Synthesis, conformation and antiproliferative activity of isothiazoloisoxazole 1,1-dioxides.

Sixteen new isothiazoloisoxazole 1,1-dioxides, one new isothiazolotriazole and one new isothiazolopyrazole have been synthesised by using 1,3-dipolar cycloadditions to isothiazole 1,1-dioxides. One sub-set of these isothiazoloisoxazoles showed low µM activity against a human breast carcinoma cell line, whilst a second sub-set plus the isothiazolotriazole demonstrated an interesting restricted rotation of sterically hindered bridgehead substituents. A thiazete 1,1-dioxide produced from one of the isothiazole 1,1-dioxides underwent conversion into an unknown 1,2,3-oxathiazolin-2-oxide upon treatment with Lewis acids, but was inert towards 1,3-dipoles and cyclopropenones. Six supporting crystal structures are included.

The relative catalytic efficiency of beta-lactamase catalyzed acyl and phosphyl transfer.

Phosphonamidates which bear a simple resemblance to penicillin type structures have been synthesised as potential inhibitors of beta-lactamases: -ethyl N-(benzyloxycarbonyl) amidomethyl phosphonyl amides, PhCH(2)OCONHCH(2)P(O)(OEt)NR(2), the amines HNR(2) being l-proline, d-proline, l-thiazolidine, and o-anthranilic acid. The proline derivatives completely and irreversibly inactivated the class C beta-lactamase from Enterobacter cloacae P99, in a time-dependent manner, indicative of covalent inhibition. The inactivation was found to be exclusive to the class C enzyme and no significant inhibition was observed with any other class of beta-lactamase. The anthranilic acid derivative exhibited no appreciable inactivation of the beta-lactamases. The phosphonyl proline and phosphonyl thioproline derivatives were separated into their diastereoisomers and their individual second order rate constants for inhibition were found to be 7.72 +/- 0.37 and 8.3 x 10(-2) +/- 0.004 M(-1) s(-1) for the l-proline derivatives, at pH 7.0. The products of the inhibition reaction of each individual diastereoisomer, analyzed by electrospray mass spectroscopy, indicate that the more reactive diastereoisomers phosphonylate the enzyme by P-N bond fission with the elimination of proline. Conversely, gas chromatographic detection of ethanol release by the less reactive proline diastereoisomer suggests phosphonylation occurs by P-O bond fission. The enzyme enhances the rate of phosphonylation with P-N fission by at least 10(6) compared with that effected by hydroxide-ion. The pH dependence of the rate of inhibition of the beta-lactamase by the more reactive diasteroisomer is consistent with the reaction of the diprotonated form of the enzyme, EH(2), with the inhibitor, I (or its kinetic equivalents EH with IH). This pH dependence and the rate enhancement indicate that the enzyme appears to use the same catalytic apparatus for phosphonylation as that used for hydrolysis of beta-lactams. The stereochemical consequences of nucleophilic displacement at the phosphonyl centre are discussed.

Cysteinyl peptide inhibitors of Bacillus cereus zinc beta-lactamase.

Several cysteinyl peptides have been synthesised and shown to be reversible competitive inhibitors of the Bacillus cereus metallo-beta-lactamase. The pH dependence of pKi indicates that the thiol anion displaces hydroxide ion from the active site zinc(II). D,D-Peptides bind to the enzyme better than other diastereoisomers, which is compatible with the predicted stereochemistry of the active site.

Synthesis and antimicrobial activity of some isoindolin-1-ones derivatives.

A range of N-substituted isoindolin-1-ones was prepared and their potential as novel antimicrobial agents was investigated. MIC values for active compounds were determined and reported.

The reactivity of beta-lactams, the mechanism of catalysis and the inhibition of beta-lactamases.

Four membered b-lactam rings do not show unusual reactivity compared with their acyclic amide analogues and there is no evidence of concerted mechanisms for nucleophilic substitution reactions at the carbonyl centre. The identity of the general base/acid catalyst in the serine b-lactamases, which catalyse the hydrolysis of b-lactams, is unknown. There are no ideal transition state analogue inhibitors for these enzymes which involve several intermediates and transition states. The class C serine b-lactamase enhances the rate of phosphonylation of its active site serine residue by a similar magnitude to the enzyme rate enhancement factor for the hydrolysis of b-lactams. Comparisons are made between the stereochemical consequences of tetrahedral and trigonal bipyramidal intermediates for hydrolysis and phosphonylation respectively. Class B zinc b-lactamases are inhibited by thiol dipeptides with a D configuration at the cysteine centre analogous to the L configuration at C6 in penicillins. The mechanism of hydrolysis catalysed by the metallo-b-lactamases probably involves a di-anionic tetrahedral intermediate stabilised by zinc(II).

Peptidase activity of beta-lactamases.

Although beta-lactamases have generally been considered as being devoid of peptidase activity, a low but significant hydrolysis of various N-acylated dipeptides was observed with representatives of each class of beta-lactamases. The kcat/Km values were below 0.1 M(-1). s(-1), but the enzyme rate enhancement factors were in the range 5000-20000 for the best substrates. Not unexpectedly, the best 'peptidase' was the class C beta-lactamase of Enterobacter cloacae P99, but, more surprisingly, the activity was always higher with the phenylacetyl- and benzoyl-d-Ala-d-Ala dipeptides than with the diacetyl- and alpha-acetyl-l-Lys-d-Ala-d-Ala tripeptides, which are the preferred substrates of the low-molecular-mass, soluble dd-peptidases. A comparison between the beta-lactamases and dd-peptidases showed that it might be as difficult for a dd-peptidase to open the beta-lactam ring as it is for the beta-lactamases to hydrolyse the peptides, an observation which can be explained by geometric and stereoelectronic considerations.

The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent beta-lactamase.

The plot of kcat/Km against pH for the Bacillus cereus 569/H beta-lactamase class B catalysed hydrolysis of benzylpenicillin and cephalosporin indicates that there are three catalytically important groups, two of pKa 5.6+/-0.2 and one of pKa 9.5+/-0.2. Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues for catalysis. These are assigned to zinc(II)-bound water and Asp-90, both with a pKa of 5.6+/-0.2. A thiol, N-(2'-mercaptoethyl)-2-phenylacetamide, is an inhibitor of the class B enzyme with a Ki of 70 microM. The pH-dependence of Ki shows similar pH inflections to those observed in the catalysed hydrolysis of substrates. The pH-independence of Ki between pH 6 and 9 indicates that the pKa of zinc(II)-bound water must be 5.6 and not the higher pKa of 9.5. The kinetic solvent isotope effect on kcat/Km is 1.3+/-0.5 and that on kcat is 1.5. There is no effect on reactivity by either added zinc(II) or methanol. The possible mechanisms of action for the class B beta-lactamase are discussed, and it is concluded that zinc(II) acts as a Lewis acid to stabilize the dianionic form of the tetrahedral intermediate and to provide a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumably, as a general acid catalyst facilitating C-N bond fission.

Enantioselective biotransformations using rhodococci.

The use of enzymes and whole cells in enantioselective biotransformation reactions is briefly reviewed. A Rhodococcus strain is shown to possess nitrile hydratase and amidase activity. The organism can be used for the enantioselective biotransformation of racemic alpha-amino amides to (S) alpha-amino acids with an enantiomeric excess (ee) of > 98%. Enantioselectivity is effectively time independent allowing easy quantitative conversion of racemic mixtures into enantiomerically pure alpha-amino amides and alpha-amino acids. The reaction is effective for a wide range of alpha-substituents. The pH-dependence of the reaction indicates that the alpha-amino amide is bound to the amidase enzyme in its neutral unprotonated form.

The energetics of intramolecular reactions and enzyme catalysis.

The relative rates of reactions should always be examined by an awareness of differential effects. The magnitude and variation of the relative rates of intramolecular reactions can be rationalized by the differences in entropy and strain energy. The relative rates of enzyme-catalysed reactions are sometimes due to groundstate effects. The beta-lactamase-catalysed hydrolysis of beta-lactam antibiotics may require a unique disposition of catalytic groups owing to an unusual process of bond fission in the four membered ring.

Clinical significance of extreme elevation of the erythrocyte sedimentation rate.

Charts of 1006 consecutive outpatients were reviewed to ascertain the cause of extreme elevation of the erythrocyte sedimentation rate (ESR) (greater than or equal to 100 mm/h) and the sensitivity of marked ESR elevation in patients with disorders commonly reported to cause ESR elevation. Prevalence of ESRs of 100 mm/h or more was 4.2%. Infection was the most common cause (33%), with malignant neoplasms and renal disease each responsible for 17% and inflammatory disorders for 14%. Only 1% of all other patients had ESRs of 100 mm/h or more. An ESR of 100 mm/h or more had low sensitivity: 36% among patients with infection, 25% among those with malignant neoplasms, and 21% among patients with noninfectious inflammatory disorders. Specificity was high, both for individual disease categories (96% for malignant neoplasms and 97% for infection) and as a "sickness" index (greater than 99%). The positive predictive value for an identifiable cause of marked ESR elevation was 90%.

Experience with tuberculosis in a public teaching hospital.

Over a five-year period, 108 patients with active tuberculosis were admitted to the Talmadge Memorial Hospital in Augusta, Georgia. The age, sex and race distribution, case-fatality ratio, and primary drug resistance data did not differ from the national experience. The high incidence of nonpulmonary infection sites (31 percent) serves to emphasize that all hospital services and medical specialities can expect to encounter active tuberculosis. The only detected cluster of cases was from the state prison. The fact that the disease was not diagnosed or managed effectively in seven of 12 inmates emphasizes its special problem. Delays in diagnosis seemed to be unjustified in 11 patients; seven of these delays resulted from remediable human error. Four delays in diagnosis were due to difficulties that could be corrected by maintaining a greater awareness of tuberculosis and broadening the differential diagnosis to include tuberculosis among patients at high risk.

Entropic contributions to rate accelerations in enzymic and intramolecular reactions and the chelate effect.

It is pointed out that translational and (overall) rotational motions provide the important entropic driving force for enzymic and intramolecular rate accelerations and the chelate effect; internal rotations and unusually severe orientational requirements are generally of secondary importance. The loss of translational and (overall) rotational entropy for 2 --> 1 reactions in solution is ordinarily on the order of 45 entropy units (e.u.) (standard state 1 M, 25 degrees C); the translational entropy is much larger than 8 e.u. (corresponding to 55 M). Low-frequency motions in products and transition states, about 17 e.u. for cyclopentadiene dimerization, partially compensate for this loss, but "effective concentrations" on the order of 10(8) M may be accounted for without the introduction of new chemical concepts or terms.