Two-dimensional NMR Spectroscopy Research Articles

Structure of the K141 capsular polysaccharide produced by Acinetobacter baumannii isolate KZ1106 that carries KL141 at the chromosomal K locus

The structure of the K141 type capsular polysaccharide (CPS) produced by Acinetobacter baumannii KZ1106, a clinical isolate recovered from Kazakhstan in 2016, was established by sugar analyses and one- and two-dimensional 1H and 13C NMR spectroscopy. The CPS was shown to consist of branched tetrasaccharide repeating units (K-units) with the following structure: [Display omitted] This structure was found to be consistent with the genetic content of the KL141 CPS biosynthesis gene cluster at the chromosomal K locus in the KZ1106 whole genome sequence. Assignment of the encoded enzymes allowed the first sugar of the K unit to be identified, which revealed that the β-d-GlcpNAc-(1→3)-d-GlcpNAc bond is the linkage between K-units formed by the WzyKL141 polymerase.

Revised structure of the polysaccharide from Acinetobacter baumannii LUH5551 assigned as the K63 type capsular polysaccharide

K63 capsular polysaccharide produced by Acinetobacter baumannii isolate LUH5551 (previously designated isolate O24) was re-examined using sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. Though previously reported as O24 consisting of linear tetrasaccharide units that include a 7-acetamido-5-acylamino form of 8-epilegionaminic acid [8eLeg5R7Ac, acylated at C5 with (S)-3-hydroxybutanoyl or acetyl (1:1)], the elucidated structure of the K63 type capsule was found to include a derivative of 5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic (legionaminic) acid, Leg5Ac7R, where R is either (S)-3-hydroxybutanoyl or an acetyl group (∼1:1 ratio). This finding is consistent with the presence of the lgaABCHIFG gene module for Leg5Ac7R biosynthesis in the KL63 gene cluster at the capsular polysaccharide (CPS) biosynthesis K locus in the LUH5551 genome. The glycosyltransferases (Gtrs) and Wzy polymerase encoded by KL63 were assigned to linkages in the linear K63 tetrasaccharide unit and linkage of the K63 units.

Структура и надмолекулярное упорядочение L- и D-аспарагината хитозана

Chitosan (CS) with a viscosity-average molecular weight of 200 kDa and a deacetylation degree of 82 mol%, produced by Bioprogress Ltd. (RF) has been used in this work. Aqueous solutions of enantiomeric salt complexes of CS with L- and D-aspartic acid (AspA) have been obtained at an equimolar CS:AspA ratio, in terms of amino groups. Powders of CS·L-(D-) AspA salts have been isolated from the corresponding solutions by evaporation of water and stored in a desiccator at zero humidity. It has been established that under such conditions a water-soluble salt form of the polymer with lamellar light beige particles 0.05–1.0 mm in size is formed. Using the methods of elemental analysis, IR and NMR spectroscopy, and X-ray diffractometry, the chemical interaction of CS with L-(D-)AspA in aqueous solution and condensed state has been evaluated, and the chemical structure and supramolecular ordering of these enantiomeric salts have been studied. It has been established that, according to the classification of K. Ogawa et al., the formula unit CS·L-(D-)AspA corresponds to non-hydrated salts of type I, in which water molecules are replaced by acid anions. IR spectroscopy confirmed the donor--acceptor polymer–acid interaction and revealed a developed system of intermolecular and intramolecular contacts. One- and two-dimensional NMR spectroscopy showed the interaction of pairs of atomic nuclei between H3–H6 of the polymer and 2Hβ or Hα of the acid, H1 or H2 and Hα, H1 and H3–H6, due to the spatial proximity of protons in repeating monomer units, in the “bend” chain segments removed along the chain, and in neighboring macromolecules. For CS·L-AspA, additional resonances have been identified between H2 and H3–H6 of the polymer, for CS·D-AspA these have been between H1 or H 2. X-ray diffractometry has revealed a highly ordered orientation of macrochains and a high crystallinity degree, untypical for CS salts. The salt complex CS·D-AspA, in contrast to CS·L-AspA, is characterized by a smaller amount of crystallization water, a more ordered supramolecular structure, and a more developed system of intermolecular and intramolecular contacts.

Obtaining the Enoxaparin Sodium Substance Equivalent to the Original Clexane® and Lovenox®. The Selection of Technological Parameters and Optimization of the “Greenness” of the Purification Stage

The aim of the study was to adjust and optimize the purification stage of crude enoxaparin sodium to obtain a substance equivalent to the original drugs Clexane® and Lovenox® according to the criteria specified by the FDA. The purification stage involves the reprecipitation of crude enoxaparin in methanol. Determining the ratio of solvents required for the reprecipitation is important for studying the correlation between the experimental conditions of the technological process and the structural characteristics of enoxaparin samples. In the study, the method of purification of enoxaparin sodium described in the patent was assessed, and the following variations of the MeOH:H2O solvent ratio were selected – 4:1; 2:1; 1:1. The obtained samples of enoxaparin sodium were analyzed according to the in-house specification developed on the basis of the pharmacopoeial monograph, as well as by non-pharmacopoeial methods, such as two-dimensional NMR spectroscopy (HSQC) and size exclusion chromatography (SEC) for detailed characterization of the molecule. Strategies of greening of the enoxaparin sodium purification stage by reducing the E-factor were also considered in the study. Considering the principles of “green” chemistry, the method of purification of crude enoxaparin sodium was optimized by the solvent regeneration. It was experimentally possible to demonstrate the effect of the solvent ratio at the stage of purification of crude enoxaparin on the composition, as well as on the number and distribution of oligosaccharide fractions in the molecule. Based on the results of the study, it can be concluded that the ratio of MeOH:H2O=1:1 allows obtaining samples that are closest to Clexane® and Lovenox® in terms of the molecular weight distribution profile and the composition profile. The E-factor was also reduced from 14 to 5.25 by solvent regeneration.

Synthesis, Spectroscopic Characterization, Catalytic and Biological Activity of Oxidovanadium(V) Complexes with Chiral Tetradentate Schiff Bases

New oxidovanadium(V) complexes, VOL1–VOL10, with chiral tetradentate Schiff bases obtained by monocondensation reaction of salicylaldehyde derivatives with 1S,2S-(+)-2-amino-1-(4-nitrophenyl)-1,3-propanediol. All complexes have been characterized using different spectroscopic methods, viz. IR, UV-Vis, circular dichroism, one- (1H, 51V) and two-dimensional (COSY, NOESY) NMR spectroscopy, and elemental analysis. Furthermore, the catalytic ability of all compounds in the epoxidation of styrene, cyclohexene, and its naturally occurring monoterpene derivatives, i.e., S(−)-limonene and (−)-α-pinene has also been studied, using two different oxidants, i.e., aqueous 30% H2O2 or tert-butyl hydroperoxide (TBHP). In addition, the biological properties of these chiral oxidovanadium(V) compounds, but also cis-dioxidomolybdenum(VI) complexes with the same chiral Schiff bases, were studied. Their cytotoxic and cytoprotective activity studies with the HT-22 hippocampal neuronal cells revealed a concentration-dependent effect in the range of 10–100 μM. Moreover, vanadium(V) complexes, in contrast to cis-dioxidomolybdenum(VI) compounds, demonstrated higher cytotoxicity and lack of cytoprotective ability against H2O2-induced cytotoxicity.

Cytotoxic Impact of Fluorinated Ligands in Equatorial Position of Trans-Configured Diam(m)inetetracarboxylatoplatinum(IV) Complexes

A series of thirty novel tetracarboxylatoplatinum(IV) complexes in trans-configuration featuring combinations of mixed ammine, methylamine, dimethylamine, and cyclopentylamine ligands as well as acetato/propanoato and trifluoropropanoato ligands was synthesised. The platinum(IV) complexes were characterised by one- and two-dimensional multinuclear NMR spectroscopy (1H, 13C, 15N, 19F, 195Pt), ESI-MS, elemental analysis, and X-ray diffraction. Additional parameters such as reduction behaviour and lipophilicity were measured via NMR spectroscopy and RP-HPLC, revealing slow reduction and a broad spectrum of log kw values in line with the respective ligand combination. In order to determine structure–activity relationships, cytotoxic activity was evaluated via the MTT assay in three human cancer cell lines (CH1/PA-1, ovarian teratocarcinoma, SW480, colon adenocarcinoma, A549, non-small-cell lung carcinoma). The induction of apoptosis and necrosis was determined in SW480 cells via the flow-cytometric annexin V/PI assay. In general, a tendency of higher lipophilicity leading to higher cytotoxicity was noticed. In contrast, lipophilicity alone plays a subordinate role for the induction of apoptosis, which strongly depends on the combination of am(m)ine and trifluoropropanoato ligands.

Butyrolactones and acid esters from plant rhizosphere soil-derived Aspergillus sp. TW01-p-15

The fungus Aspergillus sp. TW01-p-15 was isolated from rhizosphere soil of Pteris cretica L. var. nervosa in Xitoushan, Taiwan Province of China. From the ethyl acetate extract of the fungus, five new compounds including two new butyrolactones named 2-carbonic acid-ester- butyrolactone V (1), versicolactone H (2), one new butyrolactone derivative 2-hydroxy-1,3-bis(4-hydroxyphenyl) propan-1-one (3), two new acid esters named (Z)-6-methoxy-3-methyl-6-oxohex-2-enoic acid (4) and (E)-6-methoxy-3-methyl-6-oxohex-2-enoic acid (5), together with nine known compounds were isolated. Their structures were elucidated by diverse spectroscopic analysis including high-resolution ESI-MS, one- and two-dimensional NMR spectroscopy and time-dependent density functional theory electronic circular dichroism (TDDFT ECD) calculations. In the in vitro activity experiment, compounds 3 and 13 showed significant activity against Pseudomonas aeruginosa. This result was further supported by molecular docking.

Lignan and Phthalide Derivatives from the Rhizome of Ligusticum chuanxiong (Rhizoma chuanxiong) and Evaluation of Their anti-Xanthine Oxidase Activities.

The previous research results showed that the extracts of ethyl acetate of the rhizome of Ligusticum chuanxiong (Rhizoma chuanxiong) possessed significant antigout effects in model mice. To explore the active ingredients responsible for the effects, phytochemical studies were performed, which led to the isolation of three rare 8', 9-linked neolignans, ligusticumins A-C (1-3), together with two novel phthalide-phenylpropanoid heterodimers, ligusticumalides A-B (4 and 5). It is noteworthy that 4 possesses an unprecedented 7-styryl phthalide skeleton. The structures and absolute configurations of 1-5 were elucidated by one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy and electron-capture detector (ECD) spectroscopic methods. The bioassay results showed that compounds 1, 2, 3, and 5 presented moderate inhibitory activities against xanthine oxidase (XO) and 4 possessed a significant XO inhibitory effect with an IC50 value of 93.88 μM. This is the first time to investigate the anti-XO active ingredients of R. chuanxiong, which provides valuable information for searching for new antigout agents from natural products.

A rare tetrahydroimidazopyridine from the marine-derived fungus Paraconiothyrium sp. YK-03

A new tetrahydroimidazopyridine named butyl (5R,6R,7S,8S)-5,6,7,8-tetrahydro-6,7,8-trihydroxy-5-(hydroxymethyl)imidazo[1,2-a]pyridine-2-carboxylate(1), together with eight known compounds (2–9), were isolated from the fermentation broth of a marine-derived fungus Paraconiothyrium sp. YK-03. Their chemical structures were elucidated by extensive analysis of one-dimensional and two-dimensional NMR spectroscopy, HR-ESIMS and optical rotation. Among these compounds, compound 1 represented a rare tetrahydroimidazopyridine, and compounds 2–7 were isolated from the Paraconiothyrium species for the first time. A plausible biosynthetic pathway for compound 1 was proposed.

Interaction of Methylenecycloalkanes with BF3·THF Catalyzed by 2TiCl2

The Cp2TiCl2-catalyzed interaction of methylenecycloalkanes with BF3·THF in tetrahydrofuran was carried out for the first time with the formation of target 1-fluoro-1-boraspirocarbocycles and also isomerization products of a starting monomer (1-methylcycloalk-1-enes). The structure of reaction products was elucidated using one(1H, 13C Dept, 11B, 19F) and two-dimensional (COSY, HSQC, HMBC) NMR spectroscopy, mass spectrometry combined with quantum-chemical calculations of 13C NMR chemical shifts.

4a′-Hydroxy-3′,3′,5,6′,6′,7-hexamethyl-3′,4′,4a′,6′,7′,9a′-hexahydrospiro[indole-3,9′-xanthene]-1′,2,8′(1H,2′H,5′H)-trione

Pseudo-multicomponent reactions (Pseudo-MCRs) have led to a variety of compounds with interesting biological properties, especially desirable in the pharmaceutical industry. The isatin nucleus could be considered a privileged scaffold for the design of biologically active substances. Dimedone is an interesting and versatile molecule for most organic transformations, especially one-pot and multicomponent reactions. Xanthene derivatives are still an attractive research field for both academia investigations and industry. In this investigation, a simple and efficient tandem Knoevenagel–Michael protocol with subsequent cyclization for the synthesis of the previously unknown 4a′-hydroxy-3′,3′,5,6′,6′,7-hexamethyl-3′,4′,4a′,6′,7′,9a′-hexahydrospiro[indole-3,9′-xanthene]-1′,2,8′(1H,2′H,5′H)-trione was elaborated. The suggested method is based on the pseudo-MCR of 5,7-dimethylisatin and dimedone. The structure of the earlier unknown compound was proven using 1H, 13C-NMR, and IR spectroscopy, mass spectrometry, and elemental analysis. To compare the developed protocol with the existing ones, unsubstituted spiro[indole-3,9′-xanthene] was synthesized. Its structure has been proven using two-dimensional (2D) NMR spectroscopy techniques.

Isolation and Physicochemical Characteristics of Water-Soluble Polysaccharide from Locally Growing and Cultivated Basidium Raw Materials of Ganoderma lucidum

Branched polysaccharides have been isolated from basidiomycete raw materials of locally growing and cultivated Ganoderma lucidum. It has been found that the isolated fractions contain branched polysaccharides in the form of complexes with melanin. After purification of polysaccharides by ion exchange chromatography from locally growing and cultivated basidial raw materials, two fractions have been obtained: neutral polysaccharides of locally growing Ganoderma lucidum (GW-1), cultivated Ganoderma lucidum (GWL-1) with a yield of 25.71 and 29.85%, respectively, and anionic polysaccharides of locally growing Ganoderma lucidum (GW-2), cultivated Ganoderma lucidum (GWL-2), with a yield of 5.26 and 4.19%. The physicochemical properties of the obtained samples have been studied by IR and UV spectroscopies. The purity degree of fractions of branched polysaccharides has been determined. Using gas chromatography, one-dimensional (13C NMR, 1H NMR), and two-dimensional (COSY, TOCSY, HSQC, HMBC, NOESY) NMR spectroscopies, the compositions and molecular structures of the obtained polysaccharide samples have been determined. The results showed that the isolated and purified polysaccharides are β-glucan-type branched polysaccharides that have branch point (1,4,6)- and (1,3,6)-linked glucopyranose residues.

4,5-Bisnitratomethyl and Similar Representatives of the Rare Class of 2-Nitro-1,2,3-triazoles.

A [3+2]-cycloaddition toward bishydroxymethyl-1,2,3-triazole makes the title compound available through selective nitration. The obtained sensitive 2-nitrotriazole was shown to have a high density of 1.764 g cm-3 and a detonation velocity of 8590 m s-1. It can also be classified as an oxidizer with an oxygen balance toward CO of 12%. Further representatives of this rare class of 2-nitro-1,2,3-triazoles were synthesized. One- and two-dimensional 15N NMR spectroscopy, crystal structure, and elemental analysis were performed.

Duckweed pectic-arabinogalactan-proteins can crosslink through borate diester bonds

Material containing pectin and arabinogalactan-protein (AGP) was released and purified from Spirodela alcohol insoluble residues. Results of carbohydrate analyses and two-dimensional NMR spectroscopy suggest that this material is composed of apiogalacturonan and rhamnogalacturonan-I covalently attached to AGPs. 11B NMR spectroscopy indicated that some of the glycoses in this complex exist as their boric acid monoesters. Borate diesters were formed when the pectic-AGPs were allowed to react at pH above 6.2 with the boron-depleted pectic-AGPs, suggesting that in vitro two pectic-AGP molecules can crosslink to one another through borate. Borate diesters also formed when the pectic-AGPs were incubated with monomeric rhamnogalacturonan-II in the presence of Pb2+ ion at pH 9.2. This data presents evidence of the first wall polymer after rhamnogalacturonan-II to crosslink through borate diesters. We suggest that the formation of these borate-crosslinks may help Spirodela respond to high-pH condition.

Synthesis, Electrochemical and Photochemical Properties of Sulfanyl Porphyrazine with Ferrocenyl Substituents.

Ferrocene is useful in modern organometallic chemistry due to its versatile applications in material sciences, catalysis, medicinal chemistry, and diagnostic applications. The ferrocene moiety can potentially serve many purposes in therapeutics and diagnostics. In the course of this study, (6-bromo-1-oxohexyl)ferrocene was combined with dimercaptomaleonitrile sodium salt to yield a novel maleonitrile derivative. Subsequently, this compound was subjected to an autocyclotetramerization reaction using the Linstead conditions in order to obtain an octaferrocenyl-substituted magnesium(II) sulfanyl porphyrazine. Following that, both compounds-the maleonitrile derivative and the porphyrazine derivative-were subjected to physicochemical characterization using UV-Vis, ES-TOF, MALDI-TOF, and one-dimensional and two-dimensional NMR spectroscopy. Moreover, the sulfanyl porphyrazine was subjected to various photophysical studies, including optical absorption and emission measurements, as well as the evaluation of its photochemical properties. Values of singlet oxygen generation quantum yields were obtained in different organic solvents. The electrochemical properties of the synthesized compounds were studied using cyclic voltammetry. According to the electrochemical results, the presence of electron-withdrawing oxohexyl groups attached to ferrocene afforded significantly more positive oxidation potentials of the ferrocene-based redox process up to 0.34 V vs. Fc+/Fc.

GLYCIDYL METHACRYLATE AS A NON-SILANE MODIFIER IN RUBBER/SILICA COMPOSITES

ABSTRACT Glycidyl methacrylate (GMA) was grafted onto styrene butadiene rubber (SBR) and silica by a solution grafting process. Successful grafting onto rubber and silica was confirmed by Fourier-transform infrared (FTIR) spectrometry, with the peaks at 1149, 842, and 1729 cm−1 attributed to the C–O stretching, the epoxy group, and the C=O stretching vibration of the GMA monomer, respectively. After grafting onto the silica surface, the peak at 842 cm−1 in the spectra disappeared, confirming the grafting reaction through the epoxy group. Grafting onto SBR was also confirmed by proton nuclear magnetic resonance (1H NMR) spectroscopy, and the SBR-GMA interaction was shown using two-dimensional 1H NMR spectroscopy. The grafted products were further characterized by thermogravimetric analysis (TGA) and differencial scanning calorimetry. Grafting density and grafting weight percentage of GMA-grafted silica were calculated using TGA: 10 and 20% GMA-grafted SBR and 20% GMA-grafted silica compounds and their vulcanizates were prepared. Rubber–silica interaction through grafted GMA was shown using FTIR spectroscopy for both types of vulcanizates. Dispersion of pristine and GMA-modified silica onto the SBR matrix was quantified using scanning electron microscopy and atomic force microscopy. Mechanical and dynamic mechanical properties of these GMA-modified vulcanizates were studied and compared with those of previously reported 3-octanoylthio-1-propyltriethoxysilane–modified vulcanizates. The effect of the state of dispersion of silica onto the physico-mechanical properties of the vulcanizates was investigated. The properties of the non-silane–modified systems were comparable with, and in some cases superior to, that of the new mercapto silane-modified systems in the energy-efficient tire application.

Structure elucidation and biological activities of perylenequinones from an Alternaria species

The KEAP1-Nrf2/ARE pathway is a pivotal cytoprotective regulator against oxidative stress which plays an important role in the development of many inflammatory diseases and cancer. Activation of the Nrf2 transcription factor by oxidative stress or electrophiles regulates antioxidant response element (ARE)-dependent transcription of antioxidative, detoxifying, and anti-inflammatory proteins. Therefore, modulators of the KEAP1-Nrf2/ARE pathway have received considerable interest as therapeutics to protect against diseases where oxidative stress constitutes the underlying pathophysiology. In a search for fungal secondary metabolites affecting the Nrf2/ARE-dependent expression of a luciferase reporter gene in BEAS-2B cells, three new perylenequinones, compounds 1, 2, and 3, together with altertoxin-I (ATX-I), were isolated from fermentations of an Alternaria species. The structures of the compounds were elucidated by a combination of one- and two-dimensional NMR spectroscopy and mass spectrometry. Compound 1 and ATX-I exhibited strong cytotoxic effects with LC50-values of 3.8 µM and 6.43 µM, respectively, whereas compound 3 showed no cytotoxic effects up to 100 µM on BEAS-2B cells. ATX-I induced ARE-dependent luciferase expression approximately fivefold and compound 1 approximately 2.6-fold at a concentration of 3 µM in transiently transfected BEAS-2B cells. In addition, compound 1 and ATX-I exhibited strong oxidative effects, whereas compound 3 did not show significant oxidative properties. For compound 1 and ATX-I, a strong upregulation of heme oxygenase-1 could be observed on mRNA and protein level in treated BEAS-2B cells. Moreover, compound 3 significantly decreased sod3 mRNA levels after induction of oxidative stress with benzoquinone.

The structure of Klebsiella pneumoniae K108 capsular polysaccharide is similar to Escherichia coli colanic acid

The clinical isolate of Klebsiella pneumoniae 1333/P225 was revealed as containing a KL108 K. pneumoniae K locus for capsule biosynthesis. The gene cluster demonstrated a high level of sequence and arrangement similarity with that of the E. coli colanic acid biosynthesis gene cluster. The KL108 gene cluster includes a gene of WcaD polymerase responsible for joining oligosaccharide K units into capsular polysaccharide (CPS), acetyltransferase, pyruvyltransferasefive and genes for glycosyltransferases (Gtrs), four of which have homologues in genetic units of the colanic acid synthesis. The fifth Gtr is specific to this cluster. The work involved the use of sugar analysis, Smith degradation and one- and two-dimensional 1H and 13C NMR spectroscopy to establish the structure of the K108 CPS. The CPS repetitive K unit is composed of branched pentasaccharide with three monosaccharides in the backbone and a disaccharide side chain. The main chain is the same as for colanic acid but the side chain differs. Two bacteriophages infecting K. pneumoniae strain 1333/P225 were isolated and structural depolymerase genes were determined; depolymerases Dep108.1 and Dep108.2 were cloned, expressed and purified. It was demonstrated that both depolymerases specifically cleave the β-Glcp-(1→4)-α-Fucp linkage between K108 units in the CPS.

Two-dimensional solid-state NMR spectroscopy investigations of surface precipitation of phosphate onto calcite

The interaction of phosphate with typical soil minerals is important for understanding P cycling in natural and agricultural systems. We investigated the mechanisms of kinetics of phosphate uptake onto calcite using solid-state NMR spectroscopy. At a low phosphate concentration of 0.5 mM, the 31P single-pulse solid-state NMR peak revealed the formation of amorphous calcium phosphate (ACP) within the initial 30 min, which transformed to carbonated hydroxyapatite (CHAP) after 12 d. At a high phosphate concentration (5 mM), the results showed transformation from ACP to OCP, later to brushite, and eventually to CHAP. The formation of brushite is further supported by 31P{1H} heteronuclear correlation (HETCOR) spectra via a correlation of δP–31 = 1.7 ppm and the 1H peak at δH–1 = 6.4 ppm, which denotes the structure water of brushite. Furthermore, 13C NMR directly revealed both A-type and B-type CHAP. Generally, this work provides a detailed understanding of the aging effect on the phase transition scale of phosphate surface precipitation onto calcite in soil environments.

Mitigating a Bioactivation Liability with an Azetidine-Based Inhibitor of Diacylglycerol Acyltransferase 2 (DGAT2) En Route to the Discovery of the Clinical Candidate Ervogastat.

We recently disclosed SAR studies on systemically acting, amide-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) that addressed metabolic liabilities with the liver-targeted DGAT2 inhibitor PF-06427878. Despite strategic placement of a nitrogen atom in the dialkoxyaromatic ring in PF-06427878 to evade oxidative O-dearylation, metabolic intrinsic clearance remained high due to extensive piperidine ring oxidation as exemplified with compound 1. Piperidine ring modifications through alternate N-linked heterocyclic ring/spacer combination led to azetidine 2 that demonstrated lower intrinsic clearance. However, 2 underwent a facile cytochrome P450 (CYP)-mediated α-carbon oxidation followed by azetidine ring scission, resulting in the formation of ketone (M2) and aldehyde (M6) as stable metabolites in NADPH-supplemented human liver microsomes. Inclusion of GSH or semicarbazide in microsomal incubations led to the formation of Cys-Gly-thiazolidine (M3), Cys-thiazolidine (M5), and semicarbazone (M7) conjugates, which were derived from reaction of the nucleophilic trapping agents with aldehyde M6. Metabolites M2 and M5 were biosynthesized from NADPH- and l-cysteine-fortified human liver microsomal incubations with 2, and proposed metabolite structures were verified using one- and two-dimensional NMR spectroscopy. Replacement of the azetidine substituent with a pyridine ring furnished 8, which mitigated the formation of the electrophilic aldehyde metabolite, and was a more potent DGAT2 inhibitor than 2. Further structural refinements in 8, specifically introducing amide bond substituents with greater metabolic stability, led to the discovery of PF-06865571 (ervogastat) that is currently in phase 2 clinical trials for the treatment of nonalcoholic steatohepatitis.