Ultra-clean pure shift NMR with optimal water suppression for analysis of aqueous pharmaceutical samples.

Pure shift NMR experiments greatly enhance spectral resolution by collapsing multiplet structures into singlets and, with water suppression, can be used for aqueous samples. Here, we combine ultra-clean pure-shift NMR (SAPPHIRE) with two different internally encoded water suppression schemes to achieve optimal performance for small molecule and macrocyclic peptide pharmaceuticals in water and acetonitrile-water mixtures.

Quantitative nuclear magnetic resonance of chloride by an accurate internal standard approach.

Chloride is the most common counterion used to improve aqueous solubility and enhance stability of small molecule active pharmaceutical ingredients. While several analytical techniques, such as titration, HPLC with charged aerosol detection, and ion chromatography, are currently utilized to assay the level of chloride, they have notable limitations, and these instruments may not be readily available. Here, we present a generally applicable 35 Cl solution NMR method to assay the level of chloride in pharmaceutical compounds. The method uses KClO4 as an internal standard for improved accuracy in comparison with external standard methods, and it was found to be robust, linear over three orders of magnitude, precise (<3% RSD), and accurate (<0.5% absolute error).

DELTA50: A Highly Accurate Database of Experimental 1H and 13C NMR Chemical Shifts Applied to DFT Benchmarking.

Density functional theory (DFT) benchmark studies of 1H and 13C NMR chemical shifts often yield differing conclusions, likely due to non-optimal test molecules and non-standardized data acquisition. To address this issue, we carefully selected and measured 1H and 13C NMR chemical shifts for 50 structurally diverse small organic molecules containing atoms from only the first two rows of the periodic table. Our NMR dataset, DELTA50, was used to calculate linear scaling factors and to evaluate the accuracy of 73 density functionals, 40 basis sets, 3 solvent models, and 3 gauge-referencing schemes. The best performing DFT methodologies for 1H and 13C NMR chemical shift predictions were WP04/6-311++G(2d,p) and ωB97X-D/def2-SVP, respectively, when combined with the polarizable continuum solvent model (PCM) and gauge-independent atomic orbital (GIAO) method. Geometries should be optimized at the B3LYP-D3/6-311G(d,p) level including the PCM solvent model for the best accuracy. Predictions of 20 organic compounds and natural products from a separate probe set had root-mean-square deviations (RMSD) of 0.07 to 0.19 for 1H and 0.5 to 2.9 for 13C. Maximum deviations were less than 0.5 and 6.5 ppm for 1H and 13C, respectively.

Deuterated Modifiers in Sub/Supercritical Fluid Chromatography for Streamlined NMR Structure Elucidation.

Isolation and chemical characterization of target components in fast-paced pharmaceutical laboratories can often be challenging, especially when dealing with mixtures of closely related, possibly unstable species. Traditionally, this process involves intense labor and manual intervention including chromatographic method development and optimization, fraction collection, and drying processes prior to NMR analyses for unambiguous structure elucidation. To circumvent these challenges, a foundational framework for the proper utilization of supercritical carbon dioxide (scCO2) and deuterated modifiers (CD3OD) in sub/supercritical fluid chromatography (SFC) is herein introduced. This facilitates a streamlined multicomponent isolation with minimized protic residues, further enabling immediate NMR analysis. In addition to bypassing tedious drying processes and minimizing analyte degradation, this approach (complementary to traditional reversed-phase liquid chromatography, RPLC) delivers highly efficient separations and automated fraction collection using readily available analytical/midscale SFC instrumentation. A series of diverse analytes across a wide spectrum of chemical properties (acid, basic, and neutral), combined with different stationary-phase columns in SFC are investigated using both a protic organic modifier (CH3OH) and its deuterated counterpart (CD3OD). The power of this framework is demonstrated with pharmaceutically relevant applications in the context of target characterization and analysis of complex multicomponent reaction mixtures from modern synthetic chemistry, demonstrating high isolation yields while reducing both the environmental footprint and manual intervention. This workflow enables unambiguous fast-paced structure elucidation on the analytical scale, providing results that are comparable to traditional, but time-consuming, RPLC purification approaches.

Selective Plate-Based Assay for Trace EDTA Analysis via Boron Trifluoride-methanol Derivatization UHPLC-QqQ-MS/MS Enabling Biologic and Vaccine Processes.

The employment of ethylenediaminetetraacetic acid (EDTA) across several fields in chemistry and biology has required the creation of a high number of quantitative assays. Nonetheless, the determination of trace EDTA, especially in biologics and vaccines, remains challenging. Herein, we introduce an automated high-throughput approach based on EDTA esterification in 96-well plates using boron trifluoride-methanol combined with rapid analysis by ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Derivatization of EDTA to its methyl ester (Me-EDTA) serves to significantly improve chromatographic performance (retention, peak shape, and selectivity), while also delivering a tremendous enhancement of sensitivity in the positive ion mode electrospray ionization (ESI+). This procedure, in contrast to previous EDTA methods based on complexation with metal ions, is not affected by high concentration of other metals, buffers, and related salts abundantly present in biopharmaceutical processes (e.g., iron, copper, citrate, etc.). Validation of this assay for the determination of ng·mL-1 level EDTA in monoclonal antibody and vaccine products demonstrated excellent performance (repeatability, precision, and linear range) with high recovery from small sample volumes while also providing an advantageous automation-friendly workflow for high-throughput analysis.

Synthesis of L-cyclic tetrapeptides by backbone amide activation CyClick strategy.

Cyclic tetrapeptides exhibit high cellular permeability and a wide range of biological properties and thus have gained great interest in the field of medicinal chemistry. We synthesized highly strained 12-membered head to tail cyclic peptides with varying reactive amino acids, without oligomerization using the exclusively intramolecular CyClick chemistry. This occurs by a two-step process involving the low-energy formation of a 15 atom-containing cyclic imine, followed by a chemoselective ring contraction of the peptide backbone generating a highly strained 12 atom-containing cyclic tetrapeptide. This reaction exhibited high substrate scope and generated head to tail cyclic tetrapeptides with varying amino acids at the N-terminus, showing chemoselectivity without the need for side group protection.

Application of 1,1-ADEQUATE and DFT to correct 13 C misassignments of carbonyl chemical shifts for carbapenem antibiotics.

Prior to the development of sensitive proton-detected 2D NMR experiments, assigning 13 C signals could be a significant challenge, and mistakes have occurred even for prominent compound classes. In this study, 1,1-ADEQUATE data were used to unambiguously reassign the 13 C chemical shifts for the ß-lactam carbonyl at the C-7 position and the proximal carboxylate at the C-10 position of the carbapenems, meropenem and imipenem. Density functional theory (DFT) was then investigated to provide sufficiently accurate 13 C chemical shift predictions, allowing for the carbonyl signal reassignment of thienamycin.

Trapping-Enrichment Multi-dimensional Liquid Chromatography with On-Line Deuterated Solvent Exchange for Streamlined Structure Elucidation at the Microgram Scale.

At the forefront of chemistry and biology research, development timelines are fast-paced and large quantities of pure targets are rarely available. Herein, we introduce a new framework, which is built upon an automated, online trapping-enrichment multi-dimensional liquid chromatography platform (TE-Dt-mDLC) that enables: 1) highly efficient separation of complex mixtures in a first dimension (1 D-UV); 2) automated peak trapping-enrichment and buffer removal achieved through a sequence of H2 O and D2 O washes using an independent pump setup; and 3) a second dimension separation (2 D-UV-MS) with fully deuterated mobile phases and fraction collection to minimize protic residues for immediate NMR analysis while bypassing tedious drying processes and minimizing analyte degradation. Diverse examples of target isolation and characterization from organic synthesis and natural product chemistry laboratories are illustrated, demonstrating recoveries above 90 % using as little as a few micrograms of material.

Photoenzymatic Synthesis of α-Tertiary Amines by Engineered Flavin-Dependent "Ene"-Reductases.

α-Tertiary amines are a common motif in pharmaceutically important molecules but are challenging to prepare using asymmetric catalysis. Here, we demonstrate engineered flavin-dependent 'ene'-reductases (EREDs) can catalyze radical additions into oximes to prepare this motif. Two different EREDs were evolved into competent catalysts for this transformation with high levels of stereoselectivity. Mechanistic studies indicate that the oxime contributes to the enzyme templated charge-transfer complex formed between the substrate and cofactor. These products can be further derivatized to prepare a variety of motifs, highlighting the versatility of ERED photoenzymatic catalysis for organic synthesis.

A Multifunctional Reagent Designed for the Site-Selective Amination of Pyridines.

We report the development of a multifunctional reagent for the direct conversion of pyridines to Boc-protected 2-aminopyridines with exquisite site selectivity and chemoselectivity. The novel reagent was prepared on 200-g scale in a single step, reacts in the title reaction under mild conditions without precautions toward air or moisture, and is tolerant of nearly all common functionality. Experimental and in situ spectroscopic monitoring techniques provide detailed insights and unexpected findings for the unique reaction mechanism.

Highly Diastereoselective Synthesis of a HCV NS5B Nucleoside Polymerase Inhibitor.

An asymmetric synthesis of HCV NS5B nucleoside polymerase inhibitor (1) is described. This novel route features several remarkably diastereoselective and high-yielding transformations, including construction of the all-carbon quaternary stereogenic center at C-2 via a thermodynamic aldol reaction. A subsequent glycosylation reaction with activated uracil via C-1 phosphate and installation of the cyclic phosphate group using an achiral phosphorus(III) reagent followed by oxidation provides 1.

CyClick Chemistry for the Synthesis of Cyclic Peptides.

Here, we report a novel "CyClick" strategy for the macrocyclization of peptides that works in an exclusively intramolecular fashion thereby precluding the formation of dimers and oligomers via intermolecular reactions. The CyClick chemistry is highly chemoselective for the N-terminus of the peptide with a C-terminal aldehyde. In this protocol, the peptide conformation internally directs activation of the backbone amide bond and thereby facilitates formation of a stable 4-imidazolidinone-fused cyclic peptide with high diastereoselectivity (>99 %). This method is tolerant to a variety of peptide aldehydes and has been applied for the synthesis of 12- to 23-membered rings with varying amino acid compositions in one pot under mild reaction conditions. The reaction generated peptide macrocycles featuring a 4-imidazolidinone in their scaffolds, which acts as an endocyclic control element that promotes intramolecular hydrogen bonding and leads to macrocycles with conformationally rigid turn structures.

Discovery of a Photoinduced Dark Catalytic Cycle Using in Situ LED-NMR Spectroscopy.

We report the use of LED-NMR spectroscopy to study the reaction mechanism of a newly discovered photoinduced iron-catalyzed cycloisomerization of alkynols to cyclic enol ethers. By understanding on/off ligand binding to the catalyst, we were able to appropriately design reaction conditions to balance catalyst activity and stability. LED-NMR was demonstrated to be a powerful tool in elucidating reaction mechanisms of photochemical reactions. Temporal NMR spectroscopic data under visible light illumination (1) revealed the pre-catalyst activation mechanism, (2) proved that photon flux provides a unique external control of the equilibrium distribution between the pre-catalyst and active catalyst, and ultimately the rate of reaction, (3) provided information about the reaction driving forces and the turnover-limiting step, and (4) enabled both real-time structural and kinetic insights into elusive species (e.g., dissolved gases).

Jizanpeptins, Cyanobacterial Protease Inhibitors from a Symploca sp. Cyanobacterium Collected in the Red Sea.

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 3 Jcc correlations in 1,1-ADEQUATE spectra of pyrimidine analogs: Avoiding potential interpretation pitfalls.

Recently, it has been reported that large n JCC 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 3 JCC coupling constants greater than 10 Hz. Results are supported by both the experimental measurement of 3 JCC coupling constants in question using J-modulated-ADEQUATE and density functional theory calculations.

Cyclic and Lasso Peptides: Sequence Determination, Topology Analysis, and Rotaxane Formation.

A broadly applicable chemical cleavage methodology to facilitate MS/MS sequencing was developed for macrocyclic and lasso peptides, which hold promise as exciting new therapeutics. Existing methods such as Edman degradation, CNBr cleavage, and enzymatic digestion are either limited in scope or completely fail in cleavage of constrained nonribosomal peptides. Importantly, the new method was utilized for synthesizing a unique peptide-based rotaxane (both cyclic and threaded) from the lasso peptide, benenodin-1 ΔC5.

Dearomatization of Electron-Deficient Nitrogen Heterocycles via Cobalt-Catalyzed Asymmetric Cyclopropanation.

The dearomatization of a series of electron-deficient nitrogen heterocycles has been achieved through a cobalt-catalyzed asymmetric cyclopropanation reaction. This reaction proceeds with high levels of enantio- and diastereoselectivity to afford unique cyclopropanes that can be further functionalized to provide complex heterocyclic building blocks.

Photoredox-Catalyzed Hydroxymethylation of Heteroaromatic Bases.

We report the development of a method for room-temperature C-H hydroxymethylation of heteroarenes. A key enabling advance in this work was achieved by implementing visible light photoredox catalysis that proved to be applicable to many classes of heteroarenes and tolerant of diverse functional groups found in druglike molecules.

Additional pitfalls of using 1,1-ADEQUATE for structure elucidation.

1,1-ADEQUATE is a powerful and robust NMR experiment to establish carbon-carbon connectivities using modest sample quantities when cryogenic probe technology is available. Yet potential pitfalls of applying this method are not widely appreciated, such as weak or missing 1 JCC correlations in strongly coupled 13 C-13 C AB spin systems and unusually large multi-bond (n JCC ) correlations associated with particular functional groups. These large n JCC correlations observed in 1,1-ADEQUATE spectra could be mistaken for 1 JCC correlations. Copyright © 2016 John Wiley & Sons, Ltd.

Unequivocal identification of two-bond heteronuclear correlations in natural products at nanomole scale by i-HMBC.

HMBC is an essential NMR experiment for determining multiple bond heteronuclear correlations in small to medium-sized organic molecules, including natural products, yet its major limitation is the inability to differentiate two-bond from longer-range correlations. There have been several attempts to address this issue, but all reported approaches suffer various drawbacks, such as restricted utility and poor sensitivity. Here we present a sensitive and universal methodology to identify two-bond HMBC correlations using isotope shifts, referred to as i-HMBC (isotope shift detection HMBC). Experimental utility was demonstrated at the sub-milligram / nanomole scale with only a few hours of acquisition time required for structure elucidation of several complex proton-deficient natural products, which could not be fully elucidated by conventional 2D NMR experiments. Because i-HMBC overcomes the key limitation of HMBC without significant reduction in sensitivity or performance, i-HMBC can be used as a complement to HMBC when unambiguous identifications of two-bond correlations are needed.