Phosphotyrosyl peptides and analogues as substrates and inhibitors of purple acid phosphatases

Purple acid phosphatases are metal-containing hydrolases. While their precise biological role(s) is unknown, the mammalian enzyme has been linked in a variety of biological circumstances (e.g., osteoporosis) with increased bone resorption. Inhibition of the human enzyme is a possible strategy for the treatment of bone-resorptive diseases such as osteoporosis. Previously, we determined the crystal structure of pig purple acid phosphatase to 1.55A and we showed that it is a good model for the human enzyme. Here, a study of the pH dependence of its kinetic parameters showed that the pig enzyme is most efficient at pH values similar to those encountered in the osteoclast resorptive space. Based on the observation that phosphotyrosine-containing peptides are good substrates for pig purple acid phosphatase, peptides containing a range of phosphotyrosine mimetics were synthesized. Kinetic analysis showed that they act as potent inhibitors of mammalian and plant purple acid phosphatases, with the best inhibitors exhibiting low micromolar inhibition constants at pH 3-5. These compounds are thus the most potent organic inhibitors yet reported for the purple acid phosphatases.     

Rigid analogues as probes of excitatory amino acid receptors.

The classes of compounds to be discussed are based on rigid analogues of glutamic and aspartic acids. The glutamate analogue 1-amino-1,3-cyclopentane dicarboxylic acid (1,3-ACPD) exists as two enantiomeric pairs of geometric isomers. The absolute configurations were assigned and the compounds were found to differentiate between the kainic acid (KA) and N-methyl-D-aspartic acid (NMDA) receptor subtypes when applied iontophoretically to hippocampal CA1 pyramidal neurones. The results indicate a high degree of specificity for the interaction of D-cis-1,3-ACPD with the NMDA receptor, while the remaining three isomers of 1,3-ACPD were KA-like in their action. The results are augmented with binding studies and patch clamp analysis. The second class of compound is the closely related aspartate analogue 1-amino-1,2-cyclopentane dicarboxylic acid (1,2-ACPD). The geometric isomers have been examined and found to be somewhat less active than their 1,3-ACPD counterparts; however, the cis isomer does have antagonistic properties against quisqualate (QA) evoked excitation. The results indicate that while the three-dimensional arrangement of functional groups is important for the activation of receptor subtypes, other considerations must be made, including stereochemistry and receptor affinity for sterically hindered analogues of excitatory amino acids.     

Identification of a novel amino acid, o-bromo-L-phenylalanine, in egg-associated peptides that activate spermatozoa

Eight sperm-activating peptides containing a novel amino acid were isolated from the egg jelly of the sea urchin Tripneustes gratilla. Accurate mass measurement of the peptide in FAB mass spectrometry showed that the mass of the novel amino acid residue was 224.978. On the basis of the isotopic ion distribution and the degree of unsaturation, the mass value indicated that the elemental composition of the amino acid residue was C9H8O1N1Br1, suggesting that the novel amino acid was bromophenylalanine. Proton NMR spectroscopy, amino acid analysis, and RP-HPLC with three synthetic isomers of bromophenylalanine demonstrated that o-bromophenylalanine was the novel amino acid. Derivatization of the amino acid with Marfey's reagent, (1-fluoro-2,4-dinitrophen-5-yl)-L-alanine amide (FDAA), further indicated that the amino acid was the L-isomer. In other sperm-activating peptides isolated from the egg jelly of the sea urchin, both m- and p-bromophenylalanines were discovered. The presence of m-bromophenylalanine has not been previously reported in natural products, while p-bromophenylalanine is found in theonellamide F, an antifungal bicyclic peptide from a marine sponge.     

Synthesis of novel unnatural amino acid as a building block and its incorporation into an antimicrobial peptide

Considering the biological mechanism and in vivo stability of antimicrobial peptides, we designed and synthesized novel unnatural amino acids with more positively charged and bulky side chain group than lysine residue. The unusual amino acids, which were synthesized by either solution phase or solid phase, were incorporated into an antimicrobial peptide. Its effect on the stability, activity, and the structure of the peptide was studied to evaluate the potential of these novel unnatural amino acids as a building block for antimicrobial peptides. The incorporation of this unusual amino acid increased the resistance of the peptide against serum protease more than three times without a decrease in the activity. Circular dichroism spectra of the peptides indicated that all novel unnatural amino acids must have lower helical forming propensities than lysine. Our results indicated that the unnatural amino acids synthesized in this study could be used not only as a novel building block for combinatorial libraries of antimicrobial peptides, but also for structure–activity relationship studies about antimicrobial peptides.     

Rapid hydrolysis of proteins and peptides by means of microwave technology and its application to amino acid analysis

A rapid heating method of hydrolysis by the use of microwave oven has been applied to amino acid analysis of proteins and peptides. This convenient method has been compared with the conventional 6 N HCl hydrolysis at 110 degrees for 24 h. The advantages of this new method are its expedition and the accurate and comparable results as compared to the tedious conventional technique. The method provides a rapid processing of multiple samples within minutes instead of days and inexpensive access to the important data of amino acid compositions of proteins by the commonly used microwave oven. The necessary change in the design of hydrolysis vials and the safety precautions accompanying this novel use of microwave acid-digestion method are also described.     

Synthesis of amino analogues of cholic acid

Amino analogues of cholic acid were synthesized by reduction of oximes using titanium(III) chloride in the presence of sodium cyanoborohydride.     

Solid-phase synthesis of cyclic RGD-furanoid sugar amino acid peptides as integrin inhibitors

The solid-phase synthesis of cyclic RGD peptides containing either one or two furanoid sugar amino acids (SAAs) is reported. Using a cyclization-cleavage approach five peptides were successfully assembled and consecutively tested on their ability to bind to the integrin receptors alpha(v)beta(3) and alpha(IIb)beta(3). The cyclic tetrapeptide c[RGD-SAA] (1) showed the most promising activity in an inhibition assay with an IC(50) of 1.49 microM for the alpha(v)beta(3) receptor and 384 nM for the alpha(IIb)beta(3) receptor.     

Investigation of some amino acid analogues and metabolites as inhibitors of wool and hair growth

Sheep were given intravenous infusions of ethionine together with cycloleucine or reduced glutathione, in attempts to prevent the inhibition of wool growth by ethionine. Other sheep were given cycloleucine alone to measure effects on wool growth. Twenty-two compounds related to cystine, methionine, ethionine, lysine, phenylalanine and tyrosine were given as intravenous infusions to sheep to investigate their potential as depilatory agents. Nineteen of these compounds were also tested in mice during their first cycle of hair growth. The concurrent administration of cycloleucine with ethionine prevented the weakening of wool fibres caused by ethionine, but reduced glutathione was ineffective. Cycloleucine weakened wool fibres, as judged subjectively, and caused a small reduction in fibre diameter. Selenocystine and selenomethionine caused some hair loss in mice but selenocystine was also toxic. Both seleno-amino acids were toxic for sheep; selenocystine was lethal at 0.025 mmol kg-0.75 and selenomethionine at 0.09 mmol kg-0.75. Doses that permitted survival of sheep did not have depilatory effects. However, the presence of autophagic vacuoles in the cytoplasm of follicle bulb cells of sheep indicated that a toxic dose of selenocystine had potential depilatory activity. Other compounds investigated did not induce loss of wool or hair. Some compounds, notably 3-methylthiopropionic acid and S-(2-aminoethyl)-L-cysteine, were toxic to mice but not sheep. The methionine analogue, methoxinine (O-methyl-DL-homoserine), caused a substantial reduction in the strength of wool fibres and a prolonged alteration of the crimp pattern. It is suggested tentatively that cycloleucine inhibits methionine adenosyltransferase and thereby reduces or prevents the formation of S-adenosylethionine. The failure of various compounds related to methionine and ethionine to have any depilatory activity in sheep supports the view that ethionine influences wool growth via the formation of S-adenosylethionine.     

Functional arginine-containing amino acid sequences in peptides and proteins

L-Arginine is a source of nitrogen oxide and plays a great role in a number of other biochemical processes. Functions and prospects for practical application of five groups of arginine-containing amino acid sequences and synthetic polyarginine sequences are considered. The physiological characteristics of well-known arginine-containing peptides, such as RGD containing, kyotorphin, and tuftsin, are described in detail.     

Functional arginine-containing amino acid sequences in peptides and proteins

L-arginine is a source of nitrogen oxide and plays a great role in a number of other biochemical processes. Functions and prospects for practical application of five groups of arginine-containing amino acid sequences and synthetic polyarginine sequences are considered. The physiological characteristics of well-known arginine-containing peptides, such as RGD peptides, kyotorphin, and tuftsin, are described in detail. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru     

Synthesis and application of an azobenzene amino acid as a light-switchable turn element in polypeptides

The synthesis of an azobenzene amino acid (aa) for use as a photo-inducible conformational switch in polypeptides is described. The compound can be easily incorporated into an aa sequence by solid-phase peptide synthesis using standard 9-fluorenylmethoxycarbonyl methods. A reversible conformational change of the peptide backbone is induced by switching between the cis and trans configurations of the azobenzene moiety by irradiation with light of suitable wavelength. Thermal cis --> trans isomerization of this azobenzene aa is slow, enabling detailed structural investigations of the modified peptides, e.g., using NMR techniques. The total time for the synthesis of the photoswitch is typically 4 d, with an overall yield of 40-50%.     

Enzymatic tRNA acylation by acid and alpha-hydroxy acid analogues of amino acids

Incorporation of unnatural amino acids with unique chemical functionalities has proven to be a valuable tool for expansion of the functional repertoire and properties of proteins as well as for structure-function analysis. Incorporation of alpha-hydroxy acids (primary amino group is substituted with hydroxyl) leads to the synthesis of proteins with peptide bonds being substituted by ester bonds. Practical application of this modification is limited by the necessity to prepare corresponding acylated tRNA by chemical synthesis. We investigated the possibility of enzymatic incorporation of alpha-hydroxy acid and acid analogues (lacking amino group) of amino acids into tRNA using aminoacyl-tRNA synthetases (aaRSs). We studied direct acylation of tRNAs by alpha-hydroxy acid and acid analogues of amino acids and corresponding chemically synthesized analogues of aminoacyl-adenylates. Using adenylate analogues we were able to enzymatically acylate tRNA with amino acid analogues which were otherwise completely inactive in direct aminoacylation reaction, thus bypassing the natural mechanisms ensuring the selectivity of tRNA aminoacylation. Our results are the first demonstration that the use of synthetic aminoacyl-adenylates as substrates in tRNA aminoacylation reaction may provide a way for incorporation of unnatural amino acids into tRNA, and consequently into proteins.