Sept. 20, 1952
XOTES
4725
action of pure aminopterin was decreased by hydrog e n a t i ~ n . ~These facts indicate that the state of oxidation of these compounds has important influence upon the microbiological activity. Since hydrogenation at elevated temperature under pressure could conceivably cause degradafB tion of the aminopterin molecule, it seemed worthCHZ-) contains terms expressing the ‘(resonance” while to carry out the hydrogenation under milder integral 3!, and the Coulombic integral q of the conditions and to extend the study to include NIOc€ acid and A-methopterin CHz-group. For a graded series of compounds methylpteroylglutamic (N~0-methyl-4-aminopteroylglutamic acid). Acranging between 11 and benzene with various decordingly, the compound (100 mg.) to be hydrogengrees of electronically deficient groups in place of ated was suspended, without further purification, @ CHZ, the q and 0 terms diminish to zero as one ap- in water (25 ml.), and the p H was adjusted to 7.0proaches benzene, but nonetheless, a t all times, 8.0 by adding a solution of sodium hydroxide, meta-substitution is predicted to be preferred. As whereupon a homogeneous solution was obtained. a result, I1 is a good qualitative model for nitro- Platinum oxide (50 mg.) was added, and hydrogenbenzene or any similar substance where directing ation was carried out a t room temperature and atsubstituent exerts its influence by virtue of an elec- mospheric pressure. After approximately two tronically-deficient atom attached directly to the moles of hydrogen had been taken up and hydrogenation appeared to be complete, the catalyst was benzene ring. On the other hand, in the transition between 1- removed by filtration through a bed of fullers earth and IV as 0 and q diminish, a changeover between on a sintered glass plate. The entire filtrate was prediction of meta- to ortho-para orientation must subjected to freeze-drying, and this yielded a lightoccur. Since the electronically-deficient atoms colored, fluffy, solid product which was stored under of the usual ortho-para substituting derivatives of nitrogen. When compared with the spectra for VI (X = -NO*, -CO&zH5, etc.) would be expected pteroylglutamic acid and tetrahydropteroylgluc€ tamic acid,6 spectra for the initial materials and the to have smaller q and /3 values than CHZ-, i t is pos- products of hydrogenation indicated that hydrosible that such groups on detailed analysis would be genation had been accomplished. calculated to give different orientations from V. To determine the inhibitory activities of the iniIt will be interesting t o determine whether electro- tial materials and of the products of hydrogenation philic substitution of V or a suitable analog would for Streptococcus jaecalis R. the method of Mitchell actually occur in the meta-position as predicted. and Snell’ was used with slight modification, and Acknowledgment.-We are deeply indebted to folic acid was used as the growth factor. Constant Dr. W. G. Mclllillan, Jr., for advice on methods of levels of folic acid were used, and the quantity of calculation. test compound was varied. The final volume of medium per tube was 10 ml. Seventeen hours folDEPARTMENT OF CHEMISTRY AND LABORATORY FOR NUCLEAR SCIENCE AXD ENGINEERING lowing inoculation the turbidity was determined by MASSACHUSETTS INSTITUTE OF TECHXOLOGV means of a Lumitron colorimeter with a 660 mp filCAMRRIDGE 39, MASSACHUSETTS ter. The resulting data are given in Table I. I n all instances hydrogenation caused a considerable Effects of Hydrogenation upon the Microbiological decrease in inhibitory activity. Activities of NIO-MethylpteroylglutamicAcid, TABLE I Aminopterin and A-Methopterin
and para-positions and i t appears that V is not a good model for such compounds despite the fact that I1 seems t o be a reasonable analog of nitrobenzene. The disparity can be resolved in the following way. The secular equation for I1 (and the corresponding activated complex I with X =
BY GLYNNP. WHEELER,MARGARET A. XEWTON, JEANENNE S. MORROW ASD JOYCE E . HILL RECEIVED MARCH 3, 1952
It has been found that hydrogenated folic acid has a growth-promoting activity for Leuconostoc citrovorum 8081 which is intermediate between that - ~ also inof folic acid and that of l e u c ~ v o r i n ~and termediate between that of Nlo-formylfolicacid and that of l e u c ~ v o r i n . ~Also, a commercial sample of aminopterin (4-aminoptaroylglutamic acid), which inhibits the growth of Leuconostoc citrovorum 8081, was converted to a growth factor for this organism by hydrogenation under p r e ~ s u r e . ~It was later found that this growth-promoting activity was due to an impurity, but nevertheless the inhibitory (1)
W. Shive, T. J. Bardos, T. J. Bond and L. L. Rogers, THIS
JOURNAL, 71, 2817 (1950).
(2) H . P. Broquist, M. J. Fahrenbach, J. A. Brockman, Jr., E. L. R . Stokstnd and T.H. Jnkes, ibdd., 78, 3535 (1951). (3) G. P. Wheeler, unpublished data. (4) F . Weygand, A. Wacker, H.-J. Manu, E. Rowold end H. I+ettr6, 2 Nalrrforsclrun~, ab, 413 (1960).
EFFECT OF HYDROGENATIOS UPON THE INHIBITORY PROPERCERTAINFOLICACIDANTAGONISTS WITH Strcptococcus faecalis R.
TIES OF
Compounda
Inhibition inderb Folic acid (pg./lO ml.) 0.002 0.02 0.2 2.0
Nl0-Methyl PGA Hydrogenated N’O-methyl PGA Aminopterin Hydrogenated aminopterin
25 2 . 5 0.44 0.28 Less than 50% inhibition at all levels 26 2.8 1.5 0.34 Less than 50% inhibition at ’ all levels A-methopterin 25.5 2 . 5 0.25 0.04 Hydrogenated A-methopterin 45 70 26 11 a All compounds were used as the sodium salts. Inhibition index = weight of agent required for half maximal inhibition/weight of folic acid. (5) F. Weygand, A. Wacker, H.-J. Manu and E. Rowold. i b i d . , bb, 174 (1951). (6) A. Pohland, E. H . Flynn, R. G. Jones and W. SMve. THIS JOURNAL,78,3247 (1951). (7) H. K. Mitchell and B. E. Snell, Univ, T e r a s Pub]. 4137, 36 (18411,
TABLE11 ~ J R O \ \ ' I H - ~ ' R O M L ) T I S G =\CTIYII IES OF I ~ Y n R O G E Y A T F n
cc , \ I
POI-SDS
PE
For .C. f n ~ ~ n l R rr
rGA
twin
twin
0 1
100
3
loo
I O
100 68
36 2.; 2t;
IOfl 100 100
01
%I
01 YP'1
77 ,'33
94 90
'10 ml
1011 1(JOO
For .f czlroiv~riini 5051
Transmission, % HydroHydrogenated Hydro- genated "0genated Amethyl nminop- methop-
1
titi
1 0 1)
!IO 92 90
100 (1
78
I/
1 IJ
The hydrogenated materials were also tested as growth factors for Streptococcus faecalzs R. and Leuconostoc citrovorum 8081. For the former organism,
A NEW METHOD FOR THE PREPARATION OF THIO ACIDS AND APPLICATION TO PEPTIDE CHEMISTRY
Sir:
the same basal medium was used as was used for the inhibition studies but no folic acid was added. For the latter organism, the basal medium and technique of Sauberlichs were used with the exceptions that no supplementary glycine and alanine were used and a Lumitron colorimeter with a 660 r n M filter was employed. Turbidity was determined after 17 hours. Data are given in Table 11. I t is quite likely that the growth-promoting activity is due to an impurity in the original compound as suggested by Weygand. Acknowledgment.-The authors wish to thank Lederle Laboratories for supplying the folic acid, ,"r'~o-methylpteroylglutamicacid, aminopterin and A-methopterin used in these experiments. ( 8 ) I{ E. Sauherlich, J B i d . Chem.. 181, 467 (1949).
RESEARCH INSTITUTE
SOUTHERN RIRMISGHAY,
ALABAMA
C , 54.30; H, 3.19; S, 14.47. Found: C, 54.52: 13, 3.82; s, 14.211. IT'hen thiohippuric acid was wanned to 90-1 10" in diniethylformamide with d,Z-alanine in a nitrogen atmosphere, hydrogen sulfide was rapidly evolved and there was obtained a 70% yield of hippuryl-alanine, m.p. 200-201 .:io6 and giving the correct elemental analysis. Upon treatment of thiohippuric acid with Raney nickel which had been deactivated over acetone7 there was obtained in one experiment, a 30% yield oi hippuraldehyde, isolated as the 2,4-dinitrophenylhydrazone, m.p. 200-202° (Anal. Calcd. for C15HI3N606: C, 52.48; H, 3.82; X, 20.40. Found: C, 52.63; H, 3.78; N, 20.18).
Although Pawlewski' deinonstrated that thio acids were very active acylating agents, the mcthods of preparation which have been available heretofore2 have not been suitable for making the acylaminothio acids which could be useful in peptide synthesis. By passing hydrogen sulfide into a solution of the mixed anhydride^,^*^,^ RCOOCOOCtHe, in methylene chloride with an equivalent of triethylamine at -20' and warming to room temperature, we have obtained the thio acids, RCOSH. In this manner we have prepared, in addition t o thioacetic and thiobenzoic acids, p-phenylthiocli) T. Curtius a n d E. Lamhotte. . I . p v n k l . Chcm., [ 2 ] 7 0 , 114 (1904). benzoic acid, 88% yield (from the carboxylic acid), (7) G. B. Spero, A. V. McIntosh and R . H. Levin, THISJOURXAI., m.p. 90-92' (Anal. Calcd. for CI3Hl~OS:C, 72.80: 70, 1907 (19481. .(8) J. Bougault. E. Cattelain and P . Chabrier, Bull. soc. thim., [SI 5 , H, 4.71; S, 14.94. Found: C, 72.M; H, 4.83; S, 1 B99 (1938). have reported the conversion of thioacetic acid to acetalde15.09); thiohippuric acid, 70% yield, m.p hyde. (Anal. Calcd. for C Q H ~ X O ~ C, S : 55.39; H, 4.65; OF C H E M I S T R V A N D C H E M I C A L ENGINEERING N, 7.18; S, 16.40. Found: C, 55.30; H, 4.fiO: DEPARTMENT UKIVERSITY OF CALIFORNIA MARSHALL W. CRONYN N, 6.79; S, 15.99); phthaloylthioglycine, 4591, BERKELEY4, CALIFORNIA JAMES Jxr: yield, m.p. 114-116' (Anal. Calcd. for C10H7N03S: RECEIVED JUSE 23, 1952 (1) Br. Pawlewski, Ber., 31, B A l ( 1 8 % ; 34, 657 (1901); 36, 110 (1902). (2) R. Connor, "Organic Sulfur Compounds." p. 835 in Gilman'J "Organic Chemistry," Vol. I, Second Edition, John Witey and Sons, Inc., New York, N . Y., 1943; S. Sunner and T . Nilson. Pdcnrk. K c m . Tid., 64, 163 (1942) [C. A . , 38, 3249 (1944)]; B. Tchoubar a n d Letellier-Dupre, Bull. soc. chim. France, 792 (1947). (3) R. A. Boissonnas, Helv. Chim. Acfa, 84, 874 (1961); T.Wieland and H. Bernhard, Ann., 6'73, 190 (1951); J. R. Vaughan and R . L. Oaato, THISJOURNAL, 14, 678 (1952). (4) T. Wieland, W. Schafer and E. Bokelmann, A n n . , 6T8, 99 (1961), prepared R C O S C ~ Iby addition of CsHrSH to the d x e d anhydride. (6) € Adldns I. and Q. E.Thompson, THIS JOURNAL. 11,2242 (1949), prepared thiobenzoic ncid by passing HtS into dibenzoyl sulfide in pyridine.
THE SYNTHESIS AND REACTIONS OF N-ACYL THIOL AMINO ACIDS
Sir: Recent evidence that enzymatic acylations involve thiolacid derivatives as activated intermediates' has stimulated interest in similar thiol analogs of amino acids as possible participants in the physiological synthesis of peptides. By two (1) For example, acetyl coenzyme A is considered to be a key intermediate in biological acetylations; F.Lynm, E. Rdchert and L. Rueff, A n n . , 174, 1 (1951); T.C. Cbou and P.Lipmnnn. J . B i d . Chrm.. 196. 89 (1952).
