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1456
L-01. 76
(A4-3-ketone); (Anal. Calcd. for C23H3106F : C, Amax. N'jol 3.06 p (OH), 5.72 p, 5.81 p (acetylated side 65.39; H, 7.39; F, 4.52. Found: C, 65.32; H, chain), 6.11 p (A4-3-ketone); (Anal. Calcd. for 7.26; F, 4.50) was obtained in about 50% yield C Z ~ H ~ Z OC,~ S59.98; : H, 6.71; S, G.G7. Found: when A4-pregnen-9~,11~-oxido-21-ol-3,20-dione 21- C, 60.01; H, 6.73; S, 6.35). Additional data are acetate' (11) was treated with anhydrous hydrogen required to permit a satisfactory structural assignfluoride in alcohol-free chloroform a t 0"for 4.5 hours. ment for 1V.j I possessed 10.7 f 2.3 times the activity of corti(5) Although the above data are not sufficient to establish t h e sone acetate in the rat liver glycogen assay3which is structure of IV, certain structural possibilities can be ruled out on the in keeping with the relationship between activity basis of the available evidence. Thus, the reactivity of the new hyand size of the halogen atom established for the droxyl group toward acylating agents appears to exclude the presence an Ilp-hydroxyl in a conventional ring C-saturated steroid. The other halogenated derivatives. Deacetylation of I of structure, which for steric reasons might be expected allylic A~"-llp-ol with sodium methylate afforded Sa-fluorohydro- to be susceptible to acylation (or any other allylic structure) is concortisone, m.p. 260-262' (dec.) ; [ ( Y ] ' ~ D +139" sidered unlikely, since (1) IV is resistant t o oxidation witn manganese (c, 0.55 in 95% alcohol); 239 mp ( E = 17,- dioxide (cf. F. Sondheimer, C. Amendolla and G. Rosenkranz. THIS JOURNAL, 76, 5930 (1953)). and (2) the changes in molecular rotation 600); ?'A:: 3.01 p (OH), 5.54 p (20-carbonyl), 6.07 attending propionylation (+ZOO) and mesylation ( + 4 9 O ) of IV are considerably smaller than those produced by acylation of allylic alco1.1, 6.20 p (A4-3-ketone); (Anal. Calcd. for Czlhols (d. W. Klyne, H e i v . Chinz. Acta, 35, 1224 (1952)). Rotational H2905F: C, 66.30; H, 7.68. Found: C, 66.49; data likewise serve to exclude the a pviori less likely AT-115-01 strucH, 5.22). Oxidation of I with chromic acid ture, inasmuch as the average contribution of the 7,8-double bond in yielded Sa-fluorocortisone acetate (111), m.p. 254- two AM-3-ketones is -304' (cf. R. Antonucci, S . Bernstein, R. Lenhard, K. J. Sax and 1. H. Williams, J . Org. Chem.. 17, 1369 (1952)) as 25.5'; [ f f I z 3 D +155' (c, 0.45 in CHC13); A$:x. compared t o a value of +516' for the difference between IV and hydro234 mp ( E = 17,000); AZig' 2.86 p (OH), 5.72 p, cortisone acetate. Among the more likely possibilities being con5.78 p , 5.83 p (11-ketone and acetylated side chain), sidered a t the moment are structures arising from I1 by a WagnerMeerwein type rearrangement involving CP. 6.05 (A4-3-ketone); (Anal. Calcd. for Cd&&6F: INSTITUTE C, 65.70; H, 6.95. Found: C, 65.62; H, 7.19), THESQUIBB MEDICAL RESEARCH JOSEF FRIED which on deacetylation furnished the parent al- FOR NEWBRUNSWICK, NEWJERSEY EMILY I?. SARO cohol, m.p. 261-262'; [ a I z 3+144' ~ (c, 0.41 in RECEIVED FEBRUARY 3, 1954 CHCI3); A$:;. 234 mp ( E = 16,000); ?'A;: 2.88 p (OH), 5.87 p (11- and 20-keto groups), 6.08 p (A4-3-ketone); (Anal. Calcd. for CZIHZ~OF,F:C, THE STIMULATION OF SERINE BIOSYNTHESIS IN BY TETRAHYDROFOLIC 66.65; H, 7.19. Found: C, 66.50; H, 6.98). PIGEON LIVER EXTRACTS ACID I11 had 9.0 2.7 times the activity of cortisone Sir: a~etate.~ Serine biosynthesis has been postulated to occur The reaction of I1 with hydrogen fluoride afforded in addition t o I an isomer of I1 (1070 yield) (IV), by a process resembling the Mannich reactionzs3: m.p. 259-262'; [a]z3~ +272O (c, 0.53 in 9570 the a-carbon of glycine, activated by Schiff base formation between glycine and pyridoxal phosalcohol) ; 239 mp ( E = 20,200) ; ?'A:: 2.93 p , phate, combines with a condensation product of 3.03 p (OH), 5.75 p, 5.82 p (acetylated side chain), formaldehyde and tetrahydrofolic acid (THFA), N56.12 p, 6.16 p (A4-3-ketone); (Anal. Calcd. for hydroxymethyltetrahydrofolicacid. Serine is proC23H3006: C, 68.63; H, 7.51. Found: C, 68.60; duced by hydrolytic cleavage of the resulting prodH, 7.40)) in which the epoxide group has been re- uct. arranged to form a double bond and a readily acylStudies have been reported indicating that able hydroxyl group. Thus, I V on titration with pyridoxal phosphate participates in serine bioperphthalic acid consumed one mole of peracid with ~ y n t h e s i s . ~In . ~ the present communication, exthe formation of an epoxide, m.p. 213-214' (dec.) ; perimental evidence supporting a cofactor role of [ a I z 3 D +237" (c, 0.59 in CHC13); ,x:A: 237 mp THFA in this reaction is described. ( E = 16,100); $':A: 2.92 p, 3.06 p (OH), 5.73 p , Pigeon liver extracts prepared as described by 5.80 p (acetylated side chain), 6.16 p (A4-3-ketone); Berg6interconvert glycine and serine (Anal. Calcd. for C23H3007: C? 66.01; H, 7.23. CHzOHCHNHzCOOH "Ci" CHzKHzCOOH Found: 66.03; H, 7.44), on treatment with propionic anhydride and pyridine a t room temperature Formate and formaldehyde are utilized for the afforded a propionate, m.p. 261-26404; [ a I z 3 D formation of the "C1" unit. These properties are +260° (c, 0.40 in CHC13), +243O ( 6 , 0.52 in 95% lost on treatment of the liver extracts with Dowex-1 alcohol); AzC,.,, 238 mp ( E = 18,300); Xz:c' 3.03 (chloride). The ability of inactivated6 pigeon liver extracts p (OH), 5.72 p, 5.80 p (propionyl and acetylated to interconvert serine and glycine has now been side chain), 6.07 p, 6.11 p (A4-3-ketone); (A~zal. (1) This investigation has been supported b y a grant-in-aid from Calcd. for Cz~H340,: C, 68.10; H, 7.41. Found: American Cancer Society upon recommendation of the Committee C, 67.93; H, 7.36) and with mesyl chloride in the on Growth of the National Research Council. pyridine a t 0" formed a mesylate, m.p. 151-152' (2) W. Sakami, American Chemical Society Symposium on 1Carbon Compounds, Chicago, September, 1953. (dec.); [ a l Z 3+260° ~ (c, 0.54 in CHC13), +238" In the Mannich reaction formaldehyde couples either ammonia, (c, 0.35 in 95% alcohol); 237 mp ( E = 17,800); or (3) a primary or second'ary amine with an atom possessing an active
*
Xzx,
+
(3) M. L. Pabst, R. Sheppard and M. H. Kuizenga, Endocrinology, 41, 55 (1947). We are indebted t o Drs. A. Borman and F. M. Singer for the liver glycogen assay data. A detailed account of these and other assay results will be published elsewhere. (4) A mixture of I V and its propionate melted a t 236-252°.
hydrogen. (4) S. Deodhar and W. Sakami, Fedevalion Proc., 12, 195 (1983). (5) P. Berg, J. Biol. Ckem., 205, 145 (1953). (6) Extracts treated with Dowex-1 (chloride) and dialyzed for 15-16 hr. against flowing 0.1 M K-phosphate buffer, p H 7.5.
COMMUNICATIONS TO THE EDITOR
Mar. 5, 1954
1457
fully restored by the single addition of THFA. D P N is also the cofactor of the reducing system This reaction has been studied by incubating ex- involved in the conversion of folic acid to THFA.' tracts with L-serine and l-C14-glycine under hydro(8) Studies of formate utilization for purine formation by G. R. gen for 1 hr. a t 34'. Serine was quantitatively iso- Greenberg also indicate the participation of DPN in folic acid reduclated from the deproteinated incubation mixture tion (private communication). OF BIOCHEMISTRY by chromatography on Dowex-50 columns and its DEPARTMENT ROYL. KISLIUK radioactivity determined, The @-carbonof the L- WESTERNRESERVEUNIVERSITY OF MEDICINE WARWICK SAKAMI serine provided the "Cl" unit for the conversion of SCHOOL CLEVELAND, OHIO l-C14-glycineto l-C14-serine. Treated6extracts did RECEIVED FEBRUARY 4, 1954 not introduce C14into serine but after the addition of THFA produced an incorporation of g1y~ine-C'~ NEW TYPE OF METALLO-ORGANIC COMPLEX into serine 40 x that of untreated extracts and A DERIVED FROM DICOBALT OCTACARBONYL AND equal to that of untreated extracts THFA. ACETYLENES The addition of ATP, DPN, pyridoxal phosphate Sir: and homocysteine did not further increase the A recent investigation' showed that dicobalt activity found in the serine. Folic acid and leuco- octacarbonyl (I),like iron enneacarbonyl, contains vorin were not active in substituting for THFA. two types of carbonyl groups, i.e., bridge and Addition of THFA alone to the inactivated6 terminal carbonyl groups pigeon liver extracts also restored their ability t o utilize formaldehyde for serine-&carbon formation. This property has been studied by incubation of the extracts with C14-formaldehyde and glycine for 1 hr. under hydrogen a t 34" and determination // \c/ of the radioactivity of the serine. Treated6 pigeon I/ liver extracts introduced very little formalde0 \O 0// hyde-CI4 into serine. The addition of THFA resulted in an incorporation of C14 into serine 36 X It has now been found that the two bridge carthat of untreated extracts and equivalent to that bonyls in I can be replaced by acetylene and subof untreated extracts THFA. stituted acetylenes, such as C&,C=CH, CsH6These results in which a rapid interconversion of CCGHS,CHa(CHz)4eCH, C H ~ C H Z ~ C C H ~ C H ~ , serine and glycine and utilization of formaldehyde HOCHzCzCH, HOCHZC=CCH20H, H O O C C s for serine-p-carbon formation has been stimulated CC6H6. The reaction proceeds smoothly and quanin inactivated6 pigeon liver extracts by the single titatively a t room temperature according to equaaddition of tetrahydrofolic acid, are consistent with tion (1). a cofactor role of this substance in serine biosyn- RCECR' C0p(C0)8 +RCzR'Cor(C0)e 2 c d (1) thesis. I1 The utilization of formate for serine synthesis The preparation of the diphenylacetylene comin inactivated6 pigeon liver extracts was restored by the addition of THFA4,ATP, DPN, glucose-6- plex is typical: a solution of 1.38 g. (4 millimoles) of phosphate, and M n + + but not of THFA alone. dicobalt octacarbonyl in 5 ml. of petroleum ether Formate-C14 incorporated into serine, under these (35-60') is placed in an erlenmeyer flask provided conditions, was thirteen times that of the un- with a mercury seal. A solution of 0.80 g. (4.5 treated extracts and three times that of untreated millimoles) of diphenylacetylene in 15 ml. of extracts stimulated by homocysteine.6 The radio- petroleum ether is added to the flask, the mercury activity of the serine was as high as that which was seal attached and the mixture allowed to stand obtained when THFX, ATP, DPN, glucose-6- two hours. Removal of the petroleum ether in a phosphate and M n + + were added t o untreated current of nitrogen a t room temperature yields pigeon liver extracts. Serine formation did not crude 11( R = R ' = CeH.5). I n the case of C6H6Cr occur in the absence of ATP or of THFA but was CC6H6, HOCHzCZCH, and HOCHzC=CCHzOH not completely abolished by the omission of DPN the reaction product (11) was purified by crysor Mn++. When glucose-6-phosphate alone was tallization, while the product from HC=CH was omitted, formate-C14 was rapidly incorporated into purified by distillation. I1 (R = R' = C6H6), deep-purple crystals resome substance (or substances) which was not serine, methionine, cystathionine, or purine, but sembling iodine, m.p. 109.5-1 10.0" from methanol; may have been citrovorum .factor.7 When folic acid sublimes a t 90" (1 mm.). Calcd. for C Z ~ H I O O ~ C O ~ : was substituted for THFA, the formate-C14 in- C, 51.75; H, 2.17; Co, 26.40; mol. wt., 464. corporated into serine by Dowex-1 treated-dialyzed Found: C, 51.61, H. 2.22; Co 25.6; mol. wt. extracts was one-fifth that obtained with THFA. (cryoscopic in cyclohexane), 463. The compound These results are interpreted as suggesting that, in is diamagnetic (A = -0.3 + 0.3.10+ c.g.s. units/g.) the presence of ATP, formate is incorporated into and has a dipole moment in benzene solution citrovorum factor which is subsequently reduced t o of 2.1 D. I1 (R = CHzOH, R ' ,= H), orange-red needles N6-hydroxymethyltetrahydrofolic acid by a DPN enzyme system. The ability of folic acid to sub- from petroleum ether (60-68"), m.p. 52.2-52.6'. stitute for THFA in this system indicates that Calcd. for C~H407Coz: C, 31.60; H, 1.18; Co, 34.49. Found: C, 31.65; H, 1.26; Co, 34.33. (7) This compound may also be identical with the intermediate of
+
+
+
formate utilization for inosinic acid formation that has been obtained by G. R. Greenberg, private communication.
+
(1) Unpublished work. (2) R. K. Sheline and K.S. Pitzer, THISJOURNAL, '71, 1107 (1950).
