This paper summarizes recent advances in α-alkylation reactions based on hydrogen borrowing methodologies using alcohol as an alkylating agent. This review provides a summary of recent progress toward the α-alkylation of carbonyl substrates, as well as relatively unactivated substrates bearing fewer acidic α-hydrogens, such as acetonitriles, acetamides, esters, methylpyrimidines, and methylquinolines. A summary of recent improvements in α-methylation strategies based on hydrogen borrowing methodologies has also been provided. Particular emphasis has been placed on highly practical and green chemistry approaches involving modified catalytic systems, including metal-supported heterogeneous catalysts and nanoparticle-based catalysts, as well as reactions conducted in the absence of a transition-metal catalyst. A review of recent achievements in methylation strategies using methanol as a methyl source, and their application to the α-methylation of ketones using transition-metal catalyzed hydrogen borrowing me...

Recent Advances in α-Alkylation Reactions using Alcohols with Hydrogen Borrowing Methodologies

Microbial transformations of nitriles

An enantiomer-selective amidase active on several 2-aryl and 2-aryloxy propionamides was identified and purified from Brevibacterium sp. strain R312. Oligonucleotide probes were designed from limited peptide sequence information and were used to clone the corresponding gene, named amdA. Highly significant homologies were found at the amino acid level between the deduced sequence of the enantiomer-selective amidase and the sequences of known amidases such as indoleacetamide hydrolases from Pseudomonas syringae and Agrobacterium tumefaciens and acetamidase from Aspergillus nidulans. Moreover, amdA is found in the same orientation and only 73 bp upstream from the gene coding for nitrile hydratase, strongly suggesting that both genes are part of the same operon. Our results also showed that Rhodococcus sp. strain N-774 and Brevibacterium sp. strain R312 are probably identical, or at least very similar, microorganisms. The characterized amidase is an apparent homodimer of Mr 2 x 54,671 which exhibited under our conditions a specific activity of about 13 to 17 mumol of 2-(4-hydroxyphenoxy)propionic R acid formed per min per mg of enzyme from the racemic amide. Large amounts of an active recombinant enzyme could be produced in Escherichia coli at 30 degrees C under the control of an E. coli promoter and ribosome-binding site.

Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase.

The cloned 9.4-kb insert of plasmid pNHJ20L containing low-molecular-mass nitrile hydratase (L-NHase) gene from Rhodococcus rhodochrous J1 [Kobayashi, M. et al. (1991) Biochim. Biophys. Acta 1129, 23–33] was digested with various restriction enzymes, and the trimmed fragments were inserted into pUC18 or pUC19. A 1.96-kb EcoRI–SphI region located 1.9-kb downstream of the L-NHase gene was found to be essential for the expression of amidase activity in Escherichia coli; the gene arrangement of the amidase and the NHase in R. rhodochrous J1 differed from those in Rhodococcus species including N-774 and Pseudomonas chlororaphis B23. The nucleotide-deter-mined sequence indicated that the amidase consists of 515 amino acids (54626 Da) and the deduced amino acid sequence of the amidase had high similarity to those of amidases from Rhodococcus species including N-774 and P. chlororaphis B23 and to indole-3-acetamide hydrolase from Pseudomonas savastanoi.



The amidase gene modified in the nucleotide sequence upstream from its start codon expressed 8% of the total soluble protein in E. coli under the control of lac promoter. The level of amidase activity in cell-free extracts of E. coli was 0.468 unit/mg using benzamide as a substrate. This amidase was purified to homogeneity from extracts of the E. coli transformant with 30.4% overall recovery. The molecular mass of the enzyme estimated by HPLC was about 110 kDa and the enzyme consists of two subunits identical in molecular mass (55 kDa). The enzyme acted upon aliphatic amides such as propionamide and also upon aromatic amides such as benzamide. The apparent Km values for propionamide and benzamide were 0.48 mM and 0.15 mM, respectively. This amidase was highly specific for the S-enantiomer of 2-phenylpropionamide, but could not recognize the configuration of 2-chloropropionamide. It also catalyzed the transfer of an acyl group from an amide to hydroxylamine to produce the corresponding hydroxamate.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1993.tb18250.x

Amidase coupled with low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1. Sequencing and expression of the gene and purification and characterization of the gene product.

A new enantiomer-selective amidase active on several 2-aryl propionamides was identified and purified from a newly isolated Rhodococcus strain. The characterized amidase is an apparent homodimer, each molecule of which has an Mr of 48,554; it has a specific activity of 16.5 mumol of S(+)-2-phenylpropionic acid formed per min per mg of enzyme from the racemic amide under our conditions. An oligonucleotide probe was deduced from limited peptide information and was used to clone the corresponding gene, named amdA. As expected, significant homologies were found between the amino acid sequences of the enantiomer-selective amidase of Rhodococcus sp., the corresponding enzyme from Brevibacterium sp. strain R312, and several known amidases, thus confirming the existence of a structural class of amidase enzymes. Genes probably coding for the two subunits of a nitrile hydratase, albeit in an inverse order, were found 39 bp downstream of amdA, suggesting that such a genetic organization might be conserved in different microorganisms. Although we failed to express an active Rhodococcus amidase in Escherichia coli, even in conditions allowing the expression of an active R312 enzyme, the high-level expression of the active recombinant enzyme could be demonstrated in Brevibacterium lactofermentum by using a pSR1-derived shuttle vector.

Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase.

An amidase with a wide activity spectrum was purified from the Brevibacterium sp. R 312 and studied. The purification was performed by precipitating the proteins of the supernatant fraction obtained after centrifugation of sonicated cells. The resulting proteins were submitted to a gel filtration step followed by DEAE-Sephadex ion exchange chromatography and then by another gel filtration process. The purified amidase had a molecular weight of 180,000 and was composed of four subunits of the same molecular weight (43,000 + 2,000). Its isoelectric point was 3.5. This enzyme was able to hydrolyze a large number of amides into their corresponding organic acids and it also possessed an acyl transferase activity.

Purification and properties of an acylamide amidohydrolase (E. C. 3.5.1.4) with a wide activity spectrum from Brevibacterium sp. R 312

The Brevibacterium R 312 strain has an amidase with a wide substrate spectrum previously named “acetamidase”. The study of its activity showed that this enzyme was able to hydrolyze a large number of amides into their corresponding organic acids. The affinity of this enzyme for the substrates varied according to the length of the carbon chain and the spatial crowding of the molecule. The comparison of the specific rates of hydrolysis showed that propionamide was the amide substrate most quickly hydrolyzed.

Activity and regulation of an amidase (acylamide amidohydrolase, EC 3.5.1.4) with a wide substrate spectrum from a Brevibacterium sp.

The effect of different compounds on the enzymic action of the nitrile-hydratase used for the bioconversion of nitriles was studied. An excess of acrylonitrile as a substrate was shown to inhibit the activity of the enzyme. This inhibition occurred only at relatively high substrate concentrations (0.2 mol/l or more). The nitrile bioconversion products (acrylamide, propionamide) and their structural analogues (acrylic acid, thioacetamide) were shown to inhibit the enzyme competitively. The most important inhibition found was that of cyanide (Ki= 0.004 mol/l), a break down product of some nitriles. By using an acetamidase-negative mutant, amides were shown to inhibit biosynthesis of nitrile-hydratase. An identical result was obtained with thioacetamide, a non-substrate compound for acetamidase. This compound repressed the biosynthesis of nitrile-hydratase by both the wild type and the acetamidase-negative mutant to the same extent.

A note on the enzymic action and biosynthesis of a nitrile‐hydratase from a Brevibacterium sp.

The wide spectrum amidase from Brevibacterium sp. R312, which can hydrolyse many amides to the corresponding acids, was shown to transfer the acyl groups of amides, acids and esters to hydroxylamine. The transfer rates of these reactions in cytoplasmic fractions were measured and compared. The K
m and V
max were determined for different substrates in the presence of hydroxylamine. The enzyme was also shown to transfer the acyl group of the amide analogue N-methylacetamide to hydroxylamide and that of acetamide to the hydroxylamine analogue methylhydroxylamine.

/pdf/acyltransferase-activity-of-the-wide-spectrum-amidase-of-4um0uia3da.pdf

Acyltransferase activity of the wide spectrum amidase of Brevibacterium sp. R312

In the first part of this paper different microbial enzyme systems able to hydrolyze the true amide function are reviewed, then some enzyme systems are described in detail: aminopeptidases, amidases of Aspergillus nidulans and of Pseudomonas aeruginosa.

The amidases from a Brevibacterium strain: study and applications.

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