Submicro Inverse-Detection Gradient NMR:  A Powerful New Way of Conducting Structure Elucidation Studies with <0.05 μmol Samples

Abstract

Quantities of material required for structural analysis were reduced substantially following the introduction of 3 mm microinverse and microdual NMR probes in 1992. We now report the first very low-level results obtainable with a new 1.7 mm submicro-inverse-detection gradient or SMIDG NMR probe. Using this technology at 600 MHz, it was possible to fully characterize an 8% impurity contained in a 0. 55 &mgr;mol sample of cryptolepine (1) that had been standing in excess of 2 years since its initial isolation. The impurity was unequivocally identified as cryptolepinone (2) through the concerted interpretation of GHSQC, GHMBC, homonuclear TOCSY, and ROESY spectra in conjunction with APCI LC/MS and CID data acquired from a portion of the serial dilution solution used to prepare the NMR sample. Submicro-inverse-detection gradient probes offer the prospect of reducing still further the quantities of sample required for full characterization under favorable circumstances, making rare and potentially novel natural products amenable to structural determination. SMIDG NMR technology is equally applicable to a range of small samples requiring characterization such as isolated impurities from drug substances, isolates from drug degradation studies, and secondary metabolites.

Rapid acquisition of1H-13C and1H-15N heteronuclear chemical shift correlation data at the submilligram level using SMIDG (submicro inverse-detection gradient) NMR probe technology

“…SMIDG NMR probe technology provides not only the capability of rigorously characterizing extremely small samples by heteronuclear shift correlation techniques, 1,4 but also the possibility of acquiring these data very rapidly for less modest samples, as demonstrated in this paper. The latter capability makes it reasonable to consider employing heteronuclear shift correlation data as an integral part of strategies for the full characterization of species with limited solution lifetimes.…”

“…18 Our recent work using a submicro inversedetection gradient (SMIDG) NMR probe 1 -4 was focused on the rigorous heteronuclear 2D structural characterization of extremely small samples. Using this probe technology, we have been able to acquire successfully direct (GHSQC) and long-range (GHMBC) 1 H-13 C heteronuclear shift correlation spectra of a 0.04 µmol impurity (8%) in a 0.55 µmol sample of the alkaloid cryptolepine. 1 With a weekend acquisition, we were also able to acquire long-range 1 H- 15 N heteronuclear shift correlation data for 1 µmol of strychnine at natural abundance.…”

“…Using this probe technology, we have been able to acquire successfully direct (GHSQC) and long-range (GHMBC) 1 H-13 C heteronuclear shift correlation spectra of a 0.04 µmol impurity (8%) in a 0.55 µmol sample of the alkaloid cryptolepine. 1 With a weekend acquisition, we were also able to acquire long-range 1 H- 15 N heteronuclear shift correlation data for 1 µmol of strychnine at natural abundance. 3 Hence, the performance characteristics of the SMIDG probe have been well established and experimentally documented at its practical, current limits of detection.…”

Radiation damping in microcoil NMR probes

“…Traditionally, small-volume NMR probes have found application almost exclusively in the structural analysis of mass-limited samples [11,12]. However, this trend has been changing recently as reports describing the use of such probes to study proteins [13][14][15][16] and DNA [17] have appeared.…”

Low-level long-range1H-15N heteronuclear shift correlation at natural abundance using a combination of 2D and selective 1D NMR methods with submicro NMR probe technology

“…Reports have also appeared describing µCoil NMR flow cells of various designs. 7 -11 We recently reported the first results obtained using a 1.7 mm submicro inverse-detection gradient or SMIDG NMR probe, 12 which permitted the full characterization of samples of <0.05 µmol 13 using 1 H- 13 C GHSQC 14 and GHMBC 15 experiments. Subsequently, we reported the application of SMIDG NMR probe technology in the acquisition of low-level 1 H- 15 N long-range heteronuclear shift correlation data.…”