γ-Peptides of glutamic acid and some of their properties

ARCHIVES

OF BIOCHEMISTRY

Y-Peptides

AND

of Glutamic

HAG-JOACHIM From the Division

94, 32-34 (1961)

BIOPHYSICS

of Biology,

Acid

and

Some of Their

BURKHARDT California Received

AND H. K. MITCHELL

Institute March

Properties’

of Technology,

Pasadena,

The synthesis of ~-~-glutamyl-~-~-glutamyl-~-~-glutamyl-~-r~-glutamic some derivatives is reported. Some properties of r-glutamyl di-, tri-, tides are presented and compared.

r-Polyglutamic acid polymers of high molecular weights have been found to be quite common as bacterial capsular substances (1)) but the properties of short-chain derivatives of this type have been studied relatively litt’le. Since substances of this class were suspected to exist among the peptides found in Drosophila (unpublished studies in this laboratory) the di-, tri-, and tetra-yglutamyl peptides were prepared and their properties examined. The tetrapeptide has not been reported previously.

(b) Z-ylu-OBz

(L-L-I-L)

‘-Glu-OBx I-Glu-OBz l-Glu-OBz l--:JBz

EXPERIMESTAL An amount

1. SYNTHESIS

of 810 mg. (0.001 mole) of Z-Glu-OBz I Glu-OBz

(L-L-L)

I Glu-OBa (L-L-L) was dissolved in 20 ml. dioxane, 0.24 ml. (0.001 mole) tri-n-butylamine was added, and the solution was cooled to about 10” before addition of 0.095 ml. (0.001 mole) ethyl chlorocarbonate. After standing at this temperature for 30 min., 10 ml. of a cooled (IO”) dioxane solution containing 436 mg. (0.0012 mole) H-Glu-OBz.

I-Glu-OBz J-Glu-OBz A solution

acid and and tetrapep-

out as described by Sachs and Brand for the dipeptide derivative (3), and the product was purified by crystallization from ethyl acetate-petroleum ether, methanol-water and methanol. The yield of pure compound was 1.43 g. (35%); m.p. 139-142”. Anal. Calcd. for C44H4701tN3 (809.9): C, 65.2; H, 5.9; N, 5.2. Found: C, 65.2; H, 6.1; N, 5.1.

INTRODUCTION

(a) Z-Glu-OBz

California

7. 1961

of 2.95 g. (0.005 mole) of Z-Glu-OBz

I Glu-OBz. (L-L) (2) and 1.19 ml. (0.005 mole) of tri-n-butylamine in 18 ml. dioxane was cooled to about lo”, and 0.48 ml. (0.005 mole) of ethyl chlorocarbonate was added. The reaction mixture was stirred vigorously for 30 min., and then a solution of 1.66 g. (0.007 mole) of H-Glu-OBz (L) (2) in 10 ml water containing 0.97 g. (0.007 mole) K&O3 was added. Stirring was continued for about 1 hr. at room temperature. The reaction mixture was kept in the refrigerator overnight. Isolation was carried

I 0131, HCI(L) and 0.29 ml. (0.0012 mole) t,ri-n-butylamine was added. The reaction mixture was kept in the refrigerator overnight. For isolation, 190 ml. ethyl acetate and 25 ml. of 0.5 N HCl were added, and the ethyl acetate layer was removed and washed with 0.5 N HCl, 5% NaHCOB , and water. The crude compound was precipitated with petroleum ether, recrystallized from methanolwater, and dried at 100’ over P205 under high

1 This work was supported in part by the National Science Foundation grant. R 6 6328. 32

GLUTAMIC

ACID

vacuum; yield of pure compound was 660 mg. (59%) ; m.p. 148-150”. IZnal. Calcd. for C63Hs6C)15NI (1119.2): C, 67.6; H, 6.0; N, 5.0. Found: C, 67.3; H, 5.8; N, 5.1. (c)

Z-Glu-OBZ I-

for 72 hr. Yield 395 mg. (74%); [01]:’ -16.0” (0.5 1%’HCl). Anal. Calcd. for C20H30013N1 (534.5): C, 34.9; H, 5.7; N, 10.5. Found: C, 45.1; H, 5.6; N, 10.4.

2.

(L-L-L-L)

GlwOBz

I-Glu-OBz This compound was synthesized and purified in analogy to the procedure described under 1 (a). The yield of pure compound was 527,; m.p. 141143”. ilrzal. Calcd. for Cs6H6&&? (lV29.1): C, 65.4; H, 5.9; N, 5.5. Found: C, 65.4; H, 5.7; S, 5.7. (d) H-Glu-OH

(L-L-L-L)

‘-Glu-OH l-&OH ‘-Glu-OH An amount of 1119 mg. (0.001 mole) of the carbobenzoxy tetrapeptide benzylester [compound (c)] was dissolved in 40 ml. of 95% acetic acid, 200 mg. palladium black (4) was added, and a rapid stream of hydrogen was passed through the reaction mixture for 6 hr. Water (6 ml.) was added during the first 2 hr. The solvent, of the filtrate was removed by vacuum distillation, and the tetrapeptide was recrystallized from water-ethanol and dried over P?Oj at 100” under high vacuum

mixtures of 01and y isomers (6). In order t,o insure homogeneity of the synthesized yglut,amyl tetrapeptide, the T’an Slyke carboxy1 nitrogen determination (ninhydrin) (7) and amino nitrogen determinat’ion (nitrous acid) (8) which allow a distinct,ion between 01-and y-glutamyl peptides (3) were carried out,. As can be seen from Table I, the carboxyl nitrogen determination yielded just one mole of COOH nitrogen per mole of peptide while the cu-glutamyl peptides yielded none. In the amino nitrogen determinat,ion there were nearly 4 moles nit’rogen liberated (amino and pept’ide nit’rogen) . The chromat,ographic behavior of glutamic acid and the r-di-, tri- and tetrapept,ides of glutamic acid was studied in different sol-

TABLE y-PEPTIDES

data,

R, values, specific rotations, I hfOlWhI

Compound

formula

I

OF GLUT~MIC

ACID

and ninhydrin ’ Nitrogen,

Mol. wt.

/

color. .4mino

GaH,,O,S2 ’

I

1 Carboxyl

x ,q&$Fi+

R/” Lr ~~

ix.

H-(&-OH (L-L) LGlu-OH H-Glu-OH (L-L-L) LGlu-OH 4;lu-OH H-Glu-OH (L-L-L-L) &u-OH G:lu-OH &u-OH

PROPERTIES

The mixed anhydride method (5) used for t,he synthesis of the r-L-glut(amy1 tet,rapeptide is known to yield pure y-peptides in contrast to t,he synthesis via t)he carbobenzoxy anhydride (Z-Glu-0, ] and the i-i carbobenzoxy azide [Z-Glu-OH] which yield

I-Gh-OBz

Analytical

33

PEPTIDES

10.lc

CliH&,,NS

1276.310.1 1 :405.410.4

CnoH,oOlaN,

534.510.5

10.4

5.1

10.4c 3.5

S.(i

10.lr

.---11

10.4

2.6

l-2 13:; _~

_

5.1’ 5.0c0.15

8.9< 3.5

d%

3.4r0.05

t3.8~

71

208

-7.2~

75

298

71

385

2.4 iO.O’?i -16.0

I

a Propanol-water 3: 1 (v: v) ascending. * Concent.ration in per cent referred to glutamic arid 100; equal aliquots used. c These values refer to data given by Sachs and Brand (2, 3).

of equimolar

solutions

were

34

BURKHARDT

vents, and propanol-water (3: 1) was found to be one in which all components were readily separable (Table I). It was of special interest to have quantitative dat’a on the reaction of the peptides with ninhydrin before and after hydrolysis. After chromatography in the propanol-water solvent, the colored compounds found with ninhydrin were directly measured in a Spinco Analytrol on the paper2 or more accurately in solution by means of a photometer using a 570-rnp filber (9). Equimolar aqueous solutions of glutamic acid and the y-peptides were used in this experiment; the results (Table I) demonstrate that all the peptides give about the same ninhydrin values, but these are about 30% lower than t’he one found for glutamic acid. After complete hydrolysis of the di-, tri- and tet’rapeptides in 5 N HCl at 100” for 24 hr., t’he values coincided with two, three, and four equivalents of glutamic acid within the error of t,he method used. The ultraviolet spectra of all the yglutamyl peptides did not show any absorp2 H. K. Mitchell,

unpublished.

AND

MITCHELL

tion between 235 and 400 rnp but had a very strong band at 210 rnp. All the infrared spectra performed in Nujol were very similar and had strong bands at 4350, 3300, 1735, 1660, 1550, 1220, and 718 cm.-’ and weaker peaks at 2750, 2300, 1070, and 970 rm.-‘; no striking differences in these spectra were detect’able. REFERENCES in 1. BRICAS, E., ASD FROMAGEOT,A., ddvances Protein Chem. 8, 64 (1953). 2. SACHS, H., AND BRAND, E., J. ilm. Chem. Sot. 76, 1811 (1954); Ref. (4). 3. SACHS,H., AND BRAND, E., J. Am. Chem. Sot. 76, 4F08 (1953). 4. ZELINSKY, N., AND GLINKA, iV., Rer. 44, 2309

(1911). 5. B~ISSONNAS, R. A., Helv. Chin,. ilcta 34, 874 (1951). G. SACHS, H., AND BRaND, E., J. ilm. Chem. L%C. 76, 4610 (1953). 7. VAN SLYKE, D. D., DILLOK, R. T., MACFAYDEN, D. A., AND HAMILTON, P., J. Bid. Chem. 141, 627 (1941). 8. VAN SLYKE, D. D., STEELE, R., AND PLAZIN, J., J. Biol. Chem. 192, 769 (1951). 9. BOISSONXAS, R. A., Helv. Chim. Acfa 33, 1972 (1950).