COMMUNICATIONS TO THE EDITOR
May 5, 1956
2021
TABLE I reduction of proline to 6-aminovalerate by DTP,' COFACTOR REQUIREMENTS FOR THE REDUCTION OF GLYCINE- a reaction also catalyzed by this enzyme fraction, suggests that the phosphorylation associated with 2-ci4 TO ACETATE-2-C" The complete system contained tris-(hydroxymethy1)- glycine reduction is not solely the resuh of dithiol aminomethane (TRIS) buffer, (PH 8.7) 25 m o l e s ; MgSO4, oxidation. 3 pmoles; DPN, 0.2 pmole; pyridoxal phosphate, 0.006 TABLE I11 pmole; DTP, 20 pmoles; 0.2 pC 2-C14-glycine (ca. 30,000 GLYCINEREDUCTION BALANCE EXPERIMENTS cts./min.), 10 pmoles and 8 mg. protein. Reactants i t 0.5 ml. final volumes were incubated anaerobically a t 31 Reaction mixture components as in Table I1 with AMP, for two hours. 5 pmoles and K2HP04, 3-10 pmoles. The enzyme preparaAcetate-2-r-14 tion used to measure NH8 formation had been precipitated formed with satd. Na&04 and redissolved in buffer to lower its Omission cts./min'.a (NH4)tSO4 concentration. The amount of D T P oxidized ... 940 was measured in incubation mixtures reduced to one-half DPN 225 the usual volume; all components were added in proporMg++ 465 tionally smaller amounts except for enzyme and glycine. TABLE I1 THEEFFECTOF ORTHOPHOSPHATE (PO4), ARSENATE (As04) AND ADENYLATE NUCLEOTIDES ON THE CONVERSION OF GLYCINETO ACETATE In addition to the reactants each sample contained T R I S buffer (pH 8.77,20 pmoles; MgC12, 3 m o l e s ; DPN, 0.1 pmole; pyridoxal phosphate, 0.003 c o l e ; DTP, 9 m o l e s ; 0.2 pC g l y ~ i n e - 2 - C ~10 ~ , m o l e s and 3.6 mg. protein (30 to 35% satd. (NH4)2SO4 fraction), in a final volume of 0.5 ml. Incubations were carried out anaerobically a t 31' for 90 min. Experiment
Acetate-2-C14 formed, #moles8
Additions, #moles
None' 0.09 PO4 10, ADP 5, or AMP 5 or ATP 5 1.18 1.30 1 . 2 8 Po4 10, ADP 10 1.45 Po4 10, AMP 5, ATP 5 1.60 2 None" 0.25 Po4 10 0.56 ADP 1 1.01 Po4 10, ADP 1 1.87 3 Po4 5" 0.43 AS04 5 1.29 AS04 5, Po4 5 1.42 - 4 . 3 0 4 10 1.42 As04 10, AMP 5 1.40 a The enzyme preparation (not dialyzed) contained 0.88 m o l e of orthophosphate per 3.6 mg. protein employed.
1
(Table 111) shows that the reaction can be described by the equation CHtNH2COOH
+
+
+
Po4 ADP R(SH)2 CHaCOOH NHa ATP
+
+
--f
+ RSS
(1)
The most significant result of these experiments is that for each mole of glycine converted to acetic acid and ammonia there is a concomitant esterification of one mole of orthophosphate which is incorporated into ATP. Reduction of glycine in the presence of P32labelled orthophosphate and ADP (or AMP) results in a marked synthesis of P32-labelled ATP. No labelled ATP is detected chromatographically4 when glycine is omitted from the otherwise complete system or when amino acids such as citrulline, lysine or proline5 are substituted. The failure to observe any phosphorylation associated with the (3) Residual Cl4-glycine was removed by treatment with Dowex50-H+ resin at pH 1-2 and aliquots of the supernatant solutions assayed for C14 after neutralization. Identity of the radioactive product was established by steam distillations and Duclaux distillations. (4) L.V. Eggleston and R. Hems, Biochem. J . , 64, 156 (1952). (5) T.C. Stadtman, J . Bact., 67,314 (1954).
Glycines dec., #moles
p
(SH)'
PO18
oxid., equiv.
uptske, rmoles
0.71 1.07 b 2.52 1.25 b 2.30 0.98 Glycine, 5 moles, present. Not measured.
0.85 1.01
PIOmin. formed, #moles
0.89 0.98 0.89 0.98
Acetatea,g NHrlD formed, formed, rmoles rmoles
1.03" 1.01
0.77 1.23
1.21
b
1.0
b
The balance data presented, taken together with the fact that there is a compound formed that can be arsenolyzed, make it evident that the reductive deamination of glycine must involve the formation of a high energy phosphorylated intermediate. An unstable glycine derivative that is accumulated by another enzyme fraction derived from C. sticklundii may furnish a clue as to the nature of this intermediate. (6) E. C. Cocking and E. W. Yemm, Biochem. 1..68, xii (1954). (7) P. D.Boyer, THISJOURNAL, 76, 4331 (1954). (8) C. H.Fiske and Y.SubbaRow, J . Biol. Chem., 66,375 (1925). (9) Acetate was also estimated as acethydroxamate after incubation with the acetyl kinase system o f I. A. Rose, M . Grunberg-Manago, S. R . Korey and S. Ochoa, ibid.. 911, 737 (1954). (10) Ammonia was measured by direct nesslerization o f perchloric acid filtrates.,
LABORATORY OF CELLULAR PHYSIOLOGY A N D METABOLISM NATIONAL HEARTINSTITUTE THRESSA C. STADTMAN NATIONAL INSTITUTES OF HEALTH PATRICIA ELLIOTT DEPARTMENT OF HEALTH, EDUCATION A N D WELFARE BETHESDA 14, MD. RECEIVED MARCH 5, 1956
SYNTHESIS OF COMPOUNDS RELATED TO RESERPINE. CONVERSION OF AN INTERMEDIATE TO 16-METHYLYOHIMBANE
Sir: I n the course of investigating the relationship of structure to reserpine-like activity we have synthesized a number of derivatives related to reserpine having appropriate substituents a t carbon atoms 16 and 18 of the yohimbane skeleton. We now wish to report the total synthesis of 16-carbomethoxy-18-hydroxy-A16(20)-yohimbene which contains a double bond a t a position suitable for manipulating the stereochemistry of the important D/E ring junction. Treatment of methyl 2-carbomethoxy-3,4-dimethoxyphenylacetatel with chloromethyl ether and stannic chloride a t 0' gave methyl 2-carbomethoxy- 3,4-dimethoxy- 6 - chloromethylphenylacetate,m.p. 81-81.5" (found: C, 53.64; H, 5.41; C1, (1) C. SchOpf, U. J2ckh-Tettweiler, C. Mayer, H. Perrey-Fehrenhach and L. Winterbalder, A n n . , 664, 77 (1940).
TO THE EDITOR COMMUNICATIONS
2022
Vol. 78
11.12). Condensation of this chloromethyl deriva- saturated ketone. The double bond present in tive with tryptamine in tetrahydrofuran a t room this ketoacid, therefore, must have remained in temperature yielded N-(2-(3-indolyl)-ethyl)-3-0~0-the unconjugated position between C16 and GO. 5-carbomethoxy-6,7-dimethoxy-1,2,3,4-tetrahydroWhen IVa was reduced by the Wolff-Kishner isoquinoline, m. p. 168-169' (found: C, 67.71; H I method, a neutral lactam (VIa) (C~OHNONZ; 5.73; N, 6.84). Cyclization of this lactam with m. p. 277-279'; found: C, 78.22, H I 7.60; N, phosphorous oxychloride a t 100' furnished the un- 8.75; C-CH3, 3.51; A",!' 6.22 p ) was obtained. saturated base (I), isolated as the hydrochloride, The formation of VIa is compatible with the presm. p. 245-247' (found: C, 65.13; H I 5.73; C1,8.38). ence of a c 1 6 - c 2 0 double bond since, presumably, The salt (I) was subsequently reduced with platinum the lactam is formed by migration of a double bond in methanol to the saturated base (IIa), m.p. 205- to the C ~ I - N position ~ followed by the reactions 207' (found: C, 70.55; HI 6.13; N, 7.07; eq. wt. H H (perchloric acid), 398). Hydrolysis of I I a with I OH0 I aqueous ethanolic potassium hydroxide gave the -C=N@ < A -C-N< + corresponding acid (IIb) (hydrochloride, m.p. 255I OH 256'; found: C, 63.89; H I 5.58; N, 6.92; C1, H H 8.20). Reduction of the acid with sodium in liquid I HiNNHz I ammonia in the presence of isopropylalcohol resulted -C + H-N< -C=N-NH* + --CHI. II in loss of the C-17 methoxyl group and further re0 duction to the enol ether (111) (m. p. 230-232'; ' : : :A 5.85 p , 6.00 p ) Lactamization of the newly formed amine would found: C, 71.82; H, 6.35; then lead t o VIa. The structure of the latter compound was proved by reduction with lithium aluminum hydride in tetrahydrofuran to give a basic compound (VIb), m. p. 191-193' (CXIH~~N~; found: C, 81.75; HI 9.11; eq. wt. (perchloric acid), 292) which was shown to be dl-16-methylyohimbane by the identity of its infrared spectrum in -"I carbon disulfide with that of anthentic 16-methylyohimbane (m. p. 193-195') prepared by the CHtOOC method of Karrer and Saemann.2 OCH, &HI Reduction of IVa with sodium borohydride gave I Ira, R = CHI a hydroxy acid (Ivb), m. p. 255-256' (found: C, IIb, R = H 71.02; H, 6.54) which formed a y-lactone (V), m. p. 284-286' (found: C, 75.19; H I 6.37; A':", 5.65 p ) on treatment with pyridine and acetic anhydride. Since reduction of the aromatic carboxylic H acid (IIb) by sodium in liquid ammonia should lead to the equatorially oriented carboxyl group, the formation of a y-lactone indicates that the hydroxyl group a t CISin IVb is also equatorial as well as cis I I to the CIS carboxyl group. Treatment of the lacHOOCM O C H : ROOCMR~ tone (V) with sodium methoxide in methanol gave 111 IVa, R = H ; R ~=' o __ 18 - hydroxy - 16 - carbomethoxy - A16(B) - yohimbene (IVc), m. p. 214-216' (found: C, 71.57; H, 6.74). IVb, R = H;RI = C H The pharmacological properties of these comH pounds will be reported elsewhere. IVC,R a CHI; RI
-
/A
OqN, M
(2) P. Karru and R. Saemann, Halc. Chim. Acta, 81, 1832 (1852).
THESQUIBB INSTITUTE FOR MEDICALRESEARCH FRANKL. WEISENBORN NEWBRUNSWICK, N. J. HAROLD E. APPLEGATE RECEIVED APRIL2, 1956
M
H''u
THE REACTION OF RAUWOLFIA ALKALOIDS WITH MERCURIC ACETATE. CONVERSION OF 3-ISORESERPINE TO RESERPINE
Sir: I n conjunction with our work on the total synO=&--o VIa, R = 0 V VIb, R = Hn thesis of compounds related to reserpine, a method which on hydrolysis with dilute hydrochloric was needed for converting an a-oriented COacid produced the unsaturated ketoacid (IVa), hydrogen t o the generally less stable @-oriented f o r m 2 We now wish to report a method by which isolated as the hydrochloride, m. p. 251-254' ( C ~ O H B O ~ N ~ . H C ~ . ~found: H Z O ; C, 58.48; H I this transformation may be accomplished. (1) F. L. Weisenborn and H. E. Applegate, to be published. 6.44; N, 6.82; C1, 8.68). The infrared spectrum (2) (a) E. Wenkert and L:H. Llu. Expcrisslio, 11, 303 (1855); of IVa showed carbonyl absorption a t 5.84 1 but (b) C. F. Huebner, H. B. MacPhlllamy. E. Schlittler nnd A. F. St. no absorption at 6.0 p indicative of an a,@-un- AodrC, ibid., 11, 303 (1816). CHI"
