March 5, 1959
COMMUNICATIONS TO THE EDITOR
biological oxidation of 6a,9a-difluorohydrocortiwith S. roseochromogenus gave the 16ahydroxy derivative IV (m.p. 247-251’ [a]Df58’ 234 m,u, log E 4.18. Found for (dioxane), C ~ ~ H B F ~C O , 61.61; ~: H, 6.92). I n preliminary assays, these values based on thymolytic and anti-inflammatory activity were foundI2 (hydrocortisone = 1, 9a-fluoro-16a-hydroxyprednisolone = 512): I = 5; I1 = 4; I11 = 20; IV = 15. I through IV exhibited marked excretion of sodium.12 (12) All assays were carried o u t in adrenalectomized rats, the salt assays without sodium chloride load and t h e anti-inflammatory assays by cotton pellet implant. For anti-inflammatory and thymolytic activities, essentially equal for this series of compounds, the compounds were administered orally. W e wish t o thank Dr. R. I. Dorfman, The Worcester Foundation for Experimental Biology, for t h e bioassays.
J. S. MILLS A. BOWERS C. CAWS CAMPILLO SYXTEX, s. A. CARLD JERASSI APDO. POSTAL 2679 H. J. RINGOLD MEXICO,D. F. RECEIVED DECEMBER 30, 1958
1265
The key intermediate used for the synthesis of Coenzyme A itself was IV, which was prepared in 98% yield by the reaction of adenosine-2’(3‘),5’diphosphate4 with morpholine and dicyclohexylcarbodiimide. The reaction of IV with DLpantetheine-4’ phosphate in anhydrous pyridine for 15 hr. a t room temperature gave V, which was purified by ion exchange chromatography and
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RESEARCH LABORATORIES
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HO 0 V, R = Pantetheine
characterized. More directly, the synthetic mixture was treated with 0.1N hydrochloric acid to Sir: open the cyclic phosphate ring, then with 2-merSince its discovery some twelve years ago,’ captoethanol and the products then chromatoCoenzyme A (I) has been the focus of intensive graphed on an ECTEOLA cellulose column. chemical and biochemical research and the sub- Coenzyme A and its 2‘-phosphate isomer (isostance is now known to occupy a central position as Coenzyme A) were eluted together in 50% yield. a mediator of biosynthetic reactions. We wish to The product was chromatographically and electrorecord the total chemical synthesis of this complex phoretically homogeneous and identical with natsubstance. ural sulfhydryl-Coenzyme A. It had a ratio of P:Adenosine:SH of 2.98:1.00:1.06. When assayed 0 0 OHCH3 by the phosphotransacetylase system,6 the sample ll I1 \ I HSC:-I,CH*NHCCH,CH,-HN- C- C-C-CH, ”2 had 33%‘06Coenzyme A activity. This result is in the expected range in view of the fact that DLpantetheine-4’ phosphate had been used and that h T N the cyclic phosphate ring opened to give roughly I 0 equal mixture of the two isomers. // Finally, the reaction of IV with ~-pantetheine-4‘ I; R = -P-OH \ phosphate’ followed by a work-up as above gave a 11: R = H OH product which on rechromatography on ECRO OH TEOLA cellulose largely resolved into two peaks. The basic approach used for the synthesis of the The first peak (iso-Coenzyme A) had 4%‘OBenzymic pyrophosphate bond, a major problem in the activity and gave mostly adenosine-2‘,5’-diphossynthesis of I, is that which was described recently phate after digestion with crude venom. The for the synthesis of other nucleotide coenzymes.2 second peak showed 86% Coenzyme A activity However, we now wish to report that nucleoside-5’ and gave after degradation with venom mainly phosphoromorpholidates (111) are superior to the adenosine-3’,5’ diphosphate. Acknowledgment.-This work has been supunsubstituted phosphoramidates because of their high solubility in organic solvents and greater ported by grants from the Life Insurance Medical Research Fund, New York, and the National reactivity. In initial experiments, the reaction of adenosine-5’ Research Council of Canada. phosphoromorpholidate (111, R = adenine) with BRITISHCOLUMBIA RESEARCH COUNCIL J. G. MOFFATT ~~-pantetheine-4’ phosphate3 gave “dephospho”- AT THEUNIVERSITY OF BRITISHCOLUMBIA 8, B. C., CANADA H. G. KHORANA Coenzyme A (11) containing a racemic pantoyl VANCOUVER RECEIVED JANUARY 30, 1959 group in 63% yield. The product which was homogeneous by a number of criteria had the (4) Prepared in 60% yield by an improvement of the method of F. Cramer, G. W. Kenner, N. A. Hughes and A. R. Todd, ibid., 3297 correct phosphorus :adenosine :sulfhydryl ratios. THE TOTAL SYNTHESIS OF COENZYME A
(1) F. Lipmann, “Les Prix A’obel.” Stockholm, 1954, p 151. (2) I. G. Moffdtt a n d I1 G. Khorana, THISJ O U R N A L , 80, 3756 (1OS8). (3) Prepared by modification of procedure of J . Baddiley and E. M.Thain, J . Chcm. Soc., 1610 (1953).
(1957). (5) E. R. Stadtman in “Methods in Enzymology,” Voi. I, Academic Press Inc., New York, N. Y.,1955. p. 596. (6) As against a Pabst preparation which is regarded as 75% active. (7) Prepared in 44% yield by improvement of the procedure of Baddiley a n d Thain (ref. 3).
