3 8 6 ,SHORT COM.MU N ICATII ~N.'-;

nBa 33036

The syntheses and biological activities of [GlyZZ,z2,zs]-~-ACTH-(1-19)-nona- decapeptide, [GlyZ2,z3]-~-ACTH-(1-17)-heptadecapeptide amide, [Glyn,Za]-~ - ACTH-(1-17)-heptadecapeptide amide, and [Gly13]-~-ACTH-(1-17)-heptade - capeptide amide

It has previously been shown that synthetic c~-ACI'H-(I--IO)-(I5-z())-penta- decal)eptide (I) l has approx, r "0 of the adrenal-~tinmlating and melanocvte-stimu- lating activities of synthetic ~-A(;'I'H-(z-zg)-nonadecat~ei)ti(te (II1".

Ser :l'yr Set Met (;lu tlis.-l~he-Arg-'l'ry..(ily L y s - I . y s - A r g - A r g Pro I1)

Ser-Tyr-Ser-Met-(; lu--His Phe Arg-Try-( ; ly I.ys l'ro Val ( ; ly . - l .ys - l .ys -Arg .\rg--l'ro (Ill I 2 3 -| 5 f) 7 ~ ~) IO I I 12 1.~ l.~ I 5 I(~ I 7 It~ I 9

"1"o determine whether tilt' tetrapeptide sequence at positions I I-I 4 of I I is necessary for these activities merely as a bridge t)r spacing factor between the basic core at positions I5-.IS and the N-terminal decapeptide, or is necessary l)ecause of the specific intluence ,>f one or more of the missing amino acids, we have sx'nthesized four peptides in which the lysine, proline, and valinc residues in p(~sitions z z x 3 are replaced by glycine, ;rod determined their A('TH and MSH activities. The synthetic pcptides arc i(;ly ]I.]',]a -~-ACl'II-(z-z(~)-n(madecapcptide (III), i(;ly ]'-'.la -~-ACI'H- (z -17)-heptadecapeptidc amide (IX,'), (Glyl].':~i-~-ACTH-(I--I7) hcptadccapeptidc amide (V), and i(;ly t:~ -~-ACI'H-(I -I 7)-heptadecapeptide amide (V I).

The syntheses ~}f peptides I l l -VI were accoml)lished by c()nvcnti()nal methods as previously described for the sx, nthcses of I 12 and ~-AC'I't l-(z .t 7)-heptadecap~:t)tide amide (VII) s. The pertinent protected tetrapeptide acids, corresp,)nding to positions I z -I 4 in the final product, were synthesized as outlined bch)w:

Z.-CI -t- I1 (;ly ( ; l y -Gly -Gly ()II . . . . ~ Z . t ; l v (;l.v ( ; ly -Gly ()I1

Boc I~oc i

Z-l .ys--()Np :. 1t (; ly--t; ly-Gly ()H . . . . . . . Z L£'s Gly-Gly ( ; ly- ( )H

NEI'IS Z- ( ; ly .()H -- l l - ( ; ly-() l~lut . . . . ~ Z-C;ly-{; ly-OBut

(i) I l.~/l'd (2) Z-Pro- ( )H, NI'21)IS

Z- ( ; ly - l ) ro -Gly -Gly ( )II<- Z t~ro--lily (;ly ( ) lhlt (t) H.2/1M (2) Z-.(;Iy-()H, Nl.iPl.S (l) l12,.'l'd (3) CFaC( )211 "l'os

(2 )Z- l .y s - ( )Np (3) ('FsC()~H

"l'os

Z- l .ys Pro- ( ; ly -Gly . Ol t

Abbreviations: ACTII, adrenocorticotropic hormone; MSH, melanocyte-st imulating hor- mone; Z, carbobenzoxy; Boc, tert.-butyloxycarbonyl; OBut, /ert.-butyl ester; Tos, p-toluenesul- fonyl; ()Np, p-nitrophenyl ester; N E P l S , Ar-ethyl-5-phenylisoxazolium-3'-sulfonate.

Biochim. H*ophy,..-Iota, 147 (t9~,7) 3,q~-3,~8

.~ H O R T COM.MI_" X I C . \ T I O N S 387

The p r o t e c t e d t e t r a p e p t i d e acids were coupled wi th N ' - t e r t . - b u t y l o x y c a r b o n y l l y s y l -

,V ' - tort. - bu t y l o x y c a r b o n y l l y s y l - ;\ '(;- t o sy la rg iny l - N c'- t o sy l a rg iny lp ro l i ne t e r t . - b u t y l

es ter t (or ~\" - ler t . - but y l o x y c a r b o n y l l y s y l - .V'- ter t . - b u t y l o x y c a r b o n y l l y s y l - .V C; - tosy l -

a rg in ine amidO) us ing N E P I S a as the coupl ing reagent . The n o n a p e p t i d e es te r (or

hcp ta t )ep t ide amides) was purif ied hv c o u n t e r c u r r e n t d i s t r i bu t ion e i the r in toluene--

c h l o r o f o r m - m e t h a n o l - w a t e r (5 : 5 : 8 : 2, by vol.) or in ca rbon t e t r a ch lo r i de -eh i o r o f o r m - m e t h a n o l - w a t e r ( r : 3 : 3 : I , by vol.) Hydrogcno lys i s g a v e the free base pep t ides which

were coupled wi th c a r h o b e n z o x y s e r y l t y r o s y l s e r y h n e t h i o n y l - y - b e n z y l g l u t a m y l h i s t i -

d y l p h e n y l a l a n y l - N ~ ; - t o s y l a r g i n y l t r y p t o p h y l g l y c i n e ~ us ing e i the r N E P I S or N , N ' - d i -

c y c l o h e x y l c a r b o d i i m i d e ~ as the coup l ing reagent . The final p roduc t s , I I I VI, were

o b t a i n e d a f te r deb lock ing the p r o t e c t e d pep t ide first wi th t r i f luoroace t ic acid and

then wi th sod ium in l iquid a m m o n i a . Pur i f ica t ion was ach i eved by CM-cellulose

c h r o m a t o g r a p h y r as l ) reviously descr ibed I to g ive pep t ides which were hon togeneous

by e lec t rophores i s o n paper at p H 3.7. A m i n o acid analys is ~ of acid h y d r o l y z a t e s showed the e x p e c t e d con lpos i t ions ( ' fable !).

The iJ~ v i tro 3,~ s t e ro idogen ic po tenc ies and the in v i t ro '° and i n r i v e 11 mela-

n o c y t e - s t i m u l a t i n g ac t iv i t i e s of the s y n t h e t i c pep t ides are shown in Tab le I I , t o g e t h e r

wi th the ac t iv i t i e s 3 of A ( ; T I t and re la ted s y n t h e t i c pept ides . P e p t i d e s I I I - V I e x h i b i t e d

m u c h less A C I ' H a c t i v i t y than those pep t ides (II and VII ) possessing the na tu ra l

L y s - - P r o - V a l - G l y sequence a t pos i t ions I I - ~ 4 . Of pa r t i cu l a r in te res t i~ the low

A C T t I a c t i v i t y of pep t ide VI, which ind ica tes the i m p o r t a n c e of va l ine at p.~sition 13

for full s t e ro idogen ic p o t e n c y (se.c refs. I2 and r 3 fiw the reduced a c t i v i t \ effect in

ang io tens in when va l ine is r ep laced by o the r a m i n o acids). Also, a compar i son of

the A C T I I ac t iv i t i e s of VI and V, and VI and IX', sugges ts a c o n t r i b u t i m of the

lysine and prol ine res idues to s t e ro idogen ic po tency . The m e l a n o c y t e - s t m m l a t i n g

ac t iv i t i e s fol low a s imi la r t rend, excep t t h a t pep t i de VI is s ign i f ican t ly more active.

t h a n pep t ides I I I , IV, and V, ind ica t ing tha t the. va l ine at posi t ion I3 is not as v i t a l

for M S H a c t i v i t y as it is for A C T t t ac t iv i ty , and tha t b o t h lvsine and prol ine are.

"I'A BLI:~ I

A M I N O ACII) CO31POSITION OF T H E S Y N T t I E T I C P E P ' r I D E S

,q mim~ acid Heptadecapeptide tleptadecapeptide Heptadecapeptide Nonadecapeptide amide II" amide 1" amtde 1"1 l [ l

Theor. Found" Theor. Found" Theor. Found" Theor. Found"

Serine 2 1.87 2 1.73 2 1.74 2 1.6I Tvrosine 1 1 .oo ! I .oo 1 i .oo i 1 .oo 3(et hionine i 0.8 t i o.~ 7 1 o. 73 I 0.84 Glutamic acid i i.oi i l.o~) I 1.o 4 I i.oo I Iistidine 1 0.99 I 0.90 l 1 .ot l 0.97 l~henylalaninc r o.98 l I.o2 i z.o 4 i I.o 5 A rgmine 2 2. r 7 2 1.92 2 2.o~) 3 2.97 Tryptophan *" I 0.92 I 0.90 i 0.98 I 0 . 8 5

(;lycine 4 3.97 4 3 .88 3 2.80 5 4-75 Proline . . . . . r 0.99 t 0 .93 i 0.93 l.ysine 3 3.o0 " 2.o 4 3 2.75 2 2.oo

* Chromatography. ** l)etermined spectrophoton~etrically.

Biochim. Biophys. Acta, i 4 7 (1967) 3 8 6 - 3 8 8

388 SHORT COMMUNICATIONS

T A B L E II

BIOLOGICAL ACTIVITIES OF SYNTHETIC PI';PTII)E.S

Peptide

~s-ACTH

~-ACTH-(I- I9) (I1)

[Glyn,le.la[-0~-A("I't I-( i - i 9) (1II )

x-AC'I 'H-(I-I 7NH2) ( \ ' l I)

~(;lyXZ,la!-~-ACTl t-(v 17NHo) (1V)

~(;lyn,la]-~-:\( 'Tll-(l x7Nlt2) (V)

!(;lyaal-x-ACTtl-(l 17NIta) (\ 'I)

~-ACTH-(I - m)-(15 19} (1)

Steroidogenic Melanocyle-slimulating activity potency in vitro (1. U./t~mole ) In vivo In vitro

(ttmole) (units/pmole)

6x7 (917-418) * 4.4" m - s * * 2.9' IO s

82 (I29-,54} 8. 7" 1o s 3.3" los

0.3 (o . . t -o .2) 2 .5' Io a (,. .):)

88 (123- ()3) 9 .0 . iO ,5 4 .2 . lOS

I.o (r.5-o.6) 5' IO a 4" t°a

"i0.5 .5" I0 a G" 1o a

2.g (4.4--I.Q) 2. 5 " 10 I S" 10 4

0.8 (i.2-.o.t>) I- lo 'z 3.7' t°a

* Result of at least three assays; 95 °o contidcnce limits are given in parentheses. ** The dose produces a change in melanophore index in hypophysec tomized Rana pipiens

from t - to 3 + within i h.

n e c e s s a r y fo r h i g h M S H a c t i v i t y . T h e s e r e s u l t s s h o w t h a t t h e i m p o r t a n c e of t h e

t e t r a p e p t i d e s e q u e n c e a t p o s i t i o n s I I - I 4 of I I r e s i d e s n o t o n l y in i t s e f f ec t on t h e d i s -

t a n c e b e t w e e n t h e b a s i c c o r e a t p o s i t i o n s r 5 - I 8 a n d t h e N - t e r m i n a l d e c a p e p t i d e , b u t

a l so d e p e n d s on t h e spec i f i c c h a r a c t e r of t h e a m i n o a c i d s a t p o s i t i o n s I I - 1 3 ; t h a t is,

t h e i n t a c t I , y s - P r o - V a l s e q u e n c e is n e c e s s a r y fo r full A C T H a n d M S H a c t i v i t y .

T h i s w o r k w a s s u p p o r t e d in p a r t b y a g r a n t f r o m U.S . P u b l i c H e a l t h S e r v i c e

(GM-29o7) . O n e of us ( J .B . ) w a s N a t i o n a l I n s t i t u t e s of H e a l t h P o s t d o c t o r a l F e l l o w

I 9 6 5 - 6 7.

Hormone Research Laboratory, School o f Medicine,

Universi ty o f California,

San Francisco, Calif. (U .S .A . )

JAMES BLAKE

CHOH H A o IA

i C. H. IA, J. I,~AblACHANDRAN AND I). (?HUN(',, J. Am. Chem. Soc., 86 (i964) 271 i. 2 C. It. LI, D. (_'.HUNG AND J . RAMACHANDRAN, ,]..-tin. Chem. Noc., 80 (1904) 27r 5. 3 J. RAMACHANDRAN, 1). CHL'NG AND C. H. LI, J..,l~n. Chem. Soc., 87 (19(~5) 2(790. 4 R. B. WOODWARD, R. A. OLOFSON AND H..~,]AYt';R, J . Am. Chem. Soc., 83 (i90t) lOlO. .5 C. H. I,I, J. RAMACHANDRAN, l). CHUNG AND B. (.;ORUP, J. .4m. Chem. Sot., 86 (t964) 2703. 0 J. C. SHEEItAN AND (;. 1 ). HESS, J , Am. Chem. Soc., 77 ( I 9 5 5 ) 1°07" 7 15. A. PE'rERSOX AND If. A. SOm.:R, J. Am. Chem. Soc., 78 (1956 ) 75 I. 8 I). H. SPACK.XIAr,;, \V. H. STI-;IN AND S. MOORE, .4hal. Chem., 3 ° (I958) xx9o. 9 3'I. A. SAFFR,XN :XXD A. V. SCHALLY, Endocrinology, 56 (t955) 523 •

IO IX.."~HlZU.ME;, A. }3. I.ERNER ANt)T. }~J. FITZPATRICK, Endocrinology, 54 (1954) 553. If I.. T. l loGl*I.:x AND 1.). SI.o.~II,:, Pw)c. 17o3,. Soc. London, Her. H, Io8 (x931) lo. 12 E. SCHRODER AXt) K. I.UI~KI.:, The Peptides, Vol. 2, Academic Press, New York, 1066, p. 52. 13 R. SCHWVZER AXD ]l. Tt'RRIAN, Vitamins Hormones, I8 (196o) 237.

R e c e i v e d J u l y 11 th , 1967

Hiochim. 13iophys. Acta, 147 (1907)380 388