An optically pure sequence peptide, Poly.Gly.Gly.Phe.

Tetrahedron

Printed

AN

Letters

in Great

No.48,

OPTICALLY

PURE By

Department

5985-5989,

SEQUENCE

DeLos of

Florida

pp.

Britain.

F.

State

cri sequence

methods peptide

POLY.

Norman

Institute

F.

of

29

have

recently

polymers

Ltd.

GLY.

PHE.

Estrin Biophysics U.

S.

A.

1966)

been

based

GLY.

Florida,

August

Press

Molecular

Tallahassee,

(Received Efficient

and

and

University,

Peqmm

PEPTIDE,

DeTar

Chemistry

1966.

I

on

described

the

for

the

polymerization

synthesis

of

“active”

::: esters (I)

eq.

1 (1-3).

The

generality

of

the

polymerization

HBr.H.Asp(OCH3).Ser(H).Gly.ONP=%

with

respect

Poly.Asp(OCH3).Ser(H).-

Gly to

functional

number

side

potentially the

incorporated We

nique

for

carried show

has

have

racemizable

gated.

has

chains

of polymers

report

preparing

that

little

on

or

no

residue,

example

both

but

has of

from the

Furthermore,

in which

residues

an

a polymer

studies

demonstrated.

reported

C-terminal

C-terminal

now

out

been

been

the

not use

monomer

the

optical

been of

a had purity

rigorously

the

of

and

the

on

ester

sensitive

tech-

and the

a of

investi-

“active”

HBr.H.Gly.Gly.Phe.ONP

intermediates

racemization

the

polymer

have

which

phenylalanine

group

occurred. The

readily

:::

In the stands This

precise

central

upon

problem

treating

convention for makes

the

“active”

used residue:

it possible

derivative

dicyclohexylcarbodiimide.

here

being

that

esters

here

the

Gly to

is

azlactone

with

amino

bases,

acid

= -HNCHZCO-,Ser

designate

explicitly

discussed. HOPCP

ONP is

5985

formation including

abbreviation

occurs nucleophiles.

always

=-HNCH(CH2011)CO-. and

unambiguously

= p-02NC6H40-.

pentachlorophenol.

the DCC

is

No.48

5986

The

res\Jting

azlactones

the nuclt,ophile racemiz?d. proceed

example,

through

conditior,s

which

sodium

recipe

may

good

Assay

rize

glutamic

with

to

(lb)

Phenylalanine

acid,

containing

of activity. were

have

observed

cont.

hydrochloric wiihin

tively

unstable

20-25

of peptides

peptides

acid

were

which

glycine

easy for

are

with

ac-

summaa variety

along

at about

with

100°.

conditions:

times

erratic;

read

gave

both high

parts

preparation.

in a mixture

lo- 15

gradual

and low

catalysts

of one part

of dimethylformamide The

solvent is a high and reproducible 25 [MID + 89.6 with a per reading

The use of the polyfunctional advanta,geous (7).

is neither

developed

drastic

Longer

a single the extent

estimates

carried

is

on the peptide. were

and three after

purity

of samples

more

5N HCl. -

group with

been

containing

this

We selected

reliably

error

is best

require

tube with

depending

min.

number

or serine

of phenylalanine

taken

These

We

since

and phenylalanine

large

At 100° the results

Rotations

phenylalanine:

optical

acid,

Hydrolysis

at I2Oo in a sealed

for

techniques

of

of direct

azlactone.

to establish

determination. a very

extents

through

to use a “monomer”

and of polymers

2- 3% per

a variety

the phenylalanine

to be able

hydrolytic

serine,

glutamic

acid,

important

polymerization.

aspartic

values

shown

racemic

out under

in dimethylsulfoxide :;: sensitive cases.

in other

during

HowevBr,

acid,

as base

in order

of structures.

loss

to give

to differing

as the example:

of peptides

tc about

to lead

center

our experience

hrs.

of

or extensively

has been

shown

polymerization

results

and it was

active

nor routjne.

curate

is partly

can be carried

be expected

Gly.Phe.ONP

of racerrization

aspartic

so that acylation

of Bz. Phe. ONP in turn was

reaction

p-nitrophenoxide

rs.cemized

optically

and this

and of indirect

had given

HBr.H.Gly. easily

agents,

but the product

hydrolysis

azlactone

.zation

chose

acylating

(4, 5).

The polymerization

polymer

good

is not prevented, For

Bz.Phe.OH

are

advantage rotation standard

of Beyerman

may

of

and were of this value

deviation

prove

rela-

for of 2.

No.48

5987

Where of optical

applicable

purity

of these

in the synthesis

CH3OH);

(eqs.

2-4)

Z. Gly. Gly.Phe.ONP,

corrected

as described

value

above

- 1.42

and of estimates

tally

active

from

the carboxyl

(eqs.

2,

tions .)

C-terminal

the synthesis

3).

Conversion

Z.Phe.ONP 1

group,

active

HBr.H.Gly.Phe-

:::

of 86. 5.

A high degree

esters

Although

integrity

is

even with an opti-

we have found it advisable

amino

this

of rotation

to start

group as a blocking

may be used in this sequence

of a C-terminal

the

40/c low,

errors

of optical

methods,

the p-nitrophenyl

of

Upon hydrolysis,

on combined

experimental

upon hydro-

for phenylalanine

on each.

based

of “monomer”

end using

(Other

rotation

rotation

purity.

of present

Molar as follows:

t 67O (c 2 in dichloroacetic

a molar

limits

of polymer

within reach

pure.

(all L) were

2 (c 2 in CHC13);

of two determinations

showed a phenylalanine

In

optically

All the above intermediates

showed

is within permissible

clearly

test

HBr .H. Phe. ONP t 140 (c 2 in

poly.Gly.Gly.Phe.

for 95% purity).

2 I. 5, average

polymer

a rigorous

to each of the key intermediates

sh ows that all were

129 (c 2 in CH30H);

acid,

(2)

new techniques

- 6 1 (c 2 in ethyl acetate);

lysis 89.6

may provide

at 25O and 589 rnp of the intermediates

Z.Phe.ONP,

0NP3-

hydrolysis

(8).

Application

rotations

enzymic

acid to its ester

group

of reac-

according

to

+ HBrHoAC)HBr.H.Phe.ONP -2

(C2H5)J’J (3)

Z.Gly.Gly.OH

Kovacs

et al (3) prepared

method. there

t DCC t 2

However,

was loss

this polymer

the evidence

of activity

during

value

is given for the polymer,

iate,

Z.Gly.Gly.Phe.OPCP,

ancy may have arisen nique (6N HCl, results.

+Z.Gly.Gly.Phe.ONP ?

reflux)

using

the pentachlorophenyl

cited is not adequate the polymerization and the optical

is in question

in part from is similar

purity

ester

to show whether step.

No rotation

of a key intermed-

(see below).

The discrep-

the fact that the reported

assay

tech-

to one we have found to give erratic

5988

eq.

No.&8

5,

3x1the other

mechmisms tations

hand,

of these

have

been

(4)

3 +HBrHOAc,

(5)

Z.(;ly.Asp.

gives

discussed

optically

t HONP

purity

sequence Kovacs eq.

(5).

scope

The and limi-

largely

have

However,

then it may

the occurrence

in a number

examined?’

been made

ester.

and if the solubility

favorable,

of racerniza-

specific

that certain

using

probe possible

of such reactions

We call

it has ccnne to our notice

may

racemic

that

attention

to

commerical

such steps.

et al (3) prepared 2. Gly. Gly. Phe. OPCP by a route similar 25 100 [eJ589 , m.p. 112. This sample is extensively

to

5 and report

racemized. sequence coupling CHCl,).

We have similar

which

rotation

-32 t 2O (c, e jter

(lb,

a series

difficult

to separate

from

DCU.

C,H,N

showed

points

analyses

rotations

of the resulting

1 in CHC13)

product

from

to be about to those 5).

range.

rotation

that rotation

in agreement the coupling

These

for with

observed

were

measured.

gave

that which

of Z. Gly

in other

ester

.05 In

four with no

and 25.1

and

hydrolyzed A plot

a straight

the pure

of the

line with 25 is [oJ,,~

a

we found directly.

Gly. Phe . OH t HOPCP

30-400/cD and 70-6Oo/cL.

we have

NMR

as

fractions

We examined

and the expected

phenylalanine

-vs the phenylalanine

of ester

of - 7.0, - 11. 3, -16.9

in the 120-150°

and indicated

zppears

compz.rable pheno

gave

These

s melting

0, 0 intercept

DCC

by the alternate

instead

of synthesis

correct

and the rotations ester

L- Z. Gly. Gly. Phe. OPCP

2-4 using

method

rather

having

DCU peaks. varicn

to eqs.

Kovacs’

were

samples

prepared

diisopropylcarbodiimide 25 reagent (eq. 3) and find a m.p. of 149- 150: [a] - 30 (c, 589 This sample of L-ester was not very soluble in chloroform.

our hands

The

are

demonstrated

be carefully

because

polymers

ester

product.

well

must

this problem

+ DCC----_)

is not too extensive active

pure

tion is sufficiently optical

products known

(5).

(OCH3).OH

of the optically

to isolate

racemized

and the presently

HBr.H.Gly.Gly.Phe.ONP

11 the racemization perties

partly

reactions

These systems

results

are

.with p-nitro-

+

NO.18

5989

This Research

work under

was

supported

Grant

No.

by the Air

Force

Office

of Scientific

AF-AFUSR-62-279.

(1)

(a) D. F. DeTar, W. Honsberg, U. Honsberg, A. Wieland, M. Gouge, H. Bach, A. Tahara, W. S. Brinigar, and F. F. Rogers, Jr., J. Am. Chem. Sot., & 2873 (1963). Ib) Parts I-III submitted to J. Am. Chem.zc.

(2)

F. H. Stewart, Australian J. Chem., 18, 887 (1965); R. D. B. Fraser, B. S. Harrap, T. P. MacRae, F. H. Stewart, and E. Suzuki, J. Mol. Biol., 12, 482 (1965). J. Kovacs, R. 2282 (1966).

(4)

M.

(5)

D. F. Chem.

DeTar, Sot.,

(6)

D. F.

DeTar, Rogers,

(7)

H. C. Beyerman and W. M. Van den Brink, Proc. Chem. Sot. 266 (1963). H. C. Beyerman and W. M. Van den Brink, and F. Weygand, A. Prox, W. Kt)nig, L. Schmidhammer. and E. Nintz, Rec. trav. chim. s, 213 (1965).

(8)

K. Hofmann. M. E. J. Am. Chem. Sot.,

F. F.

and L. R.

g,

and A.

Levine,

Silverstein, 1024 (1966).

J.

Kapoor,

Am. and F.

J.

Chem. F.

H. Bach, and F. F. Rogers, Jr. Ph.D. thesis, Florida

Woolner, G. Splihler, 8&l, 1486 (1958).

Am.

Chem.

Sm.,

86,

Rogers,

Jr., State

and E.

Jr.,

SIX.,

88, -

(3)

Goodman

Giannotti,

C.

2918 (1964). J.

Am.

in preparation. University, 1964.

T.

Schwartz,