3980
LfUKKAY
GOODMAN AND
difference could be detected in the relative rates of decomposition of the enzymatic and synthetic nucleosides in 0.1 N potassium hydroxide as determined by the change in absorption a t 280 mp. 3. T h e various diastereoisomers, and the 5'-methylthioadenosine and L-homoserine arising from their decomposition products, had Rrs identical with those of authentic samples in the following solvent systems. A . Ethanol (300), water (loo), concd. hydrochloric acid (1). Rrs: S-adenosylniethionine, 0.12; 5'-methylthioadenosine, 0.62; t-honioserine, 0.80. B . Methyl Cellosolve (112), water (E?), acetic acid (1). Rfs: S-adenosylmethionine, 0.05; 5'-methylthioadenosine, 0.65; L-homoserine, 0.42. C . 1-Propanol (6), ammonium hydroxide (3, d . , 0.880), water (1). Rfs: 5'-methvlthioadenosine, 0.88; I,homoserine, 0.76. Configuration of S-Adenosylhomocysteie Isolated from Guanidinoacetate Methylpherase Reaction.-A reaction mixture containing potassium phosphate (30 pmoles, pH 7.4), guanidinoacetic acid (3.8 pmoles), freshly neutralized )-S-adenosyl-Dreduced glutathione (8.2 pmoles), (i methionine (4 pmoles, prepared by the methylation of Sadenosyl-D-homocysteine) and guanidinoacetate methylpherase (9 units) in a final volume of 1 mi. was incubated for 2 hr. a t 37". T h e reaction was terminated by the addition of 0.05 ml. of 30r0 perchloric acid. The precipitate was removed by centrifugation, the supernatant fluid brought to p H 6.7 and placed on a buffered Amberlite XE-63 column as for the separation of S-adenosylhomocysteine and
[ COXTRIBUTIoX FROM
THE
KEPU'NETH
c. STUEBEN
1-01. SI
S-adenosylmethionine. The control mixture contained all the components, but the enzyme was added after addition of perchloric acid. A similar pair of vessels was incubated with the corresponding cheniically synthesized ( zk)-Sadenosyl-L-methionine (3.8 pmoles). The material eluted from the column by 15 ml. of phosphate buffer 0.01 M , pH 6.7, was collected. The eluates from the incubated mixtures contained large amounts of ultraviolet absorbing material, while negligible amounts were found in the unincubated samples. I t was shown by paper chromatography in three solvent systems t h a t Sadenosylhomocysteine was the major component of this ultraviolet absorbing material. Aliquots of the eluates were assayed enzymatically for the presence of S-adenosyl-Lhomocysteine using a n enzyme which reversibly and specifically cleaves the L-form of this nucleoside.9 Adenosine liberation was measured by means of adenosine deaminase (Table V).
Acknowledgment.-The authors wish to acknowledge the sustained interest of Dr. G. L. Cantoni in the course of this work. Much help and information in the experiments with catechol 0-methyl transferase was provided by Dr. J. Axelrod and in the polarimetric measurements by Miss P. Wagner ; their cooperation is gratefully acknowledged. BETHESDA, MARVLASD
DEPARTMENT O F CHEMISTRY, P O L Y T E C H N I C INSTITUTE OD BROOKLYN]
Peptide Syntheses Via Amino Acid Active Esters1 BY MURRAY GOODMAN AKD KENNETH C. STUEBEN RECEIVED OCTOBER16, 1958 Amino acid active ester hydrobromides have been prepared. By use of these compounds, tripeptide dcrivatives have beeii synthesized in high over-all yield without isolation of a dipeptide intermediate. This has been accomplished by taking advantage of the difunctionality of the amino acid active ester as well as the difference in rate of reaction a t the amino and active ester ends of the molecule.
Since 1950, several promising methods for peptide synthesis have been published. Among these are the mixed anhydride,2-6 carbodiimide' and active ester8 approaches. These new methods form amides a t different rates. Thus, typical reaction times for peptide formation are 2-4 hours for mixed anhydrides, 5 hours for carbodiimides and 12-24 hours for the active esters. In this paper we wish to report the synthesis of difunctional amino acid derivatives of the type (where Act = R
I
HX.KH2CHCOOAct
I (1) T h i s research was supported b y a xrant from t h e National Science Foundation. NSF 0-1.571. Preyented before t h e l ? l t h Meeting of t h e American Chemical Society, Chicago, Ill., September 8-12. 1958. , 117 (1950); ( 2 ) (a) T. Wieland, W Kern and R. Sehring, A a ~ t .569, ( h ) 1'.Wieland and R . Sehring, i b i d . , 569 122 (1950). (3) R . A. Boissonnas, H d v . Chiin. Acta, 3 4 , 874 (1951). (4) (a) J. R . Vaughan, THISJ O U R N A L , 73, 3547 (19.51); ( b ) J. R. Vaughan a n d R. L. Osato, i b i d . , 7 4 , 678 (1952). (5) T . Wieland and H. Bernhard, A n n . , 573, 190 (1951). (61 J, M, Kenner. Chemistry &I~ndzrslrg, 15 (1951). (7) (a) J. C. Sheehan a n d 0 . P. Hess, THISJowRNnr.. 77, l0GT (1955); ( b ) J. C. Sheehan, &I, Goodman a n d C . P. Hess, ibid.. 7 8 , I367 (195A); ( c ) J. C. Sheehan and J. J , H l a v k a , J . Org. C h f m . , 21, 439 (19X). ( 8 ) (a) I-lformamide-ether. .4nal. Calcd. for CI1HlsSyOaBr:C . 43.57; 11, t.98; S , 9 . 2 4 . Found: C,43.63 H , 5 . 1 1 ; 9,9.41. Benzyloxycarbonylglycyl-DL-phenylalanineCarboxamido Methyl Ester (XI).-To a stirred suspension of the hydrobromide X (4.0 g . , 0.0132 mole) in 50 mi. of ethyl acetate was added dropwise over a 15-minute period 70 n i l . of an ethereal solution of benzylolcpcarbonylglycine acid clilor ide (prepared from 0.014 mole of benzyl~xycarbonylglycine). The temperature was maintained a t 0 . The gradual addition of j r i sodium bicarbonate kept the pH between 7 and 8 during the reaction. After this treatment the resultaiit solid was filtered and washed with 2 .V hydrocliloric w i d , 5'; sodium bicarbonate and water On drying in 7 ~ 7 ~ 1 4 0 the dipeptide derivative S I \vas obtained, m.p. 110.3-11l3. .in analytical sample was crystallized from acetoiie anti then from ethanol, m.p. 141-142', yield 3.6 g. (6eC:). , l m d . Calcd. for C21H,:,S306:C , fi1.00; H . 5.fiI; 1. li1.16. Found: C , 61.30; H, 5.78: N,10.36. Attempted Aminolysis of XI.-To a solutioii of S I ( 0 2 0 f i g., 0.0005 mole) in 10 ml. of acetonitrile was added glycine ethyl ester hydrochloride (0.14 g., 0.001 mole) followed b y triethylamine (0.14 ml., 0.001 mole). T h e resultant clear solution was stored at 55" for 23 hours, diluted \ acetate, washed with 2 9 hydrr~hloric acid, 5' bicarbonate, water and finally dried. Removal of the solvent i n ItaczLo gave 0.199 g. of unreacted starting inatrrial (97C; recover)-), m.p. 140-142". .L\ mixed melting point determination with original 111 gave 140-141 '. Formyl-DL-phenylalanine cyanomethyl ester (XII) was obtained by refluxing a solution of the formJ-lamino acid Ivith chloroacetoriitrile and triethylamines" in ethyl acetate. The product was crystallized from ethyl acetate-petroleunn ether, m . p . 80.5-81.5", yield 83LJ. 122) All melting points are corrected. Analyses are b y Schwarzkopf 1,ahoratories. Wcodside 77, N. 1 ' . ( 2 ' : ) T h e infrared spectrum of t h i s compound showed i i n nitrile l>caL. T h i s is in ajireenient with p r t - v , < , ' s ( r h w r v a t i i m s that ail o x y xen-ci,ntaining gro111>attached t o t h e s a m e c.irhon a i t h e nitrile ma?. result in t h e complete "qaenchinc' o f thiq i ~ a k ,I.. 1.T3ellarny. " T h e Infrared Spectra of Complex lloiecules." 2 n d H d . . John n'iley ani1 Son., I n ? . , S e w YorL, N. Y , I l l i Y . 1) 261'1.
Anal. Calcd. for CI2Hl2S2O3:C , 62.06; H. ,?.?I; S. 12.07. Found: C, 62.25; H , ,5.19; S,12.15. Formyl-L-phenylalanine cyanomethyl ester (XIII) was prepared in similar fashion except that rather than refluxing, the reactants were warmed sloivlv over four hours t o 8rl". Xfter storage at room temperature for two days the reactioii mixture was worked u p in the usual m a n n e r . Crystallization from ethyl ~rcetate-petri)leuinether gave n 76 o f colorless needles, m . p . R1-93'. The an:ilytical was crystallized from ethanol~-p~tr(ileuin ether in yield. m . p . 96..5-97.0", [N]Z~."D -3.58' ( 6 2 . 3 , etliyl acetate). .IniiZ. Calcd. for C12HI??J 12.07. Found: C, 62.32; H DL-Phenylalanine Cya ( X I V , . ~ - . l suspension of the form)-l compound XI1 (0.6 g., 0 . 0 0 3 mole) in 21 ml. of A\7 h~-drocliloricacid was heated ;it hoilirig water-bath temperaturc for 6 miriut querit sn-irling. The clear solution resulting \v extracted with three S-nil. portiozs of ethyl :ice drying of the aqueous layer afforded 0.38 g. of solid, m . p . 1j0.5-l,j9.tj", After repeated recrpstallizations froiri etllannl-ether, a n analytic,rl wrnple (yicld 2'Ic%) of the Iiydrcichlorirle X I I T , m.p. 16.i- 1&5.3", \viis ohtaincd.
Optimum c ~ ~ n d i t i o nfor s this li>-drnl>-si.:Iiavc i i i r t 11ec.11 fully ascertained. Higher coiicentrations of acid and longer contact times lead to increasing quantities of the cnrbosamido methyl ester, the infrared spectrum of wliicli wa\ identical to S. L-Phenylalanine Cyanomethyl Ester Hydrochloride (XV) . --Similar treatment of XI11 led t o the ~-isornerXV, n1.v. 1 85.,5-188.5" dec. The analytical samplfo (yicld Xf;; cr>-stallized from ethanol had m . p . 185-183.0 dec. .Innl. Calctl. for C1,HliN?O2Cl:C , 51.89; H , 5 . 4 4 ; S . 11.61. Found: C , 55.12; H , 5.31; S, 11.96. Benzyloxycarbonylamino Acid-p-nitrophenyl Esters. The intermediate p-nitrophenyl esters were prepared via reaction nf the corresponding benzyloxycarhoiiylamino acids with tris-(p-Iiitrophenosy)-phosphine in pyridine Pertinent d a t a are presented in Table 11. All ~-ieldsare fnr recrystallized materials. Amino Acid-p-nitrophenyl Ester Hydrobromides. -Removal of the betizyloxycarbonyl group from the h l w k e d :rinirio acid p-nitrophenyl esters with saturated hytlrogeii bromide in acetic acid'? gave the crystalline Iiydrohroinides in high yield. These d a t a are summarized i n Tnhle 111. All yields itre for recrystallized materials.
PEPTIDE SYNTHESES viu AMINOACID ACTIVE ESTERS TABLEI11 AMINQACID p- NITROPHEXYL ESTERHTDRORROMIDES p Nitrophenyl
No. XXI XXII XXIIT XXIV XXV XXVI XXVII XXVIII
ester of ?vI.p,,' ' C . HBr-gly 213-213.5 d. HBr-L-phe 215-216 d . HBr-L-leu 198.6-199.5 d . HBr-L-ala 182-183.5 d. HBr-L-pro 198-199d. HBr-L-cys 155-155.5 d . HBr-L-glub 155. 5-157 d . H B ~ - ? - O B Z - L - ~ I U120-120.5 ~ d.
Yield, [a]26.nD
... +40 8 O +11.4 - 2.4
2.3 2.2 2 1
-18.6 +14.6
2 Zd
,,
,
,
+26.9
%
EtCdH
2.1
.. 2.0
96 91 92 97 99 97 100 84'
CsHoh-tOaBr ClaHlsh'zO1Br CnH1Ih'zOdBr CeHnhsOaBr ClIHlaNZOaBr ClsHliNpOiSBr CllHlsNzOeBr CieHlgNzOnBr
__-_
r _ . . ~ . _ _ ~ . ~ .- Analyses, ---Calcd,---_
Ly0
C 3 4 80 4%)OD 43.25 37.13 41.66 46 49 37 84
C
4Y.21
H 3 29 4.12 5 14 3.81 1.13 4.15 3 75 4.30
N 10.13
7 (3 8.41
9.02 8.87 (i 78 8.03 ti :38
-.
~
- Found-----
:34,91 4X $17 43.27
37.41 41 94 4fi 2
