Breaking the low barrier hydrogen bond in a serine protease

The serine protease subtilisin BPN' is a useful catalyst for peptide synthesis when dissolved in high concentrations of a water-miscible organic co-solvent such as N,N-dimethylformamide (DMF). However, in 50% DMF, the k(cat) for amide hydrolysis is two orders of magnitude lower than in aqueous solution. Surprisingly, the k(cat) for ester hydrolysis is unchanged in 50% DMF. To explain this alteration in activity, the structure of subtilisin 8397+1 was determined in 20, 35, and 50% (v/v) DMF to 1.8 A resolution. In 50% DMF, the imidazole ring of His64, the central residue of the catalytic triad, has rotated approximately 180 degrees around the Cbeta-Cgamma bond. Two new water molecules in the active site stabilize the rotated conformation. This rotation places His64 in an unfavorable geometry to interact with the other members of the catalytic triad, Ser221 and Asp32. NMR experiments confirm that the characteristic resonance due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. These experiments provide a clear structural basis for the change in activity of serine proteases in organic co-solvents.     

Kinetic analysis of deactivation of immobilized alpha-chymotrypsin by water-miscible organic solvent in kyotorphin synthesis.

Two different immobilized chymotrypsin derivatives were used to synthesize kyotorphin, using N-benzoyl-L-tyrosine ethyl ester and L-arginine ethyl ester as substrates, in water-DMF media. The first was adsorbed onto Celite particles and the second was multipoint covalently attached into polyacrylamide gel. In all cases, the conversion of the carboxyl substrate was carried out in first-order reaction conditions. For the adsorbed enzyme, the reaction kinetics deviated from first-order likely due to a fast irreversible inactivation of enzyme during the reaction time even at low DMF concentration (15-20% v/v). The covalent attachment of enzyme resulted in elimination of irreversible activity loss by organic solvent up to 60% (v/v) of DMF. The catalytic activity of the covalent derivative was conserved as appropriate for performing a synthetic reaction up to 60% v/v of DMF (in comparison to 30% v/v for the adsorbed derivative), showing a clear improvement in its stability against reversible denaturation by this solvent. The selectivity of the synthetic reaction was slightly enhanced (from 40-50%) with the increase in DMF concentration to 80% v/v, but it was significantly improved (to 80%) when L-argininamide was used as nucleophile.     

Chemically stabilized trypsin used in dipeptide synthesis

Bovine pancreatic trypsin was treated with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester). Approximately 8 of 14 lysines per trypsin molecule were modified. This derivative (EG trypsin) was more stable than native between 30 degrees and 70 degrees C: T50 values were 59 degrees C and 46 degrees C, respective. EG trypsin's half-life of 25 min at 55 degrees C was fivefold greater than native's. EG trypsin had a decreased rate of autolysis and retained more activity in aqueous mixtures of 1,4-dioxan, dimethylformamide, dimethylsulfoxide, and acetonitrile. EG trypsin had lower Km values for both amide and ester substrates; its kcat values for two amides (benzoyl-L-arginine p-nitroanilide and benzyloxycarbonyl glycyl-glycyl-arginyl-7-amino-4-methyl coumarin) increased, whereas its kcat value for an ester (thiobenzoyl benzoyloxycarbonyl-L-lysinate) decreased slightly. The specific activity (kcat/Km) of EG trypsin was increased for both amide and ester substrates. EG trypsin gave higher yields and reaction rates than native in kinetically controlled synthesis of benzoyl argininyl-leucinamide in acetonitrile and in t-butanol. Highest peptide yields occurred with EG trypsin in 95% acetonitrile, where 90% of the substrate was converted to product. No peptide synthesis occurred in 95% DMF with either form of trypsin.     

Oxidation of tissue polysaccharides by periodic acid in dimethyl sulfoxide and its anhydrous and aqueous mixtures.

Periodic acid (1% w/v) solvated by anhydrous dimethyl sulfoxide (DMSO) readily induced a strong Schiff reaction in a variety of structures containing polysaccharides, but not glycogen. With the increasing amounts of water added to DMSO, glycogen was also oxidized, while the selective localization of other polysaccharides remained unimpaired. Periodate, solvated in the anhydrous acetic acid-DMSO mixture, rapidly induced concomitant oxidation of nucin and glycogen-containing structures. Sodium bisulfite addition derivatives of carbonyls, induced by periodate oxidation in DMSO, were stained meta- and orthochromatically with toluidine blue at controlled pH. Certain metachromatic tissue components were strongly birefringent in polarized light in contrast to the identical structures oxidized by aqueous periodate. Marked differences in staining reactions elicited in identical structures by periodate in DMSO as compared with aqueous periodate suggest that DMSO-periodate method considerably enhances the range of histochemical oxidations by periodate.     

Characteristics of chymotrypsin modified with water-soluble acylating reagents and its peptide synthesis ability in aqueous organic media.

Several kinds of modified chymotrypsin were prepared with water-soluble acylating reagents, and their characteristics after hydrolyzing with unmodified chymotrypsin in aqueous-N,N'-dimethylformamide (DMF) media were compared. It was found that chymotrypsin (Csin), of which a 20% amino group was modified with a benzyloxycarbonyl group (Z(20)Csin), had more favorable characteristics than unmodified chymotrypsin with regard to hydrolytic activity in an aqueous DMF media. We also investigated the Z(20)Csin-catalyzed peptide synthesis in two different solution systems. In the one-layer system containing water and DMF, Z(20)Csin catalyzed the peptide bond formation in a higher yield than that by unmodifide chymotrypsin and enabled a synthetic reaction in even an 80% (v/v) DMF media, in which the hydrolytic reaction could not be carried out. Z(20)Csin catalyzed the condensation between some N-acyl amino acids or peptide derivatives and amino acids in 90% ethylacetate, 90% hexane or 50% benzene. This latter method employs a two-layer system, and the modified enzyme may be able to reduce the number of synthetic steps when preparing acyl peptides.     

A spectrophotometric method for the microdetermination of periodate

1. A method is described for measuring the concentration of periodate over the range 0.2-20mum by adding 1,2-di-(p-dimethylaminophenyl)ethane-1,2-diol to a sample solution. Periodate cleaves this compound to from two molecules of p-dimethylaminobenzaldehyde, the extinction of which is then read at 352mmu. 2. The method has been used to follow the course of periodate oxidations of serine methyl ester, ribonuclease A and ribonuclease S-protein. Addition of the reagent stops further periodate reaction by reducing the remaining periodate to iodate. 3. The presence of protein does not interfere with the assay.     

Structures of Gibberellins A33 and A34 from Immature Seeds of Calonyction aculeatum

Several kinds of modified chymotrypsin were prepared with water-soluble acylating reagents, and their characteristics after hydrolyzing with unmodified chymotrypsin in aqueous-N,N’ -dimethylformamide (DMF) media were compared. It was found that chymotrypsin (Csin), of which a 20% amino group was modified with a benzyloxycarbonyl group (Z(20)Csin), had more favorable characteristics than unmodified chymotrypsin with regard to hydrolytic activity in an aqueous DMF media. We also investigated the Z(20)Csin-catalyzed peptide synthesis in two different solution systems. In the one-layer system containing water and DMF, Z(20)Csin catalyzed the peptide bond formation in a higher yield than that by unmodifide chymotrypsin and enabled a synthetic reaction in even an 80% (v/v) DMF media, in which the hydrolytic reaction could not be carried out. Z(20)Csin catalyzed the condensation between some N-acyl amino acids or peptide derivatives and amino acids in 90% ethylacetate, 90% hexane or 50% benzene. This latter method employs a two-layer system, and the modified enzyme may be able to reduce the number of synthetic steps when preparing acyl peptides.     

Enzymatic transesterification of purine nucleoside having a low solubility in organic medium

Enzymatic transesterification of guanosine having low solubility against organic solvent was examined. For the transesterification between guanosine and divinyl adipate catalyzed by alkaline protease from Bacillus (Bioprase), DMSO was added to DMF to increase the solublility of the nucleoside, and the conversion rate of guanosine to the vinyl guanosine ester was less than 30%. To overcome the reversible inactivation of enzyme by hydrophilic organic solvents, the reaction was carried out with 10% (v/v) water. The transesterification reaction was effectively catalyzed in DMF/DMSO in the presence of water and the conversion rate increased ca. 70% after 7 d reaction. The result shows that the water effect of Bioprase would be a useful method for the synthesis of low solublility nucleoside esters.     

The synthesis of a cobalt(II) tetracarboxyphthalocyanine- deoxyribooligonucleotide conjugate as a reagent for the directed DNA modification

The cobalt(II) tetracarboxyphthalocyanine-deoxyribonucleotide pd(TCTTCCCA) conjugate was synthesized. The phthalocyanine N-succinimide ester prepared from phthalocyanine using DCC was mixed in DMF with an aqueous solution of the oligonucleotide bearing a 1,3-diaminopropane linker at the 5'-phosphate. The resulting conjugate was tested in the intraduplex reaction with target 14-mer and 22-mer oligonucleotides containing conjugate-complementary sequences. In the presence of O2 and a thiol (2-mercaptoethanol or DTT) as a coupled reducer or H2O2, sequence-specific DNA modification was observed that caused the cleavage of the target upon treatment with piperidine.     

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.     

The role of methionine and α-chymotrypsin-catalysed reactions

1. The reaction of α-chymotrypsin with sodium periodate at pH5·0 has been investigated. The enzyme consumes 2 moles of periodate/mole, and there is a concomitant fall in enzymic activity (with respect to l-tyrosine ethyl ester) to 55% of that of the native enzyme. After 3hr. no further change is observed in periodate uptake or in catalytic activity. 2. The oxidized enzyme is a homogeneous preparation of partially active chymotrypsin. 3. In the oxidized enzyme, one of the two methionine residues in the molecule has been converted into its sulphoxide. It is this reaction only that is responsible for the loss of activity. 4. The rate constants for the enzyme-catalysed acylation and deacylation reactions are unaltered by oxidation of the enzyme, both for a non-specific substrate (p-nitrophenyl acetate), and for three specific substrates: N-acetyl-l-tryptophan ethyl ester, N-acetyl-l-tryptophanamide and N-acetyl-l-valine ethyl ester. 5. The K_m values for the aromatic substrates with the oxidized enzyme are twice those with the native enzyme. No change in Michaelis constant is seen for the non-aromatic substrate N-acetyl-l-valine ethyl ester. 6. The evidence points to the oxidized methionine residue in the modified enzyme being situated in the locus of the active site at which aromatic (or bulky) side chains of the substrates are bound.     

Enzymatic synthesis of arginine-based cationic surfactants

A novel enzymatic approach for the synthesis of arginine N-alkyl amide and ester derivatives is reported. Papain deposited onto solid support materials was used as catalyst for the amide and ester bond formation between Z-Arg-OMe and various long-chain alkyl amines and alcohols (H2N-Cn2, HO-Cn; n = 8-16) in organic media. Changes in enzymatic activity and product yield were studied for the following variables: organic solvent, aqueous buffer content, support for the enzyme deposition, presence of additives, enzyme loading, substrate concentration, and reaction temperature. The best yields (81-89%) of arginine N-alkyl amide derivatives were obtained at 25 degrees C in acetonitrile with an aqueous buffer content ranging from 0 to 1% (v/v) depending on the substrate concentration. The synthesis of arginine alkyl ester derivatives was carried out in solvent-free systems at 50 or 65 degrees C depending on the fatty alcohol chain length. In this case, product yields ranging from 86 to 89% were obtained with a molar ratio Z-Arg-OMe/fatty alcohol of 0.01. Papain deposited onto polyamide gave, in all cases, both the highest enzymatic activities and yields. Under the best reaction conditions the syntheses were scaled up to the production of 2 g of final product. The overall yields, which include reaction, Nalpha-benzyloxycarbonyl group (Z) deprotection and purification, varied from 53 to 77% of pure (99.9% by HPLC) product.     

Periodate oxidation of sodium alginate in water and in ethanol-water mixture: a comparative study

Periodate oxidation of sodium alginate in aqueous solution as well as a dispersion in 1:1 ethanol-water was examined. The oxidation proceeded smoothly in both media, and the kinetics of oxidation was surprisingly similar. Polymer cleavage was observed in both media, but it was extensive in ethanol-water. The weight-average molar mass (Mw) of the oxidized product obtained from aqueous solution showed a gradual decrease with increase in the periodate concentration, whereas, except for very high periodate equivalent, the change in Mw was not reflected with increase in concentration of periodate in ethanol-water. The oxidized alginate obtained from the ethanol-water mixture was found to be more efficient in crosslinking proteins such as gelatin, leading to hydrogels. Oxidation of a dispersion has the advantage of generating large quantities of the oxidized alginate in higher yield with one reaction using less solvent.     

Entrapment of l-tryptophan producing Escherichia coli in different matrices: activity of immobilized cells

Cells of Escherichia coli induced for l-tryptophan synthase [l-serine hydro-lyase (adding indole-glycerol-phosphate), EC 4.2.1.20] have been assayed in DMF and DMSO aqueous solvents as reaction medium. Up to 20% DMF/water, cells retained 90% of their tryptophan synthase activity. Concentrations of 20 mM indole, which did not inhibit this reactivity, could be reached with 5% DMF/water. Four matrices were compared for cell immobilization: polyacrylamide, foam particles of bovine seum albumin, alginate and κ-carrageenan. The best activity was retained with the latter matrix, and the preparations thus obtained allowed high productivity of l-tryptophan. Various systems of production of l-tryptophan with κ-carrageenan and DMF/water were studied.     

Kinetic model studies on the chemical ligation of oligonucleotides via hydrazone formation

We report on the suitability of hydrazone formation for activator-free ligation of oligonucleotides. 5'-Acyl hydrazides were synthesized using a previously described phosphoramidite modifier, whereas 3'-hydrazides resulted from a hydrazinolysis of an ester group serving as a linker to the solid support. Aromatic aldehydes could be directly introduced on the 5'-terminus via the respective phosphoramidates. Aliphatic aldehydes were generated by periodate cleavage of the corresponding 3'- and 5'-modified diol precursors. Ligation of a 3'-hydrazide-modified oligonucleotide with oligonucleotides bearing an aromatic aldehyde in 5'-position showed a fast reaction kinetics (k(1) about 10(-1) M(-1)s(-1)) [corrected] and irreversible hydrazone formation. The ligation of a 5'-hydrazide-modified oligonucleotide and a 3'-ribobisaldehyde appeared to proceed reversibly at the beginning, but became irreversible with increasing reaction time. Hydrazide-modified oligonucleotides were found to be somewhat unstable in aqueous solutions.     

Synthesis and evaluation of 2-amino-6-fluoro-9-(2-hydroxyethoxymethyl)purine esters as potential prodrugs of acyclovir

2-Amino-6-fluoro-9-(2-hydroxyethoxymethyl)purine (2) and its ester derivatives 4a–d were synthesized as potential prodrugs of acyclovir, and were evaluated for their oral acyclovir bioavailability in rats and in vivo antiviral efficacy in HSV-1-infected mice. Treatment of 2-amino-6-chloro-9-(2-hydroxyethoxymethyl)purine (3) with trimethylamine in THF/DMF (4:1) followed by a reaction of the resulting trimethylammonium chloride salt 5 with KF in DMF gave 2 in 78% yield. Esterification of 2 with an appropriate acid anhydride (Ac₂O, (EtCO)₂O, (n-PrCO)₂O, or (i-PrCO)₂O) in DMF in the presence of a catalytic amount of DMAP at room temperature produced the esters 4a–d in 90–98% yields. Of the prodrugs tested in rats, the isobutyrate 4d achieved the highest mean urinary recovery of acyclovir (51%) that is 5.7-fold higher than that of acyclovir (9%) and comparable to that of valacyclovir (50%). The prodrug 4d protected dose-dependently the mortality of HSV-1-infected mice, and the group treated with 4d at a dose of 400 mg/kg showed the longest mean survival day (14.6 ± 3.1 days) (mean ± S.D.).     

Enantioselective reduction of C=N double bond by chromium(II) complexes of natural amino acids

Asymmetric reduction of oximes was performed by chromium(II) complexes of natural amino acids in aqueous phase or in H(2)O/DMF (1:1) solvent. Medium-to-quantitative chemoselectivity (54% to >95%) and low-to-medium enantioselectivity (5-50% ee) were found.     

Conversion of 2,5-dimethylfuran to 2-hydroxy-5-hydroperoxy-2,5-dimethyldihydrofuran,a true 1O2-derived reaction in aqueous 1O2 generating systems

It has been studied whether 2,5-dimethylfuran (DMF) is a specific ¹O₂ trapping agent in aqueous system. The exposure of DMF to aqueous ¹O₂ generating system (Rose Bengal photooxygenation system) gave 2-hydroxy-5-hydroperoxy-2,5-dimethyldihydrofuran (a hydrated form of endoperoxide, ¹O₂-derived reaction product) and cis-diacetylethylene (cis-DAE), while the bromine-catalyzed autoxidation of DMF afforded only trans-DAE. In Fenton system (·OH generating system) DMF was converted in the main to cis-DAE, but not to the hydrated form of endoperoxide. The exposure of DMF to acetaldehyde-xanthine oxidase system failed to detect the hydrated form of endoperoxide, but chiefly yielded a non-specific oxidation product, cis-DAE.     

Effects of methanol on the synthesis of aspartame precursor catalysed byPseudomonas aeruginosa elastase

Summary Pseudomonas elastase was found to be efficient in catalysing the reaction betweenN-benzyloxycarbonyl-L-aspartic acid and L-phenylalanine methyl ester producing the aspartame precursor in aqueous and aqueous methanolic solutions. 25% (v/v) methanol was most favourable for the synthesis where about 100% increase in yield was obtained compared to that in aqueous solution.Abbreviations N-cbz-L-Asp N-benzyloxycarbonyl-L-Aspartic acid - L-Phe-OMe L-phenyl alanine methyl ester - HPLC high performance liquid chromatography. All the % of methanol is a volume % in water unless otherwise specified     

Use of the excluded protecting group (EPG) method for peptide synthesis.

The excluded protecting group (EPG) method has been used for the solution synthesis of several peptides including Merrifield's Model Tetrapeptide, linear antamanide and an analogue of magainin-1, [Ala(19), Asn(22)]magainin-1. In the approach reported, the C-terminal amino acid is esterified to the 2-position of cholestane as the [2s,3s]iodohydrin ester and the penultimate amino acid added to the aminoacyl-steroid as the Fmoc-pentafluorophenyl-ester. The Fmoc group is removed with Et(2)NH/DMF ( approximately 15% v/v) and, after evaporation to approximately 10 mL, the solution chromatographed on Sephadex LH-20 in DMF. The dipeptidyl-steroid elutes as the free amine well separated from other reaction mixture components. Fractions containing the dipeptide, as determined by counting and TLC, are pooled and reacted with the next Fmoc-amino acid-pentafluorophenyl ester in the sequence. Repetition of the deprotection/purification/reaction cycle yields the fully protected peptide.On completion of the synthesis, the cholestane iodohydrin ester is selectively removed by treatment with Zn degrees /AcOH to yield the peptide with intact alpha-amino and side chain protecting groups. Global deprotection is achieved with HF. All intermediates from the syntheses reported were characterized. The magainin analogue was shown to have full biologic activity. The Fmoc iodohydrin esters of 16 of the 20 proteogenic amino acids have been prepared and characterized for use as the C-terminal amino acids in other EPG syntheses.