Coupling of proteins and other amines to carbohydrate polymers via bromine oxidation and reductive amination

Carboh_v&are Research, 58 (1977) XL9-51 nc! Elseeier Sc~enuhc Pubhjhiog Company.

Amswrdam

- Printed

in Belgium

Note

Coupling of proteins and other amines to carbohydrate via bromine oxidation and reductive amination OLLE

LARM

Department

AND

ELISABETH

WHOLANDER

of Chemistry.

(Recewcd October IXh,

Polysaccharide (Sepbadex

polymers

Agriculrural

College

1976; acceplcd

polymers,

and Sepbarose,

affinity chromatography

of

Su&n.

S-TJO 07 Uppsolu 7 (SlrPi!cn)

Tar publlcarlon,

Deismber

such as cross-ilnked

respectively)

‘. Biologically

dcvtran

and cellulose, acti\,e

Xnd,

1976)

or agarose

are useful

compounds

solid

derivatives

for

supports

can be coupled

in alkaline

Polysaccharides may also be activated by periodate oxidation’, as the resulting aldehyde pouts can react with aminrs to gibe Schiff bases that can be stabilised by reduction3. media

to polysaccharide

Carbonyl

gro~lps

polymers

can

that are activated

also be generated

with

cyanogen

in polysaccharides

halides’.

by treatment

\\ith

nqueous solution’*‘. When Scphadeu. Sepharose, or cellulose were treated with aqueous bromine at pH 7.0, products !serz formed which became yellow when treated with aqueous sodium hqdrolide due to the presence of alkali-labile ketoglycosyl units6. Reduction of the bromine-ouidiscd polymers with sodium borohydride eliminated this property. Suspensions of the ovidised polysaccharidcs in \\ater at pH 6.S or in formamide under neutral conditions reacted H ith primary amines, and the resulting SchiK ‘)ascs bromine

TABLE

in

I

REDWTIWE

COLiPLWG

Polysacchoride

Sephsrose

Sephadcux

cellulose

OF

4MIXTS

TO BROWhT-OYIDIZED

POLSSUTCHARIDES

Degree of oxrdarron Corrplrng peldi (,lfof of &3rrinrol ~~~~pof.~s~cchor~~~-.-)

(UC-rghr9;) Aiburnitr

I.&Diaminohrtone

I-

0 0.25 0.50

0” l.gh 3 Sb

OC 3.s= 63C

01’

Oh

l-lb

6.gb

-

6.Eb

0 0.20 0.40

0”

-

-

1.8” 1.6b

-

-

-

-

-

-

0” I .Ob

0 -0.10

JA tenfold excess of amine was used in the coupilng rext1ons. pH 6.5. CCoupling performed in Formzmlde at nculral pH.

“Coupling

1mino&cane

performed

III

wnler a.i

250

NOTE

were

reduced

hydride’.

10 the

corresponding

The formation

formamIde

is not always

suitable

of carbonyl

groups

of reduction

pH

bzies’.‘:

at

reactions

in abater were therefore

\
the

by

hydroiysares Data

amino

for coupling in water

oxidised

smines

of C=N

are

out at room or

The

The

fair yields

specrroscopy

coupling

of products

for 3 days

degree

but

The rate

to [hat of Schiff

degradedh.

temperature

groups. n.m.r.

cyanoborosolvent,

active substances.

at pH 6.5, and

C=G

arraiysis’

acid

sodium

at pH 6-S is low compared

poiysaccharides

3nd

wirh

by the non-aqueous

biologically

performed

was carried

of reactIon

measured

gilen

> 7.

Reduction

completion

secondary

of SchifT bases is favoured

ensure

to

of substitution on

the

was

partial,

acid

I”. on ihe couplmg

in Table

of I.6-diaminohexane.

I-aminodecanc,

and albumin

3re

I’.

The method

complements

I’ for derivatising

e\istin_g methods

polyjnccharides.

EYPERIMENTAL

m-m/rods. -

Gct7~ru/

sprcrromerer.

(2.1.~.

&me-ionlssfron

N.m.r.

Spectra

performed

~3s

Jetecfor.

liiih

3

Separations

were

rscorded

Vsrian

\verc

modei

310 of OV 215 on Gas Chrom

m.s. \tas performed

kth

O.~rdutro~ or 400 ml)

addition tiere

95’.

for

adjusted (4-g

suspended

HISO,,

in water

of 0.5~1 sodium

1v:i.s consumed

CH-7 -

a/- pol~~cclrari&~~

(S g) \sere suollrn 200,

Varian

fo pH 7.0, hydroxide

in kvater. reduced

ICSh), neutralised

then

and i\ere

CI4B

suspendrd

3

(240x G.l.c.-

Sephadev

in 0.1~

pH meter). water

G-50

bromine

(100,

thereat When

(3 litres),

by the

the ovIdant and ssmplej

hydrolysed

(HOAc),

into aldirol

D

spKtrometcr.

(6 81 and

neutralised

converted

columns

maintained

Hith

HA-100 fitted with

mesh at 200’.

at 30’

E 3008

ibashed

(TusBH,).

(B&O,).

glass

Q (100-120)

rzutomatically

(hlerrohm

h), the products

a Varian

gas chromalopaph-mass Sepharose

21 h and

on

performed

0.15 cm) coniarnlng

3

\cith

2700 instrument.

(025x1

and subjected

acetates,

fo g.1.c. analysis ’ ?. The from

formation

modified

galactosyl

Sepharose

formation

indicated

from

to glucitol.

the glucos~l

modified

indicated

Cellulose

(25 g. small

product

that

oxidation

Sephadek oxidation

had

and galaclitol.

occurred

of mannitol,

allitol,

had occurred

pieces of Whatman

with 0. I M bromine

t\as slow and only a small

amount

was rhen colkctcd,

crnalysis, the formation oxidation

thar

to 3,6-snhydro@actitol

at

C-4

of

the

and galactitol,

in

at C-3.

C-3,

and C-4

rn

units.

2-I h. and then ovidrsed The

in addition

units.

The addition

of glucitnl.

Cmtplmg I ,6-diaminohexane

reaclionr.

of’ the o.tidising

icashed,

of mannitol

had occurred

3hIb! paper)

and allitol.

-

(I@ ml) adjusted

as described

in addition

(a) /,6-Diuminciiexane.

in \vatrr

above.

to glucitol,

for

The reaction

agent ~‘3s consumed

and analysed

at C-2 and C-3 in the glucosyl

(5 g) In water

u’3s swollen

(500 ml) as described

after

above.

50 h.

in this

indicated

that

units. To

a cold

(-

5”‘) solution

of

to pH 6.5 with acetic acid, oxidised

hOTE

XI

Sephaross added.

(0.5 g), or

The

mixtures

v.cre collected Samples{

Srphadek \iere

and washed

protons

arc szi\cn in Table

mamlde

acetic

IO ml).

stirred

descrlhcd

at

was calculawd

and

sodium

finally

\\lth

for

are gi\cn

O\ldiscd

sodium

60 h at room

(IO0

coupling

v~rllr! \+a5 derermlned

arc gl\sn

In Table

diicusslonj.

of human

ccllulo~e

\iaj

The

coupling

analyzed

as

products

to olldlsed

viclds

\rere

Sepharose

\+ere \\ashcd ethanol

determined

\\ith

IO”.,

(I lltrc of

as described

I. (0.5 g) itas

suspended

(I ,g). to a wlu1lon butkr 0.1 v

13 irater

of human

7. 50 ml). The

tpH

mluturr’

~a5 cc~llected. washed sodium

b\

auromaric

authors and Mr.

thank

serum

hydrogen

amino-acid

and the producr in Table

Proiewr

Rdi Andersson

supported

albumin

(1 21 and sodium

performed.

results arc given

been financial11

and

miiturz

~lth

and

added,

serum albumin \\as stirred

for

i+ater I I Iltre).

carbonare.

M sodium

analysis”.

and

the results

I.

To a solution

The

processed

Lsith ior-

The

acerntc bufT?r I pH 1
\\aler.

The

The

ivater.

0. I M sodium

rhen

(7.9 g) was coupled

in Table

(IO ml) was

prewashed

I.

a~o~c.

and the product

acid.

(0.5 g).

25. 50. 75. and 950Lv aqw;us

(I g) In phosphare accLic

35 ml). o\ldised

tube.

The results

t I g) kccrs added.

60 h. and

Sepharoje

chloride.

The reaction

( I Iltre).

signals of the

(5 21 in dry formamldc

Srpharosc

in Table

c>anoborohydr&

temperature.

aqueous

ml).

\val -r (I Ilrre)

Si\eden)

g the integrated

In I .6-diaminohe\ane’”

cvanoborohydrlde

results are gIlen

iinter

(cm) .I/brttttrtt.

aboLe.

o\idlscd

in \va[er as described

and the rewlts

togther

IO50

b) compdrln protons

!- -l~,linorf~~ca/~r. I-Amlnodecanr

acct~c acid t I lirre).

(KABI.

and

temperature

The

(0.3 p) suspended

aboLe,

the products

acetic acid ( I hrre) and water

of I .6-diaminohexanc

acid.

and

room

abole.

(h)

(I g) were

for 60 h. and

I.

with

(-lx

cyanoborohydnde

DCI t I ml) at 100‘ for I h in an n.m.r.

in cont.

and the mcrnylene

A cold ( - 5’) solurlon neutrallsed

sodium

temperature

1%ith 1036 aqueous

The degree of substitution

cxh)

91 room

IO0 ml!) \\err’ hydrolysed

csrbohydrate

was

(0.5 g), and

stirred

by KABI.

16iI0 mg) In phosphate

cvanobor~~h>drldr was processed

buffer

(500 mg’) lrerz

(pH

7.

added

and anal> jed as descrl’xd

I.

0.

Thcandcr

ior recording Stochholm.

and

Dr.

tne n.m.r.

13

Eaher ior ~~luabls

spectra.

The \\orh

has

S\\eden

REFERENCES I 2 3 -l 5 6 7 8 9 IO II 13

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