1104

Speeialia

EXPERIENTIA 32/9 Fig. 4. 'Shatter cone' with a nearly ideal cone-shaped structure in qtlartz gangue with little feidspar and muscovite from the locality 'Pos Cota', E of Lago di Tremorgio, at height ca. 1920 m. The cone diameter becomes smaller over a distance of only 20 eni, from about 17 cm (left) to nearly 0 cm (right, in the qenter of the rock). Note the feldspar (4 x 12 cm) like part of a mantle (G; some ram) at the thick end of the cone (below). Dimension of the rock 2 0 • em long. Photograph by K. PFISTER, Photograph. Institute of ETH Ziirich.

c u l a r t o each o t h e r a n d kinked. I n t h e plagioclase b e n t a l b i t e t w i n lamellae (up to 10 ~ m a y also a p p e a r a n d g r a d e i n t o o p t i c a l l y i n d i v i d u a l segregations of 5 0 • 100 # m a n d a k i n k angle u p to 25 ~ a g a i n s t t h e h o s t mineral, w h i l s t s c a l a r i f o r m l a m e l l a e p e r p e n d i c u l a r ~o t h e t w i n lamellae r e s e m b l e p l a n a r features. T h e s a m e o p t i cally g l o b u l a r segregations as in t h e q u a r t z were also obs e r v e d in plagioclase, b u t m o r e rarely. S o m e t i m e s t h e s m a l l spheres a r o u n d t h e vesicles are s t r u n g like a s t r i n g of b e a d s along planes parallel to fine fractures. I n o t h e r minerals, c a r b o n a t e s a n d o t h e r silicates, no i n d i s p u t a b l e ind i c a t i o n s for s h o c k m e t a m o r p h i s m h a v e b e e n f o u n d in t h e s a m p l e s so far collected. I n a d d i t i o n to d e t a i l e d field i n v e s t i g a t i o n s 5, v a r i o u s p h y s i c a l t e s t s f o r s h o c k m e t a m o r p h i s m 6 10 w i l l b e m a d e i n t h e n e a r f u t u r e . I n p a r t i c u l a r a s e a r c h w i l l b e m a d e for t h e v e r y h i g h p r e s s u r e m i n e r a l s c o e s i t e n,12 a n d / o r s t i s h o v i t e ~3, ~4 i n t h e q u a r t z o s e r o c k s of t h a t r e g i o n ~5.

5 K. B~/.CHTIGER, Schweiz. miiler, petrogr. Mitt., in press {1976). 6 B. M. FRENCH and N. M. SHORT, Shoe]~ Metamorphism o[ Natural Materials (Morlo Book Corporation, Baltimore 1968), p. 644. 7 D. J. McDoUGALL, Applieation o[ Thermoluminescence to Geological Problems. Proc. NATO Adv. Research Institute (Academic Press, l.ondon and New York 1968), p. 678. s K. BXCHTIOER, F. HO~MANN and F. LAVES, 9e Congr6s Internatiohale de S6dimentologie (SEDIM 75), Nice 5-14 Juillet 1975, th6me 10, p. 1. 9 K. BACHTIGER arid F. HOFMANN, Eclog. geol. Helv. 69, 204 (1976). 1o F. HOFMANN and K. BKCItTIGER, Eelog. geol. Helv. 69, 177 (1976). 11 E, C. T. CHAO, JG. M. SHOEMAKER, and B. M. NANSEN, Science 732,

220 {1960). Ie E. M. SHOEMAKER and E. C. T. CHAO, J. geophys. Res. 66, 3371 (1961). 13 E. C. Z. Ct[AO, ] . J. FAHEY, J. LITTLER and D. J. MILTON, J. geophys. Res. 67, 419 (1962). 14 E. C. T. CHAO and J. LITTLER, Geol. Soc. Am. Abstr. 1962, 27 A (1963). J~ F. BIANCONI, Beitr. geol. Karte Sehweiz ld2, 238 (1971).

S y n t h e s i s of E e l - C a l c i t o n i n and [ A s u l , 7 ] - E e l - C a l c i t o n i n : Hormonal Activity

C o n t r i b u t i o n of the Disulfide B o n d to the

T. MORIKAWA, E . ~IdNEKATA, S. SAKAKIBARA, T. NODA l, a n d M. OTANI 1

Peptide Institute, Protein Research Foundation, d76 [ha, Minoh, Osal~a 562 (Japan)," and Research Laboratory, Toyo ]ozo Co., Ltd., Ohito, Shizuoka ,110-23 (Japan), 20 A p r i l 1976.

Summary. E e l - c a l c i t o n i n a n d its [Asu 1, 7 ] _ a n a l o g , d e a m i n o - d i c a r b a - a n a l o g , were s y n t h e s i z e d b y t h e c o n v e n t i o n a l solution m e t h o d . T h e n a t u r a l - t y p e p r o d u c t s h o w e d 4300 M R C U / m g in t h e h y p o c a l c e m i c p o t e n c y w h i c h was c o m p a r a b l e to t h a t of t h e n a t u r a l h o r m o n e i s o l a t e d f r o m eel. H o r m o n a l a c t i v i t y of t h e A s u - a n a l o g was also as h i g h as 3400 M R C U / m g . I n 1974, OTANI e t el. 2 h a d s u c c e e d e d i n i s o l a t i n g a hypocalcemic factor from the pericardium and esophagus of eel, a n d t h e a m i n o a c i d s e q u e n c e w a s e l u c i d a t e d b y NODA a n d NARITA3 aS s h o w n b e l o w .

I

I

Cys- S e r - A s n - L e u - S e r - T h r - C y s - V a l - L e u - G l y - L y s - L e u - S e r - G l n Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-VaI-GlyAla-GIy-Thr-Pro-NH 2

1 Research Laboratory, Toyo Jozo Co., Ltd., Ohito, Shizuoka 410-23, Japan. 2 M. OTANI, T. NODA, H. YAMAUCI-II,S. WATANABE, T. MATSUDA, H. ORI~O and K. NARITA, Proc. 5th Parathyroid Conference, Oxford (Eds. R. V. TALMAGE, M. OWEN and J. A. PARSONS; Excerpta Medica 1974), p. 111. - M. OTANI, H. YAMAUCHI, T. MEGURO, S. I~ITAZAWA,S. ~VATANABEand I-I. ORIMO, J. Bioehem., Tokyo 79, 345 (I976). s T. NODA aild K. NARITA, J. Biochem., Tokyo 79, 353 (1976).

15.9. 1976

T h e s t r u c t u r e is q u i t e s i m i l a r to t h a t of s a l m o n calcit o n i n I4: o n l y 3 a m i n o acid residues, A s n 2~, T h r 27, a n d Ser 29, in s a l m o n c a l c i t o n i n are r e p l a c e d b y Asp, Val a n d Ala, r~spectively, in eel. A h i g h l y p u r i f i e d s a m p l e of t h e eel c a l c i t o n i n (E-CT) shows h y p o c a l c e m i c p o t e n c i e s of 3500 t o 5000 M R C u n i t s p e r m g 2, w h i c h are in t h e s a m e r a n g e of t h e r e p o r t e d v a l u e s of s a l m o n c a l c i t o n i n I ; 5000 M R C u n i t s 5 a n d 2300-3200 M R C u n i t s / m g ~ for t h e isolated samples, a n d 3500 M R C u n i t s / r a g 7 for a s y n t h e t i c sample. T h e p r e s e n t r e p o r t deals w i t h t h e s y n t h e s i s of a p e p t i d e w i t h t h e a b o v e s t r u c t u r e a n d of a n analog, in w h i c h t h e N - t e r m i n a l a m i n o g r o u p a n d t h e S S - b o n d of t h e n a t u r a l p e p t i d e were r e p l a c e d b y a h y d r o g e n a t o m a n d b y a n e t h y l e n e linkage, respectively. T h e c o n c e p t of p r e p a r i n g t h i s k i n d of a n a l o g was o r g i n a l l y i n t r o d u c e d b y JSsT a n d RUDINGER 8 a n d b y a g r o u p oi a p r e s e n t a u t h o r (S.S.) 9, i n d e p e n d e n t l y , in t h e field of n e u r o h y p o p h y s e a l hormones.

Synthesis o f the natural-E-CT was carried out by taking a route indicated in Figures I and 2 I~ All the functional groups in the amino acid residues were protected by groups which are used commonly in the solid

Boc-Thr (Bzl) -Pro-NH2 - - ~ Boc-Ala-Gly-OH ~ Boc-Val-Gly-OH - -

A~ P

BOC-ASp (OBzI) -OSu ~ Boc-Thr (Bzl) -OSu ~

IA

Aoc-Arg (Tos) -OH -' IA Boc-Thr (Bzl) -Tyr [Bzl (C12) ] -Pro-OH m Boc-Gln-ONp BOc-Leu-His-Lys [Z (CI) ] -Leu-OH Boc-GIu (OBzl) -OSu.

-

~ ~ ]

Boc-Gln-ONp

~

Boc-Lys [Z (C1) ] -Leu-Ser (Bzl) -OH Z(CI

1105

Specialia

Bzl

QBzl

Z(CI)

Bzl Bzl (C12)

B o c - L y s - L e u - S e r - G l n - G l u - L e u - H i s-L y s - L e u - G l n - T h r - T y r -

Tos Bzl OBzl Bzl Pr o-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr -Pro-NH2

(I)

Fig. 1. Synthesis of fragment I (11-32) 1~

Cys

Ser

Asn

p h a s e synthesis. E a c h f r a g m e n t was p u r i f i e d b y t h e conv e n t i o n a l procedures, a n d applied to t h e n e x t s t e p a f t e r confirming the homogeneity by thin layer chromatog r a p h y a n d b y a m i n o acid analysis. A p a r t of t h e F r a g m e n t I was t r e a t e d w i t h a n h y d r o u s H F u followed b y c o l u m n c h r o m a t o g r a p h y o n CM-cellulose to o b t a i n a free p e p t i d e , w h i c h was c o m p l e t e l y digestible b y a m i n o p e p t i d a s e M. T h e final p r o t e c t e d p e p t i d e was t r e a t e d w i t h a n h y d r o u s H F a t - 5 ~ for 1 h in t h e p r e s e n c e of anisole. T h e d e p r o t e c t e d m a t e r i a l was p u r i f i e d t w i c e b y c o l u m n c h r o m a t o g r a p h y on CM-cellulose u s i n g a linear g r a d i e n t c o n c e n t r a t i o n of a n a m m o n i u m a c e t a t e b u f f e r t0.01 to 0.15 M) a t p H 4.5 followed b y S e p h a d e x L H - 2 0 c h r o m a t o g r a p h y u s i n g 0.1 M acetic acid as a n eluent. T h e final p r o d u c t s h o w e d a single s p o t of R f 0.69 o n cellulose t h i n l a y e r c h r o m a t o g r a p h y w i t h a s o l v e n t s y s t e m of n - B u O H : A c O H : p y r i d i n e : H~O (15 : 3 : 1 0 : 1 2 v / v / v / v ) ; [~]~ - 8 6 ~ i 3 ~ (c 0.22, 0.1 M H C O O H ) ; t h e r e p o r t e d v a l u e 2 for t h e

4 H. D. NIALL, H. T. KEUTMANN, ]'). H. CoPP and J. T. POTTS, JR., Proc. natn. Acad. Sci., USA 6Z, 771 (1969). 5 R. K. O'DoR, C. O. PARKES and D. H. CoPP, Can. J. Bioehem. 47, 823 (I969). 6 H. T. }~EUTMANN, J. A. PARSONS, J. T. POTTS, JR. and R. J. SCHLUETER, J. biol. Chem. 245, 1491 (1970). 7 St. GUTTMANN, J. PLESS, R. L. H U G U E N I N , Ed. SANDRIN, H. BOSSERT and K. ZEHNDER~ Helv. chim. Acta 52, 1789 (1969). s K. J~sT and J. RUDINGER, Collect. Czech. chem. Commun. 32, 1229 (1967). 9 S. SAKAKIBARA and S. HASE, Bull. chem. Soc. Japan 41, 2816 (1968). - A. KOBAYASHI, S. HASE, R. KIYOI and S. SAKAKIBARA, Bull. chem. soc. J a p a n 42, 3491 (1969). - S. HASE, T. MORIKAWA and S. SAKAKIBARA, E x p e r i e n t i a 25, 1239 (1969). x0 Amino acids e x c e p t Gly denote the i.-configuration. Abbreviations are in accordance with the r e c o m m e n d a t i o n s of I U P A C - I U B Comrnissioi~ (J. biol. Chem. 247, 977 (1972). Z(CI), 2-Chlorobenzylo x y c a r b o n y I ; HOSu, N - H y d r o x y s u c c i n i m i d e ; Bzl(CI2), 2,6-DichlorobenzyI; MBzl, p - M e t h o x y b e n z y l ; TFA, Trifluoroacetic acid; DMF, N,N-Dimethylformamide; A, C2Hs-N C=N-(CH2) aN(CHa)2/1-Hydroxybenztriazole m e t h o d ; B, DCC/HOSu method. The Boc group in each f r a g m e n t was r e m o v e d b y TFA. 11 S. SAKAKIBARA and Y. SHIMO~ISHI, Bull. chem. Soc. J a p a n 38, 1412 (1965). - S. SAKAKIBARA, Y. SHIMONISHI, Y. KISHIDA, M. OKADA and H. SUGIHARA, Bull. chem. Soc. J a p a n 40, 2164 (1967).

Leu

Set

Thr

Cys

Val

Leu

Gly

OBzl

OBzl 1 OBzl

I Bzl

B o c ~Bzl

OH

Boc

Bzl

MBzl

i

f

Bzl t

Bzl t

MBzl

Bzl t Na/NH3

Bzl /

MBzl

OBzl

A

MBzl Bzl BOC. b S u B O C ' M9 B z l

Bzl

r

BOC

2

OBzl

Aeration

/

Boc/

!

B ~ Protected

Fragment Eel-Calcitonin HF/Anisole

Fig. 2. Synthesis of eel-ealcitonin 1~

OBzl

Eel-Calcitonin

I

OH

1106

Specialia

n a t u r a l p r o d u c t was E~x]~5 -88 ~ (c 0.1, 0.1 M H C O O H ) . The ratio of a m i n o acids in an acid h y d r o l y z a t e : Cys 2.13(2), Ser 2.86(3), Leu 5.10(5), Thr 3.48(4), Val 2.20(2), S l y 3.30(3), Lys 1.98(2), GIn 3.27(3), His 0.95(1), T y r 0.90(1), Pro 1.96(2), Arg 1.03(1), Asp 2.o7(2), Ala 0.94(1). (Average r e c o v e r y was 89.9%) The head p a r t of [Asu 1, 7]_E_CT ' which corresponds to the sequence (1-10) of the n a t u r a l hormone, was synthesized as shown in F i g u r e 3. A l m o s t the same procedure used for the synthesis of [Asu 1, 6]-oxytocin 9 was applied to cyclization of the f r a g m e n t (2-7). The cyclized p r o d u c t was further coupled w i t h Val-Leu-Gly to obtain a protected n o n a p e p t i d e w i t h a free c a r b o x y l group at t h e Cterminus. This m a t e r i a l was c o n v e r t e d to t h e N - h y d r o x y succinimide ester, which was t h e n coupled w i t h the fragm e n t I in D M F at 30~ The crude product, fully protected [Asul, V]-E-CT, was t r e a t e d w i t h a n h y d r o u s H F as in the case of the synthesis of the n a t u r a l hormone, and the liberated free peptide was purified by the use of CM-cellulose c o l u m n c h r o m a t o g r a p h y under t h e s a m e conditions as above, S e p h a d e x G-25 p a r t i t i o n c h r o m a t o g r a p h y w i t h a solvent system of n - B u O H : P y r i d i n e : 1 ~o-AcOH (5 : 3 : 11 v / v / v ) , and gel-filtration w i t h Sephadex L H - 2 0 using 0.1 M A c O H as a solvent, successively. Thus, homogeneous [Asul, 7]-E-CT was o b t a i n e d in an over-all yield of more t h a n 30% t h r o u g h t h e final coupling and purification procedures: R f 0.71 on cellulose thin layer c h r o m a t o g r a p h y using a m i x t u r e of n - B u O H : A c O H : Pyridine: H 2 0 (15: 3: 10:12 v / v / v / v ) as the solvent; [~]~ -95 ~ • 2 ~ (c 0.54, 1 M AcOH). The ratio of amino acids in an acid h y d r o l y z a t e : Lys 1.92(2), His 0.89(1), Arg 0.95(1), Asp 1.90(2), Thr 3.88(4), Ser 3.00(3), Glu 3.33(3), Pro 2.16(2), Gly 3.00(3), Ala 0.92(1), Val 2.10(2), Leu 4.95(5), T y r 0.92(1), Asu 0.99(1), (Average r e c o v e r y 90.1%). The synthetic E - C T showed a rat hypocalcemic activ i t y of 4300 MRC U / m g using a similar procedure to t h a t outlined b y KUMAR et al. ~2. This p o t e n c y was reasonably high in comparison w i t h t h a t of the n a t u r a l h o r m o n e as described above. The synthetic [Asu 1, Vj_analog showed a specific a c t i v i t y of 3400 MRC U/nag under the same bioassay system. These findings indicate clearly t h a t t h e disulfide bond in calcitonin is essential not for the biological a c t i v i t y b u t for m a i n t a i n a n c e of the specific conf o r m a t i o n b y forming an intramotecular bridge, since an open-chain analog of h u m a n CT is k n o w n to be inactive~K During the course of the final purification of the syn-

EXPERIENTIA32/9 Ser

Asn

Leu

Ser

Thr

Asu

i BOC-

~ ~3

:OMe

Bzl Boc. I

Bzl f

Bzl

Bzl l

Bzl l

OMe

Tfa-ONp Bzl BOC. J

I

I

i.

TFA

2.

Cyclization

/Bzl.

ONp /OMe

in pyridine

Bzl

~zl I O M e

,Bzl

,Bzl /NHNH 2

I NHz-NH2

i

L

. 2.

Val-Leu-Gly B

Bzl Bzl Bzl l Ser - A s n - L e u - S e r -Thr - A s u - V a l -Leu -Gly-OSu Protected

~ Fr agment I [Asu i , 7 ] - E e l - e a l c i t o n i n HF/Anisole

[Asu i , 7 ] - E e l - C a l c i t o n i n

Fig. 3. Synthesis of [Asu 1' 7~-eel-calcitoninl~

thetic n a t u r a l - t y p e E-CT, we were troubled seriously b y an u n e x p e c t e d loss of the biological a c t i v i t y ; thus, we could n o t get a reproducible yield of this c o m p o u n d in different runs. I n the case of [Asu 1, ~]_analog synthesis, no difficulty was encountered during purification in keeping the biological activity. These fact suggest t h a t the instability of s y n t h e t i c calcitonin should m a i n l y be attrib u t e d to the presenc~ of a disulfide bond, and that, if the Asu-analog were proved not toxic, the use of such an analog m u s t be m u c h more a d v a n t a g e o u s in developing a hypocalcemic drug because of its ease of manufacturing, enough p o t e n c y and higher stability during storage.

12 M. A. I~UMAR, E. SLACK, A. EDWARDS, H. A. SOLIMAN, A. BAGHDIANTZ, G. V. FOSTER and I. MACI:STYRE, J. Endocr. 33, 469

(1965). la W. R1TTEL, R. MAIER, M. BRUGGER, B. KAMBER, B. RINIKER and P. SIEBER, Experientia 32, 246 (1976).

A Modified Dipeptide from the Alga Cystoseira corniculata Hauck B. C. MAITI and R. H. THOMSON 1

Department o/ Chemistry, University o~ Aberdeen, Meston Walk, Old Aberdeen A B 9 2UE (Scotland), 22 March 1976. Summary. A modified L-Phe-L-Phe dipeptide has been isolated from the alga Cystoseira eornieulata. A l t h o u g h n m n e r o u s c o m p o u n d s h a v e been isolated from algae in recent years t h e y include, so far as we are aware, only two simple peptides 3, a W e h a v e now obtained the modified phenylalanine dipeptide 1 b y chloroform e x t r a c t i o n of Cystoseira corniculata 4 collected at Marmarls, Turkey. E x t r a c t i o n ot the dried alga (550 g) gave a thick green oil (2 g) which, after c o l u m n and repeated thin layer chrom a t o g r a p h y , afforded the dipeptide (57 rag) as needles, m.p. 185-186 ~ (from chloroform-petrol), C~H~.sN~O 4

(iV[+, 444.2048), [~D = --74"0~ (c, 0.98; CHCla); ,~maz (MeOH) 214, 227sh (log e 4.18, 4.07); CD, ~,maz (MeOH) 212 (de -1.27), 227 n m (-4.74); Vmax (KBr) 3320 (NH), 1728 (ester CO), 1662 and 1634 cm -1 ( - N H C O - ) . The 1H N M R - s p e c t r u m (CDCla) showed signals for an acetate m e t h y l group (6 2.00, s), 3 m e t h y l e n e (2.74d, 3.15 m, and 3.88 m), 2 methine (4.34 m and 4.83 q), 2 N H (6.26 d and 6.88 d, exchanged w i t h D20 ), and 15 a r o m a t i c protons. Decoupling e x p e r i m e n t s showed t h a t the methine p r o t o n at 6 4.83 was coupled to t h e m e t h y l e n e group at d 3.15