Bond Synthesis Research Articles

Oligoglyceric acid synthesis by autocondensation of glyceroyl thioester.

The autocondensation of the glyceroyl thioester S-glyceroyl-ethanethiol yielded oligoglyceric acid. The rates of autocondensation and hydrolysis of the thioester increased from pH 6.5 to pH 7.5 in 2,6-lutidine and imidazole buffers. Autocondensation and hydrolysis were much more rapid to imidazole buffers than in 2,6-lutidine buffers of the same pH. The efficiency of ester bond synthesis was about 20% for 40 mM S-glyceroyl-ethanethiol in 2,6-lutidine and imidazole buffers near neutral pH. The size and yield of the oligoglyceric acid products increased when the concentration of the thioester was increased. The relationship of these results to prebiotic polymer synthesis is discussed.

Substrate- and stereo-specificities in clostripain-catalyzed peptide synthesis

Clostripain was used for peptide bond synthesis with 18 carbobenzoxy-amino acid methyl esters as substrate and PRO-NH2 as nucleophile. Dipeptide bond synthesis was observed only for Cbz-L-ARG-OMe (72%) and Cbz-L-LYS-OMe (34%). With the exception of D-PRO-NH2, nucleophile stereospecificity is not restricted to the L isomer. D- and L-forms of PHE and TRP amides gave the same product yields.

Effect of glycerol on thermolysin-catalyzed peptide bond synthesis

The effect of glycerol on the hydrolytic activity of thermolysin (EC 3.4.24.4) has been compared with the effect on the condensation of N-benzyloxycarbonyl- l-aspartic acid with l-phenylalanine methyl ester to form N-benzyloxycarbonyl- l-aspartyl- l-phenylalanine methyl ester (Z · Asp · Phe · OMe), the precursor to the sweet-tasting compound l-aspartyl- l-phenylalanine methyl ester. Hydrolytic activity was measured by the degradation of azocasein and furylacryloyl- l-glycyl- l-leucinamide. Increasing concentrations of glycerol reversibly inhibited the hydrolytic activity of the enzyme toward both substrates. The inclusion of glycerol in the synthetic medium facilitated the production of Z · Asp · Phe · OMe in a water-soluble system but reduced the initial rate of peptide synthesis. Glycerol stabilized thermolysin against thermal denaturation.

A Computer model of a polyribonucleotide structure which may have functioned as a catalytic adaptor for peptide bond synthesis

A Computer model of a polyribonucleotide structure which may have functioned as a catalytic adaptor for peptide bond synthesis

ChemInform Abstract: Proteases as Catalytic Agents in Peptide Synthetic Chemistry: Shifting the Extent of Peptide Bond Synthesis from a "Quantite Negligeable" to a "Quantite Considerable"

Under ordinary conditions the equilibrium point in protease-catalyzed reactions is near to complete hydrolysis. Therefore, proteases are commonly known for their proteolytic rather than for their proteosynthetic activities. Nevertheless, the proteases have proved to be excellent catalysts in preparative peptide synthetic chemistry. A brief review is given of the historical development of protease-catalyzed peptide synthesis. The theoretical aspects of peptide bond formation are described and particular emphasis is given to techniques for favoring the synthesis of the desired peptide linkages. The applicability of these techniques is exemplified with selected syntheses and semisyntheses. The advantages as well as the problems associated with the enzymatic method are evaluated. A critical assessment is given of the present state of the art and the perspectives of the enzymatic approach to peptide synthetic chemistry.

The effect of inhibitors on thermolysin-catalysed peptide bond synthesis

The effect of inhibitors on thermolysin-catalysed peptide bond synthesis

Peptide bond synthesis by clostridiopeptidase B

Clostridiopeptidase B was used for peptide bond synthesis with Cbz-ARG-OMe as substrate and 17 amino acid amides as nucleophiles. Synthetic yields ranging from 75 to 98% were obtained for most of the amides.

Erratum to: Proteases as catalytic agents in peptide synthetic chemistry: Shifting the extent of peptide bond synthesis from a “quantité negligeable” to a “quantité considérable”

Under ordinary conditions the equilibrium point in protease-catalyzed reactions is near to complete hydrolysis. Therefore, proteases are commonly known for their proteolytic rather than for their proteosynthetic activities. Nevertheless, the proteases have proved to be excellent catalysts in preparative peptide synthetic chemistry. A brief review is given of the historical development of protease-catalyzed peptide synthesis. The theoretical aspects of peptide bond formation are described and particular emphasis is given to techniques for favoring the synthesis of the desired peptide linkages. The applicability of these techniques is exemplified with selected syntheses and semisyntheses. The advantages as well as the problems associated with the enzymatic method are evaluated. A critical assessment is given of the present state of the art and the perspectives of the enzymatic approach to peptide synthetic chemistry.

ChemInform Abstract: PEPSIN AND THERMOLYSIN AS CATALYSTS OF PEPTIDE BOND SYNTHESIS

The early studies on proteinase-catalyzed peptide bond synthesis are summarized as background for a report on some recent work in our laboratory on pepsin and thermolysin. The primary and secondary specificity of these two enzymes have been found to be suitable for the preparative synthesis of oligopeptides by the condensation of two hydrophobicl-amino acid residues. Kinetic data are presented showing that the rate of entry of each of the two components in a thermolysin-catalyzed condensation reaction depends on the nature of the other component, and that this effect is related to differences in the transition-state conformation of the enzyme-reactant complexes.

Proteases as catalytic agents in peptide synthetic chemistry: Shifting the extent of peptide bond synthesis from a “quantité négligeable” to a “quantité considérable”

Under ordinary conditions the equilibrium point in protease-catalyzed reactions is near to complete hydrolysis. Therefore, proteases are commonly known for their proteolytic rather than for their proteosynthetic activities. Nevertheless, the proteases have proved to be excellent catalysts in preparative peptide synthetic chemistry. A brief review is given of the historical development of protease-catalyzed peptide synthesis. The theoretical aspects of peptide bond formation are described and particular emphasis is given to techniques for favoring the synthesis of the desired peptide linkages. The applicability of these techniques is exemplified with selected syntheses and semisyntheses. The advantages as well as the problems associated with the enzymatic method are evaluated. A critical assessment is given of the present state of the art and the perspectives of the enzymatic approach to peptide synthetic chemistry.

Kinetics of trypsin catalysis in the industrial conversion of porcine insulin to human insulin.

The kinetics of coupling and transpeptidation reactions catalysed by trypsin were studied in media with low water contents to see whether the usual Michaelis-Menten kinetics applied under the unusual conditions used in peptide bond synthesis, to obtain information about the magnitude of rate constants and activation energies, and to gain insight into the mechanism of catalysis. Porcine insulin, des-(AlaB30)-porcine insulin, human insulin-ThrB30-OMe and human insulin-ThrB30(But)-OBut were used as substrates. Two threonine esters (Thr-OMe and Thr(But)-OBut) were used for transpeptidation and coupling. The reactions progressed according to first-order kinetics until about 35% conversion, and the experimental data were adequately explained by Michaelis-Menten kinetics. The rates of the coupling and transpeptidation reactions in media with low water contents were orders of magnitude below the rates of peptide bond hydrolysis by trypsin in water. It was not possible to approach saturation of the enzyme with substrate so determination of Km was impossible, but for each substrate a value larger than 0.1 M was estimated from the Lineweaver-Burk plot. The rate of release of alanine from porcine insulin depended on the type of threonine ester present; for example, Thr-OMe inhibited the reaction. Coupling occurred faster than transpeptidation. However, in the medium used, the activation energies for the two reactions were similar (about 50 kJ/mol), so the difference in reaction rates is probably due to different transmission coefficients in the activated transition states. Computer simulations enabled us to obtain quantitative descriptions of the reaction progress curves from fitted rate constants.

Uses of SiN bonds in synthesis: Easy routes to functional protected primary amines from propargylic silylamines

The readily obtained N,N-bis(trimethylsilyl)-propargylic amines are shown to be useful precursors of various functional protected primary amines. It readily gives rise to N,N-bis(trimethylsilyl)dienamines, 2-aza-1,3,5-hexatrienes, α-allenic amines, substituted allylamines and lactams.

Oligonucleotides complementary to a promoter over the region -8...+2 as transcription primers for E. coli RNA polymerase.

Primer-dependent transcription by E. coli RNA polymerase on T7 promoter A2 has been studied. Synthetic deoxyribonucleotides complementary to the promoter over the region -8...+2 were taken as primers. A ribonucleoside residue was present at the 3'-end of some of these oligonucleotides. The octanucleotide complementary to the region -8...-1 appeared to be an active primer. Oligonucleotides having lengths from 3 to 6 nucleotide residues complementary to the promoter over the region -4...+2 also exhibited primer activity. The latter was some 5-10 times greater in the case of oligonucleotides having a ribonucleoside residue at the 3'-end. Oligonucleotides which on complementary binding do not reach the center of phosphodiester bond synthesis, as well as the decanucleotides (-8...+2) and octanucleotides (-6...+2) of both the ribo- and deoxyribo-series were inactive as primers.

Acyl silicates and acyl aluminates as activated intermediates in peptide formation on clays.

Glycine reacts with heating on dried clays and other minerals to give peptides in much better yield than in the absence of mineral. This reaction was proposed to occur by way of an activated intermediate such as an acyl silicate or acyl aluminate (i.e., the anhydride of a carboxylic acid with Si-OH or Al-OH), analogous to acyl phosphates involved in several biochemical reactions including peptide bond synthesis. We confirmed the proposed mechanism by trapping the intermediate, as well as by direct spectroscopic observation of a related intermediate. The reaction of amino acids on periodically dried mineral surfaces represents a widespread, geologically realistic setting for prebiotic peptide formation via in situ activation.

Pepsin and thermolysin as catalysts of peptide bond synthesis

The early studies on proteinase-catalyzed peptide bond synthesis are summarized as background for a report on some recent work in our laboratory on pepsin and thermolysin. The primary and secondary specificity of these two enzymes have been found to be suitable for the preparative synthesis of oligopeptides by the condensation of two hydrophobicl-amino acid residues. Kinetic data are presented showing that the rate of entry of each of the two components in a thermolysin-catalyzed condensation reaction depends on the nature of the other component, and that this effect is related to differences in the transition-state conformation of the enzyme-reactant complexes.

Proteinases as catalysts of peptide bond synthesis.

Proteinases as catalysts of peptide bond synthesis.

Stable and unstable phase of memory in classically conditioned fly, Phormia regina: Effects of nitrogen gas anaesthesia and cycloheximide injection

This study examined the effects of nitrogen anaesthesia and cycloheximide injection on memory of the classically-conditioned fly, Phormia. 1 M NaCl solution was given to each fly as a conditioning stimulus and 0.5 M sucrose solution was the unconditioned stimulus that induced the proboscis extension response. The training period was as short as 2 min and testing was usually carried out 2 hr later. At varying times (0–60 min) between training and testing, flies were anaesthetized with nitrogen gas for 25 sec. When flies were anaesthetized immediately after training the effect of nitrogen gas was the greatest and few flies showed any conditioned response, but the sensitivity of memory to nitrogen gas declined as the interval between training and nitrogen treatment became longer, and such treatment had no effect on memory when the interval was longer than 30 min. The effect on memory of cycloheximide, an inhibitor of peptide bond synthesis, was also investigated. The injection of cycloheximide (0.37 μg) immediately after training diminished the memory, but when given 1 hr after training it had no effect on memory. These results show that the memory in Phormia has two phases, stable and an unstable phase, like long-term and short-term memory in vertebrates.

Specificity of pepsin-catalyzed peptide bond synthesis.

The rates of the pepsin-catalyzed synthesis of oligopeptides of the general type A-Phe-Leu-B by the condensation of A-Phe-OH with H-Leu-B have been determined by means of analytical high performance liquid chromatography. Variation of the A group led to large changes in the initial rates of the condensation reaction, and the effect of such changes was found to be similar to that previously found for the secondary specificity of pepsin in the hydrolysis of oligopeptide substrates. Replacement of the Phe and Leu residues of A-Phe-OH or H-Leu-B by other amino acid residues gave relative rates of synthesis in accord with the known primary specificity of the hydrolytic action of pepsin. Partially-acetylated pepsin, which exhibits enhanced hydrolytic activity, also catalyzed the condensation reaction more effectively. The results are discussed in relation to the potential utility and limitations of pepsin as a catalyst in the preparative synthesis of oligopeptides and to the problem of the mechanism of its action.

In vitro energy costs of Na+, K+ATPase activity and protein synthesis in muscle from calves differing in age and breed.

1. An in vitro preparation was used to measure rates of oxygen consumption, Na+, K+-ATPase-dependent respiration. [14C]phenylalanine incorporation and tyrosine release of skeletal (sternomandibularis) muscle from 10-21-d-old (three) and 7-month dairy (three) calves and control (CDM; four) and extreme double-muscled (EDM; two) calves. 2. Rates of oxygen consumption was greatest (P less than 0.001) for muscle from 10-21-d-old dairy calves and lowest (P less than 0.05) for CDM calves. 3. Ouabain (10(-6) M) caused a 40% inhibition of muscle respiration. 4. Na+, K+-ATPase-independent respiration was similar for muscle from all calf groups except 10-21-d-old dairy calves which had a value 26% greater (P less than 0.001) than than of older dairy calves. 5. Na+, K+-ATPase-independent respiration was 16% greater (P less than 0.001) for muscle from 10-21-d-old than that of older dairy calves while muscle from EDM calves had a value 11% greater than that of CDM calves. 6. The rate of [14C]phenylalanine incorporation was greater (P less than 0.05) for muscle from 10-21-d-old dairy than from older dairy calves, similar between older dairy and CDM calves, and decreased (P less than 0.05) for EDM calves. 7. Rate of tyrosine release was greatest (P less than 0.05) for muscle from CDM and EDM calves; both dairy groups had similarly low rates of muscle tyrosine release. 8. The energy estimated to be required for peptide bond synthesis accounted for 2.0-3.3% of the O2 consumption of the muscle preparations.

The use of SiC bond in synthesis: preparation of functionalised vinylsilanes from the allyltrimethylsilyl anion and some aspects of their reactivity

A general preparation of functionalised vinylsilanes is reported through the allyltrimethylsilylanion. Their reactivity is studied.