The discovery of MK-0674, an orally bioavailable cathepsin K inhibitor

MK-0674 is a potent and selective cathepsin K inhibitor from the same structural class as odanacatib with a comparable inhibitory potency profile against Cat K. It is orally bioavailable and exhibits long half-life in pre-clinical species. In vivo studies using deuterated MK-0674 show stereoselective epimerization of the alcohol stereocenter via an oxidation/reduction cycle. From in vitro incubations, two metabolites could be identified: the hydroxyleucine and the glucuronide conjugate which were confirmed using authentic synthetic standards.     

Determination of the epimeric composition of ibuprofenyl-CoA

Ibuprofen [racemic2-(4-isobutylphenyl)propionic acid] is a 2-arylpropionic acid nonsteroidal anti-inflammatory drug which undergoes unidirectional, R to S chiral inversion in vivo. It has been proposed that this chiral inversion phenomenon occurs via a coenzyme A (CoA) thioester intermediate. To characterize the formation and metabolism of this metabolic intermediate, ibuprofenyl-CoA, reference standards were needed and thus the CoA derivatives of (R)-, (S)-, and racemic ibuprofen were chemically synthesized. An HPLC assay employing a C18 reverse-phase column was developed to quantitate "total" ibuprofenyl CoA. Samples collected from this assay were then analyzed for ibuprofenyl-CoA epimeric composition by chiral chromatography employing a Chiral-AGP alpha 1-acid glycoprotein column. The applicability of these methods was demonstrated by assessing (R)- and (S)-ibuprofenyl-CoA hydrolysis and epimerization following incubation with rat liver homogenates. Rat liver homogenate catalyzed the complete and rapid epimerization of ibuprofenyl-CoA and the rate constants for (R)- and (S)-ibuprofenyl-CoA hydrolysis were equal. ATP and CoA were found to inhibit rat liver-catalyzed ibuprofenyl-CoA hydrolysis by 70-80% with no effect on epimerization. Additionally, it was demonstrated that traditional indirect ibuprofenyl-CoA assays which employ basic hydrolysis result in erroneous epimeric ratio determinations due to chemical epimerization.     

Mechanism of dihydroneopterin aldolase. NMR, equilibrium and transient kinetic studies of the Staphylococcus aureus and Escherichia coli enzymes

Dihydroneopterin aldolase (DHNA) catalyzes both the cleavage of 7,8-dihydro-D-neopterin (DHNP) to form 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde and the epimerization of DHNP to form 7,8-dihydro-L-monapterin (DHMP). Whether the epimerization reaction uses the same reaction intermediate as the aldol reaction or the deprotonation and reprotonation of C2' of DHNP has been investigated by NMR analysis of the reaction products in a D2O solvent. No deuteration of C2' was observed for the newly formed DHMP. This result strongly suggests that the epimerization reaction uses the same reaction intermediate as the aldol reaction. In contrast with an earlier observation, the DHNA-catalyzed reaction is reversible, which also supports a nonstereospecific retroaldol/aldol mechanism for the epimerization reaction. The binding and catalytic properties of DHNAs from both Staphylococcus aureus (SaDHNA) and Escherichia coli (EcDHNA) were determined by equilibrium binding and transient kinetic studies. A complete set of kinetic constants for both the aldol and epimerization reactions according to a unified kinetic mechanism was determined for both SaDHNA and EcDHNA. The results show that the two enzymes have significantly different binding and catalytic properties, in accordance with the significant sequence differences between them.     

Epimerization in peptide thioester condensation

Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C‐terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid‐phase method, no substantial epimerization of the C‐terminal amino acid was detected. Epimerization was, however, observed during a thioester–thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester–thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Suppression of epimerization by cupric (II) salts in peptide synthesis using free amino acids as amino components.

An epimerization-free system for coupling N-protected peptides with free amino acids was developed. A number of inorganic substances were tested as epimerization suppressant additives during the coupling by various methods (carbodiimide plus additives, uronium salts, Woodward's reagent-K, isobutyl-chloroformate, etc.). Some of them (ZnCl2, RbClO4, LiCl, SnCl4, AlCl3, etc.) in combination with some coupling methods can guarantee coupling with minimal epimerization (D-epimer < 1%). But only a simultaneous use of 1-hydroxybenzotriazole and Cu2+ ions as additives in carbodiimide-mediated peptide couplings appeared to give a standard result (D-epimer < 0.1%). There was no epimerization even in the case when N-methyl amino acid (sarcosine) was used as an amino component, while in the absence of Cu2+ ions an unacceptable level of epimerization was observed (D-epimer, 22% for carbodiimide with the 1-hydroxybenzotriazole method). So far it has been considered that Cu2+ ions prevent obtaining peptides in high yields (< 90%) by various coupling methods. We have found that the use of 1-hydroxybenzotriazole, CuCl2 and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of dicyclohexylcarbodiimide provides a possible method for obtaining the desired peptides in 90-99% yields without epimerization. All these results were shown by employing several model peptide couplings with free amino acids as amino components dissolved in an effective solvent system which readily dissolved them.     

Epimerization of moxalactam by albumin and simulation of in vivo epimerization by a physiologically based pharmacokinetic model

We investigated the mechanism of epimerization (R to S or S to R) of moxalactam in serum of rats, dogs, and humans. The epimerization of moxalactam occurred in the serum of these animals, but not in the serum filtrate. The albumin fraction of human serum purified by gel filtration catalysed the epimerization of moxalactam at an identical rate to serum, but other fractions (i.e., lipoproteins and globulins) showed slower epimerization. alpha 1-acid glycoprotein, which was eluted in the same fraction with albumin by G-200 gel filtration, did not epimerize moxalactam. The presence of 2 mM warfarin decreased the binding of R- and S-moxalactam and decreased the epimerization of moxalactam in human serum. These results demonstrate moxalactam was epimerized on the warfarin binding site on albumin in serum. Additionally, a physiologically based pharmacokinetic model shows that the epimerization of moxalactam after administration in dogs is simulated by the epimerization in serum.     

Chemical mechanism and specificity of the C5-mannuronan epimerase reaction

C5-mannuronan epimerase catalyzes the formation of alpha-L-guluronate residues from beta-D-mannuronate residues in the synthesis of the linear polysaccharide alginate. The reaction requires the abstraction of a proton from C5 of the residue undergoing epimerization followed by re-protonation on the opposite face. Rapid-mixing chemical quench experiments were conducted to determine the nature of the intermediate formed upon proton abstraction in the reaction catalyzed by the enzyme from Pseudomonas aeruginosa. Colorimetric and HPLC analysis of quenched samples indicated that shortened oligosaccharides containing an unsaturated sugar residue form as transient intermediates in the epimerization reaction. This suggests that the carbanion is stabilized by glycal formation, concomitant with cleavage of the glycosidic bond between the residue undergoing epimerization and the adjacent residue. The time dependence of glycal formation suggested that slow steps flank the chemical steps in the catalytic cycle. Solvent isotope effects on V and V/K were unity, consistent with a catalytic cycle in which chemistry is not rate-limiting. The specificity of the epimerase with regard to neighboring residues was examined, and it was determined that the enzyme showed no bias for mannuronate residues adjacent to guluronates versus those adjacent to mannuronates. Proton abstraction and sugar epimerization were irreversible. Existing guluronate residues already present in the polysaccharide were not converted to mannuronates, nor was incorporation of solvent deuterium into existing mannuronates observed.     

Metabolic stereoisomeric inversion of ibuprofen in mammals

Studies on the mechanism and enzymology of metabolic ibuprofen isomerization constituted the focus of this investigation. Comparative in vivo studies revealed that this biotransformation proceeded via a proton abstraction mechanism in all tested species of mammals, which is in agreement with the previous reports. Direct evidence supporting this conclusion stemmed from the in vitro epimerization of ibuprofen-CoA thioester in rat liver homogenates. Chemically synthesized (R)-ibuprofen-CoA thioester was rapidly transformed to its (S)-counterpart by subcellular hepatic preparations. Examination of this epimerase activity in various rat tissue homogenates indicated that this enzyme was highly tissue specific. This biochemical reaction mainly took place in the liver and kidney, whereas low levels of enzyme activity were associated with other tissues. Nevertheless, the liver and kidney homogenates failed to invert (R)-ibuprofen directly even in the presence of all the necessary cofactors. Presumably, the failure to characterize this bioconversion was due to the lack of enzymatic acyl-CoA synthesis in these homogenates. It is noteworthy that the '2-arylpropionyl-CoA epimerase' catalyzed the transformation from either direction and with high turnover rates. The catalytic efficiency of (S)-ibuprofen CoA epimerization appeared to be greater than that of the (R)-counterpart. These in vitro findings suggest that the step of acyl-CoA formation assume a pivotal role in controlling the stereoselectivity and efficiency of the in vivo metabolism. As the responsible acyl-CoA synthetase(s) in different species of animals may exert the reaction with different degrees of enantiomeric preference and efficiency, the resulting stereochemical outcome and metabolic rates of this bioinversion vary accordingly. Consequently, in guinea pigs, this biotransformation proceeds in both directions with nearly equal efficiency, whereas it is virtually unidirectional and slow in humans. Currently, the purification and characterization of this novel '2-arylpropionyl-CoA epimerase' from rat livers constitute the focus of this investigation.     

Mode of action of recombinant Azotobacter vinelandii mannuronan C-5 epimerases AlgE2 and AlgE4.

The enzymes mannuronan C-5 epimerases catalyze conversion of beta-D-mannuronic acid to alpha-L-guluronic acid in alginates at the polymer level and thereby introduce sequences that have functional properties relevant to gelation. The enzymatic conversion by recombinant mannuronan C-5 epimerases AlgE4 and AlgE2 on alginate type substrates with different degree of polymerization and initial low fraction of alpha-L-guluronic acid was investigated. Essentially no enzymatic activity was found for fractionated mannuronan oligomer substrates with an average degree of polymerization, DP(n), less than or equal 6, whereas increasing the DP(n) yielded increased epimerization activity. This indicates that these enzymes have an active site consisting of binding domains for consecutive residues that requires interaction with 7 or more consecutive residues to show enzymatic activity. The experimentally determined kinetics of the reaction, and the residue sequence arrangement introduced by the epimerization, were modeled using Monte Carlo simulation accounting for the various competing intrachain substrates and assuming either a processive mode of action or preferred attack. The comparison between experimental data and simulation results suggests that epimerization by AlgE4 is best described by a processive mode of action, whereas the mode of action of AlgE2 appears to be more difficult to determine.     

Synthesis and antibacterial activity of C-6 carbamate ketolides, a novel series of orally active ketolide antibiotics

A new series of antibacterial ketolides is reported, which features the use of a C-6 carbamate for tethering the arylalkyl sidechain to the macrolide core. The best members of this series display in vitro and in vivo activity comparable to telithromycin. Partial epimerization at C-2, unobserved in previously reported ketolides, was noted for this series.     

Fourier transform near-infrared vibrational circular dichroism used for on-line monitoring the epimerization of 2,2-dimethyl-1,3-dioxolane-4-methanol: A pseudo racemization reaction

Near-infrared (near-IR) Fourier transform vibrational circular dichroism (FT-VCD) spectroscopy has been used to monitor the epimerization of (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (S-DDM). The near-IR-VCD spectra display clear isolated VCD bands at the range of 4700-5050 cm(-1) resulting from the OH stretch-bend combination bands of S-DDM, which were found to decrease in intensity with increasing reaction time. The near-IR-VCD spectra of 10 reference samples obtained were subjected to partial least-squares (PLS) regression analysis, and the results were used to build predictive models for enantiomeric excess (EE) determination. Multivariate regression was carried out on three different sets of spectra, corresponding to the epimerization of S-DDM in three different solvents: methylcyclohexane, carbon tetrachloride and tetrahydrofuran. The effects of solvent in DDM epimerization are discussed in terms of the relative stabilization of the reaction intermediate of the DDM epimerization reaction. The results of these near-IR-VCD studies for the determination of EE highlights the potential of VCD for in situ real-time process monitoring of the reaction kinetics of chiral molecules in solution.     

C(6)-oxidation followed by C(5)-epimerization of guar gum studied by high field NMR

Guar gum, a beta-D-(1-->4)-linked D-mannan with alpha-D-galactopyranosyl units attached as side groups, was treated with alpha-galactosidase, an enzyme that splits off the alpha-D-galactosyl units to obtain a galactomannan with a low galactose content. The galactose-depleted polysaccharide was then selectively oxidized in C(6) position and epimerized using mannuronan C(5)-epimerases, namely AlgE1, AlgE4, AlgE6, and their mixtures, obtaining new pseudo-alginates. In this paper, we report a full high field 1D and 2D NMR study of guar gum as such and of the galactose-depleted, oxidized and epimerized compounds, respectively. From the 1H NMR spectra, the degree of epimerization, the distribution of mannuronic acid (M) and guluronic acid (G) residues and the average G-block length, N(G>1), were obtained. By means of NMR diffusion experiments, it was also shown that no significant degradation of the polysaccharide occurs as a consequence of the epimerization reactions.     

A new amino acid racemase with threonine alpha-epimerase activity from Pseudomonas putida: purification and characterization.

We have found that Pseudomonas putida ATCC 17642 cells grown in a medium containing D-threonine as the sole nitrogen source produce an enzyme that catalyzes epimerization of threonine. Proton nuclear magnetic resonance analysis of the enzyme reaction in deuterium oxide clearly showed epimerization from L- to D-allo-threonine and also from D- to L-allo-threonine. This is the first example of an enzyme that was clearly shown to epimerize threonine. The enzyme has been purified to homogeneity, which was shown by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of about 82,000 and consists of two subunits identical in molecular weight (about 41,000). The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of subunit as a cofactor, and its absorption spectrum exhibits absorption maxima at 280 and 420 nm. The enzyme catalyzes not only epimerization of threonine by stereoconversion at the alpha position but also racemization of various amino acids, except acidic and aromatic amino acids. The enzyme is similar to amino acid racemase with low substrate specificity (EC 5.1.1.10) in enzymological properties but is distinct from it in the action on threonine.     

Isoleucine epimerization ages of some Pleistocene sites near Rome

Isoleucine epimerization was used to date tooth enamel samples collected from the fossiliferous layers of several Pleistocene formations in the Rome area. To calibrate the isoleucine epimerization rate, we used teeth from the Isernia-La Pineta deposit, dated at approximately 0.70 m.a by K/Ar and Paleomagnetics. The chronological sequence obtained for the deposits is in close agreement with that indicated by biostratigraphic and geologic evidence. The isoleucine epimerization dates represent the first age determinations made directly on the fossil material from the Rome deposits.     

Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan epimerase.

Alginate is a viscous extracellular polymer produced by mucoid strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis. Alginate is polymerized from GDP-mannuronate to a linear polymer of beta-1-4-linked residues of D-mannuronate and its C5-epimer, L-guluronate. We previously identified a gene called algG in the alginate biosynthetic operon that is required for incorporation of L-guluronate residues into alginate. In this study, we tested the hypothesis that the product of algG is a C5-epimerase that directly converts D-mannuronate to L-guluronate. The DNA sequence of algG was determined, and an open reading frame encoding a protein (AlgG) of approximately 60 kDa was identified. The inferred amino terminus of AlgG protein contained a putative signal sequence of 35 amino acids. Expression of algG in Escherichia coli demonstrated both 60-kDa pre-AlgG and 55-kDa mature AlgG proteins, the latter of which was localized to the periplasm. An N-terminal analysis of AlgG showed that the signal sequence was removed in the mature form. Pulse-chase experiments in both E. coli and P. aeruginosa provided evidence for conversion of the 60- to the 55-kDa size in vivo. Expression of algG from a plasmid inan algG (i.e., polymannuronate-producing) mutant of P. aeruginosa restored production of an alginate containing L-guluronate residues. The observation that AlgG is apparently processed and exported from the cytoplasm suggested that it may act as a polymer-level mannuronan C5-epimerase. An in vitro assay for mannuronan C5 epimerization was developed wherein extracts of E. coli expressing high levels of AlgG were incubated with polymannuronate. Epimerization of D-mannuronate to L-guluronate residues in the polymer was detected enzymatically, using a L-guluronate-specific alginate lyase of Klebsiella aerogenes. Epimerization was also detected in the in vitro reaction between recombinant AlgG and poly-D-mannuronate, using high-performance anion-exchange chromatography. The epimerization reaction was detected only when acetyl groups were removed from the poly-D-mannuronate substrate, suggesting that AlgG epimerization activity in vivo may be sensitive to acetylation of the D-mannuronan residues. These results demonstrate that AlgG has polymer-level mannuronan C5-epimerase activity.     

Crystal structure of carbapenem synthase (CarC)

The proposed biosynthetic pathway to the carbapenem antibiotics proceeds via epimerization/desaturation of a carbapenam in an unusual process catalyzed by an iron- and 2-oxoglutarate-dependent oxygenase, CarC. Crystal structures of CarC complexed with Fe(II) and 2-oxoglutarate reveal it to be hexameric (space group C2221), consistent with solution studies. CarC monomers contain a double-stranded beta-helix core that supports ligands binding a single Fe(II) to which 2-oxoglutarate complexes in a bi-dentate manner. A structure was obtained with l-N-acetylproline acting as a substrate analogue. Quantum mechanical/molecular mechanical modeling studies with stereoisomers of carbapenams and carbapenems were used to investigate substrate binding. The combined work will stimulate further mechanistic studies and aid in the engineering of carbapenem biosynthesis.     

The A modules of the Azotobacter vinelandii mannuronan-C-5-epimerase AlgE1 are sufficient for both epimerization and binding of Ca2+.

The industrially important polysaccharide alginate is composed of the two sugar monomers beta-D-mannuronic acid (M) and its epimer alpha-L-guluronic acid (G). In the bacterium Azotobacter vinelandii, the G residues originate from a polymer-level reaction catalyzed by one periplasmic and at least five secreted mannuronan C-5-epimerases. The secreted enzymes are composed of repeats of two protein modules designated A (385 amino acids) and R (153 amino acids). The modular structure of one of the epimerases, AlgE1, is A1R1R2R3A2R4. This enzyme has two catalytic sites for epimerization, each site introducing a different G distribution pattern, and in this article we report the DNA-level construction of a variety of truncated forms of the enzyme. Analyses of the properties of the corresponding proteins showed that an A module alone is sufficient for epimerization and that A1 catalyzed the formation of contiguous stretches of G residues in the polymer, while A2 introduces single G residues. These differences are predicted to strongly affect the physical and immunological properties of the reaction product. The epimerization reaction is Ca2+ dependent, and direct binding studies showed that both the A and R modules bind this cation. The R modules appeared to reduce the Ca2+ concentration needed for full activity and also stimulated the reaction rate when positioned both N and C terminally.     

Measurement and characterization of C-3 epimerization activity toward vitamin D3

Recently, epimerization of the hydroxyl group at C-3 has been identified as a unique metabolic pathway of vitamin D compounds. We measured C-3 epimerization activity in subcellular fractions prepared from cultured cells and investigated the basic properties of the enzyme responsible for the epimerization. C-3 epimerization activity was detected using a NADPH-generating system containing glucose-6-phosphate, NADP, glucose-6-phosphate dehydrogenase, and Mg(2+). The highest level of activity was observed in a microsomal fraction prepared from rat osteoblastic UMR-106 cells but activity was also observed in microsomal fractions prepared from MG-63, Caco-2, Hep G2, and HUH-7 cells. In terms of maximum velocity (V(max)) and the Michaelis constant (K(m)), 25-hydroxyvitamin D(3) [25(OH)D(3)] exhibited the highest specificity for the epimerization at C-3 among 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], 25(OH)D(3), 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)], and 22-oxacalcitriol (OCT). The epimerization activity was not inhibited by various cytochrome P450 inhibitors and antiserum against NADPH cytochrome P450 reductase. Neither CYP24, CYP27A1, CYP27B1 nor 3(alpha-->beta)hydroxysteroid epimerase (HSE) catalyzed the epimerization in vitro. Based on these results, the enzyme(s) responsible for the epimerization of vitamin D(3) at C-3 are thought to be located in microsomes and different from cytochrome P450 and HSE.     

Novel biosynthesis of d-pinitol in simmondsia chinensis

Chase experiments with ¹⁴CO₂ and feeding experiments with labelled inositols showed that d-pinitol in leaves of Simmondsia chinensis arises via epimerization of d-ononitol. This finding represents an alternative pathway, since d-pinitol is formed in gymnosperms and other plants by epimerization of sequoyitol.