Reactivity and selectivity in the inhibition of elastase by 3-oxo-beta-sultams and in their hydrolysis

3-oxo-beta-sultams are both beta-sultams and beta-lactams and are a novel class of time-dependent inhibitors of elastase. The inhibition involves formation of a covalent enzyme-inhibitor adduct with transient stability by acylation of the active-site serine resulting from substitution at the carbonyl centre of the 3-oxo-beta-sultam, C-N fission, and expulsion of the sulfonamide. The lead compound, N-benzyl-4,4-dimethyl-3-oxo-beta-sultam 1 is a reasonably potent inhibitor against porcine pancreatic elastase with a second-order rate constant of 768 M(-1) s(-1) at pH 6, but also possesses high chemical reactivity with a half-life for hydrolysis of only 6 mins at the same pH in water. Interestingly, the hydrolysis of 3-oxo-beta-sultams occurs at the sulfonyl centre with S-N fission and expulsion of the amide leaving group, whereas the enzyme reaction occurs at the acyl centre. Increasing selectivity between these two reactive centres was explored by examining the effect of substituents on the reactivity of 3-oxo-beta-sultam towards hydrolysis and enzyme inhibition. The inhibition activity against porcine pancreatic elastase has a much higher sensitivity to substituent variation than does the rate of alkaline hydrolysis. A difference of 2000-fold is observed in the second-order rate constants, k(i), for inhibition whereas there is only a 100-fold difference in the second-order rate constants, k(OH), for alkaline hydrolysis within the series. The higher sensitivity of enzyme inhibition to substituents than that of simple chemical reactivity indicates a significant degree of molecular recognition of the 3-oxo-beta-sultams by the enzyme.     

Structure-reactivity relationships in the inactivation of elastase by beta-sultams

N-Acyl-beta-sultams are time dependent irreversible active site directed inhibitors of elastase. The rate of inactivation is first order with respect to beta-sultam concentration and the second order rate constants show a similar dependence on pH to that for the hydrolysis of a peptide substrate. Inactivation is due to the formation of a stable 1:1 enzyme inhibitor complex as a result of the active site serine being sulfonylated by the beta-sultam. Ring opening of the beta-sultam occurs by S-N fission in contrast to the C-N fission observed in the acylation of elastase by N-acylsulfonamides. Structure-activity effects are compared between sulfonylation of the enzyme and alkaline hydrolysis. Variation in 4-alkyl and N-substituted beta-sultams causes differences in the rates of inactivation by 4 orders of magnitude.     

Kinetic and theoretical studies on the mechanism of alkaline hydrolysis of DNA

The reaction mechanism of alkaline hydrolysis of DNA has been investigated by kinetic analysis and density-functional-theory calculation. The rates of hydrolysis of thymidine 3'-monophosphate esters (including thymidylyl(3'-5')thymidine (Tp-OT)) monotonically decrease as the leaving groups get poorer. According to the theoretical calculation in which the solvent effects are incorporated, no intermediate is formed in the course of the reaction. In the alkaline hydrolysis of the activated Tp-OT analogues having good leaving groups, the 3',5'-cyclic monophosphate of thymidine is concurrently formed through the intramolecular attack by the 5'-alkoxide ion. In the hydrolysis of the native dinucleotide, however, this side reaction does not occur, since the transition state leading to the departure of its poor leaving group cannot be formed due to conformational restraint. These arguments are supported by the theoretical analysis on the hydrolysis of both dimethyl phosphate and its O(bridging)-->S substituted analogue.     

Mechanistic study of protein phosphatase-1 (PP1), a catalytically promiscuous enzyme

The reaction catalyzed by the protein phosphatase-1 (PP1) has been examined by linear free energy relationships and kinetic isotope effects. With the substrate 4-nitrophenyl phosphate (4NPP), the reaction exhibits a bell-shaped pH-rate profile for kcat/KM indicative of catalysis by both acidic and basic residues, with kinetic pKa values of 6.0 and 7.2. The enzymatic hydrolysis of a series of aryl monoester substrates yields a Br?nsted beta(lg) of -0.32, considerably less negative than that of the uncatalyzed hydrolysis of monoester dianions (-1.23). Kinetic isotope effects in the leaving group with the substrate 4NPP are (18)(V/K) bridge = 1.0170 and (15)(V/K) = 1.0010, which, compared against other enzymatic KIEs with and without general acid catalysis, are consistent with a loose transition state with partial neutralization of the leaving group. PP1 also efficiently catalyzes the hydrolysis of 4-nitrophenyl methylphosphonate (4NPMP). The enzymatic hydrolysis of a series of aryl methylphosphonate substrates yields a Br?nsted beta(lg) of -0.30, smaller than the alkaline hydrolysis (-0.69) and similar to the beta(lg) measured for monoester substrates, indicative of similar transition states. The KIEs and the beta(lg) data point to a transition state for the alkaline hydrolysis of 4NPMP that is similar to that of diesters with the same leaving group. For the enzymatic reaction of 4NPMP, the KIEs are indicative of a transition state that is somewhat looser than the alkaline hydrolysis reaction and similar to the PP1-catalyzed monoester reaction. The data cumulatively point to enzymatic transition states for aryl phosphate monoester and aryl methylphosphonate hydrolysis reactions that are much more similar to one another than the nonenzymatic hydrolysis reactions of the two substrates.     

Reactive-site design in folded-polypeptide catalysts--the leaving group pKa of reactive esters sets the stage for cooperativity in nucleophilic and general-acid catalysis

The second-order rate constants for the hydrolysis of nitrophenyl esters catalysed by a number of folded designed polypeptides have been determined, and 1900-fold rate enhancements over those of the 4-methylimidazole-catalysed reactions have been observed. The rate enhancements are much larger than those expected from the pKa depression of the nucleophilic His residues alone. Kinetic solvent isotope effects were observed at pH values lower than the pKa values of the leaving groups and suggests that general-acid catalysis contributes in the pH range where the leaving group is predominantly protonated. In contrast, no isotope effects were observed at pH values above the pKa of the leaving group. A Hammett rho value of 1.4 has been determined for the peptide-catalysed hydrolysis reaction by variation of the substituents of the leaving phenol. The corresponding values for the imidazole-catalysed reaction is 0.8 and for phenol dissociation is 2.2. There is therefore, very approximately, half a negative charge localised on the phenolate oxygen in the transition state in agreement with the conclusion that transition-state hydrogen-bond formation may contribute to the observed catalysis. The elucidation at a molecular level of the principles that control cooperativity in the biocatalysed ester-hydrolysis reaction represents the first step towards a level of understanding of the concept of cooperativity that may eventually allow us to design tailor-made enzymes for chemical reactions not catalysed by nature.     

Regioselective Baeyer-Villiger oxidation in 4-carbonyl-2-azetidinone series: a revisited route toward carbapenem precursor

A novel synthesis of acetoxyazetidinone 2 is presented. The azetidinone ring of compounds 7 is formed by C-3/C-4 cyclization of (2R,3R)-epoxybutyramide precursors 6, N-protected with a benzhydryl group. N-Deprotection by photoactivated bromination and acidic treatment leads to compounds 10 with various CO-R substituents at C-4. Transformation of these substituents by Baeyer-Villiger oxidation gives the desired regioisomer 11 with the cyclopropyl side-group which is the only group (among R = H, Ph, t-Bu, i-Pr, c-Pr) able to satisfy both the steric requirements of the cyclization step and the electronic requirements of the oxidative rearrangement step.     

Different transition-state structures for the reactions of beta-lactams and analogous beta-sultams with serine beta-lactamases

Beta-sultams are the sulfonyl analogues of beta-lactams, and N-acyl beta-sultams are novel inactivators of the class C beta-lactamase of Enterobacter cloacae P99. They sulfonylate the active site serine residue to form a sulfonate ester which subsequently undergoes C-O bond fission and formation of a dehydroalanine residue by elimination of the sulfonate anion as shown by electrospray ionization mass spectroscopy. The analogous N-acyl beta-lactams are substrates for beta-lactamase and undergo enzyme-catalyzed hydrolysis presumably by the normal acylation-deacylation process. The rates of acylation of the enzyme by the beta-lactams, measured by the second-order rate constant for hydrolysis, kcat/K(m), and those of sulfonylation by the beta-sultams, measured by the second-order rate constant for inactivation, k(i), both show a similar pH dependence to that exhibited by the beta-lactamase-catalyzed hydrolysis of beta-lactam antibiotics. Electron-withdrawing groups in the aryl residue of the leaving group of N-aroyl beta-lactams increase the rate of alkaline hydrolysis and give a Bronsted beta(lg) of -0.55, indicative of a late transition state for rate-limiting formation of the tetrahedral intermediate. Interestingly, the corresponding Bronsted beta(lg) for the beta-lactamase-catalyzed hydrolysis of the same substrates is -0.06, indicative of an earlier transition state for the enzyme-catalyzed reaction. By contrast, although the Bronsted beta(lg) for the alkaline hydrolysis of N-aroyl beta-sultams is -0.73, similar to that for the beta-lactams, that for the sulfonylation of beta-lactamase by these compounds is -1.46, compatible with significant amide anion expulsion/S-N fission in the transition state. In this case, the enzyme reaction displays a later transition state compared with hydroxide-ion-catalyzed hydrolysis of the beta-sultam.     

Analysis of linear free-energy relationships combined with activation parameters assigns a concerted mechanism to alkaline hydrolysis of x-substituted phenyl diphenylphosphinates

A kinetic study is reported for alkaline hydrolysis of X‐substituted phenyl diphenylphosphinates ( 1 a – i ). The Brønsted‐type plot for the reactions of 1 a – i is linear over 4.5 pK_a units with β_lg=?0.49, a typical β_lg value for reactions which proceed through a concerted mechanism. The Hammett plots correlated with σ^o and σ^? constants are linear but exhibit many scattered points, while the corresponding Yukawa–Tsuno plot results in excellent linear correlation with ρ=1.42 and r=0.35. The r value of 0.35 implies that leaving‐group departure is partially advanced at the rate‐determining step (RDS). A stepwise mechanism, in which departure of the leaving group from an addition intermediate occurs in the RDS, is excluded since the incoming HO^? ion is much more basic and a poorer nucleofuge than the leaving aryloxide. A dissociative (D_N + A_N) mechanism is also ruled out on the basis of the small β_lg value. As the substituent X in the leaving group changes from H to 4‐NO₂ and 3,4‐(NO₂)₂, ΔH ^≠ decreases from 11.3 kcal mol^?1 to 9.7 and 8.7 kcal mol^?1, respectively, while ΔS ^≠ varies from ?22.6 cal mol^?1 K^?1 to ?21.4 and ?20.2 cal mol^?1 K^?1, respectively. Analysis of LFERs combined with the activation parameters assigns a concerted mechanism to the current alkaline hydrolysis of 1 a – i .     

Reactivity of amino acid nucleoside phosphoramidates: a mechanistic quantum chemical study

Recent experimental evidence (Maiti et al. Chem.-Eur. J., submitted) indicates that hydrolysis of nucleoside phosphoramidates is subjected to anchimeric influence by carboxyl moieties in the leaving group but also by the base in the nucleotide. A quantum chemical analysis of these findings is presented. First the intrinsic hydrolysis mechanism is investigated for simplified model compounds, and then both amino acid and nucleoside substituents are included. It is found that hydrolysis is assisted by the α-carboxyl group via formation of a five-membered intermediate and that the barrier for the reaction of this intermediate toward the product state can be influenced by the nucleobase. The adenine base protonated on N3 interacts with the transition state and considerably lowers the barrier for hydrolysis. The influence of several base modifications is explained by calculating the pK(a) for protonation on N3.     

Furanoside phosphite–phosphoroamidite and diphosphoroamidite ligands applied to asymmetric Cu-catalyzed allylic substitution reactions

A phosphite–phosphoroamidite and diphosphoroamidite ligand library was applied in the Cu-catalyzed allylic substitution of a range of cinnamyl-type substrates using several organometallic nucleophiles. Results indicated that selectivity depended strongly on the ligand parameters (position of the phosphoroamidite group at either C-5 or C-3 of the furanoside backbone, as well as the configuration of C-3, the introduction of a second phosphoroamidite moiety, the substituents and configurations in the biaryl phosphite/phosphoroamidite moieties), the nature of the leaving group of the substrate and the alkylating reagent. Good enantioselectivities (up to 76%) and activity combined with high regioselectivities were obtained.     

Rearrangements in the Course of Fluorination by Diethylaminosulfur Trifluoride at C-2 of Glycopyranosides: Some New Parameters

Summary.  Reaction of a series of 21 glycosides unprotected at O-2 and featuring various configurations with DAST (diethylaminosulfur trifluoride) was monitored by ¹⁹F NMR spectroscopy. By means of the diacritical set of data (shift values and coupling constants) thus obtained for each product, identification of the operative mechanisms was possible. By correlation of these findings with stereochemical details from the structure of the educts, new parameters governing the choice of the reaction paths could be deduced. This evaluation led to the result that ring contraction after attack at C-2 of the ring oxygen and entry of the fluoride at C-1 is strongly favoured over all other possibilities. Exceptions are all derivatives of the manno series as well as all members of the trans-decalin type structure as present after 4,6-O-benzylidene acetal formation with educts having their ring substituents at C-4 and C-5 in diequatorial (trans) orientation. From these, α-D-mannopyranosides generally and of the trans-decalin types those with an additional 1,2-trans-configuration are prone to 1,2-aglycon migration and, again, entry of the fluoride at C-1. Additional pathways like alkoxy group migration, substitution under retention of configuration, or orthoester formation, are possible by participation of a suitably located neighbouring group at C-3 inasmuch as an alkoxy group interferes from an antiperiplanar orientation to the leaving group at C-2 and an acyloxy functionality attacks in a diequatorial relationship to the latter. The generally intended nucleophilic substitution by fluoride under inversion of configuration is of minor importance. Received November 26, 2001. Accepted November 28, 2001     

Theoretical calculations of β-lactam antibiotics. V. AM1 calculations of hydrolysis of cephalothin in gaseous phase and influence of the solvent

A comprehensive study on the gas-phase alkaline hydrolysis of cephalosporins by using the semiempirical AM1 method was carried out. Cephalothin was the model compound used on account of the presence of a good leaving group at C(3′). According to the results obtained, the hydrolysis process takes place via a twostep reaction mechanism that involves the formation of an intermediate with a fully open β-lactam ring that still preserves the acetate group. Likewise, the exo methylene end product is chiefly formed by nucleophilic attack on the β-lactam carbonyl group of cephalosporins containing a good leaving group at C(3′). On the other hand, the alternative mechanism involving hydrolysis of the ester function in the side chain at 3′ and subsequent hydrolysis of the resulting β-lactam yieds essentially the corresponding enamine. The presence of a first solvation layer consisting of five water molecules showed that, even though some potential barriers are slightly increased, the mechanism involved is identical to that of the gas-phase hydrolysis of this antibiotic. © John Wiley & Sons, Inc.     

有机磷化合物的研究XXXIX. 溶剂性质对烷基膦酸-O, O-1,3-丙二酯及-O,O-1,4-丁二酯的碱性水解动力学的影响

本文报道烷基膦酸-O, O-1,3-丙二酯及O, O-1,4-丁二酯于50%二甲亚砜水溶液中的水解动力学, 考察了取代基结构对膦酸酯水解速率常数的影响。通过它们在50%的二甲亚砜水溶液和50%的二氧六环水溶液中碱性水解速率常数的比较, 说明在这些混合溶液中, 烷基的空间结构对水解速率常数的影响是近似平行的。同时, 正丙基膦酸-O, O-1,3-丙二酯于不同混合溶剂中碱性水解时, 溶剂分子的给质子接受质子的能力对水解反应过程有重要影响。     

Substituent dependence of the diastereofacial selectivity in iodination and bromination of glycals and related cyclic enol ethers

The stereochemical course of the electrophilic iodination and bromination of tri-O-benzyl-D-glucal under various conditions has been compared to that of substituted dihydropyrans 2-5. IN(3) addition in acetonitrile affords trans-alpha-iodoazides (80-87%), besides small amounts of trans-beta-adducts, in the presence or the absence of benzyloxy substituents at C-3 or C-4, and in agreement with bridged iodonium ion intermediates. In contrast, the diastereofacial selectivity of bromine addition in dichloroethane going through open bromo oxocarbenium ions depends strongly on the substituents. Whereas the trans-alpha-dibromides are the main (85-95%) adducts in the absence of C-4 and C-5 substituents, in their presence a moderate to exclusive selectivity for cis-alpha-addition (60-99%) is observed. The predominance of trans-alpha-addition is again observed whatever the substituents when the bromination is carried out in the same solvent but with a tribromide ion salt, supporting a concerted addition of the two bromine atoms under these conditions. Finally, bromine addition in methanol exhibits a completely different behavior with the nonselective formation of trans-alpha- and trans-beta-methoxybromides and a small dependence on the substituents. In agreement with the absence of azide trapping of any cationic intermediate, it is concluded that these brominations which do not go through an ionic intermediate are concerted additions of bromine and methanol with very loose rate- and product-determining transition states. Finally, the substituent conformation at C-4 influences drastically the stereoselectivity in all these brominations. Evidence for alpha-anomeric control of the nucleophile approach at C-1 is given by the highly predominant formation of alpha-adducts, except in the methanolic bromination. The factors determining the versatile selectivity of the electrophile approach are discussed in terms of PPFMO theory and of the special mechanisms of glycal reactions.     

Synthesis of 9-azolyl-3-(4-phenyl-4h-1,2,4-triazol-3-yl)-4h,8h-pyrano-[2,3-<Emphasis Type="Italic">f</Emphasis>]chromene-4,8-diones

The reaction of 6-ethyl-8-formyl-7-hydroxy-3-(4-phenyl-4H-1,2,4-triazol-3-yl)chromone with 2-azolyl-acetonitriles gave 8-iminopyrano[2,3-f]chromen-4-ones, whose acid hydrolysis led to pyrano-[2,3-f]chromene-4,8-diones containing azaheterocyclic substituents at C-3 and C-9.     

Kinetic study on the alkaline hydrolysis of 1-aryl-2-phenyl-2-imidazolines. Basicity-rate of hydrolysis and structure relationships

Rate constants (k_obs) of hydrolysis in boiling alkaline ethanolic solution for six 1-aryl-2-phenyl-2-imidazolines were determined. The influence of substituents in the phenyl group at N-1 upon rate of hydrolysis was studied. When the imidazoline ring is considered to be a substituent of the benzene ring at N-1, a good correlation with the Hammett equation is found. It was observed that reaction rates were enhanced by electron-releasing phenyl substituents of N-1 and reduced by electron-withdrawing groups, providing a change in the mechanism of the reaction in the first case that was not observed in the second. Agreement with the Hammett equation allowed comparison between experimental and “calculated” rate constants which are nearly equal. An equation relating the rate constants with the ionization constants of imidazolinium ions is given.     

Synthesis of Sulfur-Containing Analogues of αGalNAc (Tn-Antigen) and βGal1,3αGalNAc (T-Antigen)

Summary.  A method for the preparation of thio-analogues of the T- and Tn-antigen was developed. Thus, starting from a known N-acetamido-glucoside derivative, the epidithio analogue of the Tn-antigen was accessible in a four-step reaction sequence. The corresponding epidithio analogue and the thioanhydro derivative of the T-antigen were synthesized starting from a disaccharide derivative. For the preparation of the epidithio analogue the sulfur atoms were introduced via thiocyanates in a stepwise fashion, using mesylate as the leaving group at C-6 and triflate as the leaving group at C-4 in the reducing carbohydrate moiety. The synthesis of the thioanhydro analogue was achieved by introducing a thiocyanate group at C-6 into the glucose moiety, followed by subsequent displacement of a mesylate group at C-4 under inversion of configuration utilizing sodium methoxide. Received October 16, 2001. Accepted November 7, 2001     

Synthesis and Kinetic Evaluation of Inhibitors of the Phosphatidylinositol-Specific Phospholipase C from Bacillus cereus

Substrate analogues of phosphatidylinositol (1) were synthesized and evaluated as potential inhibitors of the bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus. The chiral analogues of the water-soluble phospholipid substrate 5 were designed to probe the effects of varying the inositol C-2 hydroxyl group, which is generally believed to serve as the nucleophile in the first step of the hydrolysis of phosphatidylinositols by PI-PLC. In the analogues 6-9, the C-2 hydroxyl group on the inositol ring of the phosphatidylinositol derivatives was rationally altered in several ways. Inversion of the stereochemistry at C-2 of the inositol ring led to the scyllo derivative 6. The inositol C-2 hydroxy group was replaced with inversion by a fluorine to produce the scyllo-fluoro inositol 7 and with a hydrogen atom to furnish the 2-deoxy compound 8. The C-2 hydroxyl group was O-methylated to prepare the methoxy derivative 9. The natural inositol configuration at C-2 was retained in the nonhydrolyzable phosphorodithioate analogue 10. The inhibition of PI-PLC by each of these analogues was then analyzed in a continuous assay using D-myo-inositol 1-(4-nitrophenyl phosphate) (25) as a chromogenic substrate. The kinetic parameters for each of these phosphatidylinositol derivatives were determined, and each was found to be a competitive inhibitor with K(i)'s as follows: 6, 0.2 mM; 10, 0.6 mM; 8, 2.6 mM; 9, 6.6 mM; and 7, 8.8 mM. This study further establishes that the hydrolysis of phosphatidylinositol analogues by bacterial PI-PLC requires not only the presence of a C-2 hydroxyl group on the inositol ring, but the stereochemistry at this position must also correspond to the natural myo-configuration. For future inhibitor design, it is perhaps noteworthy that the best inhibitors 6 and 10 each possess a hydroxyl group at the C-2 position. Several of the inhibitors identified in this study are now being used to obtain crystallographic information for an enzyme-inhibitor complex to gain further insights regarding the mechanism of hydrolysis of phosphatidylinositides by this PI-PLC.     

Vinyl Halide-Modified Unsaturated Cyclitols are Mechanism-Based Glycosidase Inhibitors

Suitably configured allyl ethers of unsaturated cyclitols act as substrates of β-glycosidases, reacting via allylic cation transition states. Incorporation of halogens at the vinylic position of these carbasugars, along with an activated leaving group, generates potent inactivators of β-glycosidases. Enzymatic turnover of these halogenated cyclitols (F, Cl, Br) displayed a counter-intuitive trend wherein the most electronegative substituents yielded the most labile pseudo-glycosidic linkages. Structures of complexes with the Sulfolobus β-glucosidase revealed similar enzyme-ligand interactions to those seen in complexes with a 2-fluorosugar inhibitor, the lone exception being displacement of tyrosine 322 from the active site by the halogen. Mutation of Y322 to Y322F largely abolished glycosidase activity, consistent with lost interactions at O5, but minimally affected (7-fold) rates of carbasugar hydrolysis, yielding a more selective enzyme for unsaturated cyclitol ether hydrolysis.     

An Efficient Method for Determination of the Degree of Substitution of Cellulose Esters of Long Chain Aliphatic Acids

The determination of the degree of substitution (DS) of fatty acid cellulose esters, representing a broad range of substituents (C₆, C₁₂, C₁₈ and C₂₂), was performed by alkaline hydrolysis of the ester groups and the quantification of fatty acids by gas chromatography-mass spectrometry (GC-MS) as their trimethylsilyl derivatives. The method was optimized and compared with established techniques for the DS determination (elemental analysis and alkaline hydrolysis/titrimetry). The results demonstrated that alkaline hydrolysis/GC-MS is a rapid, reliable and powerful method for analysis of fatty acid cellulose esters, particularly when different acyl substituents are present.