Fluorescent reagents for the labeling of glycoconjugates in solution and on cell surfaces

Vol.

92, No.

February

BIOCHEMICAL

4, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

27, 1980

FLUORESCENT

REAGENTS

FOR

SOLUTION Meir Dept.

Wilchck,

of Biophysics, The *Nematology,

Received

January

THE

LABELING

OF GLYCOCONJUGATES

AND ON CELL

Sarah

Spiegel

Weizmann Dept.

IN

SURFACES

and

Institute ARO, The

1215-1222

Yitzhak

Spiegel*

of Science, Rehovot, \Tolcani Center, Bet

Israel Dagan

and

14,198O

SUMMARY: Rhodamine and fluoresceine containing hydrazides were synthesized and used for fluorescent labeling of glycoconjugates on cell surface or in solution. The procedure involves the oxidation of the glycoconjugates with sodium metaperiodate or galactose oxidase to form an aldehyde group which reacts with the respective hydrazides. The method was applied for the modification of cell surface sialic acid and galactose residues on thymocytes and nematodes as well as for the labeling of glycoproteins and gangliosides in solution. The many possible application of these highly fluorescent compounds in the study of cell surface events is considered. INTRODUCTION In

order

to

better

biological

specificity,

components

could

where few

several

carbohydrates

that

(2,3), mild acid

groups

formed

biotinyl containing

to

introduction

are

the

both

of

which

yield

an

aldehyde

further

these

groups

for

communication and these

the highly

use

The

results

group

(2).

with

in

describe the

fluorescent

study

cell

best

known the

(5). surface

markers

there

are

reactions

has

been

the

ver? for NaB’Hh

shown

modification

compounds

of

aldehyde including

The

modified

events

by

cells affinity

(4,5:).

synthesis

periodate

It

utilized of

proteins

periodate

selective

stimulation the

residue

and

We have

a variety

to

acid

(I]

hydrazides to

determining

contrast

materials.

cells

used

of

amino

radioactive

lymphocyte we

In

- NaB3H,

dinitrophenyl

were

and

each

in

carbohydrate-containing

carbohydrates.

reaction and

of

for

require of

carbohydrates

importance.

oxidase

treatment

(4)

of

of

of

labeling

available

galactose

hydrazide

hydrazides

for

role

practical

labeling

(6,7) this

great are

the

for

cytochemistry

sccine

of

periodate

sialic

In

be

for

the

methods

reagents

reagents

method

understand

of oxidation into

rhodamine method carbohydrate

and for

fluorethe

moieties

on

Vol.

92, No.

cell

4, 1980

surface

used for

BIOCHEMICAL

and in solution.

probing

the

dynamics

These of cell

AND

BIOPHYSICAL

fluorescent surfaces

RESEARCH

glycoconjugates

COMMUNICATIONS

are being

by means of fluorescence

microscopy.

MATERIALS AND METHODS Fluorescenine isothiocyanate, Rhodamine isothiocyanate, bovine brain gangliosides and thyroglobulin were from Sigma Chemical Corp. Lissamine rhodamine B sulfonylchloride (Eastmen), Neuraminidase from Vibrio Comma and galactose-oxidase were from Behringwerke AG. Soybean agglutinin was from MilesYeda. Thymocytes from male Wistar rats were isolated as described in (8). Nematodes Tylenchulus semipenetrans were collected from infected citrus roots. Cells, ganglioside and thyroglobulin were oxidized by sodium periodate as described in (8)) Soybean agglutinin oxidation was described in (3). Chromatographs were performed on thin layer (TLC) using silica gels plates in methanol: ethylacetate (1: 1). Synthesis. hydrazide-B-alanine t-Boc-hydrazide carbobenzyloxy methanol.

The reagents were synthesized as described in Fig. 1. t-Bocwas prepared by coupling carbobenzyloxy-B-alanine with in the presence of dicyclohexylcabodiimide in ethylacetate. group was removed by reduction with Paladium on charcoal in

The

Fluoresceine-B-alanine hydrazide (I) was prepared by coupling fluorcsceine isothiocyanate with two fold excess of t Boc-hydrazido-B-alanine in a bicarbonate solution (0.2 M) over night at room temperature. A solution of citric acid (10%) was added and the product precipitated. It was filtered, washed with water and dried, Rf = 0.86. The compound was dissolved in dioxanc and a solution of HCl in dioxane (3 M) was added. After 10 minutes the product which precipitated, was filtered and washed with ether. Rf = 0.21. After reaction with acetone Rf = 0.68. Rhodamine-B-alanine hydrazide (II) was prepared by the same method as described for compound (I) using rhodamine isothiocyanate. The Rf’s for the t-Boc-derivatives, the hydrazide,acetone reaction product were 0.27, 0.05 and 0.15 respectively. We also run these products in methanol alone, the Rf’s obtained were 0.52, 0.1 and 0.32 respectively. Lissamine rhodamine-B-alanine hydrazide (III) was prepared by adding lissamine rhodamine B sulfonylchloride dissolved in dimethylformanide to a two fold excess of t-Boc-hydrazido-B-alanine in a bicarbonate solution (0.2 M) and left overnight. The reaction mixture was acidified with ice cold sulfuric acid and extracted with ethylacetate. The ethylacetate was concentrated to dryness. The compound was dissolved in dioxane and treated with HCl-dioxane solution as described above. The product contained several spots on TLC, therefore it was dissolved in water and extracted with ethylacetate. The required products remained in the water solution and was isolated by freeze drying. The product contained two isomers Rf = 0.17 and Rf = 0.49. Upon reaction with acetone two new spots were obtained Rf = 0.41 and Rf = 0.75. Lissamine rhodamine-e-a-aminobutyric hydrazide (IV) was prepared by coupling the sulfonylchloride with two fold excess of a-aminobutyric acid, overnight. The solution was acidified with hydrochloric acid. The precipitate was collected and dissolved in methanol followed by excess thionylchloride. After 24 h the solution was concentrated to dryness, dissolved in methanol and reacted with excess hydrazine overnight. After removal of the methanol water was added and the mixture was adsorbed on a Poropac Q column and eluted with ethanol. The ethanol was concentrated to dryness, ether was added and the Rf = 0.5. Upon reaction with acetone Rf = 0.73. product precipitated.

1216

Vol.

92,

No.

BIOCHEMICAL

4, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

0 N(C2H5)2 COOH

NHtNHCH2CH2CONHNH2

S02NHCH2CH2CONHNH2 III

S%NHCH2CH2CH2CONHNH2

(II)

m) I I:lc.

1. General

method

for

the

synthesis

of

reagents.

Attachment of the fluorescent labels to cells and nematodes. Rat thymocytes or nematodes were treated with periodate (1 til) for 30 min in ice, centrifuged, washed and resuspended in PBS containing (0.5 mg/ml) of rhodamine labeled reagents (III or IV) for 30 min at room temperature. Extensively washed Fuspensions were mounted on slide and examined using a Zeiss microscope modified for epifluorescence microscopy. Photographs were taken with a Pentax camera. Treatment with enzymes PBS (free of Ca?’ and b!g’+) suspended nematodes cells were treated with galactose oxidase (5 units/ml) for 30 minutes at After incubation, the nematodes or cells were washed with 0.2 Y D-galactose, three times wjth PBS and treated with the reagents as described previously. Ncuraminidase (50 units/ml) pretreatment was performed in medium containing 0.145 M NaCl, 0.003 bl CaC12 and 0.004 M NaHC03, pII 6.5 at 37°C.

or 37°C.

RESULTS Synthesis

of

fluoresccine were

as

to examined

separate. by

periodate-oxidized compound

described

loose

in

dihydrazide its

its

Fig. from

fluorescence.

I 1.

reaction

hydrazine

This

preparation

interaction

F, 11 This

the

direct

with

lymphocytes.

synthesize

of

The

isothiocyanate

difficult

groups

reagents.

respective dihyrazide

2).

rhodamine

acetone of

These approach

these

isothiocyanate may

1217

on

chosen

with

thin

layer

cause

plates

may

he

cross-linking

or

decided

prepared, avoid

which

aldehyde

we

to

and

products

poorly

were

which also

of

difficulties

compounds was

isothiocyanate

a mixture

reacted

Because

indirect

The

gave

with

(Fig.

of

to

as the

formation

insoluble

and of

two

with

Vol.

92, No.

4, 1980

BIOCHEMICAL

AND

Z-NHCH$b-COOH+NH2NH-Boc

BIOPHYSICAL

COMMUNICATIONS

i

DCC

-

RESEARCH

Z-NHCH2CH2CNHNH-Boc

I

Hz/ Pd

Dye,

0 II NH2CH2CH2CNHNH-Boc

.so;?C’ NCS

-Y Dye-NHCH2CH&ONHNH-Boc Dioxan

1 Dye-NHCH$H$ONHNH2

Z=~H&H$COBoc= t- butoxy

corbonyl-

Dye= Rhodamine

aldehyde

groups

results.

B-Alanine

Edman

is

has

these

been

shown

through

the

compounds

cell

under

evenly

all

over

modified cells

micros

reagent

no

However,

it

I Highly

membrane cells

were

before

or

treated after

may

found

with treatment

surface

of

order

the

to

the

prevent

microscope,

binding galactose

of

fluorescence

was

some reacted

oxidase neurominidase

1218

as

these

these

and

form (Fig.

highly

fluore-

distributed nematodes,

fluorescence

inside

with

111

reagent

a video

was

penetrate also

aldehyde 3).

the and

IV.

intensification

reagents

reagents to

non-permeable

(thymocytes,

became

such even

cells.

lissamine-rhodamine

cells

Lymphocytes

when

that

the

surface

contained

observed

and

the

moiety.

cell

3).

into

acid

treated

The

also

found

slightly

acid

(Fig.

II,

with

in

sulfonic

periodate

I-IV

sensitive

efficient

chain

synthesized

microscope.

be

interpretation

penetrate

containing

When

was

well

to

a sulfonic

I and

a more

hamper side

We therefore

fluorescence

with cope,

were

reagents

cell

thus

I-IV.

acid.

surfaces.

the

with but

a-amino

a fluorescence

reagents

possible

contain

with

of

and

shortest

compounds

cell

reacted

scence

surface

reagents

IV which

of

nematodes)

cell

membranes.

III,

Labeling

2.

an

that

Structure

I:i!:.

the

of

of

B. Fluoresceine

the

degradation Both

It

on

HCI

observed groups

The

the

reagents

plasma when

either did

not

Vol.

92, No.

Fig.

3.

react

with

dase

alone.

non-oxidized

with

gangliosides

can

subjected

bovine

reaction

with

upon

merely

Reaction modify

sodium Using

in

3).

[311]borohydride the

RESEARCH

conditions

The

COMMUNICATIONS

modified

ganglioside

cell

surface

or

The

other to

described

sugar

introduce above

same

conditions

by

modified

ganglio-

were

fluorescent,

the

surfaces

became

was

observed

between

cells

before were

we have

the

gangliosides

neuromini-

oxidized

followed of

fluorescence

with

NaI04

oxidation

other

gangliosides

glycoproteins. acid

or

treated

that

structure

modified

in

to

the

general

No difference

the

were

shown

periodate

lymphocytes

adsorbed

sialic

Using to

4.

which

we have

The

Fig.

that

with

cells

(8).

reagents.

incorporated

not

BIOPHYSICAL

Recently

with

indicating

with

ganglioside

shown

(Fig.

trypsin

or

hydrazides

four

incubation

containing

to

hind brain

is

fluorescent

and

cells,

glycolipids,

all

formed

and

AND

Surface fluorescence pattern of rat thymocytes and nematodes reacted liith rhodamine hydrazides. (A) Nemotadoes treated with galactose oxidase followed by reagent III. (B) Nematodes reacted with Periodate followed by reagent IV’. CC) Rat thymocyte treated with periodate tollowcd by reagent III. (U) Rat thymocyte containing incorporated rhodaminyl ganglioside (reagent IL’)

Reaction

side

BIOCHEMICAL

4, 1980

or

after

cells

treatment

with

incorporated

into

the

membrane

of

with

glycoproteins

proteins. reaction moieties,

periodate followeed

radioactive we were

1219

markers able

to

couple

by

treatment

with

is

well

known

the

fluorescent

(2,3).

Vol.

92, No.

4, 1980

BIOCHEMICAL

IPi:.

markers

to

sialic

rhodamine

was

structure

acid

residues

thyroglobulin

J.

and

18

added.

When

longer

exposure

were

fluorescent

acid,

e.g.

more

soybean

introduced

the

even

agglutinin.

into

the

by

et

glycoproteins

of

of

al

(3) do

latter

the

we not

reaction

oxidation (10

8 fluorescent

the

in The

oxidation

which

4 to

residues

amount

NaIOL,

13

introduced

precipitate.

the

Lotan

Up to be

to

on of

Between

mannose

could

depended

described into

gangliosides.

started

conditions

as

COMMUNICATIONS

thyroglobulin.

protein and

RESEARCH

modified

residues

drastic

used

labels

rodamIne

fluoresceine

controlable

reagent

BIOPHYSICAL

glycoprotein

before

efficient,

the

of

containing

molecule

very

AND

and mJ$

or

could

introduce

contain

sialic

chromophores

were

glycoprotein.

DISCUSSION The

function

dependent

of

on

their

structure.

It

is

residues

vectorially.

should

possess

penetrate tions labels the

reagents group enzymatic

to

are on

their

very

has

cell.

of

they

are

these

suggested (9)

and The

b)

It

should

It

should

latter

and interact

a)

react

be

treatment.

1220

It

under with

generated

than which

a good

cell

should under

easily They

only can

reagents

react be

glycolipids

membranes

that

requirements.

charged

specific surface.

c)

bonds,

in

develop

properties:

the

most

and

assembly to

been

following into

since

cells,

and important

It

stable

comply

glycoproteins

organization

the

form

surface

therefore

easily and

cell

be

not

very

by

on

their

surface small

more primary

label

these

reagent and

not

physiological

condiOur

penetrate mild

cells

be

can

detectable. do

may

which mild

fluorescent easily

conditions. contain chemical

into The

aldehyde or

Vol.

92, No.

Due being

BIOCHEMICAL

4, 1980

to

the

highly

as

markers

used

studies

during

also

very

and/or

fluorescent for

in

glycoproteins

We have

with

the

recently into

cell

trangliosides b

lvere

stimulated

could to

also form

cause

patches

capping

is

anti

follow

these

the

is

described does

not

Current

mentary

the

amino

method

for

fluorescent

proteins

such

as

the

the

staining

of

the

specific

isolation

the

carbohvdrate membrane

the

antibodies the

the

observed

caused

by of

cell

surface

patching the

the

and

fluorescented gangliosides

described

caused

with ‘The

by

here,

antibodies

to

the

method

glycoproteins

extended of is

1221

is

a simple

and

In

addition

to

to

other

the

being gels,

tryptic

digests

as

supply the

glyco-

fluorescent

currently

on polyacrylamide from

required1

isothiocyanates,

glycoproteins. he

not

proteins

provides

Introduction

‘:lycoprotein.s.

is

gangliosides.

respective

can

sialoglycopcptides

glycolipids

fluorescent-labeled

present of

of

synthesis

of

the

modification

of

total

structure

of

glycoproteins of

‘The on

flow

be

incorporated

modification

(i.e.

hormones.

moieties

can

gangliosides

chemical

labeling

gonadotropic

into

that

reaction

the

the

gangliosidc

the

glycolipids

preparation).

group.

here,

the (8).

that

follow

preparation

via

the

described

ganglioside

modification

fies

proteins

of

change

for

the

are

processes.

DNP-ganglioside

assume

not

reagents

ganglioside

fluoresceinated

note

sinnificantly

accomplished modi

of

flow

a simple

methods

h~~cnernllv which

is

of

fate

currently perturbation

the

containing

the

cell These

recycling

only

could

in

to

and

for

are

ligands.

IJNP antibodies

of

tie

(manuscript

here

anti

incorporated

worthwhile

flow and

We could

the

other

COMMUNlCATlONS

they

and

Thymocytes

movement

directly

markers

Jt

of

reagents

dinitrophenyl-modified

with

since

Using

WC can

that

caps.

a result

directly.

and

and

these

and

membrane

redistribution

antibodies,

UN1

of

membranes.

the

RESEARCH

fractionation,

endocytosis

shown

incorporated

of

mitogens

study

during

BIOPHYSICAL

nature

subcellular

interaction

useful

AND

labels used well of

for as

for

Vol.

92, No.

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

REFERENCES 1. Gahmberg, C. G ., and Hakamori, S. (1973) J. Biol. Chem. 5, 4311-4317. G. (1971) J. Biol. Chem. 246, 1889-1894. 2. Van Lenten. , L., and Ashwell, 3. Lotan, R., Debray, H., Cacan, M., Cacan, R., and Sharon, N. (1975) J. Biol. Chem. 250, 1955-1957. 4. Wynne, D., Wilchek, M., and Novogrodsky, A. (1976) Biochem.Biophys.Res. Commun. 68, 730-739. 5. Ravid, A., Novogrodsky, A., and Wilchek, M. (1978) Eur.J.Immunol. 8,289-294 6. Bayer, E.A., Wilchek, M., and Skutelsky, E. (1976) FEBS Lett. 68, 240-244. 7. Bayer, E.A., Skutelsky, E., and Wilchek, M. (1979) Method in Enzymology 62, 308-315. M. (1979) Proc.Nat.Acad.Sci.U.S. 8. ‘Sr;iegel, S., Ravid, A., and Wilchek, 2, 5277-5281. Biophys. Acta. @, 390-399. 9. Fladdy, A.H. (1964) Biochim.

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