New variants of the ADEQUATE experiments, by Josep Saurí, Ikenna E. Ndukwe, Mikhail Reibarkh, Yizhou Liu, R. Thomas Williamson and Gary E. Martin. Annual Reports on NMR Spectroscopy, 2019. In Press. DOI: https://doi.org/10.1016/bs.arnmr.2019.04.001
ADEQUATE experiments were first described in the mid-1990s and are a family of proton-detected NMR experiments used to establish carbon–carbon correlations at natural abundance. Applications of these experiments were initially limited by the statistical probability of two 13C nuclides in the same molecule (~ 1:10,000) and the low intrinsic sensitivity of the NMR probe technology of the day. In the intervening two decades, there have been significant advances in NMR probe technology and it is now possible, for example, to perform multiplicity-edited pure shift HSQC NMR experiments on a microgram of material or less. The enhanced NMR sensitivity offered by cryogenic NMR probes has dropped sample requirements for ADEQUATE experiments from the ~ 10 mg range to less than a milligram when working with a 1.7 mm Micro Cryoprobe™ at 600 MHz on a modern spectrometer. In parallel with advances in NMR probe technology, this chapter will detail a number of modifications of the ADEQUATE experiment that have been reported to enhance both the utility and sensitivity of these experiments. Combining newly reported variants of the ADEQUATE experiments with anisotropic NMR and Computer-Assisted Structure Elucidation methods affords an orthogonal means of verifying both the constitution and configuration of complex molecular structures that can stem the flow of incorrectly reported structures into the chemical literature.
Process Safety Considerations for the Supply of a High-Energy Oxadiazole IDO1-Selective Inhibitor, by Theodore A. Martinot, Michael Ardolino, Lu Chen, Yu-hong Lam, Chaomin Li, Matthew L. Maddess, Daniel Muzzio, Ji Qi, Josep Saurí, Zhiguo J. Song, Lushi Tan, Thomas Vickery, Jingjun Yin and Ralph Zhao. Org. Process Res. Dev. Published online (2019) DOI: https://doi.org/10.1021/acs.oprd.9b00105
The development of a stereospecific synthesis of a IDO1-selective inhibitor is described. The synthetic strategy toward enabling early discovery efforts along with additional findings pertaining to process safety that limited scalability are outlined. A convergent approach that supported the synthesis of material suitable for early preclinical and/or good laboratory practice toxicology studies and avoided the formation of key high-energy intermediates is summarized.
Structural elucidation of a dimeric impurity in the process development of ceftolozane using LC/HRMS and 2D-NMR, by Jing Liao, Huaming Sheng, Josep Saurí, Rong Xiang and Gary E. Martin. Journal of Pharmaceutical and Biomedical Analysis, 174 (2019) 242–247. DOI: https://doi.org/10.1016/j.jpba.2019.05.057
Ceftolozane is MSD’s 5th generation cephalosporin antibiotic with broad-spectrum gram-negative activity. While refining the synthetic route to the drug substance, a dimeric impurity was observed in the final active pharmaceutical ingredient (API) of ceftolozane. Through impurity enrichment and preparative isolation, the structure of the impurity was subsequently established through LC/HRMS, HRMSMS, H/D exchange and 2D-NMR studies. A kinetic study of the impurity formation was conducted to determine the best conditions to control it in the final chemical process.
Enhancing the utility of 1JCH couplings constants in structural studies through optimized DFT analysis, by Alexei V. Buevich, Josep Saurí, Teodor Parella, Nunziatina De Tommasi, Giuseppe Bifulco, R. Thomas Williamson and Gary E. Martin. Chem. Comm. 2019. Accepted Manuscript. DOI: 10.1039/C9CC02469G
Commonly used DFT methods for calculation of 1JCH coupling constants have typically required the application of ad hoc correction factors, modifications of functionals, or empirical scaling to improve the fit between predicted and experimental values. Here we demonstrate that highly accurate 1JCH coupling predictions can be obtained without such adjustments by careful selection of DFT methods for geometry optimization and J-coupling calculations (e.g. B3LYP/6-31G(d,p)(mixed)//mPW1PW/cc-pVTZ). The proposed method was cross-validated against a diverse set of 122 1JCH couplings and was successfully applied to the conformational and stereochemical analysis of strychnine and a previously unreported trachylobane diterpene natural product.
Cyanobufalins: Cardioactive Toxins from Cyanobacterial Blooms, by Haiyin He, Matthew J. Bertin, ShiBiao Wu, Paul G. Wahome, Kevin R. Beauchesne, Ross O. Youngs, Paul V. Zimba, Peter D. R. Moeller, Josep Saurí and Guy T. Carter. J. Nat. Prod. 2018. Article ASAP. DOI: 10.1021/acs.jnatprod.8b00736
Cyanobufalins A–C (1–3), a new series of cardiotoxic steroids, have been discovered from cyanobacterial blooms in Buckeye Lake and Grand Lake St. Marys in Ohio. Compounds 1–3contain distinctive structural features, including geminal methyl groups at C-4, a 7,8 double bond, and a C-16 chlorine substituent that distinguish them from plant- or animal-derived congeners. Despite these structural differences, the compounds are qualitatively identical to bufalin in their cytotoxic profiles versus cell lines in tissue culture and cardiac activity, as demonstrated in an impedance-based cellular assay conducted with IPSC-derived cardiomyocytes. Cyanobufalins are nonselectively toxic to human cells in the single-digit nanomolar range and show stimulation of contractility in cardiomyocytes at sub-nanomolar concentrations. The estimated combined concentration of 1–3 in the environment is in the same nanomolar range, and consequently more precise quantitative analyses are recommended along with more detailed cardiotoxicity studies. This is the first time that cardioactive steroid toxins have been found associated with microorganisms in an aquatic environment. Several factors point to a microbial biosynthetic origin for the cyanobufalins.
Trichophycins B–F, Chlorovinylidene-Containing Polyketides Isolated from a Cyanobacterial Bloom, by Matthew J. Bertin, Josep Saurí, Yizhou Liu, Christopher W. Via, Alexandre F. Roduit and R. Thomas Williamson. J. Org. Chem. 2018. Article ASAP. DOI: 10.1021/acs.joc.8b02070
NMR-guided isolation (based on 1D 1H and 13C NMR resonances consistent with a chlorovinylidene moiety) resulted in the characterization of five new highly functionalized polyketides, trichophycins B–F (1–5), and one nonchlorinated metabolite tricholactone (6) from a collection of Trichodesmium bloom material from the Gulf of Mexico. The planar structures of 1–6were determined using 1D and 2D NMR spectroscopy, mass spectrometry, and complementary spectroscopic procedures. Absolute configuration analysis of 1 and 2 were carried out by 1H NMR analysis of diastereomeric Mosher esters in addition to ECD spectroscopy, J-based configuration analysis, and DFT calculations. The absolute configurations of 3–6 were proposed on the basis of comparative analysis of 13C NMR chemical shifts, relative configurations, and optical rotation values to compounds 1 and 2. Compounds 1–5 represent new additions to the trichophycin family and are hallmarked by a chlorovinylidene moiety. These new trichophycins and tricholactone (1–6) feature intriguing variations with respect to putative biosynthetic starting units, halogenation, and terminations, and trichophycin E (4) features a rare alkynyl bromide functionality. The phenyl-containing trichophycins showed low cytotoxicity to neuro-2A cells, while the alkyne-containing trichophycins showed no toxicity.
Jizanpeptins, Cyanobacterial Protease Inhibitors from a Symploca sp. Cyanobacterium Collected in the Red Sea, by David A. Gallegos, Josep Saurí, Ryan D. Cohen, Xuemei Wan, Patrick Videau, Alec O. Vallota-Eastman, Lamiaa A. Shaala, Diaa T. A. Youssef, R. Thomas Williamson, Gary E. Martin, Benjamin Philmus, Aleksandra E. Sikora, Jane E. Ishmael and Kerry L. McPhail. J. Nat. Prod. 2018. Article ASAP. DOI: 10.1021/acs.jnatprod.8b00117
Jizanpeptins A–E (1–5) are micropeptin depsipeptides isolated from a Red Sea specimen of a Symploca sp. cyanobacterium. The planar structures of the jizanpeptins were established using NMR spectroscopy and mass spectrometry and contain 3-amino-6-hydroxy-2-piperidone (Ahp) as one of eight residues in a typical micropeptin motif, as well as a side chain terminal glyceric acid sulfate moiety. The absolute configurations of the jizanpeptins were assigned using a combination of Marfey’s methodology and chiral-phase HPLC analysis of hydrolysis products compared to commercial and synthesized standards. Jizanpeptins A–E showed specific inhibition of the serine protease trypsin (IC50 = 72 nM to 1 μM) compared to chymotrypsin (IC50 = 1.4 to >10 μM) in vitro and were not overtly cytotoxic to HeLa cervical or NCI-H460 lung cancer cell lines at micromolar concentrations.
Observation of untoward 3JCC correlations in 1,1‐ADEQUATE spectra of pyrimidine analogs: Avoiding potential interpretation pitfalls, by Hai-Young Kim, Josep Saurí, Ryan D. Cohen and Gary E. Martin. Magn. Reson. Chem. 2018. DOI: 10.1002/mrc.4736
Recently, it has been reported that large nJCC correlations can sometimes be observed in 1,1‐ADEQUATE spectra with significant intensity, which opens the possibility of structural misassignment. In this work, we have focused on pyrimidine‐based compounds, which exhibit multiple bond correlations in the 1,1‐ADEQUATE experiment as a consequence of 3JCC coupling constants greater than 10 Hz. Results are supported by both the experimental measurement of 3JCC coupling constants in question using J‐modulated‐ADEQUATE and density functional theory calculations.
Incorporating BIRD‐based homodecoupling in the dual‐optimized, inverted 1JCC 1,n‐ADEQUATE experiment, by Josep Saurí, Wolfgang Bermel, Teodor Parella, R. Thomas Williamson and Gary E. Martin. Magn. Reson. Chem. 2018. DOI: 10.1002/mrc.4732
1,n‐ADEQUATE is a powerful NMR technique for elucidating the structure of proton‐deficient small molecules that can help establish the carbon skeleton of a given molecule by providing long‐range three‐bond 13C─13C correlations. Care must be taken when using the experiment to identify the simultaneous presence of one‐bond 13C─13C correlations that are not filtered out, unlike the HMBC experiment that has a low‐pass J‐filter to filter 1JCH responses out. Dual‐optimized, inverted 1JCC 1,n‐ADEQUATE is an improved variant of the experiment that affords broadband inversion of direct responses, obviating the need to take additional steps to identify these correlations. Even though ADEQUATE experiments can now be acquired in a reasonable amount of experimental time if a cryogenic probe is available, low sensitivity is still the main impediment limiting the application of this elegant experiment. Here, we wish to report a further refinement that incorporates real‐time bilinear rotation decoupling‐based homodecoupling methodology into the dual‐optimized, inverted 1JCC 1,n‐ADEQUATE pulse sequence. Improved sensitivity and resolution are achieved by collapsing homonuclear proton–proton couplings from the observed multiplets for most spin systems. The application of the method is illustrated with several model compounds.
Identification of ortho-Substituted Benzoic Acid/Ester Derivatives via the Gas-Phase Neighboring Group Participation Effect in (+)-ESI High Resolution Mass Spectrometry, by William D. Blincoe, Agustina Rodriguez-Granillo, Josep Saurí, Nicholas A. Pierson, Leo A. Joyce, Ian Mangion and Huaming Sheng. J. Am. Soc. Mass Spectrom., 2018. DOI: 10.1007/s13361-017-1884-8
Benzoic acid/ester/amide derivatives are common moieties in pharmaceutical compounds and present a challenge in positional isomer identification by traditional tandem mass spectrometric analysis. A method is presented for exploiting the gas-phase neighboring group participation (NGP) effect to differentiate ortho-substituted benzoic acid/ester derivatives with high resolution mass spectrometry (HRMS1). Significant water/alcohol loss (>30% abundance in MS1 spectra) was observed for ortho-substituted nucleophilic groups; these fragment peaks are not observable for the corresponding para and meta-substituted analogs. Experiments were also extended to the analysis of two intermediates in the synthesis of suvorexant (Belsomra) with additional analysis conducted with nuclear magnetic resonance (NMR), density functional theory (DFT), and ion mobility spectrometry-mass spectrometry (IMS-MS) studies. Significant water/alcohol loss was also observed for 1-substituted 1, 2, 3-triazoles but not for the isomeric 2-substituted 1, 2, 3-triazole analogs. IMS-MS, NMR, and DFT studies were conducted to show that the preferred orientation of the 2-substituted triazole rotamer was away from the electrophilic center of the reaction, whereas the 1-subtituted triazole was oriented in close proximity to the center. Abundance of NGP product was determined to be a product of three factors: (1) proton affinity of the nucleophilic group; (2) steric impact of the nucleophile; and (3) proximity of the nucleophile to carboxylic acid/ester functional groups.