Synthesis of 2,3,4-trihydroxy-L-phenylalanine from S-methyl-L-cysteine and pyrogallol by L-tyrosine phenol-lyase

BIOCHEMICAL

Vol. 64, No. 1,1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SYNTHESIS OF 2,3,4-TRIHYDROKY-L-PHENYLALANINE S-METHYL-L-CYSTEINE

Peter

AND PYROGALLOL BY L-TYROSINE

Rap**,

Hidehiko

The Research

Institute

Uji,

Kyoto

Tamio

611,

Kumagai for

PHENOL-LYASE

and Hideaki

Yamada

Food Science,

Kyoto

**

University,

Japan

Ueno and Hiroshi

Pesticide

FROM

Research

Fukami

Institute,

Kyoto

University,

Kyoto

606,

Japan Received

March

17,1975

Summary A trihydroxy derivative of phenylalanine was synthesized from S-methyl-L-cysteine and pyrogallol by the crystalline tyrosine phenollyase (L-tyrosine phenol-lyase (deaminating) EC 4. I. 99.2 formerly known as S-tyrosinase) from Escherichia intermedia. The product was isolated as its N-acetyl-triacetoxymethylester and identified as 2,3,4-trihydroxy-L-phenylalanine by the analyses of NMR, MS spectra and optical rotation. Tyrosine functional (1, vii) (6,

2), (5) 7),

ammonia

enzyme which S-replacement and the

**

is

a pyridoxal

catalyzes (equations

reverse

to form L-tyrosine

phosphate-dependent

a,B-elimination v, vi)

(3,

of a,S-eliminations or its

derivatives

(equations 4),

racemization

(equations

viii,

from pyruvic

multii-iv) (equation ix) acid,

and phenols.

L-Tyrosine

*

phenol-lyase

+

H20 2

phenol

+

pyruvic

acid

Present address, Gesellschaft fur Molekularbiologische Forschung mbH, D-3301 Stb'ckheim iiber Braunschweig To whom requests for reprints should be addressed.

Cop.vright o 19 75 b-v Academic Press, Inc. All rights of reproduction in any form reserwd.

241

+

NH3

(G.F.R.)

(9

Vol. 64, No. 1, 1975

BIOCHEMICAL

S-Methyl-L-cysteine

+

Hz0 2

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

methylmercaptan +

L-Cysteine

+

L-Serine

Hz02

2

HzS

pyruvic

L-Tyrosine

+

+

acid

+

acid

Phenol

2

+

NH3

(iii)

(VI

phenol

resorcinol-

2,4-dihydroxy-L-phenylalanine

(vi)

phenol

D,L-alanine

pyruvic

Pyrocatechol

+

3,4-dihydroxy-L-phenylalanine

+ L(D)-Alanine

(ii)

(iv)

+ S-Methyl-L-cysteine

acid

NH3

-

pyrocatechol

pyruvic

NH3

pyruvic

+

+

acid

+

(vii)

+

pyruvic

NH3 AL-tyrosine

acid

+

+

NH3 2

Hz0

(viii)

3,4-dihydroxy-L-phenyl-

(ix)

alanine The mechanism (8),

by adopting

reactions

(9,

these

the

general

methods

for

reactions

has been studied

mechanism

proposed

preparing

were

developed

using

cells

phenol-lyase

activity

(ll),

that

L-DOPA from DL-serine of these

aromatic

corresponding During properties

amino

the

course

of tyrosine

S-methyl-L-cysteine

acids

L-tyrosine of Eruinia

some detail

pyridoxal-dependent

and its herbicola

or pyrocatechol

from

pyruvate,

related with

the production

is,

and phenol

derivative

trihydroxyphenylalanine

synthesis

for

in

10).

Microbial acids

for

amino

high

tyrosine

of L-tyrosine (12),

ammonia

or

and the production and phenol

or its

(13). of further

investigation

phenol-lyase, through

we have

on the catalytic studied

the S-replacement This

and trihydroxybenzenes.

of trihydroxy-L-phenylalanine

from

the synthesis reaction paper

of

between deals

with

S-methyl-L-cysteine

the and

pyrogallol.

Methods The enzymatic at 30°C for

synthesis

2 hr in a reaction

of trihydroxyphenylalanine mixture

242

containing

was carried 100 nmoles

S-methyl-

out

BIOCHEMICAL

Vol. 64, No. 1,1975

L-cysteine, 250 umoles line

40 umoles

of pyrogallol,

of potassium

phosphate

L-tyrosine

tyrosine grown cording

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

phenol-lyase

phenol-lyase in a bouillon

4 pmoles buffer,

peptone

to the methods

pH 8.0,

in a total

was prepared

volume

from

medium

of Kumagai

of pyridoxal

cells

and 150 ug of crystal-

of 4 ml.

Crystalline

of E. intermedia

supplemented et ~2.

phosphate,

with

A-21

L-tyrosine,

ac-

(1).

Results Chromatographic layer

(Merck,

4:2:1

(v/v))

Kieselgel

examination GF254,

and ion-exchange

of the incubation developed resin

with

(Fig.

mixture

butanol-acetic

1) showed

on thin acid-water

the presence

of a

.A..---L.‘ ~. .:..*-.-. ’ ----u==+-q.5’

._

..L l

!

--:-.L---~.-L.---.

.

! -I--.--

. -4

_ _._

_ _

.__1..,-.--

;

L~i~-.-AL-!~.

I, I_ 1 I

.i

._ Li 30 MINUTES

40

50 MINUTES

60

70

80

chromatography of an incubation mixture of Fig. 1. Ion-exchange S-methyl-L-cysteine and pyrogallol with tyrosine phenol-lyase. The chromatography was done with a Yanagimoto model LC-5s automatic amino acids analyzer. Samples were placed on a JO x 0.9 cm column of Aminex A-4 resin and eluted with 0.2 N sodium citrate buffer, pH 4.25. Samples were (I), standard (0.1 umole of S-methyl-L-cysteine and 3,4,5-trihydroxyphenylalanine); (II), incubation mixture (l-ml aliquot of a 4 ml mixture). 3,4,5-Trihydroxyphenylalanine was a kind gift from F. Hoffman-La Roche & Co. AG. Basel.

243

BIOCHEMICAL

Vol. 64, No. 1,1975

new ninhydrin-positive

compound.

the new main peak same retention the

amount

(Peak

time

compound

A),

with

(Peak

was not

amino

peak

acid

analysis,

With

(Peak B) was observed

on the

boiled

beside

The amount

reaction

enzyme,

at the

time

of

and on the

the synthesis

of the

com-

observed. was replaced

a ninhydrin

tention

a small

A) was dependent

When pyrogallol mixture,

In the

3,4,5-trihydroxyphenylalanine.

of enzyme used.

pound

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

positive

With

time.

by hydroxyhydroquinone

compound

phloroglucinol,

was observed

in the

reaction

at different

no new ninhydrin

re-

positive

spot

was

detected. The enzymatically pyrogallol

(Peak A in Fig.

mixture,

as its

carried

out with

in the

text.

compound acid

synthesized

trihydroxyphenylalanine

1) was isolated

from

from

a large

N-acetyl-tri-O-acetoxy-methylester. 40 times

Under

were

analysis

large

these

authentic

mixture

approximately

calculated

incubation

The incubation

reaction

conditions,

synthesized, using

scale

scale

from

of that

described

0.5 mmoles

the chart

was

of the

of automatic

3,4,5-trihydroxyphenylalanine

amino

as the

standard. The deproteinized x 8 column

(12.5

incubation

column then

was adjusted was washed the product

product

with

the product

to dryness

one liter

was eluted

extracted

were

from

contaminated

that

methanol inorganic

of pH 5.0. reaction

with

reduced

pressure.

agent

The

of pH 1.5 and

The elution

acid

contain-

and evaporated obtained

chloride again

of the

as by the

Fractions

The solid

and evaporated

244

acid.

analyzer.

hydrogen

out with

The pH of the

as well

hydrochloric

containing salts,

was carried

of hydrochloric

amino acid

acidified

to a Dowex 50-W

sulfite.

of the elution

with

an automatic

with

M sodium

by the ninhydrin

at 50°C under

then

0.05

by the addition

with

was followed

examination ning

containing

was applied The elution

x 2.4 cm, Na+-form).

0.6 M NaCl solution solution

mixture

was

to separate to dryness.

BIOCHEMICAL

Vol. 64, No. 1,1975

Remaining solid

water

(10 ml) acetic

treated

with

in

vacua,

in pyridine anhydride

was treated

with

the

After

obtained

peroxide

separated

the

on a silicic

acid

9:l

Further

(v/v)).

derivative

Kiesel

The chemical ally

synthesized

structure product methylester,

The UV spectrum

of the

396 (consistent

sulphone,

its

eluted of the

developed

thin

with

based

compound

the molecular

anhydride

After the

removing

reaction

mixture

acid

one

for

remained which

methylated

was easily

by chromatography

with

obtained

benzene-ethanol trihydroxyphenylalanine

layer

benzene-ethanol

of the purified

of the derivative with

derivative

was assigned

L-phenylalanine

The MS spectra

into

by a preparative

GF254,

acetic

acetic

possibly

purification

gel

vacua,

to transform

(Mallinckrodt,

was performed

with

in anhydrous

trihydroxyphenylalanine column

in

(10 ml)

of the mixture

at room temperature.

30% hydrogen

S-methyl-L-cysteine

in methanol

was treated

by evaporation

and acetylated from

syrup

The residual

benzene.

the evaporation

overnight

bath,

RESEARCH COMMUNICATIONS

(2 ml)

and pyridine

water

with

thionylchloride

(10 ml)

on the boiling

(Merck,

removed

at room temperature.

to dryness

hour

was azeotropically

was then

overnight

AND BIOPHYSICAL

derivative

chromatography 9:l

(v/v>).

of the enzymatic-

to N-acetyl-2,3,4-tri-o-acetoxyon the

following

showed

a strong

(Fig.

2) gave

formula

spectral

data.

absorbance at 262 nm. + ion peak at m/e [M + l]

+ lH,C1g

H22 NOg),

and the

OAc

OH 100

E 150 Lz

Fig. 2. MS spectrum of N-acetyl-2,3,4-tri-0-acetoxy-phenylalanine The MS spectrum methylester synthesized by tyrosine phenol-lyase. taken with a Hitachi RMS-4, 70eV, direct inlet 12O'C.

245

was

Vol. 64, No. 1,1975

successive ion

BIOCHEMICAL

loss

of three

(M+ 395-m/e

suggested

spectrum methoxy

group

of the (3H)

acetamide 3112

of m/e 139 (base

the presence

carbomethoxy

ketenes,

353 -m/e

and the appearance

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and three

acetoxy

(9H)

the

must be attributed

aromatic

ring

unequivocally

concluded

methylester, (1H) which acid

considering were

assigned

the molecular m/e 178),

trihydroxytropylium

cation,

moiety

and

revealed

and N-acetyl

signals.

I,

the NMR

N-acetyl

(3H),

From the

coupling

of the AR pattern

and consequently,

2102

As shown in Table

in CD30D also

coupling)

signals

peak),

in the molecule.

compound

from

m/e 269 -m/e

of triacetoxybenzyl

CJA33 = 9.0 Hz ortko acetoxy

and methanol

in the lowest

to 2,3 and 4 position

the structure

of the

compound

carboconstant field, of the was

to be N-acetyl-2,3,4-tri-0-acetoxy-phenylalanine the multiplet to benzyl

signals protons

at 63.00

(2H)

and a-proton

and 64.60

of the amino

respectively.

Table

I.

NMR data of N-acetyl-2,3,4-tri-0-acetylphenylalanine methylester synthesized by tyrosine phenol-lyase.

6 (assignments) 7.22, 4.60

7.12

(2H, AB, J = 9.0)

3.70

m*, -C&L) RX (3H, s , -COOCI13>

3.00

(2H, m, -ClT,-ph)

2.30, 1.90

(lH,

2.25,

2.22

(each

3H, s,

-OCOW3>

(3H, s, -A--COCIi3)

* m, multiplet

** s, silane

singlet

Table I. The NMR spectrum was taken by a Hitachi R-24 Spectrometer.

246

in

CD30D with

internal

tetramethyl

BIOCHEMICAL

Vol. 64, No. 1,1975

The absolute

configuration

from

the fact

that

plain

curve

as that

alanine

AND BIOPHYSICAL

of the compound

the OKD spectra

of this

of the authentic

RESEARCH COMMUNICATIONS

was assigned

compound

to L-series

gave the same positive

N-acetyl-3,4-di-O-acetoxy-L-phenyl-

methylester.

References 1.

2. 3. 4.

Kumagai, H., Yamada, H., Matsui, H., Ohkishi, H. and Ogata, K. (1970) J. BioZ. aem., 245, 1767-1772. Kumagai, H., Yamada, H., Matsui, H., Ohkishi, H. and Ogata, K. (1970) J. Biol. Chem., 245, 1773-1777. Kumagai, H., Matsui, H., Ohkishi, H., Ogata, K., Yamada, H., Ueno, T. and Fukami, H. (1969) Biochem. Biophys. Res. Corrrnun., 24, 266-270. Ueno, T., Fukami, H., Ohkishi, H., Kumagai, H. and Yamada, H. (1970)

Biochim. Biophys. Acta, 206, 476-479. 5.

Kumagai,

H.,

Kashima, N. and Yamada, H. (1970) Biochem. Biophys. 796-801. Kumagai, H., Kashima, N., Torii, H., Enei, H. and Okumura,

Res. Cormrmn.,2, 6. 7. 8. 9. 10.

Yamada, H., S. (1972) Biochem. Biophys. Res. Common.,66, 370-374. Enei, H., Nakazawa, H., Matsui, H., Okumura, S. and Yamada, H. (1972) FEBSLetters, 1, 39-41. Yamada, H. and Kumagai, H. "Advances in Applied Microbiology (Perlman, D Vol. 17, in press, Academic Press, New York. ed.)", BZunstein, A. E. and Shemyakin, M. M. (1953) Biokimiya, 18, 393-411. Metzler, D. E., Ikawa, M. and Snell, E. E. (1954) J. Amer. Chem. Sot.,

76, 648-652. 11.

Enei,

H.,

Yamashita,

K.,

Okumura,

S. and Yamada, H.

Agr. Biol.

(1973)

Chem., 21, 485-492. 12.

Enei,

H.,

Matsui,

H.,

Nakazawa,

H.,

Okumura,

S. and Yamada,

H. (1973)

Agr. BioZ. Chen;., 37, 493-499. 13.

Enei,

H.,

Nakazawa,

H.,

Okumura,

S. and Yamada,

Chem., 3J, 725-735.

247

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Agr. Biol.