Fluorescent enkephalin derivatives with biological activity

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 300-305

Vol. 83, No. 1, 1978 July 14, 1978 -

FLUORESCENT ENKEH-IALIN DERIVATIVES WITH BIOLOGICAL ACTIVITY M.C. FOURNIE-ZALUSKI,

G. GACEL, B.P.

ROQUES

Dgpartement de Chimie Organique Universi te’ Rene’Descartes 4, avenue de Z’Observatoire 75270 Paris, France B. SENAULT,

J.M.

LECOMTE

Centre de Recherche Lebrun 5, rue de Liibeck 75016 Paris, France B. MALFROY, J.P.

SWERTS and J.C.

SCHWARTZ

Unite’ 209 de NeurobioZogie Centre Paul Broca de 1 ‘I.N.S.E.R.M. Zter, rme d’Al&ia 75014 Paris, France Received

March

30,1978

SUMMARY: Fluorescent dansylated derivatives of enkephatins were prepared, with the dansy2 group either at the N- or C-term&a2 part of methionine-enkephaZin (Met-El or its peptidase-resistant anaZogue D-A&2-Met-E. An energy transfer, from the Tyr to the dansyZ group, was found for aZZ compoundsand spectra2 properties were dependent upon the environment pozarity. Met-E-(CH2)2 dansyZ II and D-Ala2Met-E-(CH2)2 dansyi! III exhibit bioLogicaL activities on guinea-pig ileum and binding affinities on striatu2 membranes, simiZar to Met-E. III exhibits significant anaZgesic activity in tie after i.v. administration (about 35 % that of morphine). Fluorescent enkephalins potentiaZZy represent useful probes for the exploration of opiate receptors, studies of Zigand-receptor interactions and evaluation of enkephatins cleaving peptidases in various tissues. Fluorescent mental

tools

derivatives for

ligand-receptor perties

of enkephalins

the visualization interactions.

of such compounds.

potentially

of opiate We report

receptors

here

The dansyl,

represent

useful

and .the exploration

the synthesis

as a consequence

transfer vely

with

of ligand-receptor

tyrosine

easy covalent The dansyl

(or

tryptophane)

attachment group

The fluorescence brain

medium.

has been

barrier

introduced

of all

these

the ability

was investigated

by studies

00z91x,78,083lr0300$01.00,0 0 1978

by Academic in any

of reproduction

it

allows

ii) (3)

changes particu-

the good iii)

its

energy relati-

Press, Inc.

formreserved.

its

the N- or C-terminal peptidase-resistant

compounds

were

of the D-Ala2-Met-E

administration. Copyright All rights

(1,2)

at either

(Met-E) or of (D-Ala2-Met-E).

properties Finaly,

residues

pro-

sulfonyl

to peptides.

moiety of methionine-enkephalin (4), D-Ala2-methionine-enkephalin and aqueous

interaction

of

and opiate-like

I-(5-dimethylaminonaphtalene)

has been selected as fluorescent probe in view of i) the marked group, in spectral properties induced by the modifications of its environment, larly

experi-

300

of analgesic

activity

analogue

studied

in ethanol

to cross

the blood

in mice

after

BIOCHEMICAL

Vol. 83, No. 1, 1978

MATERIAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

AND METHOD.

1. Synthesis. Met-E and D-Ala2-Met-E were prepared as previously described (5,6) by liquid phase method using terbutyloxycarbonyl (t.boc) and methylesters as protecting groups and dicyclohexylacarbodiimide (DCC) for the coupling reactions. The N-dansyl Met-E I was prepared by direct reaction of one equivalent of dansyl chloride on Met-E in 95 % EtOH at 5'C for 3 hours, in presence of one equivalent of triethylamine. The solid compound obtained by evaporation in vacua was washed by acetone in order to eliminate the triethylammonium hydrochloride ; it migrated as a single spot on silicagel glass plates (Rf=0.89 in BuOH:AcOH:H20 ; 4:l:l). The position of the dansyl group was checked by 1H NMR: presence of the phenolic proton at 9.3 ppm and the sulfamidic NH at 8.8 ppm (doublet by coupling with Tyr Ho). The synthesis of N-I-(2-dansylamino) ethyl-Met-enkephalinamide (Met-E(CH2)2 dansyl) II and of N-I-(Z-dansylamino) ethyl-D-Ala2-Met-enkephalinamide (D-Ala2-Met-E-(CH2)2 dansyl) III and of N-I-(2-dansylamino pentyl-Met-enkephalinamide (Met-E-(CH2)5 dansyl) IV were performed by coupling the t.boc protected peptides with the corresponding N-aminoalkyl dansylamides in the presence of DCC, and purified by chromatography on silicagel column using CHC13:MeOH (9:l) as eluent. N-aminoalkyl dansylamides were obtained according to NILSSON et al. (7). Deprotection was performed using trifluoroacetic acid. The structure of the compounds used as trifluoroacetates, was confirmed by NMR spectroscopy and their purity was checked by thin-layer chromatography on silicagel glass plates using n-BuOH:AcOH:H20 (4:l:l) as solvent. Met-E-(CH2)2 dansyl, Rf=0.62 ; D-Ala2-Met-E-(CH2)2 dansyl, Rf=0.80 ; Met-E-(CH2)5 dansyl, Rf=0.71. 2. Fluorescence experiments. The fluorescence spectra were recorded with a differential corrected spectra unit. ethanol, water or tris-HCl buffer (pH:7.4) 3. Biological

on a Perkin Elmer MPF 44A equiped The spectra were performed in at various concentrations.

activities.

@o-id peptide activity

was measured on the guinea-pig ileum electrically stimulated at 0.15Hz with rectangular pulses of 0.5ms (8). At least 5 different concentrations of each compound (3-6 assays for each) were tested and the specificity of the inhibition of the contractions was established by reversal with naloxone. IC5G were determined from regression analysis. Relative potencies represent the ratio expressed in percent of IC50 of the peptide/IC50 of morphine on the same preparation. Leu-E was equipotent to morphine. For binding studies, Swiss mice were decapitated and their striatum homogenized in 20 volumes of tris-HCl buffer (O.O5M, pH:7.4). After two centrifugations (18Og, 5min and IOOOg, 20min) the resulting pellet was preincubated for 15min at 30°C in fractions of 100~1 containing about 15Opg proteins. The incubations were performed for 15min in the presence of 20nM 3H-Leu-Enk (41 Ci/mmol, the Radiochemical Center, Amersham), 20pM bacitracin and increasing concentrations of the various peptides. After addition of 3ml cold buffer the membranes were separated by vacuum filtration (Whatman, GF/B) and the radioactivity counted by liquid scintillation spectrometry. The IC50 of each compound was estimated from the effects of at least 6 concentrations with triplicate assays and the Ki calculated on the assumption of a competitive inhibition. Means ? S.E.M. form 3 independent experiments. Analgesic activity of III was measured on the hot plate test (9) after administration of solutions of III (1, 3 or 10 mg/kg) or morphine 0.3, 1, 3 mg/kg) to male mice weighing from 21 to 26g (7 animals a dose). RESULTS AND DISCUSSION Spectral (CH2)2

dansyl,

properties II,

are

of all

compounds

shown in figure

have been 1. The energy

301

studied transfer

and those from

of Met-Ethe Tyr

to

BIOCHEMICAL

Vol. 83, No. 1, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

e 1. Spectral properties of Met-E-fCH2)2 dansyl at 2.10-5M in ethanol. A. A sorptzon spectum. B. Fluorescence spectrum (Xexc : 27Onm). The absorption spectrum A shows two maxima at 250 and 335nrn, corresponding to the dansyl chromophore and a shoulder at 27Onmcorresponding to the Tyr residue. Upon excitation at 27Onma strong fluorescence transfer from the Tyr (donnor) to the dansy7, (acceptor) is indicated by the band at 52Gnm.

iF=-

the dansyl tation

group,

evidenced

at one of the

compounds.

The occurrence

be demonstrated spectrum

by inspection

range

As could upon

the

to ethanol

in 0.05M of Tyr

tris (X:305nm)

re 3.).

in

contractions striatal potency

it

was necessary

and 92 % for activity

found

As regard

excitation in the are depen-

a ten-fold

towards

ileum

(IO).

to evaluate decrease

increase

the blue

Both

(8)

tests

from

compounds

derivatives

(figu-

96 % for

similar

has been

results(Table

to that

evaluated

of electrically-induced of 3H-Leu-E binding of Leu-F

In agreement with previous reporta leads to a decreased, although still C-substituted compounds, it can be noted that

302

to Met-E

dansyl.

the inhibition and the inhibition

relatively

transfer intensity

N-dansyl-Met-E,

i.e.

gave

the energy in emission

as compared

: 94 % for

of the fluorescent

was of the same magnitude). tution of the enkephalins vity.

all nicely

shown).

approximately

Met-E-(CH2)5

systems,

of guinea-pig of the various

for

can also

of the compounds

derivatives

were

biological

membranes

is

by the percent

values

The opiate-like on two different

: there

the fluorescent

dansyl

was found

excitation

(not

exci-

2.).

(pH:7.4)

The following

Met-E-(CH2)2

polarity

upon

fluorescence

absorption

properties

latter

to dansyl

shows additional

to the Tyr

studies, buffer

27Ow),

Tyr

accompan .i ed by a 45nm shift

(figure

For biological

from

which

the spec t ral

intensity

of the

(Xexc.:

transfer

II,

corresponding

environment

emission

of Tyr

at 520nm of the dansyl

dansyl,

be expected,

in fluorescence water

maxima

of energy

of Met-E-(CH2)2

wavelength dent

by fluorescence

absorption

to

I> as regard (or

Met-E (II),

which N-stib&,i-

evaluable, the

the

size

actiof the

BIOCHEMICAL

Vol. 83, No. 1, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

,

10

7

0

.lO

I-‘\ '’ i\ II II '’ II \\

\ L-4 It

’\

I

\\

I II I II I

\\ \\

-5

4

\

\

A A

Figure

600

2. Changes

in

300

nm

is crucial

dansyl, II,

for

same is

true

for

dansyl,

III,

(table

decrease

both

barrier

after at

demonstrated

but

all

that these

and a modification by Pro5

it

the criteria

strong

biological Different

specially receptors

derivatives,

to that

dansyl,

Met-E-(CH2)2

of Leu-E.

The

D-Ala2-Met-E-(CH2)2 IV,

shows a large

_

significant

So, III

duration

exhibit including

of the C-terminal

III,

the blood-brain

analgesic

at

1Omg /kg

of action

being

Met5 residue

equivalent. after

basis) It

i.v.

a D-aminoacid (14,15,16)

of

this

the same potency

on a molar

properties

contain

properties

elicits

of morphine

analgesic III,

to cross

can be

injection

in position or its

in

2

replacement

(13).

Hence meet

dansyl,

by the

the

few compounds

(12).

compound,

Met-E-(CH2)5

(35 % of the activity

test,

0

nm

and activity.

administration.

the mouse hot-plate observed

properties

Img /kg

similar

enzyme-resistant contrast,

of D-Ala2-Met-E-(CH2)2 i.~.

--

350

shown by BAJUSZ et al.

an activity

tests

In

in binding

is clearly

as morphine

on both I).

\

3. ,%rksiorZ intensity Of Tyr upon excitation at 275~ for Met-E-(CHZ) dansyl (Al and Met-E (B) at the sameconcentration (4.10-5M) in 0.05M Tris-HCZ buffer.

as also

the potentially

The ability compound

activity

exhibits

\

Figure

fluorescence

properties of Met-E-(CHZ)Z dansyZ, II, induad by modification of the solvent polarity. A. II (1.3 10 5M) in water I). B. II (1.3 lO+M,, in ethanol f--- 4. Note the shift and the increased fluorescent intensity of the band corresponding to the dansyl group.

chain

\

Lk

-0

so0

\

appears

that

both

of a fluorescent

Met-E-(CH2)2 prove,

dansyl and its D-Ala2 analogue adequate spectral properties i.e.

and

activity. applications

of these probes are in progess. These compounds, and could be utilized for the visualisation of opiate derivative,

the D-Ala 2 by an histofluorescence

technique

303

similar

to that

already

developed

BIOCHEMICAL

Vol. 83, No. 1, 1978

Table I. Inhibitory on the contractions

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

potencies of Leu-en.kephaZin and fluorescent pep-tides of guinea-pig ileum and on the binding of 'H-Leu-enkephalin

by membranesfrom mouse striatum.

Guinea-pig

3

ileum

Compound ICso

Met-E-(CH2)5

50 + 11

27 2 4

86 147

10 2 3 5+2

288 2 15

13

21 +_5

dansyl

dansyl

N-dansyl-Met-E

for

8-adrenergic

moleculesin

solution

+_ 328

(17).

The fine

analysis

can provide

interesting

data

formation (18) and could be compared the energy transfer 21). In addition, the molecule the

provides

biological

bond

(22),

inactivation

in this

provides

a convenient

actions between

index

consists dansyl

cleavage

of

monitoring

mean to evaluate

in

transfer

regarding

their

such

preferred

con-

of the molecule

the hydrolysis

as a substrate in Vitpo

activity

within

by other approaches (19,20, and the dansyl groups of

the integrity

the the

of energy

provided the Tyr

primarily

can be used

and,

and perhaps

tryptophane

the probe during

to those between

50 2 25

of the Tyr-Gly

for

the peptidases

changes

of these

: since

in

fluorescence,

enzymes

and the

of inhibitors. Finally,

provide

an indirect

Met-E-(CH2)2

involved

4

1,306

receptors

5+1

100

dansyl

D-Ala2-Met-E-(CH2)2

Potency

67 2 12

Leu-E Met-E-(CH2)2

Relative

mf)

H-Leu-enkephalin binding Ki (nM)

(8,23)

of

the receptor

molecule

or between

Tyr

and dansyl

chromophores

data of

can be extended

regarding

the peptide

to the different

at the N-terminal

the analysis

residues

interesting attachment

more interestingly,

part

of

and the dansyl

the conformational to its

recognition

endorphins

which

of energy within

changes sites. contain

transfer group

the latter, possibly

of could

occuring

Such an approach only

one Tyr

residue

the peptide.

Supported by the foZZowing institutions : CNRS(ERA 6131, DGRST(Al'P Pharmacobiochimie des Peptides) and INSERM(con&at n“77.1.106.9). Wegratefully acknowzedge the skillful technical assistance of G. ClfERET and C. FERRARO. REFERENCES 1.

Weber, G., De Carlin,

Borris, S.D., De Robertis, E., Barrantes, M., Mol. Pharmacol. 1, 530-537 (1971).

304

F.,

La Torre,

J.

and

Vol. 83, No. 1,1978

2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

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305