Synergy of synthesis, computation and NMR reveals correct baulamycin structures.

Small-molecule, biologically active natural products continue to be our most rewarding source of, and inspiration for, new medicines. Sometimes we happen upon such molecules in minute quantities in unique, difficult-to-reach, and often fleeting environments, perhaps never to be discovered again. In these cases, determining the structure of a molecule-including assigning its relative and absolute configurations-is paramount, enabling one to understand its biological activity. Molecules that comprise stereochemically complex acyclic and conformationally flexible carbon chains make such a task extremely challenging. The baulamycins (A and B) serve as a contemporary example. Isolated in small quantities and shown to have promising antimicrobial activity, the structure of the conformationally flexible molecules was determined largely through J-based configurational analysis, but has been found to be incorrect. Our subsequent campaign to identify the true structures of the baulamycins has revealed a powerful method for the rapid structural elucidation of such molecules. Specifically, the prediction of nuclear magnetic resonance (NMR) parameters through density functional theory-combined with an efficient sequence of boron-based synthetic transformations, which allowed an encoded (labelled) mixture of natural-product diastereomers to be prepared-enabled us rapidly to pinpoint and synthesize the correct structures.

Conformationally Controlled sp3 -Hydrocarbon-Based α-Helix Mimetics.

With over 60 % of protein-protein interfaces featuring an α-helix, the use of α-helix mimetics as inhibitors of these interactions is a prevalent therapeutic strategy. However, methods to control the conformation of mimetics, thus enabling maximum efficacy, can be restrictive. Alternatively, conformation can be controlled through the introduction of destabilizing syn-pentane interactions. This tactic, which is often adopted by Nature, is not a common feature of lead optimization owing to the significant synthetic effort required. Through assembly-line synthesis with NMR and computational analysis, we have shown that alternating syn-anti configured contiguously substituted hydrocarbons, by avoiding syn-pentane interactions, adopt well-defined conformations that present functional groups in an arrangement that mimics the α-helix. The design of a p53 mimetic that binds to Mdm2 with moderate to good affinity, demonstrates the therapeutic promise of these scaffolds.

Crystal Structure and NMR of an α,δ-Peptide Foldamer Helix Shows Side-Chains are Well Placed for Bifunctional Catalysis: Application as a Minimalist Aldolase Mimic.

We report the first NMR and X-ray diffraction (XRD) structures of an unusual 13/11-helix (alternating i, i+1 {NH-O=C} and i, i+3 {C=O-H-N} H-bonds) formed by a heteromeric 1 : 1 sequence of α- and δ-amino acids, and demonstrate the application of this framework towards catalysis. Whilst intramolecular hydrogen bonds (IMHBs) are the clear driver of helix formation in this system, we also observe an apolar interaction between the ethyl residue of one δ-amino acid and the cyclohexyl group of the next δ-residue in the sequence that seems to stabilize one type of helix over another. To the best of our knowledge this type of additional stabilization leading to a specific helical preference has not been observed before. Critically, the helix type realized places the α-residue functionalities in positions proximal enough to engage in bifunctional catalysis as demonstrated in the application of our system as a minimalist aldolase mimic.

Monitoring off-resonance signals with SHARPER NMR - the MR-SHARPER experiment.

We demonstrate an extension to the SHARPER (Sensitive Homogenous and Refocussed Peaks in Real Time) NMR experiment which allows more than one signal to be monitored simultaneously, while still giving ultra-sharp, homo- and hetero-decoupled NMR signals. This is especially valuable in situations where magnetic field inhomogeneity would normally make NMR a problematic tool, for example when gas evolution is occurring during reaction monitoring. The originally reported SHARPER experiment only works for a single, on-resonance NMR signal, but here we demonstrate the Multiple Resonance SHARPER approach can be developed, which in principle can acquire multiple on-/off-resonance signals simultaneously while retaining the desirable properties of the parent sequence. In practice, the case of two resonances, e.g. those of a reactant and a product, will most of the time be considered for MR-SHARPER, as illustrated here.

Prediction of 15 N chemical shifts by machine learning.

We demonstrate the potential for machine learning systems to predict three-dimensional (3D)-relevant NMR properties beyond traditional 1 H- and 13 C-based data, with comparable accuracy to density functional theory (DFT) (but orders of magnitude faster). Predictions of DFT-calculated 15 N chemical shifts for 3D molecular structures can be achieved using a machine learning system-IMPRESSION (Intelligent Machine PREdiction of Shift and Scalar information Of Nuclei), with an accuracy of 6.12-ppm mean absolute error (∼1% of the δ15 N chemical shift range) and an error of less than 20 ppm for 95% of the chemical shifts. It provides less accurate raw predictions of experimental chemical shifts, due to the limited size and chemical space diversity of the training dataset used in its creation, coupled with the limitations of the underlying DFT methodology in reproducing experiment.

3× Axial vs 3× Equatorial: The ΔGGA Value Is a Robust Computational Measure of Substituent Steric Effects.

We define ΔGGA as the free energy change for the formal equilibrium: [13]G-H + 1-X-adamantane → [13]G-X + adamantane, where [13]G-H is the C13H22 fragment of all-trans graphane with 3-fold symmetry. This compares with a situation where the group X is equatorial to three cyclohexane rings with one where it is axial to three rings. ΔGGA values vary from 2.9 (CN) to 145.7 kJ mol-1 (CCl3), and this wide range means that ΔG can be calculated with confidence. ΔGGA values for Me, Et, i-Pr, and t-Bu form a regular series, 34.9, 63.3, 101.6, and 142.0, and clearly reflect the steric size of the groups. We propose a model where the six axial hydrogens surrounding X on [13]G-X provide a nearly circular constriction on the substituent close to its point of attachment but which does not extend far above this. We compare these results with A values and with calculations on 2- and 7-substituted [1(2,3)4]pentamantanes. We show that electronic effects on ΔGGA values are negligible but that they correlate well with computed cone and solid angles subtended by the substituent.

Conformationally Controlled Linear and Helical Hydrocarbons Bearing Extended Side Chains.

Conformationally controlled flexible molecules are ideal for applications in medicine and materials, where shape matters but an ability to adapt to multiple and changing environments is often required. The conformation of flexible hydrocarbon chains bearing contiguous methyl substituents is controlled through the avoidance of syn-pentane interactions: alternating syn-anti isomers adopt a linear conformation while all-syn isomers adopt a helical conformation. From a simple diamond lattice analysis, larger substituents, which would be required for most potential applications, result in significant and unavoidable syn-pentane interactions, suggesting substantially reduced conformational control. Through a combination of computation, synthesis, and NMR analysis, we have identified a selection of substitution patterns that allow large groups to be incorporated on conformationally controlled linear and helical hydrocarbon chains. Surprisingly, when the methyl substituents of alternating syn-anti hydrocarbons are replaced with acetoxyethyl groups, the main chain of almost 95% of the population of molecules adopt a linear conformation. Here, the side chains adopt nonideal eclipsed conformations with the main chain, thus minimizing syn-pentane interactions. In the case of all-syn hydrocarbons, concurrent removal of some methyl groups on the main chain adjacent to the large substituents is required to maintain a high population of molecules adopting a helical conformation. This information can now be used to design flexible hydrocarbon chains displaying functional groups in a defined relative orientation for multivalent binding or cooperative reactivity, for example, in targeting the interfaces defined by disease-relevant protein-protein interactions.

Synthesis and pharmacological characterisation of arctigenin analogues as antagonists of AMPA and kainate receptors.

(-)-Arctigenin and a series of new analogues have been synthesised and then tested for their potential as AMPA and kainate receptor antagonists of human homomeric GluA1 and GluK2 receptors expressed in HEK293 cells using a Ca2+ influx assay. In general, these compounds showed antagonist activity at both receptors with greater activity evident at AMPARs. Schild analysis indicates that a spirocyclic analogue 6c acts as a non-competitive antagonist. Molecular docking studies in which 6c was docked into the X-ray crystal structure of the GluA2 tetramer suggest that (-)-arctigenin and its analogues bind in the transmembrane domain in a similar manner to the known AMPA receptor non-competitive antagonists GYKI53655 and the antiepileptic drug perampanel. The arctigenin derivatives described herein may serve as novel leads for the development of drugs for the treatment of epilepsy.

Carbonylative C-C Bond Activation of Aminocyclopropanes Using a Temporary Directing Group Strategy.

Temporary directing groups (TDGs) underpin a range of C-C bond activation methodologies; however, the use of TDGs for the regiocontrolled activation of cyclopropane C-C bonds is underdeveloped. In this report, we show how an unusual ring contraction process can be harnessed for TDG-based carbonylative C-C bond activations of cyclopropanes. The method involves the transient installation of an isocyanate-derived TDG, rather than relying on carbonyl condensation events as used in previous TDG-enabled C-C bond activations.

Assembly-line synthesis of organic molecules with tailored shapes.

Molecular 'assembly lines', in which organic molecules undergo iterative processes such as chain elongation and functional group manipulation, are found in many natural systems, including polyketide biosynthesis. Here we report the creation of such an assembly line using the iterative, reagent-controlled homologation of a boronic ester. This process relies on the reactivity of α-lithioethyl tri-isopropylbenzoate, which inserts into carbon-boron bonds with exceptionally high fidelity and stereocontrol; each chain-extension step generates a new boronic ester, which is immediately ready for further homologation. We used this method to generate organic molecules that contain ten contiguous, stereochemically defined methyl groups. Several stereoisomers were synthesized and shown to adopt different shapes-helical or linear-depending on the stereochemistry of the methyl groups. This work should facilitate the rational design of molecules with predictable shapes, which could have an impact in areas of molecular sciences in which bespoke molecules are required.

How Big is the Pinacol Boronic Ester as a Substituent?

The synthetically versatile pinacol boronic ester group (Bpin) is generally thought of as a bulky moiety because of the two adjacent quaternary sp3 -hydribized carbon atoms in its diol backbone. However, recent diastereoselective reactions reported in the literature have cast doubt on this perception. Reported herein is a detailed experimental and computational analysis of Bpin and structurally related boronic esters which allows determination of three different steric parameters for the Bpin group: the A-value, ligand cone angle, and percent buried volume. All three parameters suggest that the Bpin moiety is remarkably small, with the planarity of the oxygen-boron-oxygen motif playing an important role in minimising steric interactions. Of the three steric parameters, percent buried volume provides the best correlation between steric size and diastereoselectivity in a Diels-Alder reaction.

Improving the accuracy of 1 H-19 F internuclear distance measurement using 2D 1 H-19 F HOESY.

With the rise in fluorinated pharmaceuticals, it is becoming increasingly important to develop new 19 F NMR-based methods to assist in their analysis. Crucially, obtaining information regarding the conformational dynamics of a molecule in solution can aid the design of strongly binding therapeutics. Herein, we report the development of a 2D 1 H-19 F Heteronuclear Overhauser Spectroscopy (HOESY) experiment to measure 1 H-19 F internuclear distances, with accuracies of ~5% when compared with 1 H-19 F internuclear distances calculated by quantum chemical methods. We demonstrate that correcting for cross-relaxation of 1 H, using the diagonal peaks from the 2D 1 H-1 H Nuclear Overhauser Enhancement Spectroscopy (NOESY), is critical in obtaining accurate values for 1 H-19 F internuclear distances. Finally, we show that by using the proposed method to measure 1 H-19 F internuclear distances, we are able to determine the relative stereochemistry of two fluorinated pharmaceuticals.

High Resolution for Chemical Shifts and Scalar Coupling Constants: The 2D Real-Time J-Upscaled PSYCHE-DIAG.

The facile determination of chemical shift and scalar coupling constants in NMR spectra is often prevented by spectral overlap and limited resolution. Here, we present a high-resolution NMR experiment for the simultaneous detection of both resonance frequencies and coupling patterns even with small J-values. A PSYCHE-decoupled DIAG (Pure Shift Yielded by Chirp Excitation- DIAGonal) experiment, which resolves chemical shift in the indirect dimension of a 2D experiment is combined with real-time J-upscaling in order to visualize small coupling constants that would otherwise be hidden in the linewidth of a regular proton or DIAG spectrum.

Improved NOE fitting for flexible molecules based on molecular mechanics data - a case study with S-adenosylmethionine.

The use of molecular dynamics (MD) calculations to derive relative populations of conformers is highly sensitive to both timescale and parameterisation of the MD. Where these calculations are coupled with NOE data to determine the dynamics of a molecular system, this can present issues if these populations are thus relied upon. We present an approach that refines the highly accurate PANIC NMR methodology combined with clustering approaches to generate conformers, but without restraining the simulations or considering the relative population distributions generated by MD. Combining this structural sampling with NOE fitting, we demonstrate, for S-adenosylmethionine (aqueous solution at pH 7.0), significant improvements are made to the fit of populations to the experimental data, revealing a strong overall preference for the syn conformation of the adenosyl group relative to the ribose ring, but with less discrimination for the conformation of the ribose ring itself.

NMReDATA, a standard to report the NMR assignment and parameters of organic compounds.

Even though NMR has found countless applications in the field of small molecule characterization, there is no standard file format available for the NMR data relevant to structure characterization of small molecules. A new format is therefore introduced to associate the NMR parameters extracted from 1D and 2D spectra of organic compounds to the proposed chemical structure. These NMR parameters, which we shall call NMReDATA (for nuclear magnetic resonance extracted data), include chemical shift values, signal integrals, intensities, multiplicities, scalar coupling constants, lists of 2D correlations, relaxation times, and diffusion rates. The file format is an extension of the existing Structure Data Format, which is compatible with the commonly used MOL format. The association of an NMReDATA file with the raw and spectral data from which it originates constitutes an NMR record. This format is easily readable by humans and computers and provides a simple and efficient way for disseminating results of structural chemistry investigations, allowing automatic verification of published results, and for assisting the constitution of highly needed open-source structural databases.

Enabling Fast Pseudo-2D NMR Spectral Acquisition for Broadband Homonuclear Decoupling: The EXACT NMR Approach.

Pseudo-2D NMR spectroscopy provides a means of acquiring broadband homonuclear decoupled spectra useful for structural characterization of complex molecules. However, data points concatenated in the direct dimension in these experiments are acquired over incremented time periods-leading to long acquisition times with no sensitivity benefits due to the absence of signal averaging between scans. Herein, the concept of EXACT NMR spectroscopy ("burst" non-uniform sampling of data points) is explored in pseudo-2D experiments with results revealing little or no loss in spectral quality or signal intensity despite the acceleration of acquisition-up to 400 % in some cases.

Accurate measurement of long range proton-carbon scalar coupling constants.

The accuracy and practicality of measuring heteronuclear scalar coupling constants, nJCH, from modern NMR experimental methods is examined, based on F1 or F2 evolution of nJCH in HSQMBC (including EXSIDE) and HMBC experiments. The results from these methods are compared to both robust experimental data (derived from coupled 13C spectra), computed (Density Functional Theory) and literature values where available. We report on the accuracy, ease of use and time efficiency of these multi-dimensional methods and highlight their extent and limitations.

EXtended ACquisition Time (EXACT) NMR-A Case for 'Burst' Non-Uniform Sampling.

A strong case exists for the introduction of burst non-uniform sampling (NUS) in the direct dimension of NMR spectroscopy experiments. The resulting gaps in the NMR free induction decay can reduce the power demands of long experiments (by switching off broadband decoupling for example) and/or be used to introduce additional pulses (to refocus homonuclear coupling, for example). The final EXtended ACquisition Time (EXACT) spectra are accessed by algorithmic reconstruction of the missing data points and can provide higher resolution in the direct dimension than is achievable with existing non-NUS methods.

The hydrolysis of geminal ethers: a kinetic appraisal of orthoesters and ketals.

A novel approach to protecting jet fuel against the effects of water contamination is predicated upon the coupling of the rapid hydrolysis reactions of lipophilic cyclic geminal ethers, with the concomitant production of a hydrophilic acyclic hydroxyester with de-icing properties (Fuel Dehydrating Icing Inhibitors - FDII). To this end, a kinetic appraisal of the hydrolysis reactions of representative geminal ethers was undertaken using a convenient surrogate for the fuel-water interface (D2O/CD3CN 1:4). We present here a library of acyclic and five/six-membered cyclic geminal ethers arranged according to their hydroxonium catalytic coefficients for hydrolysis, providing for the first time a framework for the development of FDII. A combination of (1)H NMR, labelling and computational studies was used to assess the effects that may govern the observed relative rates of hydrolyses.