Characterization of an unusual ring-contraction degradant of the antifungal agent posaconazole.

Posaconazole, a clinically useful antifungal agent, has several known oxidative degradation products involving the piperazine ring near the center of the molecule. A novel degradant was recently isolated and characterized spectroscopically as a novel ring-contraction product incorporating a dihydroimidazolium moiety in lieu of the normally present piperazine ring.

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-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.

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-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.

Investigation of the experimental limits of small-sample heteronuclear 2D NMR.

Practical experimental performance limits for an ensemble of heteronuclear 2D NMR experiments using a state-of-the-art 600 MHz 1.7 mm Bruker TCI Micro CryoProbe are reported. In the specific case of multiplicity-edited GHSQC, it was possible to acquire data on a 540 ng sample of strychnine (1; ∼1.6 nmol), prepared by serial dilution, which was used as a model compound. The experiments discussed also included GCOSY, (13)C reference spectra, (1)H-(13)C GHMBC, IDR-GHSQC-TOCSY, 1,1-ADEQUATE, and (1)H-(15)N GHMBC.

Long-range 1H-15N heteronuclear shift correlation across wide F1 spectral windows.

Long-range (1)H-(15)N heteronuclear shift correlation experiments at natural abundance are becoming more routinely utilized in the characterization of unknown chemical structures from a diverse range of sources including natural products and pharmaceuticals. Apart from the inherent challenges of the low gyromagnetic ratio and natural abundance of (15)N, investigators are also occasionally hampered by having to deal with the wide spectral range inherent to various nitrogen functional groups, which can exceed 500 ppm. Earlier triple resonance cryoprobe designs typically provided 90° (15)N pulses in the range of 35-40 µs, which did not allow the uniform excitation of wide F(1) spectral ranges for (1)H-(15)N GHMBC spectra. We report the results obtained with a newly designed Bruker 600 MHz triple resonance TCI Micro CryoProbe™ using methyl orange as a model compound, in which the (15)N resonances are separated by >450 ppm.

Using indirect covariance spectra to identify artifact responses in unsymmetrical indirect covariance calculated spectra.

Several groups of authors have reported studies in the areas of indirect and unsymmetrical indirect covariance NMR processing methods. Efforts have recently focused on the use of unsymmetrical indirect covariance processing methods to combine various discrete two-dimensional NMR spectra to afford the equivalent of the much less sensitive hyphenated 2D NMR experiments, for example indirect covariance (icv)-heteronuclear single quantum coherence (HSQC)-COSY and icv-HSQC-nuclear Overhauser effect spectroscopy (NOESY). Alternatively, unsymmetrical indirect covariance processing methods can be used to combine multiple heteronuclear 2D spectra to afford icv-13C-15N HSQC-HMBC correlation spectra. We now report the use of responses contained in indirect covariance processed HSQC spectra as a means for the identification of artifacts in both indirect covariance and unsymmetrical indirect covariance processed 2D NMR spectra.

Multistep correlations via covariance processing of COSY/GCOSY spectra: opportunities and artifacts.

Long-range homonuclear coupling pathways can be observed in COSY or GCOSY spectra by the acquisition of spectra with larger numbers of increments of the evolution period, t(1), than would normally be used. Alternatively, covariance processing of COSY-type spectra acquired with modest numbers of t(1) increments, allows the observation of multistage correlations. In this work results obtained from covariance-processed GCOSY spectra are fully analyzed and compared to normally processed COSY and 80 ms TOCSY spectra. Multistage or 'RCOSY-type' correlations are observed when remote protons both exhibit correlations to the same coupling partner e.g. A --> B and B --> C gives rise to an A --> C correlation. In the strict sense, RCOSY-type responses are artifacts albeit providing useful information. Nonbeneficial artifact correlations are observed when protons couple to other protons that overlap or partially overlap. The origin of artifact responses is also analyzed.

Utilizing unsymmetrical indirect covariance processing to define 15N- 13C connectivity networks.

There has been considerable interest over the past decade in the utilization of direct and long-range 1H- 15N heteronuclear shift correlation methods at natural abundance to facilitate the elucidation of small molecule structures. Recently, there has also been a high level of interest in the exploration of indirect covariance NMR methods. Our initial explorations in this area led to the development of unsymmetrical indirect covariance methods, which allow the calculation of hyphenated 2D NMR spectra such as 2D GHSQC-COSY and GHSQC-NOESY from the discrete component 2D NMR experiments. We now wish to report the utilization of unsymmetrical indirect covariance NMR methods for the combination of 1H- 13C GHSQC and 1H- 15N long-range (GHMBC, IMPEACH-MBC, CIGAR-HMBC, etc.) heteronuclear chemical shift correlation spectra to determine 15N- 13C correlation pathways.

Using unsymmetrical indirect covariance processing to calculate GHSQC-COSY spectra.

GHSQC-TOCSY experiments allow sorting of proton-proton connectivity information as a function of (13)C chemical shift. GHSQC-TOCSY is a relatively insensitive 2D NMR experiment. Given two coherence transfer experiments, A --> B and A --> C, it is possible to indirectly determine B <--> C. Unsymmetrical indirect covariance processing of a (1)H- (13)C GHSQC and a GCOSY spectrum afforded a GHSQC-COSY spectrum, with an information content analogous to a GHSQC-TOCSY experiment. However, GHSQC-TOCSY is of significantly lower sensitivity and the data require considerably more time to acquire than either of the component experiments. Investigators needing access to GHSQC-TOCSY type data can, in principle, access it from more readily acquired 2D NMR data. Strychnine ( 1) was used as a model compound to illustrate this capability.

The use of unsymmetrical indirect covariance NMR methods to obtain the equivalent of HSQC-NOESY data.

We have recently demonstrated that unsymmetrical indirect covariance NMR methods can be used to mathematically calculate the equivalent of low sensitivity, hyphenated NMR experiments by combining data from a pair of higher sensitivity experiments. The present report demonstrates the application of this method to the combination of HSQC and NOESY spectra to provide results comparable to HSQC-NOESY data, albeit with greater sensitivity and with considerably less spectrometer time.

Application of unsymmetrical indirect covariance NMR methods to the computation of the (13)C <--> (15)N HSQC-IMPEACH and (13)C <--> (15)N HMBC-IMPEACH correlation spectra.

Utilization of long-range (1)H--(15)N heteronuclear chemical shift correlation has continually grown in importance since the first applications were reported in 1995. More recently, indirect covariance NMR methods have been introduced followed by the development of unsymmetrical indirect covariance processing methods. The latter technique has been shown to allow the calculation of hyphenated 2D NMR data matrices from more readily acquired nonhyphenated 2D NMR spectra. We recently reported the use of unsymmetrical indirect covariance processing to combine (1)H--(13)C GHSQC and (1)H--(15)N GHMBC long-range spectra to yield a (13)C--(15)N HSQC-HMBC chemical shift correlation spectrum that could not be acquired in a reasonable period of time without resorting to (15)N-labeled molecules. We now report the unsymmetrical indirect covariance processing of (1)H--(13)C GHMBC and (1)H--(15)N IMPEACH spectra to afford a (13)C--(15)N HMBC-IMPEACH spectrum that has the potential to span as many as six to eight bonds. Correlations for carbon resonances long-range coupled to a protonated carbon in the (1)H--(13)C HMBC spectrum are transferred via the long-range (1)H--(15)N coupling pathway in the (1)H--(15)N IMPEACH spectrum to afford a much broader range of correlation possibilities in the (13)C--(15)N HMBC-IMPEACH correlation spectrum. The indole alkaloid vincamine is used as a model compound to illustrate the application of the method.

(13)C-(15)N correlation via unsymmetrical indirect covariance NMR: application to vinblastine.

Unsymmetrical indirect covariance processing methods allow the derivation of hyphenated 2D NMR data from the component 2D spectra, potentially circumventing the acquisition of the much lower sensitivity hyphenated 2D NMR experimental data. Calculation of HSQC-COSY and HSQC-NOESY spectra from GHSQC, COSY, and NOESY spectra, respectively, has been reported. The use of unsymmetrical indirect covariance processing has also been applied to the combination of (1)H- (13)C GHSQC and (1)H- (15)N long-range correlation data (GHMBC, IMPEACH, or CIGAR-HMBC). The application of unsymmetrical indirect covariance processing to spectra of vinblastine is now reported, specifically the algorithmic extraction of (13)C- (15)N correlations via the unsymmetrical indirect covariance processing of the combination of (1)H- (13)C GHSQC and long-range (1)H- (15)N GHMBC to produce the equivalent of a (13)C- (15)N HSQC-HMBC correlation spectrum. The elimination of artifact responses with aromatic solvent-induced shifts (ASIS) is shown in addition to a method of forecasting potential artifact responses through the indirect covariance processing of the GHSQC spectrum used in the unsymmetrical indirect covariance processing.