November 1969
EXZYJlATIC DECARBOXYL.4TIOS' O F
1029
JIETHYL-0-TYROSINES
T.4BLE 1 available sites of the ring of o-tyrosine on the substrate ~ I ~ ~ M ~ C I' S. D \ I PHYSIC.\L ~ n.lT.4 O X T H E IS1.I~~RMI~~UI.\'I.I~:S *pecificity of these materials for DOPA decarboxylase. I N u TI{ E. 2 - I I r m i o s ~ i,;~riryr,~wcxyr, ~r \T,.\NINF:S I t wah found that, the 3-, -I-, and li-niethy\;l-i~-tyrnsinr~~ Yield, vould serve as substrates for this enzyme while 5Compound rr, N ] I , "(' Compositiona methyl-0-tyrosine was inert, serving as neither sub.4zlactonesb strate nor inhibitor. That the presence of a 5-CH3 3-Me 61 164-165 Ci~Hi:N03 could prevent binding t o the enzyme had been observed 4-1Ie 31 196-197 Ci~HijN03 on an earlier occasion by Lambooy6 in a study of struc5-11e 4 .i 180-181 Ci~HijN03 tural requirements of the methyl-2,4-dihydroxyphenyl6-1Ie 49 163- 168 Ci,Hi2JOa :ilanines as inhibitors for tyrosinase. The similarity Acrylic acidsc between 5-methyl-o-tyrosine and 5-methyl-2,4-dihy87 230-231 3-;lle ClsHnNO4 droxypheiiylalariiiie is obvious but since o-tyrosine is 4-Me 98 2 14-2 1 5 CisHnNOc riot an inhibitor of tyrosinase,6 the influence of the 89 2 13-2 16 ;-Me CIRHI~NO~ Propionic acidsd methyl groups in o-tyrosine on the inhibition of tyrosi3-Me 99 183 Ci8Hi9NO( nase could not be studied. 4-AIe 94 184-185 C&lg~04 Extensive studies have been made on the sub5-Me 9 3 184-1 85 ClsH19NO4 strate specificity of DOPA Sourkes, Amino acidsesh.' et aLli had shown that 2,4-dihydroxyphenylalanine was 3-Me (HCl) 87 188- 190 CioH14ClN03 R substrate for DOPA decarboxylase and a logical ex83 248-249 CioKi3KOa 4-Ne tension of our study was t o investigate the substrate %Me 79 248-249 C1oHi3N08 specificity of the three nuclear-substituted methyl-2,450+g 273-273 6-MeI CiaHi3NO3 dihydroxyphenylalanines for DOPA decarboxylase. It a All compounds were analyzed for C, H, N ; analytical results was found that the 3- and 6-methyl analogs served a i obtained for these elements or functions were within h 0 . 4 7 , of iubstrates but that the S-methyl-2,4-dihydroxyphenyl- the theoretical values. Recrystallized from PhH. c Recryst,allized from Et,OH. Recrystallized from 40% (v,'v) AcOH. :ilanine was inert, serving as neither substrate nor e See text for purification procedures. Prepared by direct coninhibitor for DOPA decarboxylase. version of the azlactone. 0 The yield was subjectively thought We are unable to offer a n explanatiori as to why the to be like the ot'hers but lack of data prevented giving a more exact value. Using the BuOH-H~O-A~OH syst,em, the Rf . X H 3 in these two qeries of compounds prevents bindvalues for the 3-, 4, 5-, and 6-Me isomers were 0.36, 0.57, 0.58, ing t o the decarboxylase other than that it is of sufficient and 0.54, respectively. Usiiig t,he 1120-saturated PhOH system, ,\ize and uniquely located to prevent an adequate apthe Ri values ill t'he same order were 0.81, 0.80, 0.80, and 0.78. proach t o the active bite of the enzyme. The ir peaks (cm-l) for the 3 - , 4-, 3-, and 6-hIe isomers were The appropriate methyl-substituted o-methoxybenz745, 773: 785, 750, 795, 820; 715, 735, 765, 815; and 740, 773, respectively: R. 31. Silverstein and G. C. Bassler, "Spectroaldehydes were converted to the azlactones by mean> metric Identification of Organic Compounds," John Wiley and of the Erlenmeyer reaction. The azlactones were con1966, p 56. Sons, Inr,, New York, X. I-., verted to the amino acids directly utilizing P-HI. The products of these reactions were relatively inas substrate were expressed as per cent of the activity soluble and a doubt existed a i to their identity. For of ~~-3,4-dihydroxyphenylalanine. this reason the 3-, 4-, and 5-methyl-substituted azlactones were converted to the beiizaniidocirinamic acids :md the latter w a i reduced to the benzoylamino acids Experimental SectionY b y meaiis of Ilaney S i catalyzed hydrogenation. The Benzaldehydes.-2-Hydroxy-3-methylbenzaldehyde was prelatter acids were converted to the amino acids by means pared by the formylation of o-cresol as described by Tieman and of concentrated " 2 1 in a sealed tube at 1313". The Schotten.lo The product (207,), bp 120-140" (15 mm), was found by gas chromatography to contain only 337, of the benzalamino acids prepared by the longer procedure were dehyde. The benzaldehyde was purified as the bisulfite addition identical with those prepared by the yhorter procedure. compoiuid to yield, on hydrolysis, mat'erial with bp 100" (23 The relative insolubility of the amino acid:, precluded mm). This prodilrt was methylated wit,h 1ZIe2SOd to form 2the usual techniques for purification. met,hoxy-3-methylbeiizaldehyde (94'5), bp 132" (28 mm) [lit,." 120" (6 mm)]. 2-hIet~hoxy-4-methylbenzoic acid was The 4-, 5-, and 6-methyl-o-tyro5inei were purified b y prepared by the methylation of 4-met,hylsalicylic acid followed the repeated formation of the soluble S a salts and by hydrolysis. The benxoic acid was converted to t.he chloride reprecipitation of the free amino acid by the addition and t)he latter was reduced by the Rosenmund reduction as of AcOH. This procedure resulted in destructive oxidadescribed for the preparat,iori of 3,3-dimethoxybenzaldehyde,12 tion of the 3-methyl-o-tyrosine. The hydrochloride of t,o yield 2-methoxy-4methylbenzaldehyde (49C&), bp 1.54' (23 mm) [lit,.13 263-264" (atni)]. This benzaldehyde was also obthii amino acid was purified b y recrystallization from tained by the methylatioit of 2-hydroxy-4-niethylbenzaldehyde, abiolute EtOH-abrolute EtzO, a procedure which could a by-prodiict obtained from the synthesis of 2-hydroxy-6not be used for the 4-, 5-, and 6-methyl-o-tyrosines. methylberiznldehyde described below. p-9Iethylanisole was The properties of the new compound5 are listed in formylated by the use of S-methyl-N-phenylformanilide and Table I. POC1, by general procedure a, described t o obt,ain 2-methoxy-.i-methylbenzaldehyde (30Yc), bp 148" (31 mm) The mammalian DOPA decarboxylase was obtained [lit.l3 139' (19 mm)]. m-Cresol was formylated by the Tieman from guinea pig kidney. The substrate activity was determined in a Warburg respirometer and all activities f
(ti) J. P . Lambooy. J . Am. Chem. Soc., 78, 771 (1956). ( i )T. Sourkes, P . Heneage, and T.Trano, Arch. Bzochem. B i o p h y s . , 40, 185 (1952). (8) R . Ferrini and A . Glasser, Biochem. PhRrmacoi., 13, 798 (1964).
(9) Melting points were determined on a Fisher-Johns melting point apparatus and are uncorrected. (10) F. Tieman and C. Schotten. Be?., 11, 767 (1878). (11) R . A . Rarnesand N. S . Gerber. J . O w . Chem.. 26, 4540 (1961). (12) J. P . Lambooy, J . Am. Chem. Soc., 76, 133 (1954). (13) "Dictionary of Organic Compounds," 4th ed, Oxford University Press, New Tork. K . Y., 1965, p 1814.
