May 5, 1952 [CONTRIBUTION FROM
SYNTHESlS OF CY- OR p-Cl4-LABELED THE
GLYCEROL
2413
DEPARTMENT OF BIOLOGICAL CHEMISTRYAND THE BIOPHYSICAL LABORATORY, HARVARD MEDICAL
SCHOOL 1
A Synthesis of a- or P-CI4-Labeled Glycerol BY L. I. GIDEZ~ AND M. L. KARNOVSKY Radioglycerol, selectively labelled in the a-position ( g l y ~ e r o l - l - ( 3 ) - Cor ~ ~the ) @-position( glycerol-2-CI4) has been prepared from easily available radioactive starting materials, such as barium carbonate, acetic acid or sodium cyanide. The over-all yield starting from barium carbonate was of the order of 30%.
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BRCOOR’ LiAlH4 + LiAl(OR’)2(0CHZR)2 Glycerol has a unique relationship to both the lipids and the carbohydrates, and methods for Thus, for each mole of the above triester, 1.5 moles making radioglycerol labelled in the a- or the p- of lithium aluminum hydride was required, and the position with CI4 should be of particular interest to products of the reaction were presumably lithium investigators studying carbohydrate and lipid and aluminum alcoholates and glyceroxides, which metabolism and fermentation mechanisms. Such were decomposed with water. Experience in this methods have recently been e l a b ~ r a t e and d ~ ~the ~ ~ ~Laboratory has shown it to be necessary to remove scheme below describes the synthesis adopted in dissolved salts from the resulting glycerol solution, these laboratories and outlined previously. Bar- especially if the product is to be distilled. This ium carbonate is shown as the ultimate starting has been accomglished by passing the solution, material for both the a- and @-labelledproducts, containing glycerol and salts, through an ion-exbut the synthesis may, of course, start at any con- change column. The dilute ion-free solution of venient stage in the scheme. glycerol could then be concentrated by distillation COP + CHaOH + of the water under reduced pressure, or by lyophilization. @-labelling The syntheses reported here were carried out on I a-labelling quantities that made addition of carrier unnecescoz iBaCOaI sary, with the object of more easily establishing the degree of “radio-purity” of the final product of the dabelling series of reactions. Our experiences lead us to beNaCK lieve that these operations may be carried out on cs smaller quantities, and carrier added where necesI sary, particularly in the final step. The over-all BrCHpCOOH + CH1 I1 yields of radioglycerol were found to be about 30y0 ‘ / 11 COOH coo from barium carbonate or acetic acid, or about 40% I11 IV based on sodium cyanide. The product was assayed by periodate oxidation COOCzH5 COOC~HS CHzOH I I and collection of the formaldehyde produced as the CH2 --+ &HOCOCHI + CHOH dimedone compound. This compound could be I I I COOCzH5 CHzOH plated and counted, and represented the a-carbons COOCzHs of the glycerol molecule. The total carbon in the V VI VI1 product was determined by wet combustion, and a-Labelled glycerol is obtained from carboxyl- the carbon dioxide formed was counted as barium labelled acetic acid, or by the use of radiocyanide, carbonate. The periodate assay and the combusand @labelled glycerol results from the use of tion data revealed some carbon contamination demethyl-labelled acetic acid. These three Starting rived, it is believed, from the ion-exchange column. materials are all commercially available, or may be Glycerol sample 1, which was passed through a synthesized by well-known method^^*^,^ and their column that had been subjected to a preliminary preparation will not be discussed. The intermedi- washing of exceptional thoroughness, proved to be ates of the above scheme (I1 - VI) were made by free of this contamination. Distillation of the slight adaptations and modifications of established product, admixed with pure commercial glycerol, methods, and representative yields are given in the removed the extraneous material. Table I sumexperimental section. The final step was the marizes the results. d sample of formaldimedone reduction of the tartronic acid ester (VI) with obtained from a-labelled glycerol was recrystallithium aluminum hydride. The stoichiometry of lized and counted. The same material was comsuch reactions has been worked outs and it has been busted, and counted as barium carbonate, and a s h o w n t h a t the reduction of an ester t o an alcohol factor obtained for the conversion of counts as formcan be accomplished as follows aldimedone to counts as barium carbonate. (1) United States Atomic Energy Commission Predoctorai Fellon-. Under the conditions of counting in these labora(2) M. L. Karnovsky and L. I. Gidez, Fed. PYOC., 10,205 (1951). tories, this factor is 1.04. (3) A. P.Doerschuk, THISJOURNAL, 73, 821 (1951). The localization of the radioactivity in the a- or (4) H.Schlenk and B. W. DeHaas, ibid., 1 8 , 3921 (1951). @-carbonsof the glycerol was established after oxi(5) “Isotopic Carbon,” John Wiley and Sons,Inc.. New York, N. Y., 1949, pp. 172-179. dation with periodate. In the case of the a(6) J. A. McCarter, THIS JOURNAL, 73. 483 (1951). labelled product, the formic acid obtained, repre(7) Reference 5, pp. 193-195. senting the @-carbon,was counted after conversion R. F. Nystrom and W G. Brown, THIS JOURNAL, 69, 1197, 2548 (8) to barium carbonate, and had a specific activity ( 1947).
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L. I. GIDEZAND M. L. KARNOVSKY
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TABLEI RADIO-GLYCEROL SAMPLES
ClIAKACTEKISTICS O F Glycerol
Sample
a-Label 1 2 3"
Activity, in GlycGlycerol ct./min. mM. proderol Total C/ X 10-6 iict, carbon, carbon, Total C Formal
