The stereochemistry of the addition of glycerol to D -galactal, catalyzed by β-D -galactosidase

Curboh~dmre Rzsemch. 58 (1977) 73-75 19 Ek\ler Sc~cntiiic Puhllshlog Comoany.

THE

STEREOCHEMISTRY

D-GALACTAL, JOCHEN

OF

CATALYZED

LEHMANN

Amsterdam

THE BY

- PrInted

In Bclglum

ADDITION

OF: GL\.CEROL

TO

/3-D-GALACTOSIDASE*

AND BERJT

Chemrsches

dcr

(Received

ILh.

Unrtersrtcr.

FrArtrp

,m Bwrsgau

iw public-atmn, January

1976;

10th.

1977)

ABSTRACT

On

incubation

N Irh B-D-Ealactosidase,

I-dcoxy,olyc?rol-I-yl it was shown moiety.

that rhe hydrogen

In contrast

phenol

with

D-palacral-24

atom

introduced

to Ihis stereospecific.

peracetylated

1, catalyzed

enzyme-carnlyzed

/?-D-Galacrosidase

from (2)

D-,oZlkICt3l

t0

EstV~ericlliu

IO

W.

the reaction

of

acid. kr hich yields phenyl was iho\! n tc entail

obsened

rcqulres.

with

to mediate

arises whether

(3).

D-@ucal’

a proton-donating

of C-2,

1s stereospccilic

‘.

borh a

(AH)

calion

the stereospecific transfer to the

Because of would

ha\ ing the fi-o-anonwic

be either

HBS shown carbon-bound 4S h. As

hydrogen

Hehre’

spectroscopy exchange applied. bound

(as

(probably

could

not

as a probe.

can or.ly

find

and

at C-7) any

when

hydrogen,

‘Uncommon

CurbuhJdr.

synthesis Results

Res.,

when

even in solution

the highly

Incu’r,ated

(5 Cl/mol)

D,O.

oiGlycosldase 65-72,

ActIon, (precedmg

sensitive.

and

technique

IV.

paper),

For

Part

III.

set J. Lehmnnn

and

is

of carbon-

me preferred

I -yl 3-deoxy-B-c-!r,.\o-hexopyranoside-~-~ Pan

for

‘H-n.m.r.

IOU and that

radiolabelllng

of high sl)ecific activity.

it

exchnnsc

in ibaler-l

usinp

to yield reaction

e.kperimenrs’,

did not
in water-r

of I -deouyglycerol-

53 (1957)

addition

we assume that the eschangc rate is estrcmely

be detected

As 2-deo\v-D-/>-so-hexopyranosides

enzymic

rhe protona-

icater or &cerol.

the overall

when

’ in

ring.

2-deo? y-D-arubitto-hexose

exchange

ha\c

probably

a + b), the ques[lon

in B). In carlb?r, qualitatwe

‘-deo\y-D-/~,xo-he~o~~

or I-yl

of ihe enzyme’

(e.g.,

acceptor

configuration’-‘.

cis (as in A) or tratts

that

reactions reaction

or not and, if ir is, whether

uon occurs from abobe or below the plane of the pyranoid 3 product

of waler

I -deouy@lycerol-

A% the

group

of a 2-deoxy@lycosyl

the formarion

the protowtion

Ihe addltlon and

Correspondins

respectively. and

fi-D-glucosidass

in rhe vicinity

’ *’ catalyzes

coii

2-deoxy-D-lr,ao-he\ose

gVcZ

2-dcoxy-j3-o-Irxo-hekopyranoside

order

addIt-on.

by p-tolusnesulfonic

analqsls.

to the a&con

and a cis additlon.

&‘COrd

been

_elycerol yielded

plus

By ‘H-n.m.r.

on C -3 is rranwelated

3,4,6-tri-O-acetvl-2-deo~y-~-o-~~~.~~-he~op~~ano~ide-_ tram

(1)

‘-deo\y-8-o-lr,xo-heuopyranosidc-I(S)-lf.

the from

E. Sshrowr.

74

J. En.-,ne

L.EHhIAI%N,

B.

ZlEGW

EKyrrw

I

I

_*-_o H

A

n

ki

-0.

‘\

a

product

___c

‘.

+

--$_

,,,2-- f4

l-4

I

--\i

H

l-l

l

H

DroalAcl

--

A-

Enzyme

Enzyme

b

(1

I and glycerol in order to elucidate B-D-galactosldase.

the stereochemistry

of the addition

catalyzed

Compound 1 !\as prepared by starting with isopropyl 2,3,~,6-tz~z-0-acetyi-aD-galactopyraooside-,3-d (4) plus its C-2 epimerg, namely, isopropyl 2,3,4,6-tetra-Uac~tyl-r-D-ta~opyraoosidc-‘-d (5). and applying the reaction sequence depicted. RC

P

k-si 0

0

OR

4 =

8-l-S

=I3“7

+

Oc$l.2~

(R

0

RO

OR

OCt.ler

H

D

I 11)

5

HBr/AcOH

tr? 1 Z”/ALOH

AC)

131 MeONa

(1)

MeGtia

12)

IR-lXdH+)

(51

AqO

I

CHaOR

Ra+OR

+

‘O&OR

D

OR 6R= BK

=

R”G

R’

H

7R=

H

1

AC

SR=

AC

2R=

R=

1OR

H,R’=

D

R’=H =

Ac,R’=

D

by

iWDITION OF GLYCEROL TO D-GALACTAL

75

Glycerol was cnzymiwlly added to 1, and the resulting, crude glycoside I -yl Z-deovy-8-o

was isolated by preparative by acetylating the crude giycoside 11, and separating the acetylated product by preparative t.l.c. The 2,3-dl-U-acetyl-I-deo.uyglycerol-l-y1 3,4,6-tri-0-acetyl-2-d~o~~-~-o-/~.~~-he~op~~3noside-~(~)-~ (12) so obtained was uniform according to analysis by g.1.c. Nonlabelled 12 (13) w3s 1. Comparison of the corresponding sections of the ‘H-o.m.r. spectrn of 12 and 13 (see Fig. I) clearly indicated thar. in compounds 1 and 2, the proton added through enzymic reaction occupies the equatorial position. Thus, it is proved that alternative pathway B is correct, and that the enzyme-catalyzed

t.1.c. Final purification

was achieved

spcz~m of 11 and 13 In chlorofarm-d.

Fig. 1. Partrd. 270-hli-iz.

‘H-n.m.r.

Fig. 2. Partul, 270-MHz.

‘l-f-n.m r. spectra of I4 and 15 In chloroform-d.

J. LEHhWNN,

76 reaction

of glycerol,

finding

corresponds

experimental rirher

and, most probably, quell with

approach,

In order to exclude

of water also, with 2 is a ~rans addition.

the results

showed

bj P-o-glucosidase

that

obtained

the reaction

or a-D-glucosidsss

the possibilily

by Hehre’,

who,

of ~-gl~cal

(Canfi’i&

with

rrupicafis),

of a substrate-induced,

chjrd

by acid-catalyzed

sdditrdn

14 ~vns compared

of pheiol

uolabellcd

acctyl-2-deouy-~-~-l~~ro-l~euopyraooside’” (see Fig. 2) indicated addition

protonation

results

presence, reacrlon

of these esperimenls

at the binding

Genm71

sample

Secliocs

of almost

of

equal

(10).

of phenyl the

Com-

3,3,6-tri-0-

‘H-n.m.r.

amounts

f,hlercb),

spectra

of cix- and frans-

-

Opttcal

points

(b.p.

data

Fourier-transform

Boehringer at 40’

in 0.1~

chloride

for

and

The

the

I paper of 3%

v,ith

6:4:3

of SE 52 on

gas. and flame-ionization

hle,Si)

obtained

\ierz

with

a

Vatian

stated.

E.xhrriclriu coli was purchased

the

suspension

purification.

brlffer

reaction

\\a

(7). -

All

(pH

6.8)

with

complete

syrup was dissolved

(K’)

A mixture

ranoside-_ 3-d (4)

(_s) NTIS stirred

reG&.tal IR-120

lsith sulfuric

as the carrier

and

phosphate

141

silica gel FZjA

(S m&ml;

enzyme that

specific

reacrions

contained

!!ere

IO-%i

IO- ‘M 2-mercsptoethanol.

f-IY-D-g3kKtOpJ

D-talop~,rsnosrdo-_$-t19 IVhen

sodium

on

No.

in glass columns

from

further

a Perkin-Elmer

by charrir.g

unless othenhise

Germany),

(6) aruf D-t&w-3-d

?.?.~,6-re[ra-0-acet~

rrith Ambrrlirc

group4

benzsz-mzthanol: B. 4:i D, ether: and E, 4:l

on Whatman

internal

\b;is used \\ithout

o-Go&low-3-d

evaporated.

the addition

actrlating.

ulth

itas effected

$-D-Galactosidase*

(hlannheim.

30 Lilmg)

Derection

spectrometer

-

.-I. I:I

(v/v):

win g nitrosen

I!7&hlHz,

(100 ml).

an

was performed

acetate-3-propsnol-water:

(\a1 conducted

(CHCI,-(/,

Eu_r:lw rearriorrr.

T.l.c.

was performed

G AIV-DhlCS, N.m.r.

napneGum

60-70’).

G.1.c.

detecuon.

performed

that can trlsper

or even

iterr: measured

systemj

ethyl

chromatography

butanol-pyridine-crater.

rotations

jol\ent

C. 25.13.7

petroleum

Chromosorb

of a group

3s ;1 binding,

are uncorrected.

\sirh tbc followng

benzenz-methanol: Paper

seves

to su,ogcst the

stem

at C-2.

blelting

ether-light

ilrith P-D-gaktosidase

site of the enzyme,

m~tl~ocis.

pofarimeter.

from

(15).

11 is a mixture

to 2. and that normally

the substltuent

actr\*iry

of C-2 in

was synthesized

products.

The

acid.

that

catalyzed

water,

is a Irons addition.

to 3,4.6-tn’-c-s~tyl-D-,oalactal-2-d

wirh an authenlic.

This

by a di!ierent,

2, phenvl 3,4,6-rri-O-acetvl-3-deouv-~-o-/~~.~~-hcsop~ranoside-~-d(ll) pound

B. ZiEGER

and

M sodium (t.l.c.,

methoxide sollent

in water,

resin for 3 days at 90-95.

(25.3 g) of isopropyl

isopropyl A),

tetrs-O-acetyl-rin abs. thz

and the solution (t.l.c.,

solvent

methanol

solution

\ias

was treated

A). Neutraliza-

ADDITION

tion

OF GLYCEROL

\\ith sodium

as a syrup,

hydroxide,

identified

77

TO D-GALACTAL

filtration,

and Iyophilization

A mixture

-

with ’ ’ sodium poured

into

of 6 and 7 (9.25 g) \ras cotxcrted

acetate (4 .2 g) and acetic anhydride ice-water

(200 ml).

stirring.

ibith

and

with

\\ater and ice-colcl ether.

as colorless

m.p.

141’ ; n.m.r.

nesdks, (m,

2 H. H-3.4),

a mixture resulting

the solid

was

Compound

data (CDCI,.

5.72-5.35

into

(,4S g). The

washed successively 4AO-4.90

6 plus 7 (9.25 g)

I I-/3-D-rc;lop~ fan092

2-d (8) atrdpettru-0-acef

Petlia-0-acet~‘l-8-D-ga~aclop~~ranose2-d (9).

yielded

by g.1.c. and paper chromatography.

-CH,-).

filtered

NZS

otT, and

8 (6 g) was obtained

60 hlHz):

(m, 3 H, H-5,

of 8 and 9 solution

T

4.16 (bs. I H. H-l),

and 7.75-8.10

(m,

15 H.

-OCOCH,). The wre

filtrate

~$35 extracted

combined,

with

dichloromethane

\iashed

~UCC~SSIV~~~

~lth

(3 x 100 ml) and water (I x 100 ml), dried (hl$O,), (8.5 g) as a crude

oil \\hich

with -KF,o HBr;glnc.

(m.

4.37-I

I5H.

95 (m.

IO give 9

-

A mixture

bromides’

2 H.

of 8 and

5.5~6.20

from silica gel \ilth

60 hlHz): (m.

3 H,

p) was

reduced ’ 3 ~irh ztnc (30 g) in

‘. \i hlch liaj

data (CDCI,,

H-3.4).

9 (l-l.5

of the corresponding

T

H-5.

3.33

(d.

-CH?-).

I H,

E

jol\ent H-l.

J,.,

and 7.55-8.10

-OCOCH,).

( 5). -

D-Ga/acta/-2-d sodrum

metholide

(sollent

.-I). The

filtered.

Solvent

(309

(IO).

i0 (3.5 2. ~X’?O); n.m.r.

I.7 Hz).

the extracts

itt motto,

and aaporated

acetic acid (160 ml) to give 10 as a crude oil. Elution

afforded

and

hbdrogencarbo-arc

acetic acid (20 ml) into a mixture

tctra-O-acctyl-r-D-he\opyr3nosgl 5Pb

jodlum

was not purified.

3,~,6-Tri-O-ac~/~,l-D-ga/ac/~/-~-d con\crted

(4 x 50 ml),

aqueous

mg. 939,“).

10 (575 mg)

Compound

in abs. methanol

(20 ml).

solution

was made

removal

left an oil lrhich

m.p.

neutral

~lth

dsacctylated

\%a

the reaction

being

Amberlite

crysrallized

from

\\ith

monltored

IR-1’0

(H’)

ethyl

acetate

0.1~

by t.1.c. resin, and to give

1

103-10-I‘.

~,3-Di-O-acer~~~-~-~i~o~~g~l~cero~-i-_~~i 3.~.6-iri-0-aC~f~~i-~-~~10.~~1-~-D-l~~O-~l~.~Op~m.utoside (13). - j3-D-Galactosidase (0.5 ml) \\as added to a solution of 2 (0.5 g) and glycerol

(5 ml) tn buffer (ZOO ml). .Aiw

(monitored

by t.1.c. in solvent

Iyophilized.

Cornpound

chromatography

@l.c.:

(3 ml) in pyrldine addition

solLent

~a< heated

as a ycllo~

C):

it itas

2-I h, the mixture

and evaporation

from

oil after repeated

then

scetglated residue

prsparati\e-layer

with

acetic

ttz racuo.

was evaporated

the oily

disappeared

to 90‘. tilrcred.cooled.and

left a product

anh>dridc Repeated which

~‘3s

D) to gl\c 13 (106 mg); n.m.r. dais: T 4.i-i (m. I H. H-lc. J,,_,,3 5.0, (m, ! H. H-3a. .135,3rr 12 Hz, JJd,:* 5.5 HA). 5.U(m.

1 H,

purified H-l),

6 was obtained

(3 m!). After

oi toluene

I4 days at -IO’. 2 had almost

C). The solution

on p.l c. plates (solvent

J ,cl,lo 8 Hz), 6.11 (m,

I H, H-S),

and 7.35-5.05

(m.

Hz.

IS H, -OCOCH,).

2,3-Di-0-acetvl-I -dco.vy-D-g/_weroII-~*/ 3,~,6-tri-O-acef_~,l-,‘-dt10.~~.-P-D-I~ \ohexop~,ranosid=--2-d (12). - Compound 12 was prepared and isolated as forcompound 13, by (I.5 quent

use

ml),

of

1 (309 mg),

temperature

ncetylation,

40’.

glycerol lncubnrlon

12 (73 mg):

n.m.r.

(3 ml)

in

butTer

time:

7 dabs,

data:

~4.74

(I20

ml),

affordins (m.

I H.

P-D-galactosidsse 11. and.

H-4).

5.0’

by subs+ (m,

I H,

78

J.

LEHhlANN,

B. ZIEGBR

H-30, Jsm 5 Hz, Jwe 3 Hz), 5.44 (m, 1 H, H-la, J,.,21 2.5 Hz, J,,2 4.3 Hz), 6.21 (m, 1 H, H-5), 8.03 (m, I H, H-2), and 7.85-8.0 (m. 15 H, -OCOCH3). Both 12 and 13 could be deacetylated Hith M sodium methoxide in abs. methanol to gi\e chromatographically homogeneous 3) and 11, respectively. PhenJd 3,4,6-tri-O-acet~~I-Z-deo.~~~-ar-D-lyxo-hexop~~ranoside-2-d (14). The synthesis of IS was performed according to a modified method by WaUenfels and acid (0.05 g) Lehmann lo. Part (75 mp) of a melt of phenol (I g) and p-tolueacsulfonic was heated with 10 (70 mg) for I 5 set OF a water bath (60”) with vigorous shaking; the melt became dark-colored. (The mivturz must not be allowed to get black.) Ice-cold, aqueous sodium hydrogencarbonate (50 ml) and chloroform (30 ml) were then added; the aqueous layer was extracted several times with chloroform. The extracts were combined. dashed ~iith Hater (2 x 50 ml), dried (higSOJ, and evaporated in racuo, to give 1-l as an 011. Cqstsllization from ethanol yielded 14 (55 mg. 5S%) in chromatogaphically pure form, m.p. 127’; n.m.r. data: T 2.79 (m, 5 H, -Ph-I-F), 4.24 (m, I H. H-I. J, ,2s I Hz, J, ,2rr3.5 Hz). 4.48 (m, I H, H-3, J,,, 3.0 Hz), 4.60 (m, 1 H, H-4), 5.71 (m, 1 H, H-5). 5.9-I (m. 2 H, -CH,-), 7.74 (m. I H, H-2x1, Jzo,Le 13 Hz, Jza,, 12.5 Hz). 7.89 (m, I H, H-S-, JZr,3 5.5 Hz. J2+,J I Hz), 7.S-1 (s, 3 H, -OCOCH,),

7.97 (s, 3 H. -OCOCH,),

and 8.07 (s, 3 H, -OCOCH,).

ACKNOWLEDGXIENTS

We rhank Prof. Dr. K. \\‘alfenfe!s

the Deutxhe

for a sample of pure P-D-gaiactosidase,

and

for financial support.

Forschungsgemrinschaft

REFERENCES 1

J.

LEHMAhN

AND

E.

SCHRoTER.

Curbohbdr.

Rex.

23 (1972) 359-365.

\~‘ALLE%FELS END G. KURZ. unpublished results. 3 E. J. HEHXE. .4bjfr. Inr. Sump. Curbo/r)dr. Chm?., I’llfh, hiadison. ll’iscotuin, 4 hl. BROCKHAUS WWD J. LEHVMN, Corboh,dr. Ra., 53 (1977) 7-l-31.

:! K.

5 K. \3’ALLEhFEm

6 K.

AND

0.

P.

~~ALHOTRA. En:.vmes,

\V%LLENFEIS AND 0. P. hf,aLHomRA, W~LLENFELS AhD G. Kmz. &o&m.

(1974) 1.58.

(2nd edn.) 4 (1960) 409<30.

Adv. Curbohrdr. Ch*m.. 16 (1961) Z., 335 (1961) 559-572.

239-295. 7 K. 5 E. J. HEHRE, personal communicstlon. 3 R. Il. LEWIEL~Y, R. A. EARL. K. JWES, AND T. L. NAGABHUSHAW, Can. J. Cncm., 51 (1373) 19-26. ill K. ~‘ALLEF.FELS AND J LEHM~P.N, _/ustus Lirbiqz Ann Chrm. 635 (1960) 166-177. 11 hl. L. WOLFROM AND A. THOWSO%, AifefhtidJ Carbohrdr. Chum.. 2 (1963) 21 I-215. 12 R U. LEWIEUS. *Veerhods Curboh@. Chem.. 2 (1963) 221-222. !3 P. A. LEVENE \hD R. S. TIPSON, 1. Bol. Chern.. 93 (1931) 631-64-t; F. SHAFIZADEH, Merhods

Carbokt.dr.

Ckrv.,

2 (1963) 409410