Tritium NMR

Other methods of sensitive detection of radiotracers have been developed more recently. Eourier transform nmr can be used to detect (nuclear spin 1/2), which has an efficiency of detection - 20% greater than that of H. This technique is useful for ascertaining the position and distribution of tritium in the labeled compound (14). Eield-desorption mass spectrometry (fdms) and other mass spectral techniques can be appHed to detection of nanogram quantities of radiolabeled tracers, and are weU suited for determining the specific activity of these compounds (15). 

Nuclear Magnetic Resonance. AH three hydrogen isotopes have nuclear spins, I 7 0, and consequently can all be used in nmr spectroscopy (Table 4) (see Magnetic spin resonance). Tritium is an even more favorable nucleus for nmr than is H, which is by far the most widely used nucleus in nmr spectroscopy. The radioactivity of T and the ensuing handling problems are a deterrent to widespread use for nmr. Considerable progress has been made in the appHcations of tritium nmr (23,24). 

Hydrogen as it occurs in nature is predominantly composed of atoms in which the nucleus is a single proton. In addition, terrestrial hydrogen contains about 0.0156% of deuterium atoms in which the nucleus also contains a neutron, and this is the reason for its variable atomic weight (p. 17). Addition of a second neutron induces instability and tritium is radioactive, emitting low-energy particles with a half-life of 12.33 y. Some characteristic properties of these 3 atoms are given in Table 3.1, and their implications for stable isotope studies, radioactive tracer studies, and nmr spectroscopy are obvious. 

The tritiated version could be prepared from tritiated formic acid which we had prepared at high specific activity (2.5 Ci mmol-1) by a metal-catalyzed hydrogen-tritium exchange procedure using T2 gas. The material can be stored either as a solid or as a solution if the latter any release of tritium by back exchange can be easily monitored by 3H NMR spectroscopy. In our experience very little exchange occurs over several weeks of storage [51]. 

The distribution of tritium in compounds 137-150 can be determined by tritium NMR spectroscopy without chemical manipulations115. The structure retention index relationship (SR IR)116 has been used for identification of unknown radioactive peaks and to differentiate by-products from radioimpurities from extraneous sources. 

The comprehensive dedicated research ultimately made it possible to decode the patterns of labelling in almost any type of tritium labelled compound at low isotopic abundance (e.g., 3 x 10 4 to 3 x 10 2 per cent. 3H per site) with the aid of 3H-NMR directly, rapidly, reliably and non-destructive analytical means. Since, 1971, the 3H-NMR spectroscopy, utilizing only millicurie (mCi) quantities of radioactivity, emerged as a most useful analytical tool for the study of tritium labelled compounds. 

The two major advantages of 3H-NMR spectroscopy based on the characteristic magnetic features of the tritium nucleus are, namely ... 

Evans, EA, DC Warrell, JA Elvidge and JR Jones, Handbook of Tritium NMR-Spectroscopy and Applications, New York, John Wiley and Sons, 1985. 

It was postulated from the comparative data in Tables I and II and other observations that angiotensin had a definite compact conformational structure even though it is a linear peptide and should be a random coil. Tritium-exchange studies (39), CD (40), and NMR evidence (41, 42) now give support to this view. NMR studies with angiotensin, oxytocin, and vasopressin thus far have not indicated conformational restriction on the benzene rings, but the thin-film dialysis data are inconsistent with any conformation in which these bulky groups are extended from the otherwise compact conformation. 

A useful variation of this technique is to measure the rate of exchange of tritium from tritiated water with the C-2 proton of the imidazole ring of a histidine.31,32 The rate constant for the exchange depends on the state of ionization of the imidazole, and is faster for the unprotonated form. This procedure is a useful adjunct to NMR experiments, which also measure the pKa s of histidine residues. ... 

A non-ribosomal biosynthetic pathway is clearly indicated for cyclosporin A, considering the uncommon structural elements MeBmt, L-a-aminobutyric acid and D-alanine as well as the plethora of isolated congeners [20,21]. Non-ribosomal biosynthesis directed by multienzyme thiotemplates have been reported for other small peptides of microbial origin, for example, gramicidin S [22] and enniatin [23]. Experimental data for cyclosporin A were obtained by feeding appropriate labelled precursors to cultures of T. inflation strains. The distribution profile of the labelled atoms in cyclosporin A was determined by 3H- or 13C-NMR spectroscopy. In preliminary trials with several tritium and carbon-14 labelled precursors, [met/y>/-3H]methionine proved to be the most suitable marker for the biosynthetic preparation of radiolabelled cyclosporin A for pharmacokinetic and metabolic studies [24],... 

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