Exo-selective intermolecular Diels-Alder reaction by PyrI4 and AbnU on non-natural substrates.

The 100-year-old Diels-Alder reaction (DAr) is an atom economic and elegant organic chemistry transformation combining a 1,3-diene and a dienophile in a [4+2] cycloaddition leading to a set of products with several stereo centres and multiple stereoisomers. Stereoselective [4+2] cycloaddition is a challenge. Here, we describe two natural enzymes, PyrI4 and AbnU performing stereospecific intermolecular DAr on non-natural substrates. AbnU catalyses a single exo-stereoisomer by 32-fold higher than the background. PyrI4 catalyses the same stereoisomer (15-fold higher) as a major component (>50%). Structural, biochemical and fluorescence studies indicate that the dienophile enters first into the ß-barrel of the enzymes followed by the 1,3-diene, yielding a stereospecific product. However, if some critical interactions are disrupted to increase the catalytic efficiency, stereoselectivity is compromised. Since it is established that natural enzymes can carry out intermolecular DAr on non-natural substrates, several hundreds of Diels-Alderases available in nature could be explored.

2-Cyano-3-(2-thienyl)acrylic Acid as a New MALDI Matrix for the Analysis of a Broad Spectrum of Analytes.

A low-cost synthetic 2-cyano-3-(2-thienyl)acrylic acid (CTA) is developed as a new MALDI matrix for the analysis of various classes of compounds such as lipids (e.g., fatty acids), peptides, proteins, saccharides, natural products (i.e., iridoids), PEGs, and organometallics in the positive-ion mode. The difficulty in the analysis of high molecular mass PEGs was overcome by using CTA as matrix even at low concentrations. Both high molecular mass proteins and peptides were successfully analyzed using CTA. The mass spectra of all of the studied analytes with CTA showed high signal-to-noise (S/N) ratios and spectral resolutions when compared to other conventional matrices such as SA, DHB, DT, and HCCA. However, in the case of peptide analysis with CTA, the resulting mass spectra are found to be similar to that of the well-established HCCA matrix. On the basis of the physicochemical properties of the analytes, the CTA works as a proton/cation or electron-transfer matrix. It proves that the CTA can be used as a common matrix for the analysis of majority classes of analytes instead of using a specific matrix for the particular class of analytes. Further, the CTA provides an advantage in the analysis of unknown samples as it rules out ambiguity in the selection of particular matrix and it may also offer a complete profile of the tissue surface in the MALDI-imaging experiments.

Identification and characterization of degradation products of indacaterol using liquid chromatography/mass spectrometry.

Indacaterol (IND), 5-[2-[(5,6-Diethyl-2,3-dihydro-1H-inden-2-yl)amino]-1-hydroxyethyl]-8-hydroxyquinolin-2(1H)-one, is an active pharmaceutical ingredient (API) which is used to treat chronic obstructive pulmonary disease (COPD). We followed the International Council for Harmonization (ICH) guide lines to study the degradation behavior of IND under various stress conditions. Stressed degradation of the drug was performed under hydrolytic (alkaline, acidic and neutral), photolytic, oxidative and thermal conditions. Identification and characterization of IND and its forced degradation products (DPs) were demonstrated by using LC-HRMS and MS/MS method. A total of three DPs (DP1-DP3) were identified and characterized. The IND was found to be stable under photolytic, oxidative and thermal conditions, whereas it produced three DPs in acidic, basic and neutral hydrolytic stress conditions.

Puromycin, a selective inhibitor of PSA acts as a substrate for other M1 family aminopeptidases: Biochemical and structural basis.

Puromycin sensitive aminopeptidase (PSA or NPEPPS) is a M1 class aminopeptidase is selectively inhibited by the natural product puromycin, an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. The molecular basis for this selective inhibition has not been understood well. Here, we report the basis for selectivity of puromycin using biochemical, structural and molecular modeling tools on four different M1 family enzymes including human PSA. Except for PSA, the other three enzymes were not inhibited. Instead, the peptide bond in the puromycin is hydrolyzed to O-methyl-L-tyrosine (OMT) and puromycin aminonucleoside (PAN). Neither of the hydrolyzed products, individually or together inhibit any of the four enzymes. Crystal structure of ePepN using crystals that are incubated with puromycin contained the hydrolyzed products instead of intact puromycin. On the other hand, intact puromycin molecule was observed in the crystal structure of the inactive mutant ePepN (E298A)-puromycin complex. Surprisingly, puromycin does not enter the active site of the mutant enzyme but binds near the entrance. Comparison of puromycin binding region in ePepN mutant enzyme and molecular modeling studies suggest that PSA might be inhibited by similar mode of binding there by blocking the entrance of the active site.

In vitroand in vivo investigation of metabolic fate of riociguat by HPLC-Q-TOF/MS/MS and in silico evaluation of the metabolites by ADMET predictor™.

Riociguat, a guanyl cyclase inhibitor, is one of its kind drug regimen approved for management of pulmonary arterial hypertension and chronic thromboembolism pulmonary hypertension. Extensive literature review indicates lack of comprehensive reports on its metabolic fate. The present study reports the in vivo and in vitro identification and characterization of metabolites of riociguat, using high-performance liquid chromatography-quadruple time-of-flight tandem mass spectrometry. In vitro studies were conducted by incubating the drug in human and rat liver microsomes in presence of respective cofactors. In vivo studies were undertaken by oral administration of suspension of drug to male Sprague-Dawley rats followed by collection of urine, feces and blood at specific intervals. A total of 18 metabolites were observed in in vivo and in vitro matrices which includes hydroxyl, N-oxide, desmethyl, defluorinated hydroxyl, glucuronides and N-acetyl cysteine conjugates. Presence of N-acetyl cysteine conjugates strongly points towards the formation of a reactive metabolite intermediate trapped through N-acetyl cysteine and can be considered a matter of concern as the reactive metabolites have been known to manifest toxicities. Their presence was mimicked in in vitro samples as well. The toxicological properties of drug and metabolites were evaluated by using ADMET Predictor ™ software.

Characterization of degradation products of macitentan under various stress conditions using liquid chromatography/mass spectrometry.

RATIONALE: Stress testing of a drug candidate is an important step in the drug discovery and development process. The presence of degradation products in a drug affects the quality as well as the safety and efficacy of drug formulation. Hence, it is essential to develop an efficient analytical method which could be useful for the separation, identification and characterization of all possible degradation products (DPs) of a drug. Macitentan (MT) is an endothelin receptor antagonist (ERA) drug used to treat high blood pressure in the lungs. Comprehensive stress testing of MT was carried out as per ICH guidelines to understand the degradation profile of the drug. METHODS: MT was subjected to various stress conditions such as acidic, basic, neutral hydrolysis, oxidation, photolysis and thermal conditions; and the resulting degradation products were investigated using liquid chromatography/diode-array detection/electrospray ionization high-resolution mass spectrometry (LC/DAD/ESI-HRMS) and tandem mass spectrometry (MS/MS) techniques. An efficient and simple ultra-high-performance liquid chromatography (UHPLC) method has been developed using an Accucore C18 column (4.6 × 150 mm, 2.6 µm) using a gradient elution of 5 mM ammonium formate and acetonitrile as mobile phases. RESULTS: MT was found to degrade under acid and base hydrolysis stress conditions; whereas it was stable under oxidation, neutral hydrolysis, thermal and photolytic conditions. MT formed nine DPs (DP1 to DP9) and one DP (DP10) under acidic and basic hydrolytic conditions, respectively. All the degradation products (DP1 to DP10) were identified and characterized by LC/MS/MS in positive ion mode with accurate mass measurements. CONCLUSIONS: MT was found to be labile under hydrolytic conditions. The structures of the DPs were characterized by appropriate mechanisms. The proposed method can be effectively used for the characterization of MT and its DPs.