Substituted aryl malonamates as new serine beta-lactamase substrates: structure-activity studies.

A series of substituted aryl malonamates have been prepared. These compounds are analogues of aryl phenaceturates where the amido side chain has been replaced by a retro-amide. Like the phenaceturates, these compounds are substrates of typical class A and class C beta-lactamases, particularly of the latter, and of soluble DD-peptidases. The effect of substituents alpha to the ester carbonyl group on turnover by these enzymes is similar to that in the phenaceturates. On the other hand, N-alkylation of the side chain amide of malonamates, but not of phenaceturates, retains the susceptibility of the compounds to hydrolysis by beta-lactamases. This reactivity is not enhanced, however, by bridging the amide nitrogen and Calpha atoms. A phosphonate analogue of the malonamates was found to be an irreversible inhibitor of the beta-lactamases. These results, therefore, provide further evidence for the covalent access of compounds bearing retro-amide side chains to the active sites of beta-lactam-recognizing enzymes.

Synthesis and beta-lactamase reactivity of alpha-substituted phenaceturates.

Beta-lactams with 6alpha (penicillins) or 7alpha (cephalosporins) substituents are often beta-lactamase inhibitors. This paper assesses the effect of such substituents on acyclic beta-lactamase substrates. Thus, a series of m-carboxyphenyl phenaceturates, substituted at the glycyl alpha-carbon by -OMe, -CH(2)OH, -CO(2)(-), and -CH(2)NH(3)(+), have been prepared, and tested for their reactivity against serine beta-lactamases. The latter two are novel substituents in beta-lactamase substrates. The methoxy and hydroxymethyl compounds were found to be poor to moderately good substrates, depending on the enzyme. The aminomethyl compound gave rise to a transiently stable (t(1/2)=4.6s) complex on its reaction with a class C beta-lactamase. The reactivity of the compounds against three low molecular weight DD-peptidases was also tested. Again, the methoxy and hydroxymethyl compounds proved to be quite good substrates with no sign of inhibitory complexes. The DD-peptidases reacted with one enantiomer (the compounds were prepared as racemates), presumably the D compound. The class C beta-lactamase reacted with both D and L enantiomers although it preferred the latter. The structural bases of these stereo-preferences were explored by reference to the crystal structure of the enzyme by molecular modeling studies. The aminomethyl compound was unreactive with the DD-peptidases, whereas the carboxy compound did not react with any of the above-mentioned enzymes. The inhibitory effects of the -OMe and -CH(2)OH substituents in beta-lactams apparently require a combination of the substituent and the pendant leaving group of the beta-lactam at the acyl-enzyme stage.

Synthesis and reactivity with beta-lactamases of a monobactam bearing a retro-amide side chain.

The monobactam sodium 3-benzylcarbamoyl-2-oxo-1-azetidinesulfonate, bearing a retro (vs classical beta-lactam)-amide side chain, has been synthesized and the kinetics of its reaction with typical beta-lactamases studied. The new compound is generally a poorer substrate than the analogous compound with a normal side chain but its formation of a transiently stable complex with a class C beta-lactamase sustains the retro-amide side-chain concept.

Benzopyranones with retro-amide side chains as (inhibitory) beta-lactamase substrates.

3-(N-Benzylcarbamoyl)-7-carboxy-3, 4-dihydro-2H-1-benzo-pyran-2-one and its 8-carboxy analogue have been synthesized and evaluated as potential (inhibitory) substrates of beta-lactam-recognizing enzymes. These compounds are bicyclic delta-lactones with retro-amide (with respect to classical beta-lactams) side chains. They were found to be comparably effective as substrates of typical class A, C and D beta-lactamases as analogous benzopyranones bearing 'normal' amide side chains. The new 8-carboxy derivative, however, formed a much more (1000-fold) tightly-bound acyl-enzyme with a class C beta-lactamase than did its 'normal' analogue, and thus provides a structural lead to new inhibitors of this class of beta-lactamase.

New substrates for beta-lactam-recognizing enzymes: aryl malonamates.

Aryl malonamates are demonstrated to be novel substrates of a broad range of beta-lactam-recognizing enzymes. These compounds are isomers of the aryl phenaceturates, which are well-known substrates of these enzymes, but the new compounds contain a retro-amide side chain. Several lines of evidence, including comparisons of steady-state kinetic parameters between enzymes and a detailed investigation of the methanolysis kinetics, solvent deuterium isotope effects, and pH-rate profile for turnover of a retro substrate by the Enterobacter cloacae P99 beta-lactamase, suggested that the new substrates are likely to be hydrolyzed by the same chemical mechanisms as "normal" substrates. Molecular modeling indicated that the retro-amide group fits snugly into the active site of the P99 beta-lactamase by hydrogen bonding to the conserved lysine-67 residue. The retro-amide side chain may represent a lead to novel mechanism-based and transition state analogue inhibitors.

The synthesis and evaluation of benzofuranones as beta-lactamase substrates.

6- and 7-Carboxy-3-phenylacetamido-3H-1-benzofuran-2-one have been synthesized as potential beta-lactamase substrates and/or inhibitors. These compounds were prepared by lactonization of the corresponding, appropriately substituted phenylglycines. The latter compounds were prepared by either the Strecker or the Bücherer-Berg method. The benzofuran-2-ones were less stable in aqueous solution than the analogous acyclic phenaceturate esters but comparably stable to analogous benzopyran-2-ones. They differed from the latter compounds however in that the C-3 hydrogen of the furan-2-ones, adjacent to the lactone carbonyl group, was distinctly acidic; 7-carboxy-3-phenylacetamido-3H-1-benzofuran-2-one exists largely as an enolate at pH 7.5. The furan-2-ones were beta-lactamase substrates with reactivity very similar to the analogous acyclic phenaceturates. They were not, however, DD-peptidase inhibitors and are thus unlikely to have antibiotic activity. The structural basis for these observations is discussed.

CIDEP effects of intramolecular quenching of singlet and triplet excited states by nitroxide radicals in oligopeptides: a potentially useful new method for investigating peptide secondary structures in solution.

Two hexapeptides, each bearing one photoactive alpha-amino acid (Bin or Bpa) and one nitroxide-containing TOAC residue, have been synthesized and fully characterized. FT-IR absorption measurements indicate that a 3(10)-helical conformation is adopted by these peptides in solution. As two amino acid units separate the photoactive residue from TOAC in the peptide sequences, the two moieties face each other at a distance of about 6 A after one complete turn of the ternary helix. Irradiation by a light pulse from an excimer laser populates the excited states localized on the chromophores. An intramolecular interaction between the singlet (Bin) or triplet (Bin and Bpa) excited states and the doublet state of the TOAC nitroxide makes a spin-selective decay pathway possible, that produces transient spin polarization. In addition, in order to determine whether the intramolecular exchange interaction occurs through-bond or through-space, we have prepared linear and cyclic TOAC-Bin dipeptide units. A CIDEP study revealed that a through-space intramolecular interaction is operative. The observation of spin polarization makes the two helical hexapeptides suitable models to test the possibility of application of this novel technique to conformational studies of peptides in solution.

High-performance liquid chromatographic separation of novel atropic alpha,alpha-disubstituted-beta-amino acids, either on different beta-cyclodextrin-bonded phases or as their 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide derivatives.

The high-performance liquid chromatographic enantioresolution of free and N- and/or C-protected derivatives of (R,S)-2',1':1,2;1",2":3,4-dinaphthcyclohepta-1,3-diene-6-aminometh yl-6- carboxylic acid (beta 2-Bin) by direct and indirect methods is reported. The direct separation was carried out on native and different derivatized beta-cyclodextrin-bonded phases. The indirect resolution was achieved by applying pre-column derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide. The effects of different parameters such as the mobile phase composition and the structures of the compounds on the enantiomeric resolution are discussed.

Synthesis, conformational study, and spectroscopic characterization of the cyclic C alpha, alpha-disubstituted glycine 9-amino-9-fluorenecarboxylic acid.

A series of terminally blocked peptides (to the pentamer level) from L-Ala and the cyclic C alpha, alpha-disubstituted Gly residue Afc and one Gly/Afc dipeptide have been synthesized by solution method and fully characterized. The molecular structure of the amino acid derivative Boc-Afc-OMe and the dipeptide Boc-Afc-Gly-OMe were determined in the crystal state by X-ray diffraction. In addition, the preferred conformation of all of the model peptides was assessed in deuterochloroform solution by FT-IR absorption and 1H-NMR. The experimental data favour the conclusion that the Afc residue tends to adopt either the fully-extended (C5) or a folded/helical structure. In particular, the former conformation is highly populated in solution and is also that found in the crystal state in the two compounds investigated. A comparison with the structural propensities of the strictly related C alpha, alpha-disubstituted Gly residues Ac5c and D phi g is made and the implications for the use of the Afc residue in conformationally constrained analogues of bioactive peptides are briefly examined. A spectroscopic (UV absorption, fluorescence, CD) characterization of this novel aromatic C alpha, alpha-disubstituted Gly residue is also reported.

A "cephalosporin-like" cyclic depsipeptide: synthesis and reaction with beta-lactam-recognizing enzymes.

The cyclic depsipeptide 8-carboxy-3-phenylacetamido-3,4-dihydro-2H-1-benzopyran-2-one, a cyclic analog of aryl phenaceturates with structural similarity to cephalosporins, has been synthesized as a potential substrate/inhibitor of B-lactam-recognizing enzymes. It was found to be a tight-binding, poor substrate of class A beta-lactamases and an irreversible inhibitor of several DD-peptidases.

Synthesis and Reactivity with beta-Lactamases of "Penicillin-like" Cyclic Depsipeptides.

Several 7-carboxy-3-amido-3,4-dihydro-2H-1-benzopyran-2-ones have been synthesized as potential beta-lactamase substrates and/or mechanism-based inhibitors. Substituted o-tyrosine precursors were prepared by the Sörensen method and then heated in vacuo to give the lactones. These compounds are cyclic analogues of aryl phenaceturates which are known to be beta-lactamase substrates. The goal of incorporating the scissile ester group into a lactone was to retain the leaving group tethered to the acyl moiety at the acyl-enzyme stage of turnover by serine beta-lactamases, in a manner similar to that during penicillin turnover. Further, in two cases, a functionalized methylene group para to the leaving group phenoxide oxygen was incorporated. These molecules possess a latent p-quinone methide electrophile which could, in principle, be unmasked during enzymic turnover and react with an active site nucleophile. All of these compounds were found to be substrates of class A and C beta-lactamases, the first delta-lactones with such activity. Generally, k(cat) values were smaller than for the analogous acyclic depsipeptides, which suggests that the tethered leaving group may obstruct the attack of water on the acyl-enzymes. Further exploration of this structural theme might lead to quite inert acyl-enzymes and thus to significant inhibitors. Despite the apparent advantage offered by the longer-lived acyl-enzymes, the functionalized compounds were no better as irreversible inhibitors than comparable acyclic compounds [Cabaret, D.; Liu, J.; Wakselman, M.; Pratt, R. F.; Xu, Y. Bioorg. Med. Chem. 1994, 2, 757-771]. Thus, even tethered quinone methides, at least when placed as dictated by the structures of the present compounds, were unable to efficiently trap a nucleophile at serine beta-lactamase active sites.

Dipeptidyl-peptidase IV-beta--further characterization and comparison to dipeptidyl-peptidase IV activity of CD26.

Dipeptidyl peptidase IV-beta (DPP IV-beta) is a novel protein which shows a peptidase activity similar to the T-cell-activation antigen CD26. To further characterize this DPP IV-beta and confirm its cell surface expression, we have developed a purification strategy using the CD26- cell line C8166. The purification process includes biotinylation of cell surface proteins before preparation of cell extracts and processing by gel-filtration, ion-exchange and lectin chromatographies. Consistent with the molecular mass of DPP IV-beta estimated by gel-filtration chromatography, the final purified fraction, manifesting a typical DPP IV activity, showed a major biotinylated 75-80-kDa band in SDS/PAGE, thus suggesting the monomeric nature of this enzyme. Kinetic parameters of DPP IV-beta and the sensitivity to a new family of irreversible DPP IV inhibitors, were studied in comparison to CD26. Both enzymes followed a Michaelis kinetics with different Km values for Gly-Pro-NH-Np (NH-Np, para-nitroanilide) hydrolysis (0.28+/-0.05 mM and 0.12+/-0.02 mM). More significant differences were observed in the sensitivity to inhibitors, which exerted a much higher activity on CD26 than on DPP IV-beta. These differences permitted us to study DPP IV-beta expression in CD26-expressing cells, showing the expression of this new enzyme in all lymphoid cells tested, and a rapid enhancement in phytohemagglutinin-stimulated or protein-A-stimulated peripheral blood mononuclear cells. Our results indicate that, although DPP IV-beta and CD26 are coexpressed and manifest a typical DPP IV activity, there are distinct features in their catalytic activities that may confer to each enzyme a complementary role in peptide processing.

Specific and irreversible cyclopeptide inhibitors of dipeptidyl peptidase IV activity of the T-cell activation antigen CD26.

The dipeptidyl peptidase IV (DPP IV) activity of CD26 is characterized by its post-proline-cleaving capacity that plays an important but not yet understood role in biological processes. Here we describe a new family of specific and irreversible inhibitors of this enzyme. Taking into account the substrate specificity of DPP IV for P2-P1><-P1' cleavage, we have designed and synthesized cyclopeptides c[(alphaH2N+)-Lys-Pro-Aba-(6-CH2-S+R2)-Glyn] 2TFA- (Aba = 3-aminobenzoic acid, R = alkyl) possessing a proline at the P1 position and a lysine in the P2 position, which allows the closing of the cycle on its side chain. These molecules show a free N-terminus, necessary for binding to the CD26 catalytic site, and a latent quinoniminium methide electrophile, responsible for inactivation. Treatment of c[alphaZ-Lys-Pro-Aba-(6-CH2-OC6H5)-Glyn], obtained by peptide synthesis in solution, with R2S/TFA simutaneously cleaved the Z protecting group and the phenyl ether function and led to a series of cyclopeptide sulfonium salts. These cyclopeptides inhibited rapidly and irreversibly the DPP IV activity of CD26, with IC50 values in the nanomolar range. Further studies were carried out to investigate the effect of the modification of the ring size (n = 2 or 4) and the nature of the sulfur substituents (R = Me, Bu, Oct). Cycle enlargement improved the inhibitory activity of the methylsulfonio cyclopeptide, whereas the increase of the alkyl chain length on the sulfur atom had no apparent effect. Other aminopeptidases were not inhibited, and a much weaker activity was observed on a novel isoform of DPP IV referred to as DPP IV-beta. Thus, this new family of irreversible inhibitors of DPP IV is highly specific to the peptidase activity of CD26.

Synthesis and inhibition of human leucocyte elastase by functionalized N-aryl azetidin-2-ones: effect of different substituents on the aromatic ring.

N-aryl-3,3-difluoroazetidin-2-ones featured by a latent electrophilic methylene quinoniminium function have been synthesized and evaluated as inhibitors of human leucocyte elastase. To promote hydrophobic interactions with the enzyme, to increase the rates of beta-lactam ring opening and of benzylic group departure, or to induce hydrosolubility, these compounds incorporate on their aromatic ring either an alkyl moiety, a methoxy substituent or a carboxylic group. Some of these beta-lactams proved to be good inactivators of human leucocyte elastase.

Liquid-crystal properties of octyl 6'-O-alkylmelibiosides.

6-O-alpha-D-Galactopyranosyl-D-glucopyranose (melibiose) derivatives with alkyl groups at the terminal 1-O and 6'-O positions have been synthesized. They show thermotropic and lyotropic liquid-crystal properties. The d-spacings of the strong inner X-ray diffraction rings correspond to approximately 0.9 times the extended length of the molecule. The molecules are therefore either extended in monomolecular layers or U-shaped in bimolecular layers.

Functionalized depsipeptides, substrates and inhibitors of beta-lactamases and DD-peptidases.

A series of derivatives of phenyl phenylacetylglycinates (aryl phenaceturates) with a carboxylate substituent meta to the oxygen of the phenoxide leaving group and a functionalized methylene group in the ortho- or para-position have been synthesized. These molecules possess a latent o- or p-quinone methide electrophile which could be unmasked during enzymic turnover and could react with an active site nucleophile. This chemistry does seem to occur in solution where a common hydrolysis product, independent of the benzylic leaving group, presumably o- or p-hydroxymethylphenol, was observed. These depsipeptides are substrates of class A and C beta-lactamases, particularly of the latter, comparable with the parent m-carboxyphenyl phenaceturate. They also have modest inhibitory activity against these enzymes and against the serine DD-peptidase of Streptomyces R61. The inhibition of a class C beta-lactamase was turnover dependent, as expected of mechanism-based inhibitor, but the small leaving group dependence of the inhibition suggested that the quinone methide, if it was in fact responsible for the inhibition, was generated in solution subsequent to release of the product phenol from the active site.

Urethane protected amino acid N-carboxyanhydrides and fluorides (U-NCAs and U2AAFs).

In order to obtain peptide analogues containing a central pyrrolide bond, as potential mechanism-based inhibitors of the HIV-1 proteinase, activated derivatives of amino acids were required. Treatment of a N,N-bis(Boc) amino acid pyridinium salt with cyanuric fluoride in dichloromethane furnished the correspondingbis(Boc) amino acid fluoride (Boc2AAF). Use of the Vilsmeier reagent in acetonitrile, instead of the cyanuric fluoride, led to a N-Boc amino acid N-carboxyanhydride (Boc-NCA). From a mixed N-Z,N-Boc amino acid salt a N-Z,N-Boc amino acid fluoride and a Z-NCA were respectively obtained. The very sensitive Young test showed that during the coupling of the N-benzoyl-L-Leucine N-carboxyanhydride or the N-benzoyl N-Boc-L-leucyl fluoride with ethyl glycinate the degrees of racemization were weak. Owing to the electronegativity and the small size of the fluorine atom, thebis(urethane) amino acid fluorides are efficient acylating agents for amines and pyrrole anions.

New mechanism-based inactivators of trypsin-like proteinases. Selective inactivation of urokinase by functionalized cyclopeptides incorporating a sulfoniomethyl-substituted m-aminobenzoic acid residue.

In order to obtain selective suicide substrates of trypsin-like proteases including plasminogen activators, plasmin, and thrombin, a series of cyclopeptides cyclo[Arg or Lys-aB(CH2X)-Gly4], in which a substituted o- or m-aminobenzoyl group constitutes a latent electrophile, have been prepared. Treatment of the corresponding phenyl ethers cyclo[P1-aB(CH2OC6H5)-Gly4] with HBr/HOAc or R1R2S/TFA gives the bromides (X = Br) or the sulfonium salts (X = +SR1R2 with R1 = R2 = Me or R1 = Me and R2 = C6H5), respectively. These water-soluble cyclopeptides behave as time-dependent inhibitors of bovine trypsin and human urokinase (u-PA) but have no effect on tissue plasminogen activator (t-PA) and no or poor effect on plasmin and thrombin. The compounds containing a m-aminobenzoic acid residue are more efficient inactivators than their anthranilic analogues. The kinetic criteria expected for a suicide inhibition are met. A mechanism of inhibition involving the formation of a quinonimmonium methide intermediate is proposed. The activity of the inhibitors is very sensitive to the nature of the X benzylic substituent. An increased efficiency for the inactivation of human urokinase is observed with the sulfonium salts. The selectivity of the inactivation of u-PA compared to t-PA could be of therapeutical significance in controlling cell proliferation and invasion.

Protection of vascular basement membrane and microcirculation from elastase-induced damage with a fluorinated beta-lactam derivative.

N-(2-chloromethylphenyl) 3,3-difluoroazetidin-2-one (AA 231-1), a specific suicide-type inhibitor of elastase which is known to suppress the lysis of chromogenic oligopeptides, elastin and elastic fibers, is effective also in preventing the degradation of the vascular basement membrane. The degradation of porcine glomerular basement membrane by purified human leukocyte elastase (HLE), was reduced in proportion of inhibitor dose (8.3 microM for 50% inhibition). It is noteworthy that there was no reduction of the inhibitory effect when the addition of AA 231-1 was delayed for 1 h after the addition of the enzyme to the substrate. In the guinea pig, reduction of the dermal microhemorrhage due to HLE was related to the dose of inhibitor and to its preincubation time with HLE before intradermal injection. The inflammatory hemorrhage associated with the Arthus skin reaction was moderately depressed by AA 231-1 in situ. A part of the vascular permeability induced by HLE also responded to the inhibitor. In spite of the tissular diffusion and the time-dependence parameters which restrict responsiveness of elastase to AA 231-1 in vivo this biochemical compound should be helpful in the study and possibly the cure of vascular injury related to elastase.