Patent Number: 051868687
Section: summary

TECHNICAL FIELD This invention involves agents useful for preparing isotopically labeled compounds. More particularly, it relates to novel site-selective deuterating and tritiating agents with high deuterium or tritium contents, methods for their preparation, and methods for using these agents to insert high levels of deuterium/tritium labels into reducible compounds. BACKGROUND ART Isotopic labeling is a useful tool for rendering organic compounds easily identifiable in analytical and biochemical schemes. The isotopic label may be detected very sensitively, especially in the case of a radionuclide. By placing the isotopic label in a specific site in a molecule, it is possible to study reactions involving the molecule and detect and delineate reaction paths. Traditionally, isotopic hydrogen (e.g., tritium or deuterium) labeling has been limited by the unavailability of adequate deuterating/tritiating agents. There are two fundamental techniques for introducing isotopic hydrogen into organic molecules. These are synthetic techniques and exchange techniques. Synthetic techniques, where tritium or deuterium is directly and specifically inserted, yield high tritium or deuterium abundance, but are limited by the chemistry required. In addition, the molecule being labeled may be changed, depending upon the severity of the synthetic reaction employed. Exchange techniques yield lower tritium or deuterium incorporation, often with the isotope being distributed over many sites on the molecule, but offer the advantage that they do not require separate synthetic steps and are less likely to disrupt the structure of the molecule being labeled. Three common synthetic methods for incorporating activity levels of tritium into target molecules have been: (1) "hydrogenation" of the target molecule using tritium gas (T.sub.2), with a catalyst; (2) tritiodehalogenation; and (3) tritiomethylation with CT.sub.3 I. Each of these methods has been heavily employed in the art to achieve high levels of isotope incorporation, yet each involves reaction conditions that can affect the integrity of the target molecule. Conversely, the use of milder "tritium exchange" methods typically involves reduction in the level of tritium incorporated into the target molecule. A fourth way of synthetically incorporating tritium into a target molecule which contains a reducible site is to contact the target molecule with a reducing agent which is capable of inserting one or more tritium atoms into the reducible site. This methodology essentially mimics reduction with hydrogen-inserting reducing agents. Metal borohydrides such as LiAlH.sub.4 and NaBH.sub.4 are widely used mild reducing agents. In contrast, lithium trialkylborohydride (superhydride) (Brown, H. C. et al., (1980) J. Org. Chem. 45:1-12) is known to be a highly reactive nucleophilic reducing agent, and is now commonly used in organic synthesis (Brown, H.C. et al., (1979) Aldrichimica Acta 12:3-11). This reagent is capable of reducing esters, hindered alkyl halides (Brown, H.C. et al., (1973) J. Am. Chem. Soc. 95: 1669-1671) and toluene-p-sulphonates, in addition to exhibiting great sterioselectivity and steriospecificity, as in the reduction of epoxides. More hindered trialkylborohydrides (such as lithium or potassium tri-sec-butyl borohydride; known as L-selectride and K-selectride) exhibit even more steric control, as in the reduction of cyclic ketones (Fortunator, J. M. et al., (1975) J. Org. Chem. 41: 2194-2200). These remarkable hydride reducing agents are generally synthesized by reaction of the appropriate alkylborane with a metal hydride (Brown, H. C. et al (1980) J. Org. Chem. 45:1-12). It is clear that the ability to produce metal deuterides and tritides with high deuterium/tritium content would give access to a large number of deuteriated/tritiated reducing agents for chemoselective, regioselective and stereoselective labeling sequences, and allow high level deuterium/tritium incorporation through established synthetic routes with these highly reactive and selective reagents. The utility of supertritide has been demonstrated (Hegde, S. et al., (1983) J. Chem. Soc. Chem. Commun., 1484-1485) by the reduction of acids, aldehydes, toluene-p-sulphonates and epoxides, but these reactions were conducted with supertritide of specific activities in the mCi/mmol range (100's of MBq/mmol). Later work (Coates, R. M. et al., (1986) Synthesis and Applications of Isotopically Labeled Compounds (Proc. 2nd Int. Symp.), 207-212) reported the synthesis of chiral methyl groups, starting with supertritide at approximately 3 Ci/mmol. This is still a factor of 10 below the theoretical maximum (one tritium atom per molecule gives a specific activity 28.72 Ci/mmol or 1063 GBq/mmol) and consequently this tritiation reagent has not been applied in those types of reactions where it is used in general chemistry. The same general statements are true for the availability and utility of LiAlT.sub.4. At this time, both LiAlD.sub.4 and LiEt.sub.3 BD are available commercially. Although the complex hydrides are very useful reagents, the preparation of the initial metal hydrides has been problematic, especially where radioisotopes are involved. Metal hydrides may be prepared from the respective elements: e.g. atomic hydrogen produced in a glow discharge tube was found to rapidly react with various alkali metals, vacuum condensed as thin films on the reaction tube walls, to form metal hydrides (Ferrell, E. et al., (1934) J. Chem. Soc. 7-8). Other means of producing atomic hydrogen (or tritons) include dissociation of molecular hydrogen (tritium) by microwave discharge activation (Cao, G. Y. et al., (1984) Trans. Am. Nucl. Soc. 45:18-19) or on the surface of a hot tungsten wire (Moser, H. C. et al., (1962) J. Chem. Phys. 66:2272-2273). The latter two methods offer the advantages of being less limiting in scale and the option of exchange of tritons with LiH, thereby avoiding the use of liquid lithium. Tritide synthesis on a large scale has also been reported under conditions of high temperature and pressure, where lithium tritide was synthesized at 98% purity in an iron crucible at 750.degree. C., in the presence of three atmospheres of tritium gas (Bowman, R. C. et al., (1988) J. Nucl. Materials 154:318-331). The severe conditions and need for excessive tritium in this procedure make this option less attractive than the others outlined above, and only usable by the nuclear/fusion industries. One other problem lies in the fact that once the hydride (deuteride or tritide) is formed by one of the above methods its chemical reactivity is reported to be low, and conversion into a complex hydride for use as a reducing reagent in organic synthesis may be sluggish. Hegde, S. et al., (1983) J. Chem. Soc. Chem. Commun., 1484-1485, reported that a typical reduction with such agents took several days at 150.degree. C. The present invention is directed to the aforementioned problems. It provides a new method of in situ synthesis to generate a highly reactive alkali metal deuteride or tritide with a large proportion of its hydrogen present as deuterium or tritium from the respective deuterium or tritium gas. This material is then converted into a desirable highly selective labeling agent. DESCRIPTION OF THE PRIOR ART Background References. Brown, H. C. and Krishnamurthy, S., (1979) Aldrichimica Acta 12:3-11 presents a good summary of the state of borane chemistry for organic reductions, including the increased ability to perform regioselective, stereoselective and chemoselective reductions of various organic functional groups. Selective Borohydride Reducing Agents. The synthesis of Superhydride is described in Brown, H. C. et al., (1980) J. Org. Chem. 45:1-12, and some of its uses are described in Brown, H. C. and Krishnamurthy, S., J. (1973) Am Chem. Soc. 95:1669-1671. L-Selectride and K-Selectride are described synthetically in Brown, H. C. et al., (1978) J. Am. Chem. Soc. 100:3343, and some of their uses are described in Fortunato, J. M. and Ganem, B. (1976) J. Org. Chem. 41: 2194-2200, and Brown, H. C. and Dickason, W. C., (1970) J. Am. Chem. Soc. 92:709. Tritium Labeling Agents. The synthesis of a low specific activity "Supertritide" (LiEt.sub.3 BT) is described in Hegde, S. et al., (1983) J. Chem. Soc., Chem. Comm. 1983:1484-1485, and see Coates, R. M. et al., in "Synthesis and Applications of Isotopically Labeled Compounds (Proc 2d Int'l Symp.)". pp. 207-212 Muccino, R. R., ed. (Elsevier Press: Amsterdam) (1986). Altman, L. J. and Thomas, L., (1980) Anal. Chem. 52:992-995 identified a high specific activity sodium borotritide (NaBT.sub.4). Reactive Lithium Hydride. Klusener, P. et al., (1986) Angew. Chem. (English Edition) 25:465, and Pi, R. et al., (1987) J. Org. Chem. 52:4299-4303 have reported a method of in situ synthesis of lithium hydride by bubbling hydrogen gas through a solution of n-butyllithium in hexane in the presence of tetramethylethylenediamine (TMEDA). The resulting hydride is a fine suspension and is highly reactive at room temperature. RELATED PUBLICATIONS In November of 1989, the present inventors and a colleague published a report of some of the work described herein in Trans. Am. Nucl. Soc. 60:34-36 (1989). SUMMARY OF THE INVENTION It is accordingly a primary object of the present invention to overcome the disadvantages of the prior art and to provide methods and reagents which are capable of tritium and deuterium labeling selected specific sites in target molecules, while achieving the labeling at high levels of tritium and deuterium insertion. Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The process of the invention is characterized as involving the in situ generation of highly selective reducing agents from alkali metal tritides or deuterides which have in turn formed from alkali metal alkyls. Thus, in one aspect, the invention provides a process for introducing tritium or deuterium label into organic compounds having a reducible site. This process involves reacting an organic solvented solution of an alkali metal alkyl with a gas which contains tritium or deuterium in the presence of an alkyl tertiary amine. This gives rise to an alkali metal tritide or deuteride. This tritide or deuteride can then be reacted in any of several manners to give rise to a reactive labeling reducing agent. In one of these subsequent reactions the alkali metal tritide or deuteride is reacted with a solution of trialkylborane thereby forming an alkali metal trialkyl borotritide or borodeuteride which can serve as the reducing agent. In another of the subsequent reactions the alkali metal tritide or deuteride is reacted with a solution of aluminum halide thereby forming a solution of alkali metal aluminum tritide or deuteride reducing agent. In a third such reaction the alkali metal tritide or deuteride is reacted with boron trifluoride thereby forming the tritium or deuterium analog of borane which can also serve as a selective reducing agent. Each of these reducing agents can then be contacted with the organic compound having the reducible site so as to directly reduce the reducible site and introduce tritium or deuterium atoms thereinto. In another aspect this invention provides the highly specific and selective deuterium and tritium labeling reagents just described, that is, the tritium and deuterium analogs of alkali metal trialkyl borohydride, the tritium and deuterium analogs of alkali metal aluminum hydride, and the deuterium and tritium analogs of borane. In particularly preferred embodiments the process and reagents provided by this invention are used at very high specific activities, often approaching the theoretical maximum. Thus, this invention can provide highly specific reagents and a process for achieving high levels of deuterium and tritium in selected sites of sensitive organic compounds.