Theoretical studies on β-lactam antibiotics. IV. Conformational analysis of novel β-lactam antibiotics and the binding specificities of crosslinking enzyme(s) and β-lactamases

Conformational-energy calculations were carried out on the new family of β-lactam antibiotics (viz., thienamycin, PS-5, 1-oxa- and 1-thiapenems, and their close analogs); these exhibit broad-spectrum antibacterial activity and stability towards β-lactamase-producing strains. The bicyclic ring system in all the compounds studied was found to be highly rigid and to favor only one conformation. This is in contrast to findings in penicillins, where the five-membered ring assumes two puckered conformations. The relative orientations of the bicyclic ring system and the nature and configuration of the substituent at C-5 position, besides nonplanarity of the lactam peptide bond, are shown to be important for biological activity. The present study, in agreement with x-ray studies, predicts that the lactam peptide bond in 1-carbapenem is more nonplanar than in 1-thiapenem. These studies also suggest that the conformational requirement of bicyclic ring system to bind to crosslinking enzyme(s) and β-lactamases is very similar.     

X-Pro peptides. A theoretical study of the hydrogen bonded conformations of (α-aminoisobutyryl-l-prolyl)n sequences

Intramolecularly hydrogen bonded conformations of (Aib-Pro)_n sequences have been analysed theoretically. Both 4→1 (C₁₀ and 3→1 (C₇ hydrogen bonded regular structures are shown to be stereochemically feasible. Conformational energies for the helical structures have been estimated using classical potential energy methods. Both C₁₀ and C₇ conformations have very similar energies. Pyrrolidine ring puckering has a pronounced effect on the energies, and only Cγ-endo puckered Pro residues can be accommodated. The theoretical calculations using spectroscopic data suggest that the recently proposed novel 3₁₀ helical conformation for benzyloxycarbonyl(Aib-Pro)₄-methyl ester is in solution, is indeed energetically and stereochemically favourable.     

4-Membered metallodithiophosphinate rings - flat or puckered? A comparison of two crystal structures with computational and literature data

Two crystalline forms of (dithiodiphenylphosphinate)(phenyl)(triphenylphosphine)-palladium(II) (C₃₆H₃₀P₂PdS₂), one without solvent, the other containing THF (C₄H₈O), are obtained after reaction of sodium diphenyldithiophosphinate with (phenyl) (bis-triphenylphosphine) palladium(II) chloride and crystallisation from two different solvent mixtures. The molecular structures, as determined by single crystal X-ray diffraction, differ in the planarity of the 4-membered palladium dithiophosphinate rings. The experimental conformations have been compared to the conformations of four-membered metal-S₂P rings reported in the Cambridge Structural Database. A flat conformation is more common than a puckered one. DFT calculations at the B3LYP level of theory indicate that the flat conformation of a model metallodithiophosphinate ring is very slightly lower in energy (1.2 kcal/mol) than the puckered conformation.     

Kinetic and microstructural characterization of immobilized penicillin acylase from Streptomyces lavendulae on Sepabeads EC-EP

Recombinant penicillin acylase from Streptomyces lavendulae was covalently bound to epoxy-activated Sepabeads EC-EP303®. Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, as shown by confocal electron microscopy. The optimal immobilized biocatalyst had a specific enzymatic activity of 26.2IUgwetcarrier^?1 in the hydrolysis of penicillin V at pH 8.0 and 40°C. This biocatalyst showed the highest activity at pH 8.5 and 65°C, 1.5 pH units lower and 5°C higher than its soluble counterpart. Substrate specificity of the derivative also showed its ability to efficiently hydrolyze other natural aliphatic penicillins such as penicillins K, F and dihydroF. The immobilized enzyme was highly stable at 40°C and pH 8.0 (t_1/2=625 h vs. t_1/2=397 h for the soluble enzyme), and it could be recycled for at least 30 consecutive batch reactions without loss of catalytic activity.     

The stereochemistry of biosynthesis of decaprenoxanthin in a cell-free system

Intact cells of Flavobacterium dehydrogenans grown on glucose or acetate did not incorporate mevalonic acid-[¹⁴C]. After treatment with lysozyme the protoplasts were lysed by sonication in a dilute medium containing mevalonic acid-[¹⁴C] and the cell-free system produced incorporated label into uncyclized C₄₀, monocyclic C₄₅ and bicyclic C₅₀ carotenoids of which decaprenoxanthin was the most abundant.With mevalonate-[2-¹⁴C,4R-4-³H₁] the ¹⁴C:³H ratios of the carotenoids showed that the hydrogen atoms at C-2 and C-6 of the ring and that at C-3 of the 1-hydroxy, 2-methyl but-2-ene-4-yl residues of decaprenoxanthin were derived from the 4-pro-R hydrogen atom of mevalonic acid.Mevalonate-[2-¹⁴C,2R-2-³H₁] and mevalonate-[2-¹⁴C,2S-2-³H₁] gave ratios which showed that the C-4 hydrogen atoms of decaprenoxanthin were derived from the 2-pro-S hydrogen atom of mevalonic acid.     

Nuclear magnetic resonance study of ring conformations of cyclic tetradepsipeptide AM-toxins and related peptides

Cyclic tetradepsipeptides, AM-toxin I and II, are the host-specific phytotoxins of Alternaria mali. In order to elucidate conformation-toxicity relationships, we analyzed the 270-MHz proton nmr spectra of AM-toxins and hydrogenated analogs, (D -Ala²)AM-toxin I (toxic) and (L -Ala²)AM-toxin I (not toxic), in (C²H₃)₂SO. These cyclic tetradepsipeptides do not contain N-substituted amino acid residues, and all the peptide and ester groups have been found to be transoid. Two conformers with very unequal populations have been found for AM-toxin I and II; the C^β?C^α? C?O conformations of the Dha² residues are nonplanar S-trans in the major conformer and nonplanar S-cis in the minor conformer. Only one ring conformation has been found for each of (L -Ala²) and (D -Ala²)AM-toxin I. (L -Ala²)AM-toxin I takes a C₄-type ring conformation; all the C?O groups and C^α-H bonds are oriented to the same side of the ring. (D -Ala²)AM-toxin I takes a new ring conformation; the side chain and C?O group of the L -Amp¹ residue are oriented to the same side of the ring. This new conformation is also found for the major conformers of AM-toxin I and II and thus appears to be required for the toxicity. The ring conformations of Tyr(OCH₃)¹-bearing analog tetradepsipeptides have been found to be much the same as those of Amp¹-bearing depsipeptides. Furthermore, on the basis of the two distinct conformations of (D -Ala²) and (L -Ala²)AM-toxin I, an empirical rule is proposed for the stable ring conformations of cyclic tetra-D ,L -peptides, not containing N-substituted amino acid residues.     

Molecular structure of the cis-syn photodimer of d(TpT) (cyanoethyl ester)

The three-dimensional structure was determined by x-ray crystallography for d(T[p](CE)T), a uv photoproduct of the cyanoethyl (CE) derivative of d(TpT), having the cis-syn cyclobutane (CB) geometry and the S-configuration at the chiral phosphorus atom. The crystals of C₂₃H₃₀N₅O₁₂P · 2H₂O belong to the orthorhombic space group P2₁2₁2₁ (Z = 4), with cell dimensions a = 11.596 Å, b = 14.834 Å, and c = 15.946 Å, containing two water molecules per asymmetric unit. The CB ring is puckered with a dihedral angle of 151°. The two pyrimidine bases are rotated by –29° from the position of direct overlap of their corresponding atoms. This represents a major distortion of DNA, since in DNA adjacent thymines are rotated by +36°. The pyrimidine rings are puckered with Cremer–Pople parameters for T[p] and in parentheses [p]T: Q: 0.24 Å (0.31 Å); θ: 123° (120°); ?: 141° (86°). These represent half-chairs designated as ⁶H₁ (T[p]) and ₆H⁵ ([p]T). The CB and pyrimidine ring conformations are interrelated, and we postulate that they execute a coupled interconversion in solution. The T[p] segment has the syn glycosyl conformation, a ²T₃ sugar pucker, and gauche^? conformation at C4′-C5′; the [p]T segment is anti, ³T₄, trans. The C5′-O5′ torsion of the [p]T unit is –124.5°, and the C3′-O3′ torsion of the T[p] unit is –152.9°. Bond angles and bond lengths involving the phosphorus atom are similar to those of other phosphotriesters. The P-O3′ and P-05′ torsion angles are –138.1° and 58.6°, respectively. Several intermolecular (but no intramolecular) hydrogen bonds are found in the crystal.     

Effect of the sulfur atom on S2 and S4 positions of the uracil ring in different DNA:RNA hybrid microhelixes with three nucleotide base pairs

The effect of the sulphur atom on the uracil ring was analyzed in different DNA:RNA microhelixes with three nucleotide base-pairs, including uridine, 2-thiouridine, 4-thiouridine, 2,4-dithiouridine, cytidine, adenosine and guanosine. Distinct backbone and helical parameters were optimized at different density functional (DFT) levels. The Watson-Crick pair with 2-thiouridine appears weaker than with uridine, but its interaction with water molecules appears easier. Two types of microhelixes were found, depending on the H-bond of H2′ hydroxyl atom: A-type appears with the ribose ring in ³E-envelope C_3′-endo, and B-type in ²E-envelope C_2′-endo. B-type is less common but it is more stable and with higher dipole-moment. The sulphur atoms significantly increase the dipole-moment of the microhelix, as well as the rise and propeller twist parameters. Simulations with four Na atoms H-bonded to the phosphate groups, and further hydration with explicit water molecules were carried out. A re-definition of the numerical value calculation of several base-pair and base-stacking parameters is suggested.     

Conformational energy calculations of enzyme-substrate complexes of lysozyme. I. Energy minimization of monosaccharide and oligosaccharide inhibitors and substrates of lysozyme

Conformational energy calculations were performed on monosaccharide and oligosaccharide inhibitors and substrates of lysozyme to examine the preferred conformations of these molecules. A grid-search method was used to locate all of the low-energy conformational regions for N-acetyl-β-D -glycosamine (NAG), and energy minimization was then carried out in each of these regions. Three stable positions for the N-acetyl group have ben located, in two of which the plane of the amide unit is normal to the mean plane of the pyranosyl ring. Nine local energy minima were located for the —CH₂OH group. The positions of the two vicinal cis —OH groups are determined predominantly by interactions with either the —CH₂OH or the N-acetyl group. The most stable conformations of β-N-acetylmuramic acid (NAM) were determined from the study of the low-energy conformations of NAG. In the two stable orientations for the D -lactic acid side chain, the O—C—C′ plane (C′ being the carbon atom of the terminal carboxyl group) was found to be normal to the mean plane of the pyranosyl ring. The low-energy positions for the COOH group of NAM are determined mainly by interactions with neighboring groups. The conformational preferences of the α-anomers of NAG and NAM were also explored. The calculated conformation of the N-acetyl group for α-NAG was quite close to that determined by X-ray analysis. Two of the three lowest energy conformations of α-NAM are similar to the corresponding conformations of the β-anomer. A third low-energy structure, which has a hydrogen bond from the NH of the N-acetyl group to the C?O of the lactic acid group, corresponds very closely to the X-ray structure of this molecule. The preferred conformations of the disaccharides NAG–NAG, NAM–NAG and NAG–NAM were also investigated. Two preferred orientations of the reducing pyranosyl ring relative to the nonreducing ring were found for all of these disaccharides, both of which are close to the extended conformation. In one of these conformations, a hydrogen bond can form between the OH group attached to C₃ of the reducing sugar and the ring oxygen of the preceding residue. Each conformation can be stabilized further by a hydrogen bond between the CH₂OH (donor) of residue i + 1 and the C?O of residue i (acceptor). The interactions that determine conformations for all oligosaccharides containing both NAG and NAM are shown to be exclusively intraresidue and nearest neighbor interactions, so that it is possible to predict all stable conformations of oligosaccharides containing NAG and NAM in any sequence.     

Desacetoxycephalosporin C synthetase: Importance of order of cofactor/ reactant addition

The ring expansion enzyme (desacetoxycephalosporin C synthetase) of cephalosporin C biosynthesis in Cephalosporium acremonium has been purified 40-fold, using gel filtration on LKB Ultrogel AcA54. Purity was about twice that previously attained. The purified enzyme showed an almost absolute requirement for α-ketoglutarate, and most other acids were inactive or only weakly active. The order of addition of the cofactors and reactants was found to exert a major effect on enzyme activity. The major negative effect was caused by α-ketoglutarate or penicillin N being added to the enzyme first. The most effective order of addition to the enzyme was found to be the simultaneous addition of Fe²⁺, ascorbate and ATP, followed by α-ketoglutarate and then penicillin N. The negative effect of the required reactant, α-ketoglutarate, when added too early, coincides with observations made about other α-ketoglutarate-dependent dioxygenases even though, in the case of the synthetase, one atom of oxygen does not end up in the product, desacetoxycephalosporin C.     

Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position α188, α189, or β24 improved the K_m for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.     

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C₁₈ solid-phase extraction column. After elution with 500 μl of elution solution, the penicillins are derivatized with 500 μl of 1,2,4-triazole—mercuric chloride solution at 65°C for 30 min. The penicillin—mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C₁₈ column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.     

Application of CNDO/2 theoretical calculations to interpretation of the chemical reactivity and biological activity of the syn and anti diolepoxides of benzo[a]pyrene

CNDO/2 molecular orbital theoretical calculations performed on the anti and syn diolepoxides (1 and 2) of the potent carcinogen benzo[a]pyrene provide insight into the molecular structure and reactivity of these mutagenic and carcinogenic hydrocarbon metabolites. Hydrogen-bonded interaction between the 7-HO proton and the epoxide oxygen atom of 2 is shown to be absent in the normal semichair conformation of the tetrahydro ring, (H…O bond DISTANCE = 2.7 Å), but is energetically favored in a somewhat distorted puckered structure (H…O bond DISTANCE = 1.7 Å). Unexpectedly, internal H-bonding alters the relative electron density at C₉ and C₁₀, leading to prediction of the former as the more electrophilic center. Since all reactions of 2 take place exclusively at C₁₀, transannular H-bonding is concluded not to contribute significantly to the structure of 2. Diolepoxide reactions with both weak and strong nucleophiles and with DNA are discussed and the mechanisms interpreted in terms of molecular structure as determined by the theoretical calculations.     

Crystal and molecular structure of benzyloxycarbonyl-α-aminoisobutyryl-L-prolyl methylamide: The observation of an X2-Pro3 Type III β-Turn

The crystal and molecular structure of N-benzyloxycarbonyl-α-aminoisobutyryl-L -prolyl methylamide, the amino terminal dipeptide fragment of alamethicin, has been determined using direct methods. The compound crystallizes in the orthorhombic system with the space group P2₁2₁2₁. Cell dimensions are a = 7.705 Å, b = 11.365 Å, and c = 21.904 Å. The structure has been refined using conventional procedures to a final R factor of 0.054. The molecular structure possesses a 4 → 1 intramolecular N-H—O hydrogen bond formed between the CO group of the urethane moiety and the NH group of the methylamide function. The peptide backbone adopts the type III β-turn conformation, with ?₂ = ?51.0°, ψ₂ = ?39.7°, ?₃ = ?65.0°, ψ₃ = ?25.4°. An unusual feature is the occurrence of the proline residue at position 3 of the β-turn. The observed structure supports the view that Aib residues initiate the formation of type III β-turn conformations. The pyrrolidine ring is puckered in C^γ-exo fashion.     

Application of CNDO2 theoretical calculations to interpretation of the chemical reactivity and biological activity of the syn and anti diolepoxides of benzo[a]pyrene

$\text{CNDO}\text{2}$ molecular orbital theoretical calculations performed on the anti and syn diolepoxides (1 and 2) of the potent carcinogen benzo[a]pyrene provide insight into the molecular structure and reactivity of these mutagenic and carcinogenic hydrocarbon metabolites. Hydrogen-bonded interaction between the 7-HO proton and the epoxide oxygen atom of 2 is shown to be absent in the normal semichair conformation of the tetrahydro ring, (H…O bond distance = 2.7 Å), but is energetically favored in a somewhat distorted puckered structure (H…O bond distance = 1.7 Å). Unexpectedly, internal H-bonding alters the relative electron density at C₉ and C₁₀, leading to prediction of the former as the more electrophilic center. Since all reactions of 2 take place exclusively at C₁₀, transannular H-bonding is concluded not to contribute significantly to the structure of 2. Diolepoxide reactions with both weak and strong nucleophiles and with DNA are discussed and the mechanisms interpreted in terms of molecular structure as determined by the theoretical calculations.     

Flexibility of the pyrrolidine ring in proline peptides

A survey of over 50 crystal structures indicates that both imino acid and peptide derivatives of proline populate ring conformers consistent with the torsional potentials about single bonds. In both cases, lower barriers for rotation about C? N bonds relative to those about C? C bonds favor smaller values for dihedral angles about the former bonds. In peptides a minimum in the torsional potential about C? N bonds occurs at zero dihedral angle, further favoring small angles. The pyrrolidine-ring dihedral angles of the proline compounds in the solid state obey a cyclopentane-type pseudorotation function. Thus the puckering of the five-membered ring can be quantitatively described by two parameters. Consistent with small dihedral angles about C? N bonds, C^β and/or C^γ are puckered out of the mean plane of the ring in nearly all of the nonstrained compounds. Utilizing the consistent force-field method of Lifson and coworkers [see A. Warshel, M. Levitt, and S. Lifson (1970) J. Mol. Spectrosc. 33 , 84] the intramolecular energy of five proline peptides was minimized with respect to all internal coordinates. In addition, the energy surface near minima was explored by constraining a particular dihedral angle and reminimizing the energy with respect to all remaining variables. In linear peptides two types of pyrrolidine-ring conformers have identical predicted energies. In the cyclic dipeptide cyclo (Pro-Gly) one of the ring conformers is favored by about 3 kcal/mol, while the cyclic tripeptide cyclo(Pro-Gly-Gly) favors the other conformer by a comparable margin. In agreement with observations in the solid state and in solution, C^β and/or C^γ are puckered in the predicted conformers. A correlation between proline Φ and the details of the puckered conformation was predicted and found to match precisely conformers observed in crystals. For the diamides N-acetyl-L -proline-N′-methyl-amide and N-acetyl-L -proline-N′,N′-dimethylamide (AcProMe₂A) 30% and 60% cis acetyl peptide bonds were predicted in good agreement with observations in nonpolar solvents for the respective compounds. The conformational distributions with respect to proline Ψ are also in accord with experimental observations. For AcProMe₂A, a model for a -Pro-Pro-sequence in a peptide chain, this study is the first to predict stable conformers for proline Ψ either ca. ?50° or 140° for both cis and trans peptides.     

Structure-activity relations and beta-lactamase resistance

beta-Lactam antibiotics resistant to beta-lactamase degradation can be produced by many chemical modifications, but often at the expense of antibacterial activity. Substitution onto several positions in the molecule produces different and often selective resistance; for instance, heavily sterically hindered acyl groups give staphylococcal beta-lactamase resistance to penicillins, and resistance to some enzymes from Gram-negative pathogens to both penicillins and cephalosporins. 6-alpha- or 7-alpha-substituents respectively confer a broad spectrum of resistance (e.g. cefoxitin), but changes at positions 2 or 3 have only a minor influence on enzyme susceptibility. Changes in the ring condensed with the beta-lactam, such as changing ceph-3-em to ceph-2-em may greatly enhance stability. Small improvement can occur when the nuclear sulphur atom is oxidized, but a much better effect is obtained when it is replaced by another atom such as oxygen, as in clavulanic acid. This compound appears to have broad spectrum resistance which is actually due to susceptibility and subsequent produce inhibition.     

Interaction between monobactams and model d-alanyl-d-alanine-cleaving peptidases

Abstract Several monobactams reacted with the serine dd -peptidases of Streptomyces R61 and Actinomadura R39 in a manner similar to that of bicyclic penicillins and cephalosporins. The dissociation constants of the Michaelis complexes formed between the R61 enzyme and sulfazecin (32 μM) and between the R39 peptidase and SQ 26324 (0.35 μM) had the lowest values ever observed with any β-lactam compound, suggesting an excellent fit of these two monobactams with the active sites of the respective enzymes. Azthreonam had a very poor inactivating potency, confirming its high selective reactivity towards the penicillin binding protein No. 3 of Escherichia coli . The Zn²⁺ dd -peptidase (from Streptomyces albus G) had a high intrinsic resistance to β-lactam compounds whether they possessed a mono- or a bicyclic structure.     

Mixed conformation in Cα,α-disubstituted tripeptides: X-ray crystal structures of Z-Aib-Dph-Gly-Ome and Bz-Dph-Dph-Gly-Ome

We report here the synthesis and molecular structure in the solid state of fully protected tripeptides containing C^α,α-diphenylglycine (Dph), namely Z-Aib-Dph-Gly-OMe (Aib: C^α,α-dimethylgrycine) and Bz-Dph-Dph-Gly-OMe. The molecular conformation around the Dph residue, containing two bulky substituents, is fully extended, while the Aib residue, containing two smaller groups on the C^α atom, adopts the typical 3₁₀/α-helical conformation. Gly residues, without substituents on the C^α atom, show different conformational preferences. Each residue seems to behave, from a conformational point of view, independently from the presence of the other residues, and thus mixed local conformations (folded and extended) are present in the crystals. The nonconventional peptide synthesis, using the Ugi reaction, is also reported. © 1994 John Wiley & Sons, Inc.     

Cephalosporin C acylase and penicillin V acylase formation by Aeromonas sp. ACY 95

Aeromonas sp. ACY 95 produces constitutively and intracellularly a penicillin V acylase at an early stage of fermentation (12 h) and a cephalosporin C acylase at a later stage (36 h). Some penicillins, cephalosporin C and their side chain moieties/analogues, phenoxyacetic acid, penicillin V and penicillin G, enhanced penicillin V acylase production while none of the test compounds affected cephalosporin C acylase production. Supplementation of the medium with some sugars and sugar derivatives repressed enzyme production to varying degrees. The studies on enzyme formation, induction and repression, and substrate profile suggest that the cephalosporin C acylase and penicillin V acylase are two distinct enzymes. Substrate specificity studies indicate that the Aeromonas sp. ACY 95 produces a true cephalosporin C acylase which unlike the enzymes reported hitherto hydrolyses cephalosporin C specifically.The authors are with Research and Development, Hindustan Antibiotics Limited, Pimpri. Pune 411 018, India