Amino Acid Hydroxamates Research Articles

MgO-Mediated Synthesis of Fmoc-Protected Amino Acid Hydroxamates

Key words magnesium oxide - amino acids - hydroxamates - hydroxylamine hydrochloride - Fmoc acid chlorides

Structures of the Peptidoglycan N-Acetylglucosamine Deacetylase Bc1974 and Its Complexes with Zinc Metalloenzyme Inhibitors.

The cell wall peptidoglycan is recognized as a primary target of the innate immune system, and usually its disintegration results in bacterial lysis. Bacillus cereus, a close relative of the highly virulent Bacillus anthracis, contains 10 polysaccharide deacetylases. Among these, the peptidoglycan N-acetylglucosamine deacetylase Bc1974 is the highest homologue to the Bacillus anthracis Ba1977 that is required for full virulence and is involved in resistance to the host's lysozyme. These metalloenzymes belong to the carbohydrate esterase family 4 (CE4) and are attractive targets for the development of new anti-infective agents. Herein we report the first X-ray crystal structures of the NodB domain of Bc1974, the conserved catalytic core of CE4s, in the unliganded form and in complex with four known metalloenzyme inhibitors and two amino acid hydroxamates that target the active site metal. These structures revealed the presence of two conformational states of a catalytic loop known as motif-4 (MT4), which were not observed previously for peptidoglycan deacetylases, but were recently shown in the structure of a Vibrio clolerae chitin deacetylase. By employing molecular docking of a substrate model, we describe a catalytic mechanism that probably involves initial binding of the substrate in a receptive, more open state of MT4 and optimal catalytic activity in the closed state of MT4, consistent with the previous observations. The ligand-bound structures presented here, in addition to the five Bc1974 inhibitors identified, provide a valuable basis for the design of antibacterial agents that target the peptidoglycan deacetylase Ba1977.

Glycine hydroxamate inhibits tyrosinase activity and melanin contents through downregulating cAMP/PKA signaling pathways.

Among the eight amino acid hydroxamates tested, Glycine hydroxamate (GH) was the best inhibitor of mushroom tyrosinase (TYR). With L-tyrosine as substrate, the GH inhibition of the monophenolase activity of the mushroom TYR was noncompetitive. GH decreased not only TYR protein expression, but also melanin content, tyrosinase-related protein (TRP)-1, TRP-2, and microphthalmia-associated transcription factor (MITF) expression in B16F10 melanoma cells while in the presence of α-melanocyte-stimulating hormone (α-MSH). GH also significantly decreased the isobutylmethylxanthine (IBMX)-induced increase in melanin content, which was not prevented by the ERK inhibitor PD98059. These results suggest GH has the potential for use in cosmetic hypopigmentation.

Single Molecule Tracking Inside Individual Living Bacterial Cells

We have developed a single-molecule fluorescence assay to directly observe the stringent response in individual living E. coli cells. For this purpose, we have created chromosomal fusions of both RelA and three ribosomal proteins with a photo-activatable fluorescent protein.The stringent factor RelA binds to a small fraction of ribosomes, where it synthesizes the global transcriptional regulator ppGpp in response to amino acids deprivation. Our objective is to study the binding kinetics of individual RelA molecules to the ribosome in living cells and to observe how its kinetics changes during a nutritional down-shift.While E. coli contains on average about 100 RelA molecules and 20000 ribosomes, using a photo-activatable fluorescent probe we can activate only a few fluorescent molecules per cell at any given time. We induce stringent response by rapid addition of amino acid hydroxamates. Since our fluorescent tag is photoconvertible, we can repeat tracking experiments many times in the same E. coli cell.We record trajectories of individual RelA molecules diffusing in living E. coli cells with a laser exposure of 1 millisecond, a frame time of 5 milliseconds, and a spatial precision of 50 nanometers. The high resolution of the experiments makes it possible to characterize RelA binding kinetics under varying growth conditions.When the cell grows exponentially, RelA trajectories closely resemble trajectories of fluorescently tagged ribosomal proteins. After nutritional downshift, RelA binding kinetics changes rapidly. Results suggest that under amino-acid starvation, RelA is only transiently bound to the ribosome. The data is consistent with an order of magnitude drop in affinity to the ribosome.

Computational Study of the Sulfonylated Amino Acid Hydroxamates Binding to the Zinc Ion within the Active Site of Carbonic Anhydrase

The B3LYP/6–311+G(d,p) method and three ONIOM extrapolation methods ONI-OM (B3LYP/6–311+G(d,p): AM1); ONIOM(B3LYP/6–311+G(d,p): MNDO); ONIOM (B3LYP/6–311+G(d,p): HF/3-21G(d)) were used to characterize the complexes of Zn2+ cation with anionic sulfonylated amino acid hydroxamates (RSO2NH-AA-CON(-)OH), possessing an unsubstituted RSO2NH—amino acyl moiety. According to the R moiety we distinguish between pentafluorophenyl and 4-methoxyphenyl derivates. The amino acid hydroxamates included in the study were the Gly, Ala, and Leu derivates. Of the inhibitors investigated, the weakest zinc affinity exhibits the pentafluorophenyl derivate with Gly amino acid and the strongest affinity the 4-methoxyphenyl derivate with Leu amino acid. The inhibitors form bidentate coordination bonds with the zinc cation by means of the sulfonyl oxygen and the ionized hydroxamate nitrogen atoms, respectively. The zinc affinities computed using the B3LYP/6–311 +G(d,p)//HF/6–31 +G(d,p) method are in very good agreement with the full density functional theory (DFT) B3LYP/6–311+G(d,p)//B3LYP/6- 311+G(d,p) method and this method can be adopted to model larger complexes of inhibitors with the active site of carbonic anhydrase.

Design and Evaluation of Hydroxamate Derivatives as Metal-Mediated Inhibitors of a Protein Tyrosine Kinase

Protein tyrosine kinases use two Mg(2+) ions as cofactors in catalysis, one as the ATP-Mg complex (M1) and the other as an essential activator (M2). The M2-binding site has high affinity for transition metal cations such as cobalt and zinc. Taking advantage of this high affinity, we examined hydroxamates as metal-mediated inhibitors against C-terminal Src kinase (Csk), a protein tyrosine kinase. Of a small group of amino acid hydroxamates, tyrosine and phenylalanine hydroxamates inhibited Csk activity only in the presence of Co(2+). Four classes of phenylalanine and tyrosine hydroxamate derivatives were synthesized and evaluated as metal-mediated inhibitors of Csk, leading to improved inhibition and a better understanding of the structure-activity relationships. This study suggests that hydroxamates may serve as a general scaffold for developing metal-mediated inhibitors against protein tyrosine kinases. To the best of our knowledge, this is the first report of designing metal-mediated inhibitors against a protein tyrosine kinase by targeting a metal binding site.

Monohydroxamates of aspartic acid and glutamic acid exhibit antioxidant and angiotensin converting enzyme inhibitory activities.

Two monohydroxamates of l-aspartic acid beta-hydroxamate (AAH) and l-glutamic acid gamma-hydroxamate (GAH) were used for testing antioxidant and angiotensin converting enzyme (ACE) inhibitory activities in comparison with those of asparagine and glutamine, respectively. The half-inhibition concentrations, IC(50), of scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) were 36 and 48 microM and against superoxide radicals were 18.99 and 6.33 mM, respectively, for AAH and GAH. However, no activities of asparagine and glutamine were found. AAH and GAH also exhibited activities against peroxynitrite-mediated dihydrorhodamine 123 oxidations and hydroxyl radical-mediated DNA damage. For ACE inhibitory activities, the IC(50) values were 4.92 and 6.56 mM, respectively, for AAH and GAH. The ACE hydrolyzed products on the TLC chromatogram also confirmed the inhibitory activities of the two amino acid hydroxamates on ACE. When 1.23 mM AAH was added, AAH showed competitive inhibitions against ACE, and the apparent inhibition constant (K(i)) was 2.20 mM.

Direct Synthesis of Fmoc‐Protected Amino Acid Hydroxamates from Acid Chlorides Mediated by Magnesium Oxide.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Direct synthesis of Fmoc protected amino acid hydroxamates from acid chlorides mediated by magnesium oxide

The synthesis of Fmoc protected amino acid hydroxamates using Fmoc-amino acid chlorides and magnesium oxide is described. The method is simple and efficient, results in complete conversion, and gives good yields and satisfactory purity.

Hydroxyurea is a carbonic anhydrase inhibitor

The interaction of hydroxyurea with the cytosolic isozymes of carbonic anhydrase (CA), hCA I and hCA II has been investigated by means of kinetic and spectroscopic techniques. Hydroxyurea acts as a weak, non-competitive inhibitor of both isozymes, for the 4-nitrophenyl acetate esterase activity, with inhibition constants around 0.1 mM for both isozymes. The spectrum of the adduct of hydroxyurea with Co(II)-hCA II is similar to the spectra of tetrahedral adducts (such as those with sulfamide, acetazolamide or cyanamide), proving a direct interaction of the inhibitor molecule with the metal center of the enzyme, whose geometry remains tetrahedral. Based on the X-ray crystal structure of the adducts of hCA II with ureate and hydroxamate inhibitors, the hypothetical binding of hydroxyurea is proposed to be achieved in deprotonated state, with the nitrogen atom coordinated to Zn(II), and the OH group of the inhibitor making a hydrogen bond with Thr 199. This binding may be exploited for the design of both CA as well as matrix metalloproteinase (MMP) inhibitors, since hydroxyurea is the simplest compound incorporating a hydroxamate functionality in its molecule. Indeed, such inhibitors of the sulfonylated amino acid hydroxamate type have been generated, with potencies in the low nanomolar range for both type of enzymes, CAs and MMPs.

A Comparative QSAR Study on Carbonic Anhydrase and Matrix Metalloproteinase Inhibition by Sulfonylated Amino Acid Hydroxamates

A quantitative structure-activity relationship (QSAR) study is made on the inhibition of a few isozymes of carbonic anhydrase (CA) and some matrix metalloproteinases (MMPs), both zinc containing families of enzymes, by sulfonylated amino acid hydroxamates. For both enzymes, the inhibition potency of the hydroxamates is found to be well correlated with Kier's first-order valence molecular connectivity index 1χv of the molecule and electrotopological state indices of some atoms. From the results, it is suggested that while hydroxamate-CA binding may involve mostly polar interactions, hydroxamate-MMP and hydroxamate-ChC (ChC: Clostridium histolyticum collagenase, another zinc enzyme related to MMPs) bindings may involve some hydrophobic interactions. Both MMPs and ChC also possess some electronic sites of exactly opposite nature to the corresponding sites in CAs. A group such as C 6 F 5 present in the sulfonyl moiety is shown to be advantageous in both CA and MMP (also ChC) inhibitions, which is supposed to be due to the interaction of this group with Zn 2+ ion present in the catalytic site of both families of enzymes.

Carbonic Anhydrase and Matrix Metalloproteinase Inhibitors: Sulfonylated Amino Acid Hydroxamates with MMP Inhibitory Properties Act as Efficient Inhibitors of CA Isozymes I, II, and IV, and N-Hydroxysulfonamides Inhibit Both These Zinc Enzymes

Carbonic Anhydrase and Matrix Metalloproteinase Inhibitors: Sulfonylated Amino Acid Hydroxamates with MMP Inhibitory Properties Act as Efficient Inhibitors of CA Isozymes I, II, and IV, and <i>N</i>-Hydroxysulfonamides Inhibit Both These Zinc Enzymes

Carbonic anhydrase and matrix metalloproteinase inhibitors: sulfonylated amino acid hydroxamates with MMP inhibitory properties act as efficient inhibitors of CA isozymes I, II, and IV, and N-hydroxysulfonamides inhibit both these zinc enzymes.

The 14 different carbonic anhydrase (CA, EC 4.2.1.1) isozymes as well as the 23 different matrix metalloproteinases (MMPs) isolated up to now in higher vertebrates play important physiological functions in these organisms. Unsubstituted sulfonamides act as high-affinity inhibitors for the first type of these enzymes, whereas hydroxamates strongly inhibit the latter ones. Since the active site geometry around the zinc ion in these two types of metalloenzymes is rather similar, we tested whether sulfonylated amino acid hydroxamates of the type RSO(2)NX-AA-CONHOH (X = H, benzyl, substituted benzyl; AA = amino acid moiety, such as Gly, Ala, Val, Leu) with well-known inhibitory properties against MMPs and Clostridium histolyticum collagenase (ChC, another zinc enzyme related to the MMPs) might also act as CA inhibitors. We also investigated whether N-hydroxysulfonamides of the type RSO(2)NHOH (which are effective CA inhibitors) inhibit MMPs and ChC. Here we report several potent sulfonylated amino acid hydroxamate CA inhibitors (with inhibition constants in the range of 5-40 nM, against the human isozymes hCA I and hCA II, and 10-50 nM, against the bovine isozyme bCA IV), as well as preliminary SAR for this new class of non-sulfonamide CA inhibitors. Some N-hydroxysulfonamides also showed inhibitory properties (in the micromolar range) against MMP-1, MMP-2, MMP-8, MMP-9, and ChC. Thus, the SO(2)NHOH group is a new zinc-binding function for the design of MMP inhibitors. Both CA as well as MMPs are involved, among others, in carcinogenesis and tumor invasion processes. On the basis of these findings, we suggest that the mechanism of antitumor action with some hydroxamate inhibitors might also involve inhibition of some CA isozymes (such as CA IX, CA XII, and CA XIV) present only in tumor cell membranes, in addition to collagenases/gelatinases of the MMP type. Our data also suggest that it should be possible to develop dual enzyme inhibitors that would strongly inhibit both these metalloenzymes, CAs and MMPs, based on the nature of the R, AA, and X moieties in the above formula. Compact X (such as H) and AA (such as Gly) moieties favor CA over MMP inhibition, whereas bulkier X (benzyl, substituted benzyl, etc.) and AA (such as Val, Leu) moieties and substituted-aryl R groups are advantageous for obtaining potent MMP and ChC inhibitors, which show lower affinity for CA.

Substrate analogues and inhibition of ACC oxidase: conversion of D-valine to iso-butanal

Amino acids and their hydroxamates were studied as inhibitors of purified recombinant 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (tomato). Neither L-valine, L-alanine, L- nor D-isoleucine displayed inhibition, but D-valine and D-alanine were weak inhibitors of ACC oxidase catalysed conversion of ACC to ethylene. GC-MS analysis showed that D-valine was converted to iso-butanal. In contrast to studies using native ACC oxidase (apple), inhibition of recombinant ACC oxidase (tomato) by amino acid hydroxamates appears to result (at least predominantly) from chelation of free iron in solution.

Lethal factor active-site mutations affect catalytic activity in vitro.

The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093-1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase.

ChemInform Abstract: A Convenient Procedure for the Preparation of Amino Acid Hydroxamates from Esters.

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A Convenient Procedure for the Preparation of Amino Acid Hydroxamates from Esters

A Convenient Procedure for the Preparation of Amino Acid Hydroxamates from Esters

Volatile BSTFA Derivatives of Amino Acid Hydroxamates

Abstract N,O-bis (trimethylsilyl) trifluoracetamide (BSTFA) was used to derivatize D,L-α-amino-n-butyric acid hydroxamate, D,L-valine hydroxamate, and L-leucine hydroxamate. A gas chromatogram of the silylated derivative was obtained. The structures of the disubstituted derivatives were confirmed by NMR.

Statistical Mixture Design Applied to RP-HPTLC Chromatographic Separations of Amino Acid Hydroxamates

Abstract Mixture Design and a Chromatographic Response Function were used to study the effect of different organic solvents as modifiers on the Reversed Phase (C-18) High Performance Thin Layer Chromatographic separation of three amino acid hydroxamates (Arginine hydroxamate, Threonine hydroxamate, and Histidine hydroxamate). Both 3 solvent (methanol, acetonitrile and tetrahydrofuran) and 4 solvent (methanol, acetonitrite, tetrahydrofuran and acetone) systems were studied.

N-Peptidyl- O-carbamoyl amino acid hydroxamates: Irreversible inhibitors for the study of the S 2′ specificity of cysteine proteinases

A series of new inhibitors for cysteine proteinases with the general structure Z-Phe-Gly-NHO-CO-Aa (Aa = amino acids) was synthesized and tested as inhibitors of papain-like enzymes (cathepsins S, L, B and papain). Like N-peptidyl- O-acyl hydroxamates the inhibitors inactivate cysteine proteinases by a sulfenamidation of the active site cysteine residue. The most effective inhibitors display second order-rate constants of inactivation in the range of 10 3–10 4 M −1·s −1. Since the structure of the N-peptidyl- O-carbamoyl amino acid hydroxamates allows the variation of the leaving group this class of inhibitors was used as a new tool for the evaluation of the S 2′ specificity of cysteine proteinases.