Basic α-aminoalkylphosphonate Derivatives

Peptidyl derivatives of diesters of α-aminoalkylphosphonic acids with basic substitutents, their use in inhibiting serine proteases with trypsin-like specificity and their roles as anti-inflammatory agents, anticoagulants, and anti-tumor agents.Georgia Tech Research Corp

Propargyloxycarbonyl as a protecting group for the side chains of serine, threonine and tyrosine

Propargyloxycarbonyl group is used as a protecting group for the hydroxyl groups of serine, threonine and tyrosine. The propargyloxycarbonyl derivatives of these hydroxy amino acids are stable to acidic and basic reagents commonly employed in peptide synthesis. The deprotection of the O-Poc derivatives using tetrathiomolybdate does not affect commonly used protecting groups such as N-Boc, N-Cbz, N-Fmoc, methyl and benzyl esters. The di-and tripeptides synthesized using O-Poc derivatives of serine, threonine and tyrosine are stable, isolable compounds and give the hydroxy peptides in good yields when treated with tetrathiomolybdate

Preparation of O-Alkyl-N-acyl-DL-serines

N-Phthaloyl amino acids have proved to be very useful for the syntheses of peptides, diazoketones, homologous optically active B-amino acids, optically active a-amino aldehydes, aminoalkylglyoocals and vinylogs of naturally occurring amino acids. As no description has hitherto been given of the N-phthaloyl derivatives of O-alkyl-DL-serine which would enable the above mentioned syntheses to be carried out with serine, we prepared O-methyl-N-phthaloyl-DL-serine by reacting O-methyl-DL-serine and O-ethyl-DL-serine respectively with phthalic anhydride. Further more, O-ethyl-N-p-tosyl-DL-serine and N-carbobenzoxy-O-ethyl -DL-serine were also prepared

Phospholipides containing amino acids other than serine. I. Detection

In view of the widespread occurrence of the amino acid-containing lipides and the unique course of their metabolism during development of Drosophila, we have carried out extensive investigations concerned with their isolation and chemical nature. The present report is concerned primarily with techniques and procedures developed to insure removal of non-lipide contaminants from preparations of these lipides

Bioengineered Nisin A Derivatives with Enhanced Activity against Both Gram Positive and Gram Negative Pathogens

peer-reviewedNisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazakii. This is the first report of derivatives of nisin, or indeed any lantibiotic, with enhanced antimicrobial activity against both Gram positive and Gram negative bacteria.This work was supported by the Irish Government under the National Development Plan, through Science Foundation Ireland Investigator awards (10/IN.1/B3027) and (06/IN.1/B98) (http://www.sfi.ie)

Intensive Mutagenesis of the Nisin Hinge Leads to the Rational Design of Enhanced Derivatives

peer-reviewedNisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid ‘hinge’ region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.This work was financed by a grant from the Irish Department of Agriculture, Food and the Marine through the Food Institutional Research Measure (08/RD/C/691) and with Science Foundation Investigator award (10/IN.1/B3027)

A comparative study of an aminopeptidase from lactic acid bacteria: a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Aminopeptidase enzymes from the proteolytic systems of S.salivarius subsp.thermophilus Lactococcus lactis subsp.cremoris and Lactococcus lactis subsp.lactis have been investigated. An aminopeptidase was purified to near homogeneity from a crude cell free extract of S.thermophilus 5109. The enzyme had a native molecular weight of approximately 96kDa determined by gel-filtration, and a subunit molecular weight of 98kDa, determined by denaturing polyacrylamide gel electrophoresis, showing the native enzyme to be a monomer. The aminopeptidase activity was optimal at pH 7.0 and 35°C. The enzyme was inactivated by p-chloromercuribenzoic acid, iodoacetic acid,the chelating agents EDTA and 1,10-phenanthroline and the divalent cations Cu2+, Zn2+ and Co2+. The aminopeptidase was not inhibited by the serine protease inhibitor PMSF and only minor inhibition occured with the inhibitor No:-p-tosyl-L-lysine chloromethyl ketone (TLCK). The aminopeptidase was capable of hydrolysing several amino-acyl amido methyl coumarin (AMC) and p-nitroanilide (pNA) derivatives, particularly those of lysine, arginine and leucine. The enzyme showed greatest activity with lysyl derivatives (and is therefore referred to in this thesis as a lys-aminopeptidase). The enzyme was able to degrade several oligopeptides by progressive cleavage of the peptide bond but did not hydrolyse peptides containing a proline or aspartic acid residue in the second position. The aminopeptidase activity was dependent on the size of the peptide in that generally only peptides with more than three amino acids were degraded. The aminopeptidase had no endopeptidase or dipeptidase activity. Five different amino-acyl p-nitroanilides derivatives and two amido methyl coumarin derivatives were used to determine the kinetic parameters of the aminopeptidase. The Km values obtained for all the substrates tested were similar, with the exception of ala-pNA, for which the Km value was significantly higher. On the basis of the distribution of activity between different cell-fractions the lys­ aminopeptidase appears to be localised intracellularly. An aminopeptidase was also partially purified from cell-free extracts from Lactococcus lactis subsp.cremoris AM2 and Lactococcus lactis subsp.lactis ML3. The aminopeptidase from L.cremoris AM2 was shown to have a molecular weight of 106kDa and was a monomer. It showed optimal activity at a pH of 7.0 and 450c. The aminopeptidase activity was inhibited by metal-chelators, SH group inhibitors and TLCK. The aminopeptidase hydrolysed lysyl-, arginyl- and leucyl-p-nitroanilide derivatives, but had little or no activity with other pNA substrates. The aminopeptidase from L.lactis ML3 had a molecular weight of 100-105kDa and was monomeric. The optimal activity for the aminopeptidase was at pH of 7.0 and 40°C. The enzyme was inactivated by metal-chelators, sulphydryl inhibitors and by TLCK. Like the aminopeptidases from the other two strains the ML3 aminopeptidase was very specific hydrolysing lysyl-, leucyl- and arginyl-pNA but with very little or no activity with other amino-acyl derivatives

Method of treating endothelial dysfunction comprising administration of a thrombin peptide derivative

Endothelial dysfunction (ED) is associated with a number of diseases and disorders. Agonists of the non-proteolytically activated thrombin receptor can be used in methods to treat ED or ED-related diseases and disorders.Board of Regents, University of Texas Syste

Mutation in the pssZ Gene Negatively Impacts Exopolysaccharide Synthesis, Surface Properties, and Symbiosis of Rhizobium leguminosarum bv. trifolii with Clover

Rhizobium leguminosarum bv. trifolii is a soil bacterium capable of establishing a nitrogen-fixing symbiosis with clover plants (Trifolium spp.). This bacterium secretes large amounts of acidic exopolysaccharide (EPS), which plays an essential role in the symbiotic interaction with the host plant. This polymer is biosynthesized by a multi-enzymatic complex located in the bacterial inner membrane, whose components are encoded by a large chromosomal gene cluster, called Pss-I. In this study, we characterize R. leguminosarum bv. trifolii strain Rt297 that harbors a Tn5 transposon insertion located in the pssZ gene from the Pss-I region. This gene codes for a protein that shares high identity with bacterial serine/threonine protein phosphatases. We demonstrated that the pssZ mutation causes pleiotropic effects in rhizobial cells. Strain Rt297 exhibited several physiological and symbiotic defects, such as lack of EPS production, reduced growth kinetics and motility, altered cell-surface properties, and failure to infect the host plant. These data indicate that the protein encoded by the pssZ gene is indispensable for EPS synthesis, but also required for proper functioning of R. leguminosarum bv. trifolii cells.Polish National Science Centre grant no. DEC-2012/07/B/NZ1/0009