May 20, 1954
COMMUNICATIONS TO
THE
2845
EDITOR
TABLE I
TABLE I1
STOICHIOMETRY OF THE CONDENSATION OF OROTIC ACID AND PRPP The reaction mixture (24 ml.) contained 0.48 ml. of tris(hydroxymethy1)-aminomethane (TRIS)buffer (1 M , PH 8.0), 0.48 ml. of MgCla (0.1 M), 1.20 ml. of PP (0.01 M, pH 8.5), 1.44 ml. of NaF (0.5 M ) 0.48 ml. of 4,7-C%odium orotate (0.01 M , 186,000 c.p.m./pmole), 0.40 ml. of P R P P (2.85 X 10-8 M , 112,000 c.p.m./pmole) and 4.8 ml. of enzyme fraction I (containing 20.6 mg. of protein). One-half of the reaction mixture was immediately placed in a boiling water-bath for 0.5 min.; the remainder was incubated a t 30" for 40 minutes and then heated in n boiling water-bath for 0.5 min.
STOICHIOMETRY OF THE PYROPHOSPHORLYSIS O F 0 5 P
7
0 min.
pmoles 40 min.
7
A
Oroticacid" 2 . 4 0 ( 2 . 3 8 ) ' 1 . 8 4 (1.87) - 0 . 5 6 ( - 0 . 5 1 ) PRPP* 0 . 5 7 ( 0 . 5 2 ) 0 . 0 3 ( 0 . 0 0 ) -0.54 ( - 0 . 5 2 ) U5P .oo (0.00) .55 (0.55) .55 (+0.55) .60 .60 COzd PP (0.09) (0.48) ( + O . 39)' DeDetermined spectrophotometrically a t 295 mp. termined spectrophotometrically by the removal of orotic Determined spectrophotometrically acid (see equation 1 ). Estimated by trapping in KaOH the C"O2 rea t 260 mp. leased enzymatically from orotic acid and measuring the radioactivity. e Values in parentheses were determined after chromatography on Dowex 1 anion-exchange resin; orotic acid and U5P were estimated by optical density measurements at 280 and 262 mp, respectively; P R P P and PP were estimated by radioactivity measurements. f This value is low because of inorganic PPase activity; 0.22 pmole was recovered as 0.44 pmole of inorganic orthophosphate.
+
+
phate by 5'-n~cleotidase.~The specific activity of the U5P (24,700 c.p.m./pmole) was one-half that of the orotic acid. The accumulation of 0 5 P was demonstrated with a more purified enzyme preparation (11) which was free of decarboxylase and relatively poor in PPase. I n an experiment in which 0.27 pmole of 4,7-C14-orotic acid was consumed, 0.26 pmole of 0 5 P was isolated by ion-exchange chromatography. The 0 5 P was identified by its absorption spectrum (identical with that of orotidine7: peak a t 266 mp, X Z ~ O / X ~ ~= O 0.66 at pH 7), and its enzymatic decarboxylation (0.26 pmole C1402 liberated; equation 2 ) . The synthesis of larger amounts of 0 5 P is made difficult by the rapid decline in reaction rate due to the accumulation of 0 5 P and PP. The stoichiometry of the 0 5 P pyrophosphorylase reaction (equation 1) with 0 5 P (prepared by phosphatase transfers) and PP as substrates was observed with the results shown in Table 11. In the presence of larger amounts of PP the reaction proceeds rapidly to completion. Thus, in a I-ml. incubation mixture containing partially purified 0 5 P pyrophosphorylase (0.02 mg. of protein), 0.036 pmole of 0 5 P and 0.30 pmole of PP, 0.037 pmole of orotic acid was formed in 17 minutes. No reaction occurred in the absence of added PP. (6) L. A. Heppel and R. J. Hilmoe, J . Bid. Chem., 188, 665 (1951). (7) A. M . Michelson, W. Drell and H. K . Mitchell, Proc. Nuil. Acud. Sci., 97, 396 (1951). (8) A compound having an absorption spect-um identical to 0 5 P synthesized by the enzymatic condensation of orotic acid and P R P P has been prepared from orotidine b y phosphate transfer from phenylphosphate with a malt phosphatase (G. Brawerman and E. Chargaff, THISJOURNAL, 7 6 , 2020 (1953)). It was further identified as 0 5 P by its properties on ion-exchange chromatography, b y its enzymatic decarboxylation to U 5 P (0.061 rmole yielded 0.061 rrmole of U 5 P estimated spectrophotometrically), and by the molar ratios of orotic nrd:pentoqe:phosphate of 1.08: 1.04:1.06.
The reaction mixture (21.6 ml.) contained 0.36 ml. of TRIS buffer (1 M , pH 8.0), 0.36 ml. of MgC12 (0.1 M), 1.81~11.of NaF (0.05 M ) , 1.8 ml. of P P (1.2 X l O - 3 M . 750,000 c.p.m./prnole), 13.2 ml. of 05P (9.8 X X ) and 3.6 rnl. of the enzyme fraction I1 (0.42 mg. of protein). One half of the reaction mixture was immediately placed in a boiling water-bath for 0.5 min.; the other half was incubated a t 30" for22min. andthenplacedinaboiling watei-bath for0.5min. 0 min.
pmoles 22 min.
7
A
05P" 0.65 ( 0 . 5 8 ) d 0 . 3 9 ( 0 . 2 9 ) - 0 . 2 6 ( - 0 . 2 9 ) PP (1.04) (0.84) (-0.20) Orotic acid* .OO (0.00) .32 (0.30) .32(+0.30) PRPP' . O O (0.00) .22 (0.16) .22(+0.16) a Estimated spectrophotometrically a t 266 mp. * Determined by the decrease in optical density a t 280 mp in the presence of dihydroorotic dehydrogenase and reduced diphosphopyridine nucleotide (I. Lieberman and A. Kornberg, Biochim. Biophys. Acta, 12, 223 (1953)). See footnote b, Table I. See footnote e, Table I. The relatively low value a t 22 min. for P R P P and the high value for P P at 22 min. suggest the destruction of P R P P to yield PP.s
+ +
0 5 P pyrophosphorylase did not convert adenine or uracil to their respective nucleotides in detectable amounts (8-C14-adenine and 2-C14-uracil of high specific activities were used in these experiments). It is noteworthy that another yeast enzyme has been purified which catalyzes the condensation of adenine with P R P P to form A5P9 and which is inactive in forming 0 5 P from orotic acid. 0 5 P decarboxylase also is specific for 05P, attacking neither orotidine nor orotic acid. (9) A. Kornberg, I. Lieberman and E. S. Simms, unpublished results.
DEPARTMENT OF MICROBIOLOGY IRVING LIEBERMAN WASHINGTON UNIVERSITY SCHOOL OF MEDICINE ARTHURKORNBERG ST. LOUIS10 MISSOURI ERNESTS. SIMMS RECEIVED APRIL 12, 1954
STRUCTURE OF A NEW AMINO ACID OBTAINED FROM ROSEOTHRICIN'
Sir: Roseothricin (H-277) is a streptothricin-like antibiotic obtained from Streptomyces roseochromogenus.2 Though fairly toxic, it possesses high antibacterial activity. The hydrolysis of roseothricin hydrochloride with 20% hydrochloric acid in a sealed tube for 48 hours a t 100' gave two ninhydrin-positive products, which were separated by solubility differences of their picrates. One was @,e-diaminohexanoicacid, an amino acid also present in streptothricin, streptolin,5 f 4 and viomycin,3.5 and characterized by American ~ o r k e r s . ~The ,~ other substance, for which the trivial name of "roseonine" is designated, has now been shown to be 2-arnino-4(or 5)- (1-carboxy-1-hydroxy-2-amino) (1) An imidazoline structure was postulated at the 6th Annual Meeting, Chemical Society of Japan, Kyoto, April, 1953. (2) S. Hosoya, el al., JaP. J . E z g t l . &fed., 20, 121 (1949): 20, 481, 683, 771 (1950). (3) H . E. Carter, et al., Abstracts of Papers 120th Meeting, American Chemical Society, New York, N. Y.,September, 1951, p. 3L. (4) E. E. Smissman, el ul., Abstracts of Papers 121st Meeting, American Chemical Society, Milwaukee, Wisconsin, April, 1952, p. 80. (5) T.H. Haskell, e1 al., THIS JOURNAL,74, 599 (1952). (6) H . E. Carter, e l a/., i b i d . , 74, 5704 (1962). (7) E. E. van Tamelen and E. E. Smissman, i b i d . , 74, 3714 (1959).
COMMUNICATIONS TO THE EDITOR
2846
Yol. 76
ethyl-2-imidazoline
(I). Roseonine, C&&r\T4, m.p. 190' dec. (Calcd. for C6H1104N3: C, 3S.00: has been characterized H , 5.S6; N, 22.21. Found: C, 37.77; H, 5.62; YOOH through the following N, 19.95; negative Iran Slyke nitrogen after seven I derivatives : dipicrate minutes, positive periodate test). Heating V I in a. (11), m.p. 237' dec. sealed tube with hydroiodic acid and phosphorus OFT (calcd. for C1~H1801~Nlo: gave a substance with one C-methyl group, positive S H A4 C. :{3.4.5: 13, '7.81: N. Pauly and bromine test" and negative N-methyl (or I1 I 22.09, iiiol. wt., G4G.40. ethyl) ; nionopicratc, ni.1). IS2O. (Calcd. for el1Found: C, 33.48; H , I11207N6: C, 3S.S3; €1, c'3.56; N, 24.70; mol. wt., :3.17; N, 22.09; mol. wt., G34*); dihydrochloride 340.23. Found: C, 39.03; €1, 3.72; N, 23.91; (III), m.p. 21j0, [ a j i 3+31.0' ~ (H20) (Calcd. for mol. wt., :15G8). Hence the structure of this coniCsH1403N4Cl?: C, 27.65; H , 5.41; N, 21.46; C1, pound is apparently that represented by VII. The 27.16. Found: C, 27.31; H, 5.40; K,19.SS; C1, conversion of VI to \.I1 could be explained by cle27.97) ; methyl ester dipicrate (IV), 1n.p. 202" hydration of the tertiary hydroxyl group, rearrange(Calcd. for C19H~oOl,Nlo:C, 34.56; H , 3.05; N, ment to the stable imidazole ring, and decarboxyla21.21. Found: C, 34.G6; H , 3.40; N, 21.39); tion a t some stage. PK;, 2.4, 9.3 and 11.9. The S a k a g ~ c h ib, ~i a ~ e t y l , ~ No other structure except that represented by I Pauly and a-amino acid reactions as well as the can account for the described facts satisfactorily. Kuhn-Roth and X-methyl determinations on I Details of the present structural studies will be rewere negative. ported in a Japanese journal shortly. Permanganate oxidation of 111 a t room temperaThe authors are greatly iiitlebted to their collahture gave guanidine and a small amount of glycine. orator, Prof. S. Hosoya, Tokyo University. \Then treated with periodate, it consumed one mole (11) Imidazoles *ith a tree nu;le.ir m e t h i n e "up d e c o l i m z e l > t < > of oxygen in 15 minutes with the formation of one mine, wnereas imidazolines do not. mole each of ammonia and formaldehyde, and CHEMICAL INSTITUTE KOJI S A K A N I S l i I coupling this with the formation of glycine, it was NAGOYA Tosmro 11.0 USIVERSITY apparent that the grouping >C(OH).CH&Ht was CHIKUSA,SAGOTA, JAPA.S Y O S H I M A S A I IIKATA present. Another mole of periodate was consumed RECEIVED APRIL 17. 1954 after 20 hours, a behavior identical to that of serine. __ These results, together with the pk': COO€I values1osuggested the existence of A NEW SYNTHETIC METHOD OF PROTEIN ANAI LOGS HAVING PERIODIC ARRANGEMENT OF AMINO --C-CH?SII? the grouping V. ACIDS I Thc Van Slyhe aiiiiiio nitrogen OII deteriiiinatioii gave one inole after Sir: lseven minutes. -4 second mole was The only method for preparing the polypeptides detected after 30 minutes, the behavior of this having several ten thousandths molecular weight of second amino group being exactly similar to that of natural protein orders is the method of a-amino-Nu'2-amino-2-imidazoline. I11 was converted into 2- carboxylic acid anhydride found by H. Leuchs' amino-4(or 5)-etliylirnitlazole (VI) through the and established by II'oodward and Schrainm.2 IVe sequences recently found a new synthetic method of protein analogs having molecular weight of protein order, COOH by polynierizing w-amino acids, such as polypeptide I AgSOr NF-C-CH,OH HI, P consisting of some kinds of amino acids, p-alanine I11 '! I and e-aminocapronic acid, as well as a-amino acids.3 150°, 8 hr. OK S-Carbothiophenyl-a-amino acids4 expel1 thiophesH/c\ Y/ no1 and carbon dioxide to be polymerized in polyH VI peptides having high niolecular weight by melting or by heating in the proper solvents such as dioxane only or benzene, having a small quantity of pyridine. The free amino group -NH2 is the initiator of the polymerization in the a-amino acid-N-carboxylic acid anhydride method, and, on the other The action of one mole of silver nitrite gave VI, hand, the free carboxylic acid group -COOH is in this latter reaction. ( 8 ) Obtained from t h e extinction coeficient by the method of K. G. Cunningham, ef a l . , J . Chcm. S o r . , 2305 (1951). Leuch's method : (9) T h e negativity of these color reactions for structure I is in ac-
S-p I ,
,
I
~
-
-
cord with theobservations of J. D.Llold, er al., THISJ O U R N A L , 76, 6321 (1953). (10) Since t h e value 9.3 (NII! +) is considerably lower t h a n the value 10.28 assigned t o t h e e-amino in hydroxylysine (D. V a n Slyke, cf ai., J. Bioi. Chem., 133, 287 (1940)), t h e neighboring effect of a n additional substituent was anticipated. Placing a carboxyl as shown in V t o make a n isoserine residue resulted in favorable agreement with existing d a t a : i . c . , isoserine (2.78. 9.27), 0. H. Emerson, et al., J. Biol. Chem., 92,449 (1931), and D,L-a-methylisoserine (2.7, 9,151, E. H. Flynn, cf al., TRISJOURNAL, 76, 5867 (1953). However, t h e first constant of roseonine (2.4) is slightly lower, a n d this might be caused by an additional neighboring positive group, which in this case proved t o be t h e 8guanido group.
"-CO
!
c1-1I
C
2+
R
R (1) H. Leuchs, Bcr., 39, 857 (1906). (2) R. B. Woodward a n d C. H. Schramm, THIS JOURNAL, 69, I551 (1947). (3) J. Noguchi, J . Chcm. Soc. of J a p a n , 74, 961 (1953). (4) G. C. H. Ehrensvard, Nafzrre, 159, 500 (1947); A. Lindemann, N. H. Khan and K. Hofmann, THISJOURNAL, 74, 476 (1952): J. Pioguchi. J Chem. Soc. of J a p a n , 74, 963 (1953)
