3 mm Micro-NMR probes

Samples of the degradant were isolated by preparative reversed-phase chromatography for structural characterization. There was sufficient sample available for the NMR measurements to be performed in a conventional 3-mm micro-NMR probe. 

Several applications of 3 mm micro-NMR probe technology were reported in 1994. The first utilized both a 3 mm micro-inverse probe as well as a heteronuclear Nano-probe to acquire the NMR data necessary for the characterization of a 30 pg (0.07 pmol) metabolite sample.59 Following the acquisition of a 13C reference spectrum using a heteronuclear Nano-probe the 40 pL sample from the nano-cell was quantitatively transferred to a 3 mm NMR tube and diluted to a total sample... 

There were a several additional studies of members of the indoloquinoline family of alkaloids isolated from Cryptolepis sanguinolenta reported that utilized 3 mm micro-NMR probe capabilities reported in 1995. The elucidation of the structure of quindolinone (16) was reported next and also utilized 3 mm micro-probe technology for the acquisition of a HMQC spectrum on an 800 pg sample of the... 

In 1998, Serianni and co-workers88 again resorted to 3 mm micro-NMR probe technology in a report dealing with the detection and quantitation of the cyclic and acyclic forms of 13C-labeled aldopentoses. In a very interesting biosynthetic application, Rowe and co-workers89 investigated the conversion of <5-(L-amino-adipoyl)-L-cysteinyl-D-valine (30) to isopenicillin-N (31), an important step in the biosynthesis of penicillins and cephalosporins. The authors utilized 3 mm micro-NMR probe capabilities in their work in the analysis of the biosynthetic products of mutants of the enzyme isopenicillin-/V synthase, the enzyme normally responsible for the conversion of 30 to 31. 

Finally, in a report in tetrahedron, Ferreira and co-workers157 employed the capabilities of 3 mm micro-NMR probes in a study that established the absolute configuration, conformation, and chiral properties of flavanone-( 3 - 8")flavone biflavonoids from Rheedia acuminate. The general structure of these compounds is shown by 77. As noted by the authors, the stereochemistry at the 2- and 3-positions remained undetermined and was established for the first time in this study. 

Nano-probes were developed nearly in parallel with the now prevalent 3 mm micro-NMR probes. For small-sample applications, Nano-probes have an advantage in that they can locate 100% of a precious sample in the rf coils of the probe. 

Schwartz and co-workers, also at Merck, reported the isolation and elucidation of the structure of pramanicin (12) and a related fatty acid that were discovered during a search for novel antifungal agents.60 The authors reported using a 500 MHz 3 mm micro-inverse probe for the acquisition of the heteronuclear 2D-NMR data to characterize the molecule, including a HMQC spectrum. 

Later in 1996 the first example of a dimeric indoloquinoline alkaloid, crypt-omisrine (22), was isolated and its structure elucidated.73 Inverse-detected 2D-NMR spectra were recorded using a 3 mm micro-inverse probe 13C chemical shift data were recorded for the sample using a 3 mm micro-dual probe. In addition to the mass spectral data, a 0.9% nuclear overhauser effect (NOE) observed between the HI and H9 protons was used to confirm the structure. 

Fig. 1. HMQC spectrum of a 12 pig (0.05 pmol) sample of the indoloquinoline alkaloid cryptolepine (1) dissolved in 145 pL of d6-DMSO. The data were recorded overnight (16 h) at 500 MHz using a 3 mm micro-inverse-detection NMR probe.6 Both a proton reference spectrum (top trace) and the projection through the Fi frequency domain (bottom trace) are shown above the contour plot. (Reprinted with permission from Ref. 6. Copyright 1992, American Chemical Society and American Society of Pharmacognosy.)...

Fig. 10. Comparative overnight 13C spectra of an 200 pg sample of a secondary fungal metabolite fumonisin B (6) in D20. The data shown in the top trace were acquired with the sample dissolved in 140 pL of solvent in a 3 mm NMR probe. The data were acquired overnight at 125 MHz using a Nalorac MD-500-3, 3 mm micro-dual NMR probe. The s/n ratio of the experiment was 5.5 1. A ketone carbonyl was expected in the 200-230 ppm region of the spectrum based on mass spectrometric fragmentation data and was not observed in the 13C reference spectrum. In an effort to observe the carbonyl 13C resonance to complete the work, the sample was washed from the 3 mm NMR tube, concentrated to 20pL and transferred quantitatively to a 40 pL nano-cell. The overnight data acquisition was repeated using a Varian 500 MHz heteronuclear Nano-probe to afford the spectrum shown in the bottom trace. The s/n ratio in the bottom spectrum was 11 1, with the expected carbonyl resonance observed at 219.5 ppm. (Reprinted with permission from Ref. 20. Copyright 1995, American Chemical Society and American Society of Pharmacognosy.)...

In 1993 shortly after 3 mm micro-probes became commercially available, an application of the use of 3 mm probe technology was reported by Hensens and coworkers at Merck.58 The enediyne antibiotic neocarzinostatin (9) is activated for duplex DNA cleavage by > 1000-fold by thiol reagents. Hensens and co-workers utilized both micro-inverse and micro-dual 3 mm NMR probes to characterize the structure and stereochemistry of the novel post-activated drug (10). 

Fu and Schmitz80 reported the isolation and characterization of a new brominated diphenyl ether from an unidentified specie of Dysidae sponge collected at Satawan Atoll in Micronesia in 1996. In addition to four known brominated diphenyl analogs for which 13C chemical shift data were reported, a new minor metabolite (25) was isolated and characterized using a 3 mm micro-dual NMR probe. 

The author and colleagues employed 3 mm micro-inverse-detection NMR probe capabilities in an effort to assign the 15N resonances of the cyclic thiopeptide antibiotic sulfomycin-I (39)." Acquiring the long-range HMBC data in a 3 mm... 

Logusch and co-workers105 further explored the capabilities of 3 mm NMR probe technology in a study of the enzymatic synthesis of a rodent metabolite the preemergence herbicide alachlor. Lipopolysaccharides from Chlamydia trachomatis serotype L2 were the subject of a study using a 2.5 mm Bruker micro-NMR probe reported by Rundt et al.106 This work represents the first complete structural characterization of a lipopolysaccharide from a Chlamydial sp. 

In 2001, Skibo et al.131 reported using a 3 mm micro-dual NMR probe for the acquisition of 13C spectra in a study of recognition and cleavage reactions in the major groove of DNA. Silverman and co-workers utilized 3 mm micro-probes for... 

In 1995, the author and co-workers reported a comparison of the 13C-NMR spectra acquired for an 100 pg sample of the alkaloid cryptolepicarboline (13) using both a 3 mm micro-dual and heteronuclear Nano-probes.61 The two probe technologies gave roughly the same s/n ratios with the Nano-probe allowing the acquisition of the spectra in roughly one-third of the time required in a conventional 3 mm probe. Sample volumes were 40 and 140 pL, respectively. 

Martin, G.E. Hadden, C.E. Comparison of 1.7mm submicro and 3 mm micro gradient NMR probes for the acquisition of H- C and H- N heteronuclear shift correlation data. Magn. Reson. Chem. 1999, 37, 721-729. 

The author and a co-worker later exploited 3 mm NMR probe capabilities in a study of the thermal degradation products of the oxazolidinone antibiotic Zyvox (linezolid, 41) based on the use of H-15N heteronuclear shift correlation experiments.127 In a study of the structure-function relationships of a new growth hormone-releasing peptide, ghrelin, Bednarek and co-workers128 at Merck utilized micro-probe capabilities in the characterization of the structures of the minimum sequence of ghrelin necessary for activity. As a result of these efforts, a small spiroindan, MK-0677 (59) with oral bioavailability was found to be one of the most potent synthetic analogs with this activity. 

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