Novel carbon-carbon bond formations for biocatalysis

Carbon-carbon bond formation is the key transformation in organic synthesis to set up the carbon backbone of organic molecules. However, only a limited number of enzymatic C-C bond forming reactions have been applied in biocatalytic organic synthesis. Recently, further name reactions have been accomplished for the first time employing enzymes on a preparative scale, for instance the Stetter and Pictet-Spengler reaction or oxidative C-C bond formation. Furthermore, novel enzymatic C-C bond forming reactions have been identified like benzylation of aromatics, intermolecular Diels-Alder or reductive coupling of carbon monoxide.     

Polyunsaturated fatty acid biosynthesis: a microsomal-peroxisomal process.

The synthesis of 22-carbon fatty acids, with their first double bond at position 4, requires the participation of enzymes in both peroxisomes and the endoplasmic reticulum as well as the controlled movement of fatty acids between these two cellular compartments. It has been observed that there is generally an inverse relationship between rates of peroxisomal beta-oxidation vs those for the microsomal esterification of fatty acids into 1-acyl-sn-glycero-3-phosphocholine. With a variety of different substrates it was found that when a fatty acid is produced in peroxisomes, with its first double bond at position 4, its preferred metabolic fate is to move to microsomes for esterification rather than to serve as a substrate for continued degradation. The required movement, and the associated reactions, in peroxisomes and microsomes is not restricted to the synthesis of 4,7,10,13,16-docosapentaenoic acid and 4,7,10,13,16,19-docosahexaenoic acid. When microsomes and peroxisomes were incubated with NAD, NADPH and malonyl-CoA it was found that 6,9,12-octadecatrienoic acid was metabolized to linoleate. Collectively our findings suggest that there may be considerably more recycling of fatty acids between peroxisomes and the endoplasmic reticulum than was previously recognized.     

Synthesis of different types of dipeptide building units containing N- or C-terminal arginine for the assembly of backbone cyclic peptides.

Different types of dipeptide building units containing N- or C-terminal arginine were prepared for synthesis of the backbone cyclic analogues of the peptide hormone bradykinin (BK: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). For cyclization in the N-terminal sequence N-carboxyalkyl and N-aminoalkyl functionalized dipeptide building units were synthesized. In order to avoid lactam formation during the condensation of the N-terminal arginine to the N-alkylated amino acids at position 2, the guanidino function has to be deprotected. The best results were obtained by coupling Z-Arg(Z)2-OH with TFFH/collidine in DCM. Another dipeptide building unit with an acylated reduced peptide bond containing C-terminal arginine was prepared to synthesize BK-analogues with backbone cyclization in the C-terminus. To achieve complete condensation to the resin and to avoid side reactions during activation of the arginine residue, this dipeptide unit was formed on a hydroxycrotonic acid linker. HYCRAM technology was applied using the Boc-Arg(Alloc)2-OH derivative and the Fmoc group to protect the aminoalkyl function. The reduced peptide bond was prepared by reductive alkylation of the arginine derivative with the Boc-protected amino aldehyde, derived from Boc-Phe-OH. The best results for condensation of the branching chain to the reduced peptide bond were obtained using mixed anhydrides. Both types of dipeptide building units can be used in solid-phase synthesis in the same manner as amino acid derivatives.     

Stereochemistry of the acyl dihydroxyacetone phosphate acyl exchange reaction

The fatty acid of acyl dihydroxyacetone phosphate can be exchanged enzymatically for another fatty acid. It has been shown that this reaction proceeds by cleavage of the oxygen bound to C-1 of the dihydroxyacetone phosphate (DHAP) moiety rather than by the more common cleavage at the acyl to oxygen bond. In the present study, the stereochemistry of this reaction was defined further; using deuterated substrates and fast atom bombardment-mass spectrometry, it was shown that the fatty acid exchange involves the stereospecific labilization of the pro-R hydrogen at C-1 of the DHAP moiety of acyl DHAP. The mechanism of ether bond formation, in which acyl DHAP is converted to O-alkyl DHAP, also proceeds via labilization of the pro-R hydrogen and cleavage of the fatty acid at the C-1 to oxygen bond. In addition, other workers have provided evidence that the enzyme responsible for the exchange reaction is O-alkyl DHAP synthetase. Therefore, the present results support the hypothesis that the acyl exchange is the reverse reaction of the first step in O-alkyl DHAP synthesis; in both of these reactions the pro-R hydrogen of C-1 of the DHAP moiety of acyl DHAP and the fatty acid moiety are labilized with cleavage of the fatty acid at the DHAP C-1 to oxygen bond.     

Simultaneous Post-cysteine(S-Acm) Group Removal Quenching of Iodine and Isolation of Peptide by One Step Ether Precipitation

The S-acetamidomethyl (Acm) protecting group is widely used in the chemical synthesis of peptides that contain one or more disulfide bonds. Treatment of peptides containing S-Acm protecting group with iodine results in simultaneous removal of the sulfhydryl protecting group and disulfide formation. However, the excess iodine needs to be quenched or adsorbed as quickly as possible after completion of the disulfide bond formation in order to minimize side reactions that are often associated with the iodination step. We report a simple method for simultaneous post-cysteine (Acm) group removal quenching of iodination and isolation. Use of large volumes of diethyl ether for direct precipitation action of the oxidized peptide from the 90 or 95% aqueous acetic acid solution affords nearly quantitative recovery of largely iodine-free peptide ready for direct purification. It was successfully applied to the synthesis of various peptides including human insulin-like peptide 3 analogues. Although recovery yields were comparable to the traditionally used ascorbic acid quenching method, this new approach offers significant advantages such as more simple utility, minimal side reactions, and greater cost effectiveness.     

Non-coded amino acids as acyl donor substrates for peptide bond formation catalyzed by thermoase in toluene

The peptide bond formation of N-protected non-coded amino acids having different structures as acyl donor substrates that is catalyzed by thermoase in organic media was investigated. In these reactions, N-protected l--non-coded amino acids, including l-Orn, l-Cit, -aminobutyric acid (l--Abu) and phenylalanine homologues, were used as the acyl donors and phenylalanine derivatives were used as the acyl acceptors. This kind of enzymatic reactions cannot be carried out in an aqueous buffer due to the rigid specificity of proteases to coded amino acids in water. The results demonstrated that the substrate specificity of proteases could be broadened in organic solvents. In addition, the factors that influenced these protease-catalyzed reactions, including structures of the substrates, water content and the bases used, were systematically studied. Our work provided important evidence for broadening the application of protease in organic synthesis.     

Oligomerization of Thioglutamic Acid: Encapsulated Reactions and Lipid Catalysis

For cellular life to begin on the early Earth, a permeation mechanism would be required to allow polar solutes to enter a membrane-bounded compartment. A second process - internal polymerization of peptides from amino acid – would also be an essential step toward the first compartmented metabolic pathways leading to biosynthesis. Here we report a study of amino acid permeation into lipid vesicles, in which thioglutamic acid penetrated lipid bilayer membranes at rates sufficient to support internal polymerization reactions. We also investigated spontaneous non-enzymatic polymerization reactions of thioglutamic acid to form oligopeptides. We found that oligomers up to 11mers are produced in the reaction mixture, and conclude that certain lipid surfaces can act as catalysts in promoting an oligomerization reaction. These observations are pertinent to understanding processes by which peptide bond synthesis could take place in primitive cellular life on the early Earth. H. H. Zepik and S. Rajamani contributed equally to the paper.     

Pepsin as a catalyst for peptide synthesis: formation of peptide bonds not typical for pepsin substrate specificity.

Porcine pepsin in water solutions containing 15-28% of dimethylformamide at pH 5 and 20-37 degrees C catalysed the formation of peptide bonds between Z-Ala-Ala-Phe-OH and various amino acid or peptide derivatives. Substrate binding subsite S1' of pepsin demonstrated broad specificity in these reactions but revealed a certain preference for hydrophobic amino acid residues, including non-proteinous homophenylalanine, p-nitrophenylalanine, S-methylcysteine, as well as for those that contained, in addition to the hydrophobic elements, a group capable of donating a hydrogen bond, e.g. o-nitrotyrosine. This observation increases the range of peptides that might be prepared by pepsin-catalysed synthesis.     

Specific formation of arachidonic acid and eicosapentaenoic acid by a front-end Delta5-desaturase from Phytophthora megasperma

The biosynthesis of arachidonic acid (20:4(Delta5Z,8Z,11Z,14Z)) from linoleic acid in plants by transgenic means requires the sequential and specific action of two desaturation reactions and one elongation reaction. Here, we describe the isolation of a specific acyl-lipid-desaturase catalyzing the formation of the double bond at position 5 from a cDNA library from Phytophthora megasperma. The isolated full-length cDNA harbors a sequence of 1740 bp encoding a protein of 477 amino acids with a calculated molecular weight of 53.5 kDa. The desaturase sequence contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. For functional identification, the cDNA was expressed in Saccharomyces cerevisiae, and the formation of newly formed fatty acids was analyzed. The expression of the heterologous enzyme resulted in the formation of arachidonic acid after di-homo-gamma-linolenic acid supplementation and in the formation of eicosapentaenoic acid synthesis from omega3-arachidonic acid. Results presented here on the substrate specificity identify this expressed protein as a classical Delta5-acyl-lipid-desaturase, capable of specifically introducing a double bond at the Delta5 position solely in 20-carbon-atom chain length fatty acids containing a double bond at position Delta8. Detailed analysis of the different lipid species showed a preferential occurrence of the desaturation reaction for fatty acids esterified to phosphatidylcholine.     

Optimization of solid-phase synthesis of difficult peptide sequences via comparison between different improved approaches

Summary. Different approaches are applied to avoid the strong aggregation of the difficult peptide sequences, which is considered as the main reason for incomplete acylation and deprotection reactions hindering the synthesis of these sequences. Temporary protection of amide nitrogen of peptide bond using 2-hydroxy-4-methoxybenzyl (Hmb) and 2,4,6-timethoxybenzyl (Tmob) amino acid derivatives, introduction of D-Ala or Pro residues in the peptide chain sequences and utilization of microwave energy are proved to be useful methods in the enhancement of solubility and in the hindrance of the aggregation during the solid-phase synthesis of oligoalanine. Oligoalanine is chosen to demonstrate the difficult sequences and to compare the efficiencies of these methods.     

Poly-N-acrylylpyrrolidine. A new resin in peptide chemistry.

Entirely beaded poly-N-acrylylpyrrolidine-co-bisacrylyl-1,2-diaminoethane-co-N-acrylyl-1,6-diaminohexane.HCl(PAP), a new resin on which to perform peptide chemistry, has been prepared by reverse phase suspension polymerization in quantitative yield. In addition to being a superior support to polystyrene, albeit readily adaptable to current techniques of peptide synthesis, its versatility has been furthur extended by the introduction and use of new peptide-to-polymer linking groups, which allow the use of the bidirectional approach to peptide chemistry. One such linkage, which connects the side chain of cysteine to PAP via an acid resistant S-carbamoyl bond, was used in a bidirectional synthesis of deamino-oxytocin. PAP solvates and swells in solvents with wide-ranging polarities, including aqueous media. Thus, peptide coupling reactions were performed in organic media of high and of low polarity as well as in aqueous solution.     

Microwave‐assisted solid‐phase peptide synthesis of neurosecretory protein GL composed of 80 amino acid residues

We recently identified a novel cDNA encoding a small secretory protein of 80 amino acid residues, termed neurosecretory protein GL (NPGL), from the chicken hypothalamus. Homologs of NPGL have been reported to be present in mammals, such as human and rat. NPGL is amidated at its C‐terminus, contains an intramolecular disulfide bond, and is hydrophobic in nature. In this study, we have optimized the synthesis of the entire 80‐amino acid peptide sequence of rat NPGL by microwave‐assisted solid‐phase peptide synthesis. NPGL was obtained with a 10% yield when the coupling reactions were performed using 1‐[Bis(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium‐3‐oxid hexafluorophosphate (HATU) at 50 °C for 5 min, and Fmoc deprotections were performed using 40% piperidine containing 0.1 M HOBt. Furthermore, the disulfide bond of NPGL was formed with 20% yield with the use of glutathione‐containing redox buffer and 50% acetonitrile. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Steroids and related products. 38. 11-Aza steroids. 3. The synthesis of 11-aza 9 -steroids. I

The synthesis of 3,20-dioxygenated 11-azapregnanes, with a “normal” (a) configuration.in position 9, is described. The saturation of the 8,9-double bond of previously described unsaturated precursors can be effected not only by reduction of 9,11-iminium salts, but also catalytically, under pressure, in the presence of perchloric acid, It is shown that the 7-position of 8,9-unsaturated 11-aza steroids can be functionalized with the chromic acid-pyridine complex, the double bond of the resulting 7-ketone being prone to Birch reduction. Other, direct, pathways from saturated 11-nor 9,12-seco steroids to saturated 11-aza 9α-steroids have been explored. The crucial difficulty inherent in this approach stems from the fact that in displacement reactions involving an axial 9α-substituent, elimination competes successfully with substitution.     

Depsipeptide analogues of elastin repeating sequences: synthesis

Depsipeptide analogues of peptide sequences can help in elucidating the role of specific hydrogen bonds in determining the conformation in peptides. The repeating pentapeptide and hexapeptide sequences of elastin have been suggested to contain a type II beta-turn with a 4----1 hydrogen bond. Depsipeptide analogues of the repeating sequences of elastin in which this 4----1 hydrogen bond cannot exist were synthesized. A fragment condensation approach was employed in which the depsipeptide ester bond was introduced early in the synthesis. This approach proved to be effective, although the increased lability of the depsipeptide ester bond resulted in side products and low yields in some reactions.     

Novel formation of alpha-amino acids from alpha-oxo acids and ammonia in an aqueous medium

In the course of a study of possible mechanisms for chemical evolution in the primeval sea, we found the novel formation of alpha-amino acids and N-acylamino acids from alpha-oxo acids and ammonia in an aqueous medium. Glyoxylic acid reacted with ammonia to form N-oxalylglycine, which gave glycine in a 5-39% yield after hydrolysis with 6N HC1. Pyruvic acid and ammonia reacted to give N-acetylalanine, which formed alanine in a 3-7% overall yield upon hydrolysis. The pH optima in these reactions were between pH 3 and 4. These reactions were further extended to the formation of other amino acids. Glutamic acid, phenylalanine and alanine were formed from alpha-ketoglutaric acid, phenylpyruvic acid and oxaloacetic acid, respectively, under similar conditions. N-Succinylglutamic acid was obtained as an intermediate in glutamic acid synthesis. Phenylacetylphenylalanineamide was also isolated as an intermediate in phenylalanine synthesis. Alanine, rather than aspartic acid, was produced from oxaloacetic acid. These reactions provide a novel route for the prebiotic synthesis of amino acids. A mechanism for the reactions will be proposed.     

Comparison of Three Phytochrome-mediated Processes in the Hypocotyl of Mustard

Anthocyanin synthesis, hair formation, and the synthesis of ascorbic acid oxidase are all phytochrome-mediated reactions occurring in the hypocotyl of mustard (Sinapis alba L.), controlled by phytochrome actually located in the hypocotyl. A comparison of these three reactions showed that in certain respects they differ greatly in their response to light. The ability of the seedling to respond to light by showing the three responses was strongly influenced by the state of development of the seedling. White light given very early after seed imbibition was unable to evoke any of the three reactions. By 50 hours after imbibition, all systems were fully inducible by light. The addition of actinomycin D to a fully competent seedling coincident with illumination strongly inhibited the development of all three responses. In contrast, the addition of cordycepin at this time inhibited the synthesis of anthocyanin and ascorbic acid oxidase but had no effect on hair formation. Cycloheximide inhibited all three responses when given up to several hours after light. This suggests the necessity for RNA and protein synthesis for light-induced expression of these reactions, and that the RNA species involved in the three reactions may have differing degrees of polyadenylation. The lag period between the onset of light and the first display of the response was 3 hours for anthocyanin and ascorbic acid oxidase synthesis, and about 5 hours for hair formation. Amounts of light sufficient to give large increases in the levels of ascorbic acid oxidase and hair formation gave a much smaller increase in anthocyanin synthesis. Hair formation and ascorbic acid oxidase synthesis showed a much greater sensitivity to induction at early stages of seedling development than did anthocyanin synthesis. Following an inductive light period, anthocyanin synthesis was sensitive to far red light inhibition for a period twice as long as the other two reactions. The differences in the response of the three reactions to light suggest that the phytochrome-mediated reactions which control their development also differ.     

Studies on griseolic acid derivatives. III. Synthesis and biological activities of the adducts of griseolic acid and their base exchanged derivatives

Addition reactions across the double bond of griseolic acid were investigated. Dihydrogriseolic acid was obtained by a reduction of the adduct having halogen at 4' position. The ring juncture of the two five membered rings of the dihydro derivatives was all "cis" configuration. An acetolysis of the protected dihydro derivative gave corresponding 1'-acetoxy sugar. A glycosidation of this sugar derivative with silylated bases gave base exchanged derivatives of the dihydrogriseolic acid. The influence of the base moiety and the double bond to the PDE inhibitory activity was investigated. As a result, we found that this type of compounds had a weaker inhibitory activity than the corresponding compounds which had an original double bond or a dihydro bond that made the ring juncture of the two five membered ring "trans".     

Stimulation of peptide elongation by thyroxine.

This study suggests that thyroxine stimulates peptide elongation in a cell-free rat liver polyribosome system. The thyroxine effect persists in the presence of sufficient aurintricarboxylic acid to prevent polyuridylic acid-stimulated peptide initiation. In addition, thyroxine stimulates elongation of pre-existing polyphenylalanine chains providing conclusive evidence that the effect does not depend on peptide initiation. Thyroxine does not stimulate release of nascent peptides from ribosomes into the supernatant phase of the reaction mixture. Therefore in this protein-synthesis system the thyroxine effect is expected to occur at one or more of the reactions of peptide chain elongation, which include aminoacyl-tRNA binding, peptide bond synthesis and translocation.     

The synthesis of poly-4-thiouridylic acid and its effect on protein synthesis in a cell free system derived from rat liver

The synthesis of poly-4-thiouridylic acid by thiolation of polycytidylic acid is described. A quantitative thiolation was achieved without any cleavage of the phosphodiester bond. The inhibitory effect of the poly-4-thiouridylic acid on protein synthesis in a cell free system derived from rat liver was investigated.     

Effect of intramolecular interactions on circular dichroism of ortho-substituted 1-phenethylamines

The synthesis of ortho-substituted (S)-1-phenethylamines via ortho-lithiation of its N,N-dimethyl derivative followed by reactions with different reagents is described. Circular dichroism spectra of synthesized compounds were measured. It is shown that the observed short-wave Cotton effects greatly depend on the existence of cyclic structure due to possible interactions such as hydrogen bond or electrostatic interaction.