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
