Applications of new peptide protective groups 1-- a synthesis of bis -s-acetamidomethyl dihydrosomatostatin

Tetrahedron Letters,Vo1.22,No.46,pp Printed in Great Britain

0040-4039/81/464575-O48o2.oo/O 01981 Pergamon Press Ltd.

4575-4578,1981

APPLICATIONS OF NEW PEPTIDE PROTECTIVE GROUPS I-A SYNTHESIS

OF BIS-S-ACETAMIDOMETHYL

D.S. Kemp*,

DIHYDROSOMATOSTATIN

David R. Bolin, and Marc E. Parham Department

Massachusetts

of Chemistry

Institute

Cambridge,

of Technology

Massachusetts

02139

Abstract: A synthesis of bis-S-Acetamidomethyl dihydrosomatostatin is reported in which the C-terminal carboxyl funcsn is protected by the Maq ester function and the N-terminal amine as well as two e-amino groups of lysine residues are protected as Tcroc urethanes. Clean removal of these groups is reported. Proof of reliability trouble-free

we have described reductively

(Tcroc)

dihydrosomatostatin.

of other peptide syntheses

hormones

much analog work directed

by straight-chain

is inert to branched-chain

papers

from their

amines

primary

in this series, we will report

amines.

tetradecapeptlde

somatostatin

growth

toward discovery

2 is widely distributed

factor which blocks the release hormone,

glucagon,

4575

of

in

of a varletv

and insulin. 3 A number of

and the molecule

of therapeutic

We wish form of

syntheses

groups which we have developed.

have been reported,4

such as

of an S-blocked

other protective

including

Recently

(Maq) ester group ,$ which is cleaved 1 and the 6-methylene-2-trifluoro-

is cleaved

and acts as an inhibitory

of somatostatln

results

to large size range.

of these groups to a synthesis

In subsequent

that employ

tissues

but which

application

The disulfide-bridged mammalian

$ which

groups

in the medium

of mild conditions

urethane

or by hydrazine

to report successful

this substance

protective

many peptides

the 2-methyleneanthraquinone

under a variety

methylchromone propylamine

for new peptide

use in preparing

has been the SubJect of

agents that can act selectively

4516

to inhibit control

release

of hormones

of diabetes.

S-deblockinq testing

such as glucagon.

Such agents may be very useful

Nearly all of the reported

and oxidation

new protective

dihydrosomatostatln, 5 coworkers.

of a bis-S-blocked

syntheses

in the

involve as final steps

dlhydrosomatostatin.

For the purpose

of

groups we sought to prepare the -bls-S-acetamldomethyl

$ which

has been characterized

and reported

(bis-Acm) by Meienhofer and

9 1 H-Ala-Gly-Cys-Lys-Asn-Phe2-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH 3 Q

Somatostatin

Acm

Acm

I

H-Ala-Gly-Cys-Lys-Asn-Phe2-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH 4 bls-Acm 'L-

dlhydrosomatostatin

am The tetrapeptides fragment

condensation

H-Asn-Phe-Phe-Trp-OH

and Boc-Thr-Phe-Thr-Ser-OH

(amino acid derivative

+ dipeptlde

-f tetrapeptide)

Z-ethyl-7-hydroxybenzisoxazolium

cation as the amide-forming

in the latter

from the water-solubllity

synthesis

the dipeptide

H-Thr-Ser-OH.

by a variety Double derivative

resulted

Both tetrapeptldes

were prepared

reagent.6

by a

using the Some inconvenience

and hygroscopic

were fully characterized

character

of

and homoqeneous

of HPLC and TLC assays. use was made of the N-hydroxysuccinimlde

2.

blocked

lysine

The e-Tcroc

derivative of lysine was prepared from Tcroc chloride' and the 7 Careful buffering was required to obtain satisfactory of lysine.

copper

(II) complex

yields

for the introduction

group 1s easily

cleaved

of the a-Boc

_

in the next step, since the Tcroc This is the sole limitation

group in the course of the somatostatln Tcroc di-t-butyldlcarbonate

1. Tcroc-Cl H-Lys-OH

functionally

at high pH in aqueous media.

have found for this protective

synthesis.

t

Boc-L&-OH 5 Q

DMSO, Et3N, 1 HR

2. H2S

that we

Tcroc

H-L&-OH

Cu(II)

Formation

ester of the doubly

(90%)

(74%)

of the Maq ester $ of Acm-cysteine

salt procedure 8 is

by the cesium

straight-forward: Acm Boc-bys-OH

Acm

1. cs2c03

HCl-dloxane

Boc-bys-OMaq

,-p 2. Maq-Br,

DMF

____)

Acm HCl H-dys-OMaq 95%

82%

$

24h SJfl&,,&,&$ The synthetic selective

sequence

for the preparation

removal of Boc groups were carried

for t-butyl

cations

and proceeded

of ,$ is outlined

out in the presence

with complete

retention

in Scheme 2. of anisole

of Tcroc

The

as SCaVanCPr

and Maq groups,

as

4571

SCHEME r?l_A’GLY

CYC

LYS

I!

ASN.Pt!E*PHE*TRP

LYS

THP*PHE-THR.:;H

CYS

TCROC

ACM

BdCSu

H

OH

TCROC 4 BOC ’ ACM Boc’-OSu

60%

BocLOSu OH

1.

H-LINAC

DCC-HOBT

2. HCL ACM ’ @llAC

1

H

82%

TCRDC

&Cc4

BmJ

74% TCRCC 1

1

CH 47%

TCRCC

OH

TCROC 4 TI=A H’

ACM Boc j

BOC

’

TCROC

A

TFA

1

HJ

OH

98%

PCM

’

8

ACM Ew

+.w

TCROC 1

’ ACM

TCROC -OH

DCC-HOBT

TCROC

TFA

I

H’

70-80%

TcRoc I

1 )

TCROC

’

ACM

TCROC

I

TCROC

80-90X

TCROC ,

c

Oi\!AG

ACM ’ OMAG Actd

7 DCC-HOBT

OhilAQ

mm

ACM l

OFi;AQ

10

fragments

8 and 9 were homogeneous

characterized doublets

and threonine

criterion

for inhomogeneous

for product

The blocked

methyls

acetonitrile.

Deblocking dithionite

g

not completely

resonances

was carried

was purified

l.Oml/min,280nm)

component

are

a valuable

for alanine

from DMF with

by HPLC, this substance

and threonine

resonances

out by treatment

of l$with

buffer

for 5-8 minutes

by HPLC

(DMF-H20)

25 equivalents

of sodium

at 25'C, followed

Immediate

(Waters u Rondapak

groups.

Material

from a sinqle HPLC preparative

to a reference

and for

of the Acm, Maq, and Tcroc groups.

in all chromatographic

was identical

impurities

evaporation

C18, 50% MeOH/0.02!

of three peaks, two of which could be attributed

of the protective

in 70% yield

for the benzylic

for these resonances

by precipitation

free of minor

(to 50% v/v) at 25'C for 35 minutes.

which qave a simple profile

products

multlplets

clean

purity.'

in a bicarbonate

of propylamine

species were also

and hlqh field NMR thus provides

a clean 250 MHz proton NMR spectrum

the characteristic

These

showed characteristic

and clean resonances

Unexpected

samples,

tetradecapeptide

Though

acid 7 and the two hexapeptide

NMR spectra which

of the Tcroc and Maq groups.

often observed

pH4.5,

The dipeptide

by TLC and HPLC criteria.

by their 250 MHz proton

for the alanlne

methylenes

showed

studies.lyc

from our earlier

expected

assays

corresponding run.

by addition

gave a residue Et3N*HOAc

to cleavage

to the third peak was isolated

This substance

and gave a satisfactory

moved as a single amino acid analysis. ID

sample of 4, supplied by Dr. J. Meienhofer, 11 solvent systems.

by HPLC, by 250

MHz IH NMR, and by TLC in three By several

criteria,

this synthesis

use of the Maq ester and Tcroc groups. practical

test of the stability

has materially The operations

increased

our confidence

of Scheme 2 have provided

of these qroups to the acidic

conditions

required

in the a to

It

4518

remove

the t-butyl-derived

during

normal

the reactions function.

protective

amide-forming of Scheme

Yields

steps.

of Maq and Tcroc

their

2 in which Tcroc was replaced

identified

susceptible

results

from model

carbonyls

of the Maq ester function

Financial

support

to a derivative

as well as for is

reaqents

of 2 which

N-propylamide.

evidently

to has been

The

render it unusually

of Health

(GM 13453)

S. Sofuku,

is

and J.

assistance.

0. Kemp and J. Reczek,

2.

D. Kemp and G. Hanson, J. Org. Chem.,

3.

P. Brazeau, Science,

for his gift of a reference

Institutes

We wish to thank Drs. C. Hoyng, G. Hanson,

acknowledged.

for technical

(Hoffmann-LaRoche)

from the National

1.

4.

screening

to reverse the order of deblockinq

in clean conversion

suggesting

'H NMR resonances

to the use of these groups

studies as the C-terminal

We wish to thank Dr. J. Meienhofer

Samanen

carbobenzoxy

The characteristic product

for

to aminolysis.

sample of t. gratefully

by the familiar

for preliminary

Attempts

first remove Tcroc from E

problems.

of free amines

out in preparation

for the two syntheses,

The sole restriction

homogeneity.

lack of orthogonality.

electronegative

no unusual

have proved valuable

product

tentatively

was carried

and product Rf values were similar

that the Tcroc group presents

establishing

groups as well as to the presence

A fore-synthesis

Tetrahedron

W. Vale, R. Burgus,

1973, 179,

Letters,

1977, 1031.

submitted

for publication.

N. Ling, M. Butcher,

J. Rivier,

and R. Guillemin,

77.

J. Rivier,

P. Brazeau,

Guillemin,

Compt.

W. Vale, N. Ling, R. Burgus,

Rend.,

5.

A. Felix, M. Jimenez,

6.

0. Kemp,

7.

J.P. Greenstein

C. Gilon, J. Yardley,

and R.

1973, 276, 2737.

C-T. Wang, and J. Meienhofer,

Int. J. Peptide

Protein

Res.,

1980, 15, 342. S-W. Wang, Z. Bernstein,

Sons, 1961,

and M. Winitz,

and J. Rebek, Tetrahedron,

"Chemistry

of the Amino Acids",

Su-Sun Wang, B.F. Gisin, D.P. Winter,

9.

Meienhofer, J. Org. Chem., 1977, 42, 1286. 1 H NMR 250 MHz; Maq:(DMSO-d6)65.35(s,2), 7.94(m,3), (DMSO-d6)65.17(s,2), B.OB(s(br),l,J=1.7 Amino

11.

7.07(s,l),

Phe 2.87(3);

Rf = 0.73

(rep 0.745)

nBuOH-AcOH-H20(4:l:5

in

7.80(d,J=8.8

Hz),

I.D. Kulesha,

8.22(m,4);

C. Tzougraki,

Tcroc:

7.89(m,l,J=8.8,1.7

Asp 0.96(l);

Thr 2.01(2);

Lys 2.06(2);

Cys (as cystelc

USA

Ser

1.09(l); acid)

Hz),

Gly 1.04(l);

4 May

1981)

Ala

1.96(2).

in nPrOH-Pyridine-H20-AcOH_EtOH(5:4:6:1:4); upper phase);

(4:7:1:1)5. (Received

R. Makofske,

Hz).

acid analysis:

1.01(l);

Vol. 2, John Wiley &

p 1059.

8.

10.

1974, 30, 3969.

Rf = 0.22 in

Rf = 0.33 in nBuOH-0.2 M_ AcOH-EtOH-Pyridine

and J.