A structure-taste study of arylsulfonyl(cyclo)alkanecarboxylic acids

A number of sweeteners contain a sulfonyl group. In our current search for new glucophores several new compounds containing such group were obtained. A series of novel 1-phenylsulfonylcyklohexanecarboxylic acids and 2-arylsulfonylalkanecarboxylic acids was obtained and evaluated for their sweet taste quality. It has been found that methyl substituents are of the key importance for the activity of these compounds.     

Preparation and utilization of fluorescent synthetic peptides

In this study, several methods for controlled labelling of synthetic peptides by the use of fluorescent compounds (fluorescein isothiocyanate and dimethylaminonaphthalene sulfonyl chloride) were investigated. The first reagent yielded monofluoresceinated, active compounds only when the peptides lacked lysine residues. Monolabelling of peptides in solution with dimethylaminonaphthalenesulphonyl chloride was hindered by the broad reactivity of the reagent, but was achieved by reacting the fluorochrome on protected resin-bound peptides in solid-phase synthesis. The remarkable stability of the linkage allowed the cleavage of the peptide from the resin and deprotection of side-chain functions without hydrolysis of the labelled group. The binding of antipeptide antibodies to the labelled fragments was then estimated using different techniques.     

Immunochemical and biological properties of synthetic peptide fragments from the 124-144 sequence of human leukocyte interferon alpha2]

Three peptides corresponding to the sequences 124-144, 124-138, 129-144 of the human leukocyte interferon alpha 2 (IFN-alpha 2) were synthesized. The synthesis was performed by DCC-HOBT coupling of protected peptide segments in solution. The segments were obtained by the active ester coupling methodology using base-labile 2-[4-(phenylazobenzyl)sulfonyl]ethyl (Pse) group as carboxyterminal protection. After complete deprotection with 1 M methanesulphonic acid in trifluoroacetic acid--thioanisol--m-cresol mixture the peptides were purified by reversed-phase chromatography. The studies of interaction of the peptides with rabbit antiserum against IFN-alpha 2 revealed at least one minor antigenic determinant within the 124-144 region of IFN-alpha 2 amino acid sequence. Rabbit antisera developed against peptides 124-138 and 129-144 showed ability of binding recombinant IFN-alpha 2 and neutralizing its antiviral activity. Free peptides or their conjugates with bovine serum albumine did not display antiviral activity, neither could they inhibit the activity of IFN-alpha 2.     

‘Sulfo‐click’ for ligation as well as for site‐specific conjugation with peptides,fluorophores, and metal chelators

The ‘sulfo‐click’ reaction, which is a chemoselective amidation reaction involving the reaction of an aminoethane sulfonyl azide with a thio acid, encompasses a new approach for ligation and conjugation. Detailed protocols are provided for decorating biologically active peptides or dendrimers with biophysical tags, fluorescent probes, metal chelators, and small peptides by using this reaction as a novel, metal‐free ‘sulfo‐click’ approach. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of methyl 2- and 4-pyridyl disulfide from 2- and 4-thiopyridone and methyl methanethiosulfonate

In 1982, methyl 2-pyridyl disulfide was reported as a new reagent for the titration of thiol groups in peptides and proteins and for their temporary blocking with the thiomethyl group [T. Kimura et al. (1982) Anal. Biochem. 122, 274-282]. We have synthesized this compound (and its 4-pyridyl isomer) by a rapid and convenient procedure which is preferable to that in the original report. Our method involves the thiomethylation of the appropriate thiopyridone by methyl methanethiosulfonate.     

Analogue-based design, synthesis and molecular docking analysis of 2,3-diaryl quinazolinones as non-ulcerogenic anti-inflammatory agents

In our effort to identify potent gastric sparing anti-inflammatory agents, a series of methyl sulfanyl/methyl sulfonyl substituted 2,3-diaryl quinazolinones were designed by analogue-based design strategy and synthesized for biological evaluation. Subsequently, the compounds were evaluated for both cyclooxygenase inhibitions by ovine COX assay and carrageenan-induced rat paw edema assay. All the methyl sulfonyl substituted quinazolinones were exhibited promising anti-inflammatory activity. In particular, 6-bromo-3-(4-methanesulfonyl-phenyl)-2-phenyl-3H-quinazolin-4-one, 7-chloro-3-(4-methanesulfonyl-phenyl)-2-phenyl-3H-quinazolin-4-one, 3-(4-methanesulfonyl-phenyl)-2-(4-methoxy-phenyl)-3H-quinazolin-4-one and 6-bromo-3-(4-methanesulfonyl-phenyl)-2-(4-methoxy-phenyl)-3H-quinazolin-4-one emerged as the most active compounds in the series. The results of ulcerogenic activity assay suggest that these compounds are gastric safe compared to indomethacin. The molecular docking analysis was performed to understand the binding interactions of these compounds to COX-2 enzyme. The results from the present investigation suggests that 2,3-diaryl quinazolinones as a promising template for the design of new gastric safe anti-inflammatory agents, which can be further explored for potential anti-inflammatory activity.     

Conformational effects of chiral alpha,alpha-dialkyl amino acids. I. C-terminal tetrapeptides of emerimicin containing alpha-ethylalanine

The syntheses and the crystal structures of the C-terminal tetrapeptide fragments of emerimicin IV and III, Boc-R-EtA-Hyp(Bzl)-Ala-Phol and Boc-R-EtA-Hyp(Bzl)-MeA-Phol, containing the chiral alpha,alpha-dialkyl amino acid, R-alpha-ethylalanine (R-EtA) are reported. The two peptides are isomorphous and assume a 3(10)-helical conformation in the crystal. A comparison of the crystal data on alpha,alpha-dialkyl amino acids indicates that alkyl substituents larger than a methyl group do not preclude peptides containing these amino acids from assuming the conformations associated with minima which have been well characterized for alpha-methylalanine.     

Protein-protein recognition via short amphiphilic helices; a mutational analysis of the binding site of annexin II for p11.

Annexin II (p36) interacts with its ligand p11 via the short stretch of 12 amino acids (Ac-S-T-V-H-E-I-L-C-K-L-S-L) situated at the N-terminus. We have now synthesized some 37 tetradecapeptides, which differ from the original p11 binding sequence (Ac1-14) by single amino acid substitutions. The relative affinity of each peptide for p11 was determined by fluorescence spectroscopy using a competitive binding assay. The binding behaviour of the different peptides confirms the model of an amphiphilic alpha-helix induced upon binding to p11. The apparent affinities delta delta Gbind of the mutant peptides revealed that the N-acetyl group of serine 1 and the hydrophobic side chains at positions 3, 6, 7 and 10 contribute most to the binding. The observed destabilization of the complex upon removal of signal methyl groups from the hydrophobic side of the helix is comparable with the destabilization of proteins in which methyl groups have been removed from the inner core. We conclude that upon binding to p11 the hydrophobic side of the amphiphatic alpha-helix becomes fully buried.     

Demonstration in vitro of two intracellular inactivators of nitrate reductase from Neurospora.

Two different inactivators of nitrate reductase have been found in cell free preparations of Neurospora. The first (Inactivator I) is very active at pH 9, is inhibited by disodium ethylene diamine tetraacetate (EDTA) and is present in all mycelia incubated under all conditions tested; the second (Inactivator II) is very active at pH 5, is repressed by ammonia or by a metabolic product of ammonia and derepressed by nitrogen starvation, cannot be derepressed by nitrogen starvation in strain nit-2, in which a number of "ammonia-represible" enzymes are permanently repressed, and is sensitive to phenyl methyl sulfonyl fluoride. Crude extracts of mycelia contain inhibitor(s) of both inactivators.     

Crystal structures of HLA-A*0201 complexed with antigenic peptides with either the amino- or carboxyl-terminal group substituted by a methyl group

The crystal structures of class I major histocompatibility complex (MHC) molecules complexed with antigenic peptides revealed a network of hydrogen bonds between the charged amino- and carboxyl-termini of the peptides and conserved MHC residues at both ends of the peptide binding site. These interactions were shown to contribute substantially to the stability of class I MHC/peptide complexes by thermal denaturation studies using synthetic peptides in which either the amino- or carboxyl-terminal group is substituted by a methyl group. Here we report crystal structures of HLA-A*0201 complexed with these terminally modified synthetic peptides showing that they adopt the same bound conformation as antigenic peptides. A number of variations in peptide conformation were observed for the terminally modified peptides, including in one case, a large conformational difference in four central peptide residues that is apparently caused by the lattice contact. This is reminiscent of the way binding a T-cell receptor changed the conformation of central residues of an MHC-bound peptide. The structures determined identify which conserved hydrogen bonds are eliminated in terminally substituted peptides and suggest an increased energetic importance of the interactions at the peptide termini for MHC-peptide stability. Proteins 33:97–106, 1998. © 1998 Wiley-Liss, Inc.     

Demonstration in vitro of two intracellular inactivators of nitrate reductase from Neurospora

Two different inactivators of nitrate reductase have been found in cell free preparations of Neurospora. T he first (Inactivator I) is very active at pH9, is inhibited by disodium ethylene diamine tetraacetate (EDTA) and is present in all mycelia incubated under all conditions tested; the second (inactivator II) is very active at pH 5, is repressed by ammonia or by a metabilic product of ammonia and derepssed by nitrogen starvation, cannot be derepressed by nitrogen starvation in strain nit-2, in which a number of “ammonia-repressible” enzymes are permanently repressed, and is sensitive to phenyl methyl sulfonyl fluoride.Crude extracts of mycelia contain inhibitors(s) of both inactivators.     

The biosynthesis of methylated amino acids in the active site region of methyl-coenzyme M reductase

The global production of the greenhouse gas methane by methanogenic archaea reaches 1 billion tons per annum. The final reaction releasing methane is catalyzed by the enzyme methyl-coenzyme M reductase. The crystal structure of methyl-coenzyme M reductase from Methanobacterium thermoautotrophicum revealed the presence of five modified amino acids within the alpha-subunit and near the active site region. Four of these modifications were C-, N-, and S-methylations, two of which, 2-(S)-methylglutamine and 5-(S)-methylarginine, have never been encountered before. We have now confirmed these modifications by mass spectrometry of chymotryptic peptides. With methyl-coenzyme M reductase purified from cells grown in the presence of L-[methyl-D(3)]methionine, it was shown that the methyl groups of the modified amino acids are derived from the methyl group of methionine rather than from methyl-coenzyme M, an intermediate in methane formation. The D(3) labeling pattern was found to be qualitatively and quantitatively the same as in the two methyl groups of the methanogenic coenzyme F(430), which are known to be introduced via S-adenosylmethionine. From the results, it is concluded that the methyl groups of the modified amino acids in methyl-coenzyme M reductase are biosynthetically introduced by an S-adenosylmethionine-dependent post-translational modification. A mechanism for the methylation of glutamine at C-2 and of arginine at C-5 is discussed.     

Enzymatic inactivation of human serum proteinase inhibitors by snake venom proteinases

Snake venoms of the Crotalid, Viperid, and Colubrid families possess proteinases which catalytically inactivate purified human α_α-proteinase inhibitor. Incubation with these venoms also results in the gradual loss of all detectable trypsin and chymotrypsin inhibitory activity present in human serum. The venom proteinases involved are metal dependent and are unaffected by phenyl methyl sulfonyl fluoride. The Elapid and Hydrophid snake venoms tested were devoid of this activity.     

Immunogold localization of a developmentally regulated,tapetal-specific, 15 kDa lily anther protein

Summary We have confirmed that the LLA-15 polypeptide ofLilium longiflorum is (a) tapetum specific with some expression possible in the adjacent middle layer cells and (b) relatively abundant as evidenced by the high density of gold particles localized to the tapetal cells. We have established that the protein is cytoplasmic and not associated with organelles, membranes, extracellular matrix or wall. We also report an amino acid composition of the molecule and a partial sequence which bears no resemblance to any protein yet described.Abbreviations BSA bovine serum albumin - PMSF phenyl methyl sulfonyl fluoride     

Introduction of the dansyl group into histidine and tyrosine residues in peptides and proteins

Two dansyl derivatives: 1-(5-dimethylaminonaphthalene) sulfonyl (4-amino)-benzyl amine and 1-(5-dimethylaminonaphthalene) sulfonyl beta(4-aminophenyl) ethylamine, have been recently synthesized. Reaction of these compounds with nitrous acid lead to the corresponding dansyl-bearing diazonium salts. The latter derivatives can couple, under mild basic conditions, to the imidazole moiety of histidine, the phenolic ring of tyrosine and to the epsilon-amino function of lysine. The applicability of the two reagents was tested in the modification of several peptides, including [D-Phe6]LHRH, [D-Gln6]LHRH, Leu-enkephalin and Tyr-tuftsin, and proteins such as calmodulin, bovine serum albumin and nerve growth factor.     

Synthesis and antifungal activity of substituted salicylaldehyde hydrazones,hydrazides and sulfohydrazides

Efficient synthetic procedures for the preparation of acid hydrazines and hydrazides were developed by converting the corresponding carboxylic acid into the methyl ester catalyzed by Amberlyst-15, followed by a reaction with hydrazine monohydrate. Sulfohydrazides were prepared from the corresponding sulfonyl chlorides and hydrazine monohydrate. Both of these group of compounds were condensed with substituted salicylaldehydes using gradient concentration methods that generated a large library of hydrazone, hydrazide and sulfohydrazide analogs. Antifungal activity of the prepared analogs showed that salicylaldehyde hydrazones and hydrazides are potent inhibitors of fungal growth with little to no mammalian cell toxicity, making these analogs promising new targets for future therapeutic development.     

New reagents for the introduction of the thiomethyl group at sulfhydryl residues of proteins with concomitant spectrophotometric titration of the sulfhydryls: Methyl 3-nitro-2-pyridyl disulfide and methyl 2-pyridyl disulfide

Two new reagents for the titration of sulfhydryl groups in peptides and proteins and for their temporary blocking with the thiomethyl group have been developed. The sulfhydryl groups in cysteine, glutathione, and papain react quantiatively under mild conditions with these reagents, methyl 3-nitro-2-pyridyl disulfide and methyl 2-pyridyl disulfide, with concomitant methanethiolation and without the need to employ a large excess of reagent. Because of the chromophoric properties of the 3-nitro-2-thiopyridone and 2-thiopyridone products, spectrophotometric titration of the sulfhydryl groups can be carried out, accompanying their methanethiolation. The modification of the sulfhydryl groups in peptides and proteins with thiomethyl is rapidly and completely reversible upon addition of thiols such as l-cysteine.     

4-N-Hydroxy-4-[1-(sulfonyl)piperidin-4-yl]-butyramides as HDAC inhibitors

A series of N-substituted 4-alkylpiperidine hydroxamic acids, corresponding to the basic structure of histone deacetylase (HDAC) inhibitors (zinc binding moiety-linker-capping group) has been previously reported by our group. Linker length and aromatic capping group connection were systematically varied to find the optimal geometric parameters. A new series of submicromolar inhibitors was thus identified, which showed antiproliferative activity on HCT-116 colon carcinoma cells. We report here the second part of the strategy used in our research group to find a new class of HDAC inhibitors, namely the SAR study for the compounds bearing a sulfonyl group on the piperidine nitrogen. In the present work, we have considered both sulfonamides and sulfonyl ureas.     

Enzymatic methyl esterification of synthetic tripeptides: structural requirements of the peptide substrate. Detection of the reaction products by fast-atom-bombardment mass spectrometry

Eukaryotic protein carboxyl methyltransferase catalyzes a two-substrates reaction in which the methyl group of S-adenosylmethionine is transferred to the free carboxyl group of D-aspartyl and L-isoaspartyl-containing peptide or protein substrates. It has been previously shown that at least three binding sites are required for the interaction of adenosylmethionine with the enzyme and/or the protein substrate [Oliva A., Galletti P., Zappia V., Paik W. K. & Kim S. (1980) Eur. J. Biochem. 104, 595-602], while very little is known concerning the structural requirements of the protein substrate. In this study several synthetic tripeptides were selected in order to elucidate the structural requirements of the methyl-accepting substrates. The results obtained with this series of peptides suggested that: (1) three residues appear to be the minimal length, so far identified, required for a productive enzyme-substrate interaction, several dipeptides being ineffective as substrates [McFadden P. N. & Clarke S. (1986) J. Biol. Chem. 261, 11,503-11,511]; (2) the isoaspartyl residue is not recognized unless its alpha-amino group is involved in a carboamide bond; (3) an hydrogen atom on the amide linkage following the isoaspartyl residue is essential for both recognition and catalysis; (4) oligopeptides containing both D-aspartyl and D-isoaspartyl residues are not recognized by this methyltransferase. On the basis of these results, interaction sites between the peptide substrate and the enzyme molecule have been proposed. This paper also reports the first application of fast-atom-bombardment mass spectrometry to the detection of the products of the enzymatic methyl esterification reaction. By this soft ionization technique, the methyl-esterified peptides as well as the corresponding cyclic imides generated during the spontaneous demethylation process have been identified.