Tritiation procedures method

A procedure for determining the absolute configuration of the tritiated succinic acid had already been developed, which involved partial oxidation on succinate dehydrogenase [45]. Applying this method it was then possible to show that the tritiated succinic acid of interest had the (S) configuration, and this in turn established the (5S) configuration of the tritiated /3-lysine from which it had been derived. Inspection of Fig. 28 reveals that the /3-lysine mutase reaction takes place with inversion at the C-5 of the substrate. 

Three types of radiation-induced procedures have been used including (a) recoil tritiation, (b) Wilzbach gas irradiation7 with its modifications, and (c) radiation synthesis.8 All these methods utilize high-energy ionizing radiation to achieve tritium incorporation. Wilzbach exchange7 is the most satisfactory of the three and has been the most widely used. Radiation synthesis is a relatively recent development of limited but valuable specificity. 

A value of kH/kD = 1.4 was obtained [114] for the rate of proton transfer compared with deuteron transfer from chloroform to hydroxide ion and this result is similar to the values determined earlier for several haloforms [164, 166]. In the most recent work [171(b)] a value kH /kD = 1.11 0.05 was determined for chloroform. These values are close to those observed for reaction of cyanocarbon acids (though a different base catalyst is involved) and in Sect. 4.3 it was argued that isotope effects as low as these are expected for a transition state in which proton transfer is almost complete. The isotope effect for proton transfer from chloroform was measured using a new and useful method [114]. It can be shown that the ratio of initial rates of uptake of tritium for the first ten per cent of reaction from tritiated water into CHC13 and CDC13 is identical to the primary isotope effect for proton loss (feH /fcD). The procedure can be used for measuring isotope effects on proton transfer from carbon acids to hydroxide ion or buffer catalysts and is more convenient than other methods. Other methods which have been used, for example, involve the comparison of rates of detritiation and dedeuteration or the comparison of rates of bromination for isotopically different acids (RCH and RCD) [113]. 

Certainly, the best method of sample preparation is the so called combustion technique, which was derived from the Schoni-ger oxygen-flask method, and has been modified and improved in many ways. Besides manual procedures there are now semiauto-mated and fully-automated oxidizer models commercially available. In an oxygen atmosphere biological specimens labelled with H, or can be combusted to tritiated water,... 

Hydroboration followed by protonolysis of the resulting alkylborane can be used as an alternative method for hydrogenation of alkenes, although catalytic hydrogenation (Section 7.12) is the more common procedure. Reaction of alkylboranes with deuterated or tritiated acetic acid also provides a very useful way to introduce these isotopes into a compound in a specific way. 

Exchange reactions may be carried out using tritiated solvent or tritiated gas, as in the Wilzbach method [19]. In the former procedure the sample is heated in a sealed tube with a hydrogen transfer catalyst and a tritiated solvent. In the Wilzbach procedure to which recourse is made only when... 

There are three major methods used to control self-decomposition. First, dilution of the labelled material with the same unlabelled compound or carrier reduces the problem of absorption of the radiant energy. Sudi a procedure, however, lowers the specific activity of the compound but provided it is not diluted beyond the lowest level required for the experiment this is an approved method. Second, the rate of self-radiolysis is often reduced by cooling the sample to as low a temperature as possible. This is advantageous for compounds labelled with carbon-14 or with sulphur-35 but for tritiated compounds the situation is not so clear. In at least once case, that of tritiated thymidine, storage below 0 °C accelerated the rate of self-radiolysis [33-35]. The third procedure for minimising self-decomposition has emanated from... 

Another procedure for dealing with samples insoluble in counting solution is to support them on a medium such as paper strips, filter discs, glass fibre or DEAE cellulose prior to adding them to a counting vial [235-237]. As indicated earlier, use of this has been in chromatography where the spot has been cut out of the paper or scraped from the plate. Although useful for materials insoluble in scintillator fluid, self-absorption for tritiated samples may constitute a major drawback in this technique, just as it does for the suspension methods. It should be evident why the recent developments in in combustion procedures are so important in the problem of sample preparation. 

The methods that have been developed for tritiating small organic molecules do not lend themselves very readily to the tritiation of large macromolecules such as proteins although there are a few examples where Myasoedov s HSCIE procedure proved successful. It is not surprising therefore that very little work has been reported on, for example, the NMR of proteins. The polymer area, however, has seen more activity, mainly because it has been much easier to tritiate such compounds - hydrogenation with H2 gas of a suitable monomer followed by polymerization leads to a specifically tritiated product. 

Stoichiometric C-deprotonation of a compound results in a carbanion which can be quenched by addition of tritiated water or another tritium source, either in situ or after some manipulation of the deprotonated substrate. This method can have advantages over thermodynamic exchange procedures, including greater chemo- or regioselectivity, the achievement of higher specific activity or economy in the use of tritium. 

Methods for the preparation of tritiated methyl iodide in the laboratory use variations of the sp tritiodehalogenation approach. Palladium-catalyzed tritiodechlorinations of chloro-methyl ester 113 and of chloromethyl sulfide 115 ° have been developed as less hazardous alternatives to the older laboratory procedure for C HH2l at 15-22 Ci/mmol starting from bis(chloromethyl)ether °. The initially formed nonvolatile monotritiated 114 and 116 can be conveniently isolated, purified and finally subjected to ester cleavage with either lithium iodide or hydriodic acid, or, in the second case, by thermolysis of the intermediately generated sulfonium salt 117. 

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