The equilibrium position becomes more favorable as the solvent polarity decreases, with K(eq) increasing 38 times between 2-butanone and 1,1,1-trichloroethane. The more apolar solvents also give faster kinetics. Addition of cosolvents (up to 10% dimethylformamide or 20% acetonitrile) does not affect the rate but does substantially reduce the equilibrium yield, presumably also by making the organic phase more polar. With trichloroethane as solvent the enzyme appears to be kinetically saturated with 1M phenylethanol. Doubling this concentration also does not cause the expected increase in equilibrium conversion, probably again because K(eq) is reduced in the more polar organic phase. Increased temperature raises the reaction rate as expected but has little effect on the equilibrium. (c) 1992 John Wiley & Sons, Inc.     

The oxidation of chiral alcohols catalyzed by catalase in organic solvents

The catalytic properties of bovine liver catalase have been investigated in organic solvents. In tetrahydrofuran, dioxane, and acetone (all containing 1% to 3% of water), the enzyme breaks down tert-butyl hydroperoxide several fold faster than in pure water. Furthermore, the rate of catalase-catalyzed production of tert-butanol from tert-butyl hydroperoxide increases more than 400-fold upon transition from aqueous buffer to ethanol as the reaction medium. The mechanistic rationale for this striking effect is that in aqueous buffer the rate-limiting step of the enzymatic process involves the reduction of catalase's compound I by tert-butyl hydroperoxide. In ethanol, and additional step in the reaction scheme becomes available in which ethanol, greatly outcompeting the hydroperoxide, is oxidized by compound I regenerating the free enzyme. In solvents, such as acetonitrile or tetrahydrofuran, which themselves are not oxidizable by compound I, catalase catalyzes the oxidation of numerous primary and secondary alcohols with tert-butyl hydroperoxide to the corresponding aldehydes or ketones. The enzymatic oxidation of some chiral alcohols (2,3-butanediol, citronellol, and menthol) under these conditions occurs enantioselectively. Examination of the enantioselectivity for the oxidation of 2,3-butanediol in a series of organic solvents reveals a considerable solvent dependence. (c) 1995 John Wiley & Sons, Inc.     

Glucose-sensing carbon paste electrode containing polyethylene glycol-modified glucose oxidase

Polyethylene glycol-modified glucose oxidase (PEG-GOD) was prepared. Carbon paste (CP) containing PEG-GOD retained enzyme activity of 0·02 U cm⁻². Anodic and cathodic peak currents of modified GOD in CP matrix were observed on the differential pulse voltammograms at the potential of −0·36 and −0·36 V vs. Ag/AgCl, respectively. The addition of glucose to a test solution brought about an increase in the anodic current on the PEG-GOD-based electrode at the potential as low as 0·0 V vs. Ag/AgCl. The current increase was proportional to the concentration of glucose up to 50 mM.     

Retinyl esters synthesis by polyethylene glycol-modified lipase in benzene

Summary Using polyethylene glycol-modified lipase we have succeeded in synthesizing retinyl palmitate through ester exchange reaction between retinyl acetate and palmitic acid in a transparent benzene solution. The product had much lower peroxide value than the one obtained by a conventional organic synthesis. As much as 85% of a substrate, retinyl acetate, was converted to the product in a day at 25°C. We have also succeeded in ester synthesis of retinyl oleate with very small peroxide value, although both substrates have double bonds and tend to be oxidized easily.     

Peptide delivery systems

Efficient delivery of peptide drugs to the desired site is very important. There are anumber of barriers that may limit using peptides as potential drugs, some of theseobstacles include poor biomembrane permeability, enzymatic degradation and lowpH. To improve peptide drug efficiency a selective drug delivery system is required.Here we review some of the delivery systems available for peptides and we will alsobriefly discuss peptides that have been used as delivery systems.     

Peptide delivery systems

Summary Efficient delivery of peptide drugs to the desired site is very important. There are a number of barriers that may limit using peptides as potential drugs, some of these obstacles include poor biomembrane permeability, enzymatic degradation and low pH. To improve peptide drug efficiency a selective drug delivery system is required. Here we review some of the delivery systems available for peptides and we will also briefly discuss peptides that have been used as delivery systems.