Short communication

Synthesis and pharmacological properties of

a new fluorescent opioid peptide analog

Monika Lukowiak1,2, Piotr Kosson1, Wim E. Hennink2,

Andrzej W. Lipkowski1

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Abstract:

Biphalin, is a palindromic peptide [(Tyr-D-Ala-Gly-Phe-NH-)�] in which two opioid pharmacophores are connected “tail-to-tail.”

This peptide displays a broad affinity for all opioid receptors (�, � and �) as well as exceptionally high antinociceptive activity. Pre-

vious structure-activity studies demonstrated that one of the biphalin pharmacophores could be substituted with a hydrophobic group

without significant loss of receptor affinity. This paper reports the pharmacological properties of a new analog in which one pharma-

cophore of biphalin was replaced with fluorescent 7-succinylamido-4-methyl-coumarin. The resulting compound displays an affin-

ity for � opioid receptors that is a � opioid receptor comparable to biphalin but with an affinity that is over a hundred times lower.

This � opioid selective fluorescent peptide analog could be applied in pharmacokinetic and pharmacodynamic studies of biphalin re-

lated analogs.

Key words:

biphalin, opioid receptors, fluorescence

Introduction

Biphalin is a dimeric peptide [(Tyr-D-Ala-Gly-Phe-

NH-)2] in which two enkephalin-type pharmacopho-

res are connected “tail-to-tail” by a hydrazine bridge

[7]. The affinity of biphalin for � opioid receptors is

similar to that of morphine, while the affinities to �

and � opioid receptors are significantly higher [8, 15].

As a result of a synergic interaction between opioid

receptors, the peptide induces over a thousand times

higher level of analgesia than morphine when applied

directly to structures in the central nervous system

[4–6]. Biphalin expresses a wide spectrum of addi-

tional positive properties such as minimal dependence

formation, low tolerance, and a synergic effect with

AIDS drug treatments [16] that make this compound

an excellent candidate for further development as new

analgesic drug. Structure-activity studies of biphalin

have indicated that the full palindromic sequence is

not required for high opioid potency and that one of

the “arms” of this peptide can be reduced to a hydro-

phobic amino acid [9] or a peptidomimetic [12]. This

creates the possibility for developing a series of ana-

logs in which the opioid peptide pharmacophore is

hybridized with hydrophilic components that express

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biological synergic properties [3] or/and physico-

chemical markers. Among the analogs in this series is

the compound AA2016 (Fig. 1) in which the dansyl

moiety plays a dual role since it is both a hydrophobic

element and a marker [10]. These types of molecules

that have both fluorescent properties and that are se-

lective to certain receptor types could prove to be ex-

tremely useful tools in a variety of pharmacological

studies. Fluorescent ligands can replace radioligands

in both in vitro as well as in vivo studies concerning

receptor binding, metabolism, permeability distribu-

tion, and the penetration of biological barriers includ-

ing the blood brain barrier. Therefore, the develop-

ment of new fluorescent opioid peptide analogs with

various types of receptor selectivity is one important

avenue of current structure-activity studies. Similar to

the parent molecule, biphalin, the previously synthe-

sized compound AA2016 displays a broad spectrum

of affinities for opioid receptors. This paper describes

the properties of a new compound in which the re-

placement of a fluorescence marker with aminocou-

marin resulted in an increased affinity for the � opioid

receptor type.

Materials and Methods

Synthesis of the fluorescent peptide analog

N-(Tyr-D-Ala-Gly-Phe), N’-(7-succinylamido-4-

aminocoumarin) hydrazide, AA3270

Boc-Tyr-D-Ala-Gly-Phe-NH-NH2

The Boc tetrapeptide hydrazide was synthesized by

classical Merrifield solid-phase peptide synthesis us-

ing the Boc-strategy, the Boc-amino acid and the

DCC/HOBt coupling system. The peptide was

cleaved from the resin with hydrazine following

a procedure described previously by Ohno and Anfin-

sen [13]. After evaporation of the solvent, the Boc-

tetrapeptide hydrazide was precipitated with 10% cit-

ric acid. After filtering off, the crude protected pep-

tide was crystallized from methanol.

Succinylamido-4-methyl-coumarin

An equimolar amount of N,N-diisopropylethylamine

(DIPEA) was added to a solution of 7-amino-4-me-

thylcoumarin in dimethylformamide (DMF) while

stirring, followed by the addition of an equimolar

amount of succinic anhydride. The reaction mixture

was stirred overnight at room temperature. Next, the

product was precipitated from the reaction by adding

a mixture of 1 M HCl in 5% NaCl. The product was

filtered and washed with water. The purity of the

compound was confirmed with TLC and HPLC, and

only pure product was used for subsequent experi-

mentation.

N-(Tyr-D-Ala-Gly-Phe-), N’-(7-succinylamido-4-

amino-coumarin) hydrazide, AA3270

The Boc-protected tetrapeptide hydrazide and succinyl-

amido-4-methylcoumarin were coupled overnight in

DMF using DCC/HOBt. After filtering off the formed

DCU, the product was precipitated with 5% NaHCO3

and washed with NaHCO3, H2O and 5% citric acid.

After desiccation, the N�-Boc-protecting group was

removed with 4 M HCl in acetic acid in 10 min reac-

tion. The product was precipitated with ethyl ether,

purified by preparative RP-HPLC and analyzed by

analytical HPLC and FAB-MS. The synthesis scheme

is detailed in Figure 2. The fluorescence emission

spectrum of the peptide dissolved in water was re-

corded on an Edinburgh FS 900 CDT spectrofluo-

rimeter. The absorption/emission spectrum is pre-

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sented in Figure 3. In the biological studies, the pep-

tide was used as its hydrochloride salt.

Receptor binding assays

Receptor binding affinities to the � and � opioid re-

ceptors were evaluated with competitive assays using

standard radiolabeled ligands as previously described

[2]. The radioligands used were [3H]DPDE and

[3H]DAMGO for the � and � opioid receptors, re-

spectively.

Antinociceptive assay

All experimental procedures were approved by the local

animal research committee. Adult male Sprague-Dawley

rats (225–250 g) were housed in groups of 3 rats per cage

and maintained on a 12 h light/12 h dark cycle. Animals

had free access to food and water at all times.

Intrathecal catheterization

For spinal drug administration, rats were implanted

with chronic indwelling intrathecal (it) catheters ac-

cording to a modification [3] of the method described

by Yaksh and Rudy [17]. Briefly, through an incision in

the atlanto-occipital membrane, silastic tubing was in-

serted for a distance of 7.5 cm, thereby positioning the

tip at the T13-L1 spinal level. To facilitate threading

the catheter through the intrathecal space, a stylet was

inserted into the silastic tubing. After catheter place-

ment, the stylet was removed and the catheter was se-

cured by sutures. After surgery, animals were housed

individually. Rats were given 2 days to recover from

surgery, during which time they were habituated daily

to the laboratory environment and analgesic testing ap-

paratus. Each experimental group consisted of six rats

and each rat was tested only once. All intrathecal injec-

tions were made with the same volume (10 �l followed

by 10 �l of saline for flushing the dead space of the

catheter). All drugs were dissolved in sterile 0.9% sa-

line solution. AA3270 hydrochloride was administered

at doses of 0.2, 0.5, and 1 nmol. Morphine hydrochlo-

ride (M.W. 322) was administered intrathecally to the

rats at a dose of 7.8 nmol according to the same experi-

mental protocol as an active control.

Tail-flick test

To measure thermal antinociception, a tail-flick test

was applied. Light intensity was adjusted to yield

a mean latency baseline of approximately 3.5 s with

automatic cutoff at 7 s to avoid tail damage. Analgesic

measurements were performed before drug admini-

stration and 5, 15, 30, 60 and 120 min after the ad-

ministration of either the drug or the 0.9% saline solu-

tion (control group). The degree of analgesia was ex-

pressed as a percent of the maximum possible effect

(%MPE) according to the following equation:

%MPE =posttreatment latency – baseline latency

cut -off time – baseline latency100×

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Fluorescent opioid peptide������ ������� � ��

Fig. 2. ������ �� ������� �� ����

wavelenght (nm)

Ab

so

rptio

n/e

mis

sio

nin

ten

sity

(arb

un

its)

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Data are expressed as the area under the curve (AUC)

of % MPE versus time. Data were analyzed by means

of ANOVA.

Results and Discussion

In the receptor binding assays, the AA3270 displayed

a � receptor binding affinity in the range of biphalin

and morphine. However, the compound is much more

� receptor-selective, because its affinity to the � re-

ceptor was over a hundred times lower than biphalin

and ten times lower than morphine (Tab. 1). Interest-

ingly, the previously synthesized fluorescent analog

AA2016, like biphalin, expresses an equal affinity for

� and � receptors [10] and differs only in the fluores-

cent group (Fig. 1).

Biphalin is over a thousand times more potent than

morphine after intrathecal application [5]. This po-

tency has been interpreted as being a result of the syn-

ergic interaction between � and � receptors. The

antinociceptive activity of AA3270 in the same test

showed that its activity is both naloxone-reversible

(not shown) and dose dependent (Fig. 4). However,

the antinociceptive activity was on the nanomolar

level and in the same range as that of morphine [5].

Additionally, the data obtained from binding and

antinociception experiments with AA3270 confirm

the previously presented interpretation of the high

antinociceptive activity of biphalin.

The compounds AA3270 and AA2016 are � selec-

tive fluorescent analogs of biphalin with broad affini-

ties for opioid receptors. They create a complemen-

tary pair of opioid ligands with promising applica-

tions in pharmacological and biochemical studies of

opioid peptides.

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Tab. 1. ������� �������� �� �� ����� ��� �� �������� ������

Compound Binding K� (nM)

� �

Morphine 9.8 ± 3.5 112.2 ± 6

Biphalin 1.4* 2.6*

AA2016 1.1* 2.0*

AA3270 5.1 ± 3.6 389 ± 6

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0.2 nmol

0.5 nmol

1.0 nmol

5 15 30 60 120Time (min)

100

80

60

40

20

0

%M

PE

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Acknowledgments:

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Received:

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