Leptochelins A-C, Cytotoxic Metallophores Produced by Geographically Dispersed Leptothoe Strains of Marine Cyanobacteria.

Metals are important cofactors in the metabolic processes of cyanobacteria, including photosynthesis, cellular respiration, DNA replication, and the biosynthesis of primary and secondary metabolites. In adaptation to the marine environment, cyanobacteria use metallophores to acquire trace metals when necessary as well as to reduce potential toxicity from excessive metal concentrations. Leptochelins A-C were identified as structurally novel metallophores from three geographically dispersed cyanobacteria of the genus Leptothoe. Determination of the complex structures of these metabolites presented numerous challenges, but they were ultimately solved using integrated data from NMR, mass spectrometry and deductions from the biosynthetic gene cluster. The leptochelins are comprised of halogenated linear NRPS-PKS hybrid products with multiple heterocycles that have potential for hexadentate and tetradentate coordination with metal ions. The genomes of the three leptochelin producers were sequenced, and retrobiosynthetic analysis revealed one candidate biosynthetic gene cluster (BGC) consistent with the structure of leptochelin. The putative BGC is highly homologous in all three Leptothoe strains, and all possess genetic signatures associated with metallophores. Postcolumn infusion of metals using an LC-MS metabolomics workflow performed with leptochelins A and B revealed promiscuous binding of iron, copper, cobalt, and zinc, with greatest preference for copper. Iron depletion and copper toxicity experiments support the hypothesis that leptochelin metallophores may play key ecological roles in iron acquisition and in copper detoxification. In addition, the leptochelins possess significant cytotoxicity against several cancer cell lines.

Unequivocal structure confirmation of a breitfussin analog by anisotropic NMR measurements.

Structural features of proton-deficient heteroaromatic natural products, such as the breitfussins, can severely complicate their characterization by NMR spectroscopy. For the breitfussins in particular, the constitution of the five-membered oxazole central ring cannot be unequivocally established via conventional NMR methods when the 4'-position is halogenated. The level of difficulty is exacerbated by 4'-iodination, as the accuracy with which theoretical NMR parameters are determined relies extensively on computational treatment of the relativistic effects of the iodine atom. It is demonstrated in the present study, that the structure of a 4'-iodo breitfussin analog can be unequivocally established by anisotropic NMR methods, by adopting a reduced singular value decomposition (SVD) protocol that leverages the planar structures exhibited by its conformers.

Unequivocal determination of caulamidines A and B: application and validation of new tools in the structure elucidation tool box.

Ambiguities and errors in the structural assignment of organic molecules hinder both drug discovery and total synthesis efforts. Newly described NMR experimental approaches can provide valuable structural details and a complementary means of structure verification. The caulamidines are trihalogenated alkaloids from a marine bryozoan with an unprecedented structural scaffold. Their unique carbon and nitrogen framework was deduced by conventional NMR methods supplemented by new experiments that define 2-bond heteronuclear connectivities, reveal very long-range connectivity data, or visualize the 35,37Cl isotopic effect on chlorinated carbons. Computer-assisted structural elucidation (CASE) analysis of the spectroscopic data for caulamidine A provided only one viable structural alternative. Anisotropic NMR parameters, specifically residual dipolar coupling and residual chemical shift anisotropy data, were measured for caulamidine A and compared to DFT-calculated values for the proposed structure, the CASE-derived alternative structure, and two energetically feasible stereoisomers. Anisotropy-based NMR experiments provide a global, orthogonal means to verify complex structures free from investigator bias. The anisotropic NMR data were fully consistent with the assigned structure and configuration of caulamidine A. Caulamidine B has the same heterocyclic scaffold as A but a different composition and pattern of halogen substitution. Caulamidines A and B inhibited both wild-type and drug-resistant strains of the malaria parasite Plasmodium falciparum at low micromolar concentrations, yet were nontoxic to human cells.

Computer-Assisted 3D Structure Elucidation (CASE-3D) of Natural Products Combining Isotropic and Anisotropic NMR Parameters.

A computer-assisted structural elucidation (CASE-3D) strategy based on the use of isotropic and/or anisotropic NMR data is proposed to elucidate relative configuration and preferred conformation in complex natural products. The methodology involves the selection of conformational models through the use of the Akaike Information Criterion and scoring of the different configurations. As illustrative examples, the methodology furnished the correct configuration of the already known compounds artemisinin (1) and homodimericin A (2). Revised structures (5 and 6), including their absolute configuration, for the recently reported curcusones I (3) and J (4) are proposed.

HMBC-1,n-ADEQUATE spectra calculated from HMBC and 1,n-ADEQUATE spectra.

Unsymmetrical and generalized indirect covariance processing methods provide a means of mathematically combining pairs of 2D NMR spectra that share a common frequency domain to facilitate the extraction of correlation information. Previous reports have focused on the combination of HSQC spectra with 1,1-, 1,n-, and inverted (1)J(CC) 1,n-ADEQUATE spectra to afford carbon-carbon correlation spectra that allow the extraction of direct ((1)J(CC)), long-range ((n)J(CC), where n ≥ 2), and (1)J(CC)-edited long-range correlation data, respectively. Covariance processing of HMBC and 1,1-ADEQUATE spectra has also recently been reported, allowing convenient, high-sensitivity access to (n)J(CC) correlation data equivalent to the much lower sensitivity n,1-ADEQUATE experiment. Furthermore, HMBC-1,1-ADEQUATE correlations are observed in the F1 frequency domain at the intrinsic chemical shift of the (13)C resonance in question rather than at the double-quantum frequency of the pair of correlated carbons, as visualized by the n,1, and m,n-ADEQUATE experiments, greatly simplifying data interpretation. In an extension of previous work, the covariance processing of HMBC and 1,n-ADEQUATE spectra is now reported. The resulting HMBC-1,n-ADEQUATE spectrum affords long-range carbon-carbon correlation data equivalent to the very low sensitivity m,n-ADEQUATE experiment. In addition to the significantly higher sensitivity of the covariance calculated spectrum, correlations in the HMBC-1,n-ADEQUATE spectrum are again detected at the intrinsic (13)C chemical shifts of the correlated carbons rather than at the double-quantum frequency of the pair of correlated carbons. HMBC-1,n-ADEQUATE spectra can provide correlations ranging from diagonal ((0)J(CC) or diagonal correlations) to (4)J(CC) under normal circumstances to as much as (6)J(CC) in rare instances. The experiment affords the potential means of establishing the structures of severely proton-deficient molecules.

Structure revision of asperjinone using computer-assisted structure elucidation methods.

The elucidated structure of asperjinone (1), a natural product isolated from thermophilic Aspergillus terreus, was revised using the expert system Structure Elucidator. The reliability of the revised structure (2) was confirmed using 180 structures containing the (3,3-dimethyloxiran-2-yl)methyl fragment (3) as a basis for comparison and whose chemical shifts contradict the suggested structure (1).

InChIKey collision resistance: an experimental testing.

InChIKey is a 27-character compacted (hashed) version of InChI which is intended for Internet and database searching/indexing and is based on an SHA-256 hash of the InChI character string. The first block of InChIKey encodes molecular skeleton while the second block represents various kinds of isomerism (stereo, tautomeric, etc.). InChIKey is designed to be a nearly unique substitute for the parent InChI. However, a single InChIKey may occasionally map to two or more InChI strings (collision). The appearance of collision itself does not compromise the signature as collision-free hashing is impossible; the only viable approach is to set and keep a reasonable level of collision resistance which is sufficient for typical applications.We tested, in computational experiments, how well the real-life InChIKey collision resistance corresponds to the theoretical estimates expected by design. For this purpose, we analyzed the statistical characteristics of InChIKey for datasets of variable size in comparison to the theoretical statistical frequencies. For the relatively short second block, an exhaustive direct testing was performed. We computed and compared to theory the numbers of collisions for the stereoisomers of Spongistatin I (using the whole set of 67,108,864 isomers and its subsets). For the longer first block, we generated, using custom-made software, InChIKeys for more than 3 × 1010 chemical structures. The statistical behavior of this block was tested by comparison of experimental and theoretical frequencies for the various four-letter sequences which may appear in the first block body.From the results of our computational experiments we conclude that the observed characteristics of InChIKey collision resistance are in good agreement with theoretical expectations.

1J(CC)-edited HSQC-1,n-ADEQUATE: a new paradigm for simultaneous direct and long-range carbon-carbon correlation.

Establishing the carbon skeleton of a molecule greatly facilitates the process of structure elucidation, leaving only heteroatoms to be inserted, heterocyclic rings to be closed, and stereochemical features to be defined. INADEQUATE, and more recently PANACEA, have been the only means of coming close to the goal of totally defining the carbon skeleton of a molecule. Unfortunately, the extremely low sensitivity and prodigious sample requirements of these experiments and the multiple receiver requirement for the latter experiment have severely restricted the usage of these experiments. Proton-detected ADEQUATE experiments, in contrast, have considerably higher sensitivity and more modest sample requirements. By combining experiments such as 1,1-ADEQUATE and 1,n-ADEQUATE with higher sensitivity experiments such as GHSQC through covariance processing, sample requirements can be further reduced with a commensurate improvement in the s/n ratio and F(1) resolution of the covariance processed spectrum. We now wish to report the covariance processing of an inverted (1)J(CC) 1,n-ADEQUATE experiment with a non-edited GHSQC spectrum to afford a spectrum that can trace the carbon skeleton of a molecule with the exception of correlations between quaternary carbons.

HMBC-1,1-ADEQUATE via generalized indirect covariance: a high sensitivity alternative to n,1-ADEQUATE.

1,1-ADEQUATE and the related long-range 1,n- and n,1-ADEQUATE variants were developed to provide an unequivocal means of establishing (2)J(CH) and the equivalent of (n)J(CH) correlations where n = 3,4. Whereas the 1,1- and 1,n-ADEQUATE experiments have two simultaneous evolution periods that refocus the chemical shift and afford net single quantum evolution for the carbon spins, the n,1-variant has a single evolution period that leaves the carbon spin to be observed at the double quantum frequency. The n,1-ADEQUATE experiment begins with an HMBC-type (n)J(CH) magnetization transfer, which leads to inherently lower sensitivity than the 1,1- and 1,n-ADEQUATE experiments that begin with a (1)J(CH) transfer. These attributes, in tandem, serve to render the n,1-ADEQUATE experiment less generally applicable and more difficult to interpret than the 1,n-ADEQUATE experiment, which can in principle afford the same structural information. Unsymmetrical and generalized indirect covariance processing methods can complement and enhance the structural information encoded in combinations of experiments e.g. HSQC-1,1- or -1,n-ADEQUATE. Another benefit is that covariance processing methods offer the possibility of mathematically combining a higher sensitivity 2D NMR spectrum with for example 1,1- or 1,n-ADEQUATE to improve access to the information content of lower sensitivity congeners. The covariance spectrum also provides a significant enhancement in the F(1) digital resolution. The combination of HMBC and 1,1-ADEQUATE spectra is shown here using strychnine as a model compound to derive structural information inherent to an n,1-ADEQUATE spectrum with higher sensitivity and in a more convenient to interpret single quantum presentation.

Blind trials of computer-assisted structure elucidation software.

BACKGROUND: One of the largest challenges in chemistry today remains that of efficiently mining through vast amounts of data in order to elucidate the chemical structure for an unknown compound. The elucidated candidate compound must be fully consistent with the data and any other competing candidates efficiently eliminated without doubt by using additional data if necessary. It has become increasingly necessary to incorporate an in silico structure generation and verification tool to facilitate this elucidation process. An effective structure elucidation software technology aims to mimic the skills of a human in interpreting the complex nature of spectral data while producing a solution within a reasonable amount of time. This type of software is known as computer-assisted structure elucidation or CASE software. A systematic trial of the ACD/Structure Elucidator CASE software was conducted over an extended period of time by analysing a set of single and double-blind trials submitted by a global audience of scientists. The purpose of the blind trials was to reduce subjective bias. Double-blind trials comprised of data where the candidate compound was unknown to both the submitting scientist and the analyst. The level of expertise of the submitting scientist ranged from novice to expert structure elucidation specialists with experience in pharmaceutical, industrial, government and academic environments. RESULTS: Beginning in 2003, and for the following nine years, the algorithms and software technology contained within ACD/Structure Elucidator have been tested against 112 data sets; many of these were unique challenges. Of these challenges 9% were double-blind trials. The results of eighteen of the single-blind trials were investigated in detail and included problems of a diverse nature with many of the specific challenges associated with algorithmic structure elucidation such as deficiency in protons, structure symmetry, a large number of heteroatoms and poor quality spectral data. CONCLUSION: When applied to a complex set of blind trials, ACD/Structure Elucidator was shown to be a very useful tool in advancing the computer's contribution to elucidating a candidate structure from a set of spectral data (NMR and MS) for an unknown. The synergistic interaction between humans and computers can be highly beneficial in terms of less biased approaches to elucidation as well as dramatic improvements in speed and throughput. In those cases where multiple candidate structures exist, ACD/Structure Elucidator is equipped to validate the correct structure and eliminate inconsistent candidates. Full elucidation can generally be performed in less than two hours; this includes the average spectral data processing time and data input.

Elucidating 'undecipherable' chemical structures using computer-assisted structure elucidation approaches.

Structure elucidation using 2D NMR data and application of traditional methods of structure elucidation are known to fail for certain problems. In this work, it is shown that computer-assisted structure elucidation methods are capable of solving such problems. We conclude that it is now impossible to evaluate the capabilities of novel NMR experimental techniques in isolation from expert systems developed for processing fuzzy, incomplete and contradictory information obtained from 2D NMR spectra.

HSQC-1,1-ADEQUATE and HSQC-1,n-ADEQUATE: enhanced methods for establishing adjacent and long-range 13C-13C connectivity networks.

1H-13C GHSQC and GHMBC spectra are irrefutably among the most valuable 2D NMR experiments for the establishment of unknown chemical structures. However, the indeterminate nature of the length of the long-range coupling(s) observed via the (n)J(CH)-optimized delay of the GHMBC experiment can complicate the interpretation of the data when dealing with novel chemical structures. A priori there is no way to differentiate 2J(CH) from (n)J(CH) correlations, where n ≥ 3. Access to high-field spectrometers with cryogenic NMR probes brings 1,1- and 1,n-ADEQUATE experiments into range for modest samples. Subjecting ADEQUATE spectra to covariance processing with high sensitivity experiments such as multiplicity-edited GHSQC affords a diagonally symmetric 13C-13C correlation spectrum in which correlation data are observed with the apparent sensitivity of the GHSQC spectrum. HSQC-1,1-ADEQUATE covariance spectra derived by co-processing of GHSQC and 1,1-ADEQUATE spectra allow the carbon skeleton of molecules to be conveniently traced. HSQC-1,n-ADEQUATE spectra provide enhanced access to correlations equivalent to 4J(CH) correlations in a GHMBC spectrum. When these data are used to supplement GHMBC data, a powerfully synergistic set of heteronuclear correlations are available. The methods discussed are illustrated using retrorsine (1) as a model compound.

HSQC-1,n-ADEQUATE: a new approach to long-range 13C-13C correlation by covariance processing.

Long-range, two-dimensional heteronuclear shift correlation NMR methods play a pivotal role in the assembly of novel molecular structures. The well-established GHMBC method is a high-sensitivity mainstay technique, affording connectivity information via (n)J(CH) coupling pathways. Unfortunately, there is no simple way of determining the value of n and hence no way of differentiating two-bond from three- and occasionally four-bond correlations. Three-bond correlations, however, generally predominate. Recent work has shown that the unsymmetrical indirect covariance or generalized indirect covariance processing of multiplicity edited GHSQC and 1,1-ADEQUATE spectra provides high-sensitivity access to a (13)C-(13) C connectivity map in the form of an HSQC-1,1-ADEQUATE spectrum. Covariance processing of these data allows the 1,1-ADEQUATE connectivity information to be exploited with the inherent sensitivity of the GHSQC spectrum rather than the intrinsically lower sensitivity of the 1,1-ADEQUATE spectrum itself. Data acquisition times and/or sample size can be substantially reduced when covariance processing is to be employed. In an extension of that work, 1,n-ADEQUATE spectra can likewise be subjected to covariance processing to afford high-sensitivity access to the equivalent of (4)J(CH) GHMBC connectivity information. The method is illustrated using strychnine as a model compound.

HSQC-ADEQUATE: an investigation of data requirements.

Utilizing (13)C-(13)C connectivity networks for the assembly of carbon skeletons from HSQC-ADEQUATE spectra was recently reported. HSQC-ADEQUATE data retain the resonance multiplicity information of the multiplicity-edited GHSQC spectrum and afford a significant improvement in the signal-to-noise (s/n) ratio relative to the 1,1-ADEQUATE data used in the calculation of the HSQC-ADEQUATE spectrum by unsymmetrical indirect covariance (UIC) processing methods. The initial investigation into the computation of HSQC-ADEQUATE correlation plots utilized overnight acquisition of the 1,1-ADEQUATE data used for the calculation. In this communication, we report the results of an investigation of the reduction in acquisition time for the 1,1-ADEQUATE data to take advantage of the s/n gain during the UIC processing to afford the final HSQC-ADEQUATE correlation plot. Data acquisition times for the 1,1-ADEQUATE spectrum can be reduced to as little as a few hours, while retaining excellent s/n ratios and all responses contained in spectra computed from overnight data acquisitions. Concatenation of multiplicity-edited GHSQC and 1,1-ADEQUATE data also allows the interrogation of submilligram samples with 1,1-ADEQUATE data when using spectrometers equipped with 1.7-mm Micro CryoProbes ™.

HSQC-ADEQUATE correlation: a new paradigm for establishing a molecular skeleton.

Various experimental methods have been developed to unequivocally identify vicinal neighbor carbon atoms. Variants of the HMBC experiment intended for this purpose have included 2J3J-HMBC and H2BC. The 1,1-ADEQUATE experiment, in contrast, was developed to accomplish the same goal but relies on the (1) J(CC) coupling between a proton-carbon resonant pair and the adjacent neighbor carbon. Hence, 1,1-ADEQUATE can identify non-protonated adjacent neighbor carbons, whereas the 2J3J-HMBC and H2BC experiments require both neighbor carbons to be protonated to operate. Since 1,1-ADEQUATE data are normally interpreted with close reference to an HSQC spectrum of the molecule in question, we were interested in exploring the unsymmetrical indirect covariance processing of multiplicity-edited GHSQC and 1,1-ADEQUATE spectra to afford an HSQC-ADEQUATE correlation spectrum that facilitates the extraction of carbon-carbon connectivity information. The HSQC-ADEQUATE spectrum of strychnine is shown and the means by which the carbon skeleton can be conveniently traced is discussed.

Enhanced automated structure elucidation by inclusion of two-bond specific data.

The availability of cryogenically cooled probes permits routine acquisition of data from low sensitivity pulse sequences such as inadequate and 1,1-adequate. We demonstrate that the use of cryo-probe generated 1,1-adequate data in conjunction with HMBC dramatically improves computer-assisted structure elucidation (CASE) both in terms of speed and accuracy of structure generation. In this study data were obtained on two dissimilar natural products and subjected to CASE analysis with and without the incorporation of two-bond specific data. Dramatic improvements in both structure calculation times and structure candidates were observed by the inclusion of the two-bond specific data.

Empirical and DFT GIAO quantum-mechanical methods of (13)C chemical shifts prediction: competitors or collaborators?

The accuracy of (13)C chemical shift prediction by both DFT GIAO quantum-mechanical (QM) and empirical methods was compared using 205 structures for which experimental and QM-calculated chemical shifts were published in the literature. For these structures, (13)C chemical shifts were calculated using HOSE code and neural network (NN) algorithms developed within our laboratory. In total, 2531 chemical shifts were analyzed and statistically processed. It has been shown that, in general, QM methods are capable of providing similar but inferior accuracy to the empirical approaches, but quite frequently they give larger mean average error values. For the structural set examined in this work, the following mean absolute errors (MAEs) were found: MAE(HOSE) = 1.58 ppm, MAE(NN) = 1.91 ppm and MAE(QM) = 3.29 ppm. A strategy of combined application of both the empirical and DFT GIAO approaches is suggested. The strategy could provide a synergistic effect if the advantages intrinsic to each method are exploited.

The application of empirical methods of (13)C NMR chemical shift prediction as a filter for determining possible relative stereochemistry.

The reliable determination of stereocenters contained within chemical structures usually requires utilization of NMR data, chemical derivatization, molecular modeling, quantum-mechanical (QM) calculations and, if available, X-ray analysis. In this article, we show that the number of stereoisomers which need to be thoroughly verified, can be significantly reduced by the application of NMR chemical shift calculation to the full stereoisomer set of possibilities using a fragmental approach based on HOSE codes. The applicability of this suggested method is illustrated using experimental data published for a series of complex chemical structures.