Relative configuration of micrograms of natural compounds using proton residual chemical shift anisotropy

Abstract

3D molecular structure determination is a challenge for organic compounds or natural products available in minute amounts. Proton/proton and proton/carbon correlations yield the constitution. J couplings and NOEs oftentimes supported by one-bond ¹H,¹³C residual dipolar couplings (RDCs) or by ¹³C residual chemical shift anisotropies (RCSAs) provide the relative configuration. However, these RDCs or carbon RCSAs rely on 1% natural abundance of ¹³C preventing their use for compounds available only in quantities of a few 10's of µgs. By contrast, ¹H RCSAs provide similar information on spatial orientation of structural moieties within a molecule, while using the abundant ¹H spin. Herein, ¹H RCSAs are accurately measured using constrained aligning gels or liquid crystals and applied to the 3D structural determination of molecules with varying complexities. Even more, deuterated alignment media allow the elucidation of the relative configuration of around 35 µg of a briarane compound isolated from Briareum asbestinum.

Fig. 1. Configuration analysis of strychnine using ¹H RCSA Q/Q_CSA factors in a PMMA gel.

a Structure of strychnine (1). b Quality factors of ¹H RCSAs derived from stretched PMMA gel of strychnine: the Q_CSA factors (red bar) for the two closest configurations RSSRRS and RSRRRS are 0.257 ± 0.006 and 0.275 ± 0.006, respectively. The Q factors are reported with the blue bars. The two configurations RSSRRS and RSRRRS were also the two with the lowest Q factors in the ¹³C RCSA analysis. The error bars are standard deviations.

Fig. 2. 1D ¹H NMR spectra of microgram amount of strychnine in gels.

a 1D ¹H NMR spectrum of 300 μg strychnine in protonated PMMA gel. The spectrum was acquired with 256 scans. Note that only few peaks from the analyte are visible (indicated by the blue arrows) and many signals are masked by the polymer signals. b 1D ¹H NMR spectrum of 80-μg strychnine in deuterated PMMA gel. The spectrum was acquired with 800 scans. Blue arrows on the inset in (b) highlight some strychnine resonances that become visible due to the removal of proton signals of the polymer. Both spectra were recorded in a Bruker 800 MHz NMR spectrometer equipped with a TCI cryoprobe. Stretching devices with an inner diameter of 4.0 mm for minimum and 3.2 mm for maximum alignment were used. c 1D ¹H NMR spectrum of 10-μg strychnine acquired with 8192 scans in deuterated PMMA gel under maximum alignment condition. For clarity, expansions for some of the proton signals (H4, H22, H8, and H20a) are also shown.

Fig. 3. Q/Q_CSA factors of retrorsine.

a Structure of retrorsine (2). b The Q factors (blue bar) and Q_CSA factors (red bar) for the eight possible relative configurations for the retrorsine. The error bars are standard deviations.

Fig. 4. Constitution of the diterpene briarane B-3.

The constitutions of briarane B-3 (3) is shown.

Fig. 5. Absolute configuration of 35-μg briarane B-3.

a Marine gorgonian Briareum asbestinum (continuous red line) collected in the waters off the Yucatan Peninsula in Mexico, from which briarane B-3 (3) was isolated. b The four possible diastereomers for the structure of 3 of which the SRRSRSSSR (3c) (red) is found to be the correct one. c Calculated ECD spectra of SRRSRSSSR (red line) and enantiomer RSSRSRRRS (black line) versus experimental ECD spectrum (green line) of 3. Note that the ordering of the stereocenters is C6, C7, C8, C9, C17, C1, C2, C10, C11.