Sept. 5, 1953
COMMUNICATIONS TO THE EDITOR
4749
and acetic anhydride. The substance gave an acetonide (11) (m. p. 258-259', found : C , 70.40; H, 7.43) by reaction with acetone-perchloric acid, (8) Visiting Scientist at the National Institutes of Health on leave indicating that the new hydroxyl group was situated of absence from the Weizmann Institute, Rehovoth, Israel. a t C-18 and cis to the C-17 hydroxyl function. NATIONAL INSTITUTE OF ARTHRITIS The presence of a 1,2-glycol system was evidenced AND METABOLIC DISEASES ABRAHAM PATCHORNIK~ NATIONAL INSTITUTES OF HEALTH WILLIAM B. LAWSON by the formation of a bis-2,4-dinitrophenylhyBETHESDA 14, Md. BERNHARD WITKOP drazone derivative after treatment of I b with perRECEIVED JUNE23, 1958 iodic acid.
hope of obtaining chemical "peptidases" selective than enzymes.
more
MICROBIOLOGICAL TRANSFORMATION OF RAUWOLFIA ALKALOIDS
Sir: I n recent years the study of the actions of microorganisms on steroids has led to the discovery of many new transformations difficult to carry out by chemical means, and to production of new important pharmacologically active compounds. A study of the action of microorganisms on alkaloids particularly of the indole type, appeared especially attractive since the susceptibility of the indole moiety to attack by many chemical oxidative processes limits changes that may be made in other parts of the molecule.1 We now wish to report the 18a-hydroxylation of yohimbine and a-yohimbine by Streptomyces aureofaciens (ATCC 11834) and Streptomyces rimosus (NRRL 2234). The Streptomyces cultures were grown in a medium containing 1.5% soybean meal, 2.5% glucose, 0.25% calcium carbonate and 0.5 mg./ml. of yohimbine hydrochloride. After incubation a t 25' on a rotary shaker set a t 280 r.p.m. with a 2-inch stroke for 1-2 weeks, the conversion could be demonstrated by subjecting the fraction extractable with chloroform a t PH 9-10 to paper chromatographic analysis using the solvent system i-amyl alcohol-carbon tetrachloride-propionic acid (75 :60 :2) against water vapor equilibrated Whatman No. 1 paper.2 The product appeared as a spot detectable by fluorescence, ultraviolet absorption and ferric ferricyanide spray, with an Rfvalue of 0.15 (Rfof yohimbine, 0.50). A control fermentation with no added yohimbine and a n uninoculated medium containing yohimbine did not give rise to this product. By using the same extraction and chromatographic procedure, the chloroform extractable material from 4.5 1. of fermentation broth (45 flasks) was separated on twelve sheets of paper 10.5 inches wide. The appropriate band was eluted with methanol, and the eluate was crystallized from ethyl acetate-acetone to give 117 mg. of colorless cubes, m.p. 252-252.5', [ c y ] ~ + 3 7(meth~ anol. The new substance was assigned structure I b on the basis of the evidence: analysis gave the composition, CzlHz604Nz (found: C, 67.93; H, 7.07; CH30, 8.7; eq. wt. (perchloric acid), 367), corresponding to the addition of one oxygen atom t o yohimbine (Ia). T h a t the oxygen was present as a secondary hydroxyl group was shown by the formation of a diacetate (IC) (m. p. 307-307.5", [ a ] ~ - 3 9 O (chloroform); found: C, 66.44; H, 6.72; CHaCO, 19.7) on treatment with pyridine (1) Since completion of this work W. 0. Godtfresen, el al., Exprrir n f i a , 14, 88 (19581, have reported the microbiological hydroxylation of apoyohimbine, 3-epiapoyohtmbine and @-yohimbinemethyl ether. (2) W. T Sokolski, S. U1lm.m H. Vuiller and P. A. Tetrault, Antibiofics a n d Chemotherapy, 4, 1057 (1967).
, :0
CHSOOC'
I
0%
R Ia, R=OH, R l = H Ib, R = R i = O H IC,R= Ri= CHsCOO-
I:
'/H
H CH300Cv ' - R 1 R IIIa, R = O H , R 1 = H IIIb, R = OH, R1=OH IIIc, R = R i = CH3COO-
When the same fermentation procedure was applied using a-yohimbine3 (IIIa) as substrate, a new substance moving with an Rf of 0.17 (Rfof ayohimbine 0.45) was detected. The product was isolated from the fermentation in the manner described above for 18a-hydroxyyohimbine. The hydrochloride crystallized in colorless needles from methanol-hydrochloric acid, m. p. 288-290" (GIH2604N2.HC1.0.5H20, found: C, 60.39; H, 7.00). It was shown to be 13a-hydroxy-ayohimbine (IIIb) since i t formed a diacetate (IIIc), map. 278-279', ( [ a ] ~14.5' (chf.), found C, 65.86; H, 6.58; CH3C0, 19.47), an acetonide, m. p. 144-146', and reacted with periodic acid t o give a bis-2,4-dinitrophenyIhydrazone. (3) A. LeHir, M. M. Janot and R.Goutarel, Bull. sac. chim. Prance, 20, 1027 (1953).
THESQUIBB INSTITUTE FOR S. C. PAN MEDICAL RESEARCH FRANKId. WEISENBORN h'sw BRUNSWICK, NEWJERSEY RECEIVED AUGUST1, 1958 STRUCTURE OF A NEW ANTIBIOTIC, PYOLUTEORIN
sir: A new antibiotic, pyoluteorin, has been isolated from cultures of Pseudomonas aeruginosa, T 359 and IF0 3455,l and shown to have a powerful antibacterial activity in vitro.2 As was previously described, pyoluteorin (I) (C1~H7OaNCl2) has these physical properties : m.p. 174-175"(dec.); 255 mp (E 4,200), 310 (E 13,000); 122'3.02 p (OH,NH), 6.14(conj. C=O); found: C, 48.48; H, 2.86; N, 5.11; C1, 25.82; mol.wt. 268 (Rast method), no C-CHs, no N-CHs, (1) Inst. for Fermentation, Osaka, List of Cultures, 108 (1956) (2) a. Takedo, J . Ferm*nlaizon Technology, Osaka, in press.
optically inactive. Acetylation of I with acetic A;:?' 2.83 (OH), no coiij. C-=O band; not anaanhydride in benzene gave an O,O'-diacetate (11) lyzed due to its instability). LVhen V I 1 was oxi(ri1.p. 208'; AEjo' 3.09 p (NH), 5.68, 5.'iS(ester dized with chromium trioxide in acetic acid, tlic C=O), 6.10 (conj. C=O); found: C, 50.53; H, product was N-methyl-2,6-dimethoxybenzoylforni 3.07; N, 3.83; C1,20.00; COCH3.23.Sl). Methyl- amide (XI) (1ii.p. 143"; A?:' 3.07 p (NH), 5.77, ation of I either with diazomethane or with di- 5.93, 5.99(conj. C=O), G.X(NH); found: C, iiiethyl sulfate gave a trimethyl compound (111) 59.34; H, 5.79; S , ii.33), which was also obtained (n1.p. 135-136"; A?;:' 6.0!) I* (conj. C=O), no OH- by the similar treatment of 111. X I was hydrolyzed band, no NH-band; found: C, 53.38; II, 4.15; with aqueous potassium hydroxide to the cor9,1.63; C1, 22.51; OCH3, 10.39; S-CH3, 4.44). responding keto acid and the latter was further coiiO n catalytic reduction of I1 i n the presence of rerted with hydrogen peroxide to 2,G-diniethoxy~~alladirrn~-charcoal a dechloro compound (IV) benzoic acid (XII) (n1.p. 187") ; which was identical (in.p. 110'; A:?' 3.07 p ( N H ) , 5.70(ester C=O), with a synthetic sample. In view of the results 6.28(conj. C-0); found: C, 62.55; H, 4.63; obtained above, pyoluteorin must have the conN, 4.90) IYas obtained, which, on hydrolysis with stitution I. The positions of the two chlorine potassium hydrogen carbonate, gave dechloro- atoms on the pyrrole ring will be clarified later. pyoluteorin (V) (m.p. 142-143", Ai:?' 2.95, 3.18 p INSTITUTE FOR FERMENTATION ATTACHED TO THE TAKEDA (OH, NH), 6.2s (conj. C=O), Found: C, 64.75; PHARMACEUTICAL INDUSTRIES LTD. ROKURO TAKEUA H, 4.29; K, 6.67). The compound V was treated OSAKA,JAPAN RECEIVED JULY 14, 1958 with diazomethane t o yield an 0,O'-dimethyl derivative (VI) (m.p. 187-1S8"; Agd' 3.18 p (NH), 6.18 (corij. C-=O); found: C, 67.35; OF ZINC FROM CARBOXYPEPTIDASE PI, 5.45; N , 6.33; OCHS, 26.55). I11 on hydro- T H E RELEASEAND ITS REPLACEMENT genation over palladium-charcoal yielded trimethyl- Sir: dechloropyoluteorin (VII) (1n.p. 137'; A:,"?' Carboxypeptidase is a zinc metalloenzyme and 6.14 p (conj. C=O), no OH-band, no NH-band; found: C, 6S.GS; H, 6.10; N , 5.9s; OCH3, 25.01; the metal is functional in enzymatic 1,lO-Phenanthroline (OP) 3 inhibits enzymatic acK-CHa,3.S2). Fusion of VI1 with potassiurn hydroxide resulted tivity and removes zinc from the protein'; spectral in thc formation of 1.3-dimethoxybenzene (VIII) changes can be observed under such condition^.^ jb.p.23105"; found: C, 69.82; H, 7.07) and N- We have now observed that zinc can be progresmethylpyrrole-2-carboxylic acid (IX) (m.p. 135O ; sively removed by dialysis of carboxypeptidase a t pH values between 5.5 and 3.4. As pH is lowered, found: C, 57.32; H, 5.42; N, 11.23). The fact that alkali fusion furnished a product increasing amounts of zinc are removed a t an achaving a carboxyl group suggests that the car- celerating rate. Activity is abolished a t a similar bonyl group is linked directly to both the benzene rate (CGP3 as substrate). The correlation coarid the pyrrole rings. This was confirmed by re- efficient between metal content and activity is 0.90. ducing VI1 with lithium aluminum hydride, which Pertinent data are given in Table I. led to 2,6-dimethoxyphenyl-N-methyl-2'-pynylTABLE I methanol (X) (Xz",",f 241 nip ( E 6,600) for the pyr- THELOSS O F Z I N C AND P E P T I D A S E ACTIVITY FROM CARBOXYrole ring, 271 rnp ( e 1,800) for the benzene ring; PEPTIDASE IN ACIDSOLUTIOS Ii, = Rz = I
