Proton Satellite Method

Proton Satellite Method,—In the conventional C-labelled method, degrada-tive procedures for the isolation of all labelled carbons in a metabolite may be precluded by formidable structural complexities or the presence of unreactive carbons such as quaternary carbons or carbons in an aromatic framework. The complexity of the chemical degradative procedure is greatly dependent on the molecular structure of the labelled compound. Complex structures require not only complicated, time-consuming procedures, but also tedious procedures for isolation and purification of the molecular fragments. Moreover, since contamination with radioactive impurities influences the experimental results, much care must be taken in the purification steps. 

Structures of many new natural products isolated today are determined by advanced instrumentation techniques, without recourse to chemical reactions. Because of this lack of knowledge of chemical reactivity of new metabolites, detailed degradative studies must be initially undertaken, with considerable 

The recent availability of materials enriched with carbon-13 (a non-radio-active magnetic isotope of carbon) has greatly simplified biosynthetic studies of microbial metabolites. The use of specifically C-labelled precursors and and n.m.r. studies of the labelled products permits detection and identification of the labelled sites without need for chemical degradation. 

Spin-spin coupling between a C nucleus and a directly bonded proton produces satellite bands on both sides of the main proton signal in the H n.m.r. spectrum. The characteristic satellite coupling constants range from 125 to 250 Hz and depend on the carbon hybridization and substituents. They also show a near linear dependence on the percentage of s-character of the —H 

The degree of enrichment of into the metabolite over the natural abund- 

---

Dorsey, et al., have described a proton nmr method for EEW determination which uses a measured amount of sym-tetrachloroeth-ane as an internal standard ( ). They report results on five resins with EEW s below 300. Hammerich and Willeboordse have analyzed the precision of proton nmr EEW determinations ( ). Some of the problems in accurate area measurement they point out, such as car-bon-13 satellites and insufficient separation of main peaks, are not of concern in the carbon-13 nmr EEW determination. 

The most rewarding procedure has involved the feeding of specifically Relabelled possible precursors to an appropriate culture foUowed by isolation of the metabolite(s). Screening of the product for radioactivity affords a preliminary indication of the precursors. Subsequent repetition with appropriately C-labelled precursors and examination of the product by NMR provides data on the exact location in which the precursors have been incorporated without the necessity for degradative studies. This information is adequate in many cases to deduce the metabolic pathway. In carbon-13 studies, both proton NMR using the satellite method, and C-NMR have been employed. 

At the time of writing, most of the metabolites examined by the C satellite method also contained labelled carbons not directly bonded to protons, and these labelled nuclei cannot be detected by the satellite method. In some cases, this problem may be circumvented by chemical transformation. For example, in fusaric acid, protons were chemically introduced on to a labelled carbon by reduction to the alcohol i.e COjH CH OH). This procedure could, perhaps, be applied to other labelled carbonyl groups and olefinic carbons ... 

Another limitation of the C-satellite method is the obscuring of satellite peaks by complex n.m.r. spectra in which the proton resonances either are not well resolved or exhibit a narrow range of chemical shifts. Investigation of the biosynthesis of steroids and carbohydrates would be particularly difficult because of this limitation. 

Since natural-abundance peaks in the labelled radicinin were not readily visible in the number of spectral scans employed to make a comparison, the enrichment value of 17% was determined by integration of the C-proton satellite bands of the methyl groups C-11 and C-14, 7i3c-h = 126 Hz, which were evident in the proton 60 MHz spectrum. Combined application with the C-satellite method can thus result in a better quantitative approach to carbon enrichments. 

Subsequently, Uhrinova et al.29 reconsidered the problem using both proton-and carbon-detected experiments. For example, couplings of anomeric carbons were measured from the 13C satellites in proton NMR spectra. The critical factor in these methods is the suppression of signals from protons bound to, 2C atoms. In the pulse-sequence proposed, these protons were selectively inverted by a BIRD (Bilinear Rotation Decoupling) pulse,30 and the spin-echo method introduced by Bendall et al.31 was used. 

An SPT experiment requires identification of at least one 29Si satellite in the H NMR spectrum non-selective experiments (INEPT, DEPT) and JCP require for optimum performance an estimate of the coupling constant, and the correct estimate of the number of coupled protons improves the performance of INEPT and DEPT experiments. While an SPT experiment achieves enhancement through one selective irradiation, the other methods use series of pulses. The group of experiments that are referred to here as non-selective use a series of properly timed pulses, and ICP, which is also non-selective, differs from them in that it uses a train of pulses known as a spin-lock. 

If the groups R1 and R2 are identical, the H NMR vicinal coupling constant between two methine protons cannot be obtained from the routine NMR spectrum because of the same chemical shift. In such a case, the H NMR13C satellite band method is useful to determine the Jvlv value.72,74... 

An interesting method, which employs the PFT technique, has been proposed (80) for extracting 15N satellites from the proton spectra of compounds with 15N at natural-abundance in order to obtain the values of 15N- H coupling constants. The method consists of alternatively generating I5N-decoupled and 15N-undecoupled proton free induction decays which are subsequently subtracted from one another. Thus, at least theoretically, the accumulated free induction decay should give, after a Fourier transformation, only the satellite peaks. This technique... 

A recently developed technique of subtracting, 3N-decoupled from 13N-undecoupled proton spectra (81, 221) permits the measurement of larger, 3N-H couplings, mostly /(N-H), from the very weak 13N satellites observed in proton spectra. A number of /(N-H) values for amides have been measured by this method (Table XXXII, note b). 

Figure 6.8. A comparison of signal suppression methods used in proton-detected heteronuclear correlation experiments (see descriptions in text). Spectrum (a) is taken from a conventional ID proton spectrum without suppression of the parent resonance and displays the required satellites. Other spectra are recorded with (b) phase-cycling, (c) optimised BIRD presaturation, and (d) pulsed field gradients to remove the parent line. All spectra were recorded under otherwise identical acquisition conditions and result from two transients. Complete suppression can be achieved with gradient selection, but at some cost in sensitivity in this case (see text).

---