3-0 -Caffeoylshikimic acid (dactylifric acid) and its isomers, a new class of enzymic browning substrates

Vol. 14, No. 2, 1964

BIOCHEMICAL

AND BIOPHYSICAL

3-g-CAFFEOYLSHIKIMIC ITS

ISOMERS,

A NEW V. P.

October

22,

CLASS

Maier,

Fruit

Received

ACID

chromatographic

dactylifera)

studies

acid

browning

(Maier

substrates

acid

and purified

by column

zation

from

water

calcd.

for

browning

SUBSTRATES’ Huber

1963).

We have

of a crystalline extracted

structures

Hanson

and Zucker,

in dates which

estdblished

(Phoenix

we named

its

and synthesis.

enzymic

structure

This

with

methanol on silicic

a] kt124O

345,

calcd.

acid. fresh

green

After

recrystalli-

C 57.50.

H 4.80%,

acid.

(decomp.);

H 4.790/0;[ equivalent

from

to be

is the first

hydroxycinnamoylshikimic

m. p. 224-225”

and neutral

but their

It is one of the main

now

chromatography

C 57.16,

of several

compound,

‘by degradation

was

1961;

evidence

substrates

crystalline

and Metzler,

it gave

C16H1608:

ethanol)

a new

acid

and synthesis Dactylifric

aqueous

F.

reported,

and Hergert,

on enzymic

in dates.

3-c-caffeoylshikimic isolation

has been

(Goldschmid

we isolated

dactylifric

and A.

and UV spectral

acids

not elucidated During

Metzler

BROWNING

AND

U. S. Department of Agriculture and Vegetable Chemistry Laboratory’ 263 S. Chester Avenue Pasadena, California

hydroxycinnamoylshikimic

1963).

M.

ACID)

1963

Previously,

were

(DACTYLIFRIC

OF ENZYMIC

D.

RESEARCH COMMUNICATIONS

(c=l.

dates

16 in 62.5%

336.

‘Presented in part at the 144th American Chemical Society Meeting, Los California, April, 1963; work supported in part by Date Administrative Committee, Indio, California. LA laboratory of the Western Agricultural Research

Utilization Service.

Research

124

and Development

Angeles

Division,

Vol. 14, No. 2, 1964 The EtOH

X

UV spectra

max hmax

moiety

in which

349,

1956;

anthocyanase acid

shikimic

acids

was

various

t NaOAc

A max

spectra

group

obtained

identified

reactions.

Caffeic

acid

was

with

silicic

also

acid

confirmed

mu ; EtOH

the car’boxyl compound

acid

group

is

with

and shikimic

authentic

t NaOAc

of a caffeic

acids.

and base hydrolysis.

by comparison

and electrophoresis,

as follows:

the presence

caffeic

acid

were

~304

of the

yielded

on both

331,

whereas

hydrolysis

1962)

reagents

show

is free

Enzymic

et al.,

also

with

These

1957).

were

chromatography

Caffeic

samples

using

chromatostrips,

and

by UV and JR spectra

and paper

color

and mixed

point. Hydrogenation

10% Pd-C

of 5 mg of dactylifric

catalyst

a molecular

The

EtOH

mu.

B (Corner

Shikimic

1.01

mu;

-306

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

compound

the o-dihydroxy

(Jurd,

melting

of the

-303

332’

H3B03

‘blocked

BIOCHEMICAL

in me,thanol

weight

moles/mole hydrogen

hydrogen caffeic

Under

as expected

and

of shikimic

data

Treatment acetone amorphous etograms

acids establish

for

white the

solid

acid.

essentially

identical

Since

caffeic

Thus,

acid

gave

a single

On paper

acid

with

derivative

treatment

125

the sum

of of

had a different

the caffeic of shikimic

HCl

acid. in dry

gave

an

and electrophor-

of the isopropylidene acid

moles/mole

moles/mole.

chromatograms

spot which

of dactylifric Similar

for

to be due

is a monocaffeoylshikimic

of dactylifric

the UV spectrum

absor’bed

moles/mole.

the 2.33

of an isopropylidene yield.

(assuming

appears

that found

2.40

mole acid

1.39

of one mole

with

i. e.,

per

absor’bed

group.

milligrams

in high

to that

unchanged.

acid

dactylifric

the formation

compound

dactylifric

was

that

conditions,

agrees

of 10 mg of

of hydrogen

in excess

separately,

of several

to test

shikimic

acid

in the presence

moles

the same

hydroxyl

up by dactylifric

and shikimic

2.33

acid

of the allylic

taken

These

molecule

of 336).

uptake

to hydrogenolysis

used

acid

Rf

value

derivative acid

portion

acid

is known

than was

of the to yield

t

Vol. 14, No. 2, 1964 only

4, 5-g-isopropylidene

the 3-hydroxyl acid

BiOCHEMlCAL shikimic

is trans.

yielded

caffeic

comparison

hydrolysis

with

derivative

shows

acid

dactylifric

(Fischer

1937),

of isopropylidene

samples.

shikimic

acid,

The

acid

is 3-O-caffeoylshikimic

since

dactylifric

shikimic

authentic of the

RESEARCH COMMUNICATIONS

and Dangschat,

and 4,5-0-isopropylidene

4, 5-(J-isopropylidene that

acid

Enzymic

acid

chromatographic

AND BIOPHYSKAL

as

formation

portion

identified

by

of a

of dactylifric

acid

acid:

O* ,C-CH=CH 0

Dactylifric

The

structure

Acid,

of dactylifric

acid

Diphenylmethylenedioxycaffeoyl shikimic with

acetic

acid

and mixed chloride

acid

melting with

methyl

the isopropylidene caffeoyl) phoretically

shikimate. identical

- HCl

yielded

purification.

point.

In addition,

with

aqueous

This

compound

with

dactylifric

of the ‘blocking

was

electrophoresis, condensation

acetic was acid

4,5-z-isopropylidene

3-z-caffeoylshikimic

compound

(4,5-g-isopropylidene) group

with

Hydrolysis

crystalline

by chromatography,

‘by synthesis.

condensed

1937).

This

Acid

confirmed

was

and Dangschat,

chromatographic dactylifric

was

chloride

(Fischer

aqueous

after with

acid

3-0-Caffeoylshikimic

shown

acid to .be identical

UV and IR spectra of 3,4-dimethoxycinnamoyl

shikimate

followed

acid

methyl

gave

chromatographically methylated

groups

with

by hydrolysis 3-i-(di-z-methyland electro-

diazomethane.

of

BIOCHEMICAL

Vol. 14, No. 2, 1964 A second than

crystalline

dactylifric

acid

on hydrolysis,

band

These

has an R

compounds

components,

(Metzenberg

acid

concluded

and ,that neodactylifric When

sprayed

paper

with

brown

colors

Each These

shikimic

establish isolation

acid cinnamic

substituted plants 4 Ratio

the date

now

cinnamic

acid

they may

of effluent

extract

volume

based esters

gave

a negative

of the

and its

or with

isomers

mushroom

in acetate

pH

‘buffer,

at room

temperature.

enzymic

‘browning

of a crystalline of a new

acid.

to that

group

‘be difficult

of caffeic

were 5. 1,

substrates. caffeoyl-

It is possible will

are tyrosinase

acids

acid

test.

acid.

to any appreciable

127

a positive

and isodactylifric

as new

compound

gave

test

is 4-CJ-caffeoylshikimic

acid

the existence

not accumulate

periodate

acid acid

10 minutes

of shikimic

and the names

and the

of dates

on shikimic

acid

and 0. 1N

hydroxyls

determination

establishes

acid

identical

of the phenolic

dactylifric

acids

respectively.

and minor

Dactylifric

after

a major

the major

and tyrosinase

and structural

esters

‘because

case.

1963)

for

neodactylifric

extract

the material

UV spectra

in 10% acetic

suggested

acid

of pure

the dactylifric

acid

are

whereas

have

acids

a neutral

and 2. 12,

is 5-O- caffeoylshikimic

color

firmly

acid,

(Hanson,

bands

slowly

and shikimic

of dactylifric

methylation

enzyme

in every

a yellow-‘brown

tests The

tuted

with

gave

a crude

develop

treated

isomers

more

When

of 2.38

Both

acid

chromatograms

either

caffeic

column

that isodactylifric

acid

column

of hydrogen/mole.

isodactylifric 1954)

the

dactylifric

Rf values

therefore

After

it is tentatively

with

of 1.83.

and neodactylifric

and Mitchell,

only

partitioning

and similar

are

diazomethane

It yields

R caf4 values

value

caf

respectively. with

with

acid

from

identical

acid

RESEARCH COMMUNICATIONS

elutes

2. 26 moles

o’bserved

acid

car.boxyl

which

isolated.

silicic

of dactylifric

isodactylifric

also

are

acid

that

Thus,

also

on an analytical

Dactylifric

HCl.

was

of 341 and absor’bs

and minor

with

material

has a UV spectrum

equivalent is tested

AND BIOPHYSICAL

extent acid.

of substithat

the

to detect

in

due to high

BtOCHEMlCAL

Vol. 14, No. 2, 1964 metabolic

activity.

aromatic

compounds

acids,

flavonoids,

Whether

The such

with

of the

RESEARCH COMMUNtCATIONS

of shikimic

in the ‘biosynthesis

as tyrosine,

coumarins

compounds

or associated

importance

AND BIOPHYSICAL

phenylalanine,

and liguin

(Brown,

caffeoylshikimic

any of these

acid

acid

biosynthetic

the substituted 1961)

type

pathways

suggests are

remains

cinnamic

this

directly

of

possibility.

involved

in

to be determined.

ACKNOWLEDGEMENTS We would their

many

helpful

like

to thank

Drs.

Ro’bert

M.

Horowitz

and David

L.

Dreyer

for

suggestions. REFERENCES

Brown, Corner,

S. A. Science 134, 305 (1961). J. J., Har’bouG, J. B. s Humphries, S. G., and GU.is, W. D. Phytochem. i, 73 (1962). Fischer, H. 0. L. and Dangs chat, G. Helv. Chim. Acta 20, 705 (1937). Goldschmid, 0. and Hergert, H. L. Tappi 44, 858 (1961)F Hanson, R. H. and Zucker, M. J. Biol. Chem. 238, 1105 (1963). Jurd, L. Arch. Biochem. Biophys. 63, 376 (195K Jurd, L. Arch. Biochem. Biophys. 6X 284 (1957), Maier, V. P. and Metzler, D. M. Amer. Chem. Sot. Abet. 144th Meeting, IC (1963). Metzenberg, R. L. and Mitchell, H. K. J. Amer. Chem. Sot. -76, 4187 (1954).

128