Nazumamide A, a thrombin-inhibitory Tetrapeptide, from a marine sponge, theonella sp.

Tetrahedron

Letters. Vol.32,

No.48, pp 7073-7074,

1991

0040.4039/91

Printed in Great Britain

Pergamon

Nazumamide

Thrombin-Inhibitory

a Marine

from Nobuhiro

a

A,

Fusetani,*

Laboratory

of

The

Sponge,

Youichi

Marine

University

Biochemistry,

of

Tokyo,

Tetrapeptide,

Theonella

Nakao

sp.1

and

Shigeki

Faculty

of

Bunkyo-ku,

Matsunaga

Agriculture,

Tokyo

JAPAN

Nazumamide A, a thrombin-inhibiting linear tetrapeptide, was isolated from Abstract. of nazumamide A was determined by interpretation Theonella sp. The structure sponge, NMR data and by chemical degradation as 2,5-dihydroxybenzoyl-L-arginyl-L-prolyl-L-isoleucyl-La-aminobutyric acid.

In the course invertebrates,

of our program

we

isolated

several

cyclotheonamidesz

from

a marine

extract

sponge

of the same

nazumamide A.3 The EtOH extract was further LH-20

extracted

(MeOH),

and Asahipak

led

potential peptides,

sponge,

was partitioned with

between

n-BuOH.

ODS [HZO-MeOH

(stepwise

biomedicinals including sp.

from

Hz0

Further

gradient)

and Et20, layer

marine

examination

and the aqueous

was

fractionated

to yield

of the

linear

and H20-MeCN-TFA

(90:10:0.05)]

a marine of 2D

thrombin-inhibitory

of a thrombin-inhibitory

The n-BuOH

GS 320 [HZO-MeCN-TFA

concentration

Theonella

to the isolation

as a colorless amorphous powder Nazumamide A inhibits thrombin

peptide, phase

on Sephadex (77:23:0.05)],

1.5 mg of nazumamide

(1, 1.0 x 10-S % yield based with an ICso of 2.8 ug/mL,4

on wet weight). but not trypsin

A

at a

of 100 ug/mL. A [ [CL]+

Nazumamide 240 (11300),

to discover new

cyclic

$3 00 + .I0

Press plc

325 (4400)]

+87.1” (c=O.O75, MeOH); had a molecular

formula

UV (MeOH)

hmax

of C28H43N7Og

212 nm (E 36000), as determined

by

Though negative to ninhydrin reagent, NMR and NMR data.5 Interpretation of the COSY and HOHAHA6 spectra spectra indicated 1 to be a peptide. in CD30H disclosed the presence of Pro, Be, Arg, and cx-aminobutyric acid (Aba)

FABMS

[ml, 606

residues,

which

(M+H)+]

was

supported

by standard

hydrolysate. Beside these amino for a 1,2,4_trisubstituted benzene 2,5-dihydroxybenzoyl The sequencing and

ROESYa

data.

(DHB)

amino

group

by HMQC7

of the above-mentioned There

acid

analysis

of the

acid

acid residues, the 1H NMR spectrum included signals (6 6.75, 6.84, 7.29) which was easily identified as a and HMBC7

units

were three sequential

was done

HMBC

spectra. by a combination

crosspeaks,

of HMBC

viz., Aba NH/ Be CO;

Be NH / Pro CO; Arg NH / DHB CO. Furthermore, the sequence of Arg and Pro was established by a ROESY crosspeak between Arg a-H and Pro &Hz, thereby completing the sequence

of DHB-Arg-Pro-Ile-Aba.

Absolute 7073

configuration

of all amino

acid

7074

residues

was

determined

derivatization

with

Nazumamide

is

the

HPLC

analysis

comments.

first

naturally-occurring

terminus.

The

We thank

This

work

39

Kato, 2.

peptide

hydrolysate

possessing

is known

as,

inter

P. J. Scheuer,

the

University

acid

Professor

was partly

of Education,

Part

acid

after

reagent.9

Science

supported

and

Culture

by a Grant-in-Aid of

alia,

the

N-2,5-

a constituent

of

of

Y.;

Fusetani,

the

bioactive

Hirota,

H.

N.;

marine

metabolites

submitted

Matsunaga,

for

S.;

for

of

Hawaii,

Scientific

for

editorial

Research

from

the

Japan.

References 1.

of the

griseofalvum.lo

Acknowledgment: Ministry

Marfey’s

A

dihydroxybenzoate

Penicillium

as L by

and

Notes

series.

Part

38,

Matsunaga,

S.; Fusetani,

N.;

publication. H.;

Matsumoto.

H. .I. Am.

Takebayashi,

Chem.

Sot.

1990,112,

70.53-7054. 3.

The

name

4.

Sevendsen,

5.

lH NMR

was

coined

after

L.; Blombtick, (CD30H):

8.91 (d, 7.4; a-NH),

the

the

M.;

4.96 (ddd, Pro

collection

Olsson,

6.2, 6.8, 7.4; a),

4.53

site,

Nazumado.

P. I. Thromb.

Res. 1972,

I,

X7-278.

Hz; H3), 6.84 (dd, 3.0, 8.7; H4), 7.29 (d, 3.0; H6); Arg

unit 6 6.75 (d, /=8.7

1.81 (m; p), 1.99 (m; p), 1.73 (2H, m; y), 3.22 (ZH,

(dd, 4.6, 8.4; a). 2.01 (m; /3), 2.25 (m; p), 2.03 (m; r), 2.08 (m; y),

3.71 (m; S), 3.87 (dt, 10.0, 7.1; 6); Ile 7.4, 9.2,

of

B.; Blomblck,

DHB

m; 6). 7.39 (m; S-NH);

name

8.07 (d, 7.9; NH), 4.20

(t. 7.6; a),

1.88 (m; fl), 1.21 (qdd,

13.6; y), 1.60 (m; y), 0.92 (3H. t, 7.4; 6). 0.97 (3H, d, 6.8; y’); Aba

7.83 (d, 7.4; NH), 4.21

(dt, 5.4, 7.2; a), 1.72 (m; p), 1.87 (m; p), 0.91 (3H, t, 7.4; y). 13C NMR(CD30H):

DHB unit 6 118.7

(s; Cl), 152.9 (s: C2), 119.1 (d; C3), 122.6 (d; C4), 151.4 (s; C5), 116.3 (d; C6), 169.3 (s; CO); Arg 52.8 Cd; a), 30.3

(t; p), 26.1 (t; y), 42.9 (t; 6), 159.2 (s; N=C), 172.5 (s; CO); Pro 62.2 (d; a), 31.4 (t;

p), 26.6 (t; r), 49.5 (t; 6), 174.8 y’), 173.3

(s; CO): Aba

6.

Edwards,

M. W.; Bax,

7.

Summers,

8.

Bothner-By,

A. A.; Stephens,

1984,106,

811-813.

9.

Marfey,

10.

Raistick,

(Received

in Japan

H; Simonart,

23 August

He 61.5 (d; a), 38.8 (d; p), 26.5 (t; y), 12.1 (q; I?), 16.5 (q;

56.6 (d; a), 26.9 (t; p), 10.9 (q; r), A. 1. Am.

M. F.; Marzilli,

P. Carlsberg

(s; CO);

Chem.

L. G.; Bax,

Res.

Commun.

1986,108,

A. J. Am,

R. L.; Lee,

P. Biochem.

1991)

Sot.

Chem.

J.; Warren,

1984, J. 1933,

4Y, 27,

176.6

(s; CO).

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Sot.