Inverse probe

The size of sample required has been reduced by a number of technical developments including micro inverse probes and micro cells (references in Martin et al. 1998), and has been reduced even further using a newly developed 1.7-mm submicro inverse-detection gradient probe (Martin et al. 1998). The combined use of inverse detection probes with solenoid microcoils has also been developed to reduce sample volumes for NMR (Subramanian and Webb 1998). 

NMR experiments are performed on an NMR spectrometer using a 5-mm broadband inverse probe. 

Hardenbol P, Baner J, Jain M, Nilsson M, Namsaraev EA, Karlin-Neumann GA, Fakhrai-Rad H, Ronaghi M, Willis TD, Landegren U, Davis RW. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat Biotechnol 2003 21 673-678. 

The most common probe head is a switchable probe head, which can be used to observe H and all NMR-active nuclei from the low-frequency limit up to the frequency of 31P. The proton coil can be tuned for the observation of 19F. The switch-able probe head is designed for either direct or inverse observation. The direct observation probe head is most sensitive for 1-D experiments on 13C and 31P. The inverse probe head in turn is most sensitive for the direct observation of H and indirect detection, for example of 31P, in 2-D experiments, taking advantage of polarization-transfer phenomena. 

ID ll spectrum, and crosspeaks are arranged symmetrically around the diagonal. There is only one radio frequency channel in a homonuclear experiment, the H channel, so the center of the spectral window (set by the exact frequency of pulses and of the reference frequency in the receiver) is the same in If and F (Varian tof, Bruker ol). The spectral widths should be set to the same value in both dimensions, leading to a square data matrix. Heteronuclear experiments have no diagonal, and two separate radio frequency channels are used (transmitter for F2, decoupler for F ) with two independently set spectral windows (Varian tof and dof, sw, and swl, Bruker ol and o2, sw(If), and sw(I )). Heteronuclear experiments can be further subdivided into direct (HETCOR) and inverse (HSQC, HMQC, HMBC) experiments. Direct experiments detect the X nucleus (e.g., 13C) in the directly detected dimension (Ff) using a direct probe (13C coil on the inside, closest to the sample, H coil on the outside), and inverse experiments detect XH in the To dimension using an inverse probe (XH coil on the inside, 13C coil outside). 

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... 

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. 

HMQC spectra using H as the detector (/) spin can be recorded on a conventional two-chaimel NMR spectrometer with a normal broad band, or selective dual, probe head. However, better signal-to-noise will be achieved using an inverse probe head due to the better filling factor. 

To illustrate this point, a comparison of the results obtained with a conventional 3 mm gradient inverse triple resonance and 5 mm cryogenic gradient inverse probe technology for the acquisition of long-range data was performed. Using a 2 mg sample of the... 

Heteronuclear NMR experiments, which can be performed with the standard equipment of practically all modern spectrometers, require in general three separate radiofrequency (RF) channels for both spectrometer and probe head. The first two channels deliver the H (for decoupling) and "X frequencies to the sample, and the third channel is commonly tuned to D and operates the field frequency lock. In most standard probe heads, these three frequencies are delivered via two concentric coils. The inner coil with the higher Q factor is generally used for detection, the outer one only for the application of pulses and decouphng. TWo general designs are in use in normal or forward probe heads, which are optimized for direct detection of X nuclei, the inner coil is a tuneable X coil and the outer coil is normally double tuned to and the lock frequency, while in inverse probe heads which are optimized for indirect detection of "X resonances via H, this order is reversed. 

Figure 3.68. The proton solvent presaturation test. The data were acquired on a 400 MHz spectrometer equipped with a dual H C inverse probe and the sample was 2 mM sucrose and 0.5 mM DSS in 90% H2O/10% D2O.

The adoption of the inverse approach also has implications for the design of the NMR instrument. Conventional probes have been constructed so as to optimise the sensitivity for observation of the low-y X-nucleus, which entails placing the X-nucleus coil closest to the sample and positioning the proton coil outside this. Inverse probes have this configuration switched such that the proton coil sits closest to the sample for optimum sensitivity, thus providing a greater filling factor. However, even with conventional probes, the proton detected experiments can still be performed, albeit with less than optimum sensitivity, and may still provide a faster approach than the former X-observe experiments. 

Hardenbol P, et al. (2005). Highly multiplexed molecular inversion probe genotyping Over 10,000 targeted SNPs genotyped in a single tube assay. Genome Res. 15(2) 269-275. 

Hardenbol P, et al. (2003). Multiplexed genotyping with sequence tagged molecular inversion probes. Nat. Biotechnol. 21 673-678. 

When modern NMR was in its infanq, few if any scientists realized that they would ever require a two-coil NMR probe in which the coil lies inside the broadband coU. It was not until the demonstration that experiment time can be drastically reduced for the acquisition of 2-D heteronuclear correlation NMR data sets by employing H detected methods and a two-coil NMR probe with the coil configurations inverted (relative to the normal coil configuration described above) that the inverse probe really caught on. Now the inverse probe (a probe with the coil closer to the sample than the broadband coil) is a staple in nearly every modern NMR laboratory. 

An ffMQC or ffSQC experiment carried out with an inverse probe will provide the same information as a ffETCOR experiment carried out with a normal probe, but will do so in far less time. Even carrying out the ffMQC or ffSQC experiment on a normal probe is more efficient than the ffETCOR experiment. This advantage stems from the higher signal-to-noise ratio we obtain when we detect signal from the ff (with its higher gyromagnetic ratio) instead of from C or... 

A typical 2-D NOESY spectrum collected from a 20 mM sample in a 5 mm inverse probe at 500 MHz will take one to two hours. 

General [a]o were measured with a Perkin-Elmer 241 MC polarimeter. NMR spectra of 1 were obtained on Varian VXR 200 and Varian Unity 500 spectrometers. NMR spectra of 2-5 were obtained on a Jeol JNM-LA 600 spectrometer. Chemical shifts were reported in S (ppm), with residual C3D3N signals (7.24/123.5) and CDCI3 signals (7.25/77.0) as internal standards. A Nalorac inverse probe of 5 mm was used for all NMR experiments on 2-5 except for C NMR measurements (Joel probe). D/CI mass spectra were recorded on a Finnigan MAT TSQ 700 instrument. P-Glucosidase from almonds (1000 lU/mg) was purchased from Fluka AG (No. 49290). 

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