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Highly potent fluorescent analogues of the opioid peptide [Dmt1]DALDA
Peptides 24 (2003) 1195–1200
Highly potent fluorescent analogues of the opioid peptide [Dmt1]DALDA Irena Berezowska a , Nga N. Chung a , Carole Lemieux a , Bogumil Zelent b , Hazel H. Szeto c , Peter W. Schiller a,∗ a
Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 b Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6058, USA c Department of Pharmacology, Joan and Sanford I. Weill Medical College of Cornell University, New York, NY 10021, USA Received 18 April 2003; accepted 3 July 2003
Abstract H-Dmt-d-Arg-Phe-Lys-NH2 (Dmt = 2 ,6 -dimethyltyrosine) ([Dmt1 ]DALDA) is a highly potent and selective opioid peptide agonist capable of producing an antinociceptive effect after systemic administration. Fluorescent analogues of [Dmt1 ]DALDA containing either -dansyl-l-␣,-diaminopropionic acid [Dap(dns)] or -anthraniloyl-l-␣,-diaminopropionic acid [Dap(atn)] in place of Lys4 were synthesized. Both analogues retained subnanomolar opioid receptor binding affinity, very high opioid agonist activity in the guinea pig ileum assay and extraordinarily high antinociceptive activity in the mouse tail-flick test (intrathecal administration). The maxima of the fluorescence emission spectra recorded in Tris–HCl buffer (pH 6.6) indicated a completely aqueous environment of the fluorophore in both peptides. The high fluorescence quantum yield (ϕ = 0.358) of the [Dap(atn)4 ] analogue was particularly remarkable. These fluorescent [Dmt1 ]DALDA analogues represent valuable pharmacological tools for various applications, including studies on the binding to receptors and other biopolymers, cellular uptake and intracellular distribution, and tissue distribution. © 2003 Elsevier Inc. All rights reserved. Keywords: [Dmt1 ]DALDA; Opioid peptides; Fluorescent opioid peptide analogues; Fluorescence spectroscopy; Fluorescence quantum yield; Opioid activity profile in vitro; Antinociception
1. Introduction Structural modification of the N-terminal tetrapeptide segment of dermorphin led to a number of potent and selective opioid agonists, including the tetrapeptides H-Tyr-d-Arg-Phe-Lys-NH2 (DALDA) [16] and H-Tyr-d-Arg-Phe--Ala-NH2 [12]. Replacement of the Tyr1 residue in these tetrapeptides with 2 ,6 -dimethyltyrosine (Dmt) produced a further increase in agonist potency and receptor selectivity. Thus, the tetrapeptides H-Dmt-d-Arg-Phe-Lys-NH2 ([Dmt1 ]DALDA) [17,18] and H-Dmt-d-Arg-Phe--Ala-NH2 [13] are among the most potent and most selective opioid agonists reported to date. Like the DALDA parent, [Dmt1 ]DALDA carries a high positive net charge (3+). In the mouse tail-flick assay, [Dmt1 ]DALDA displayed extraordinary potency (ED50 = 1.2 pmol) when given intrathecally (i.t.), being 833 times more potent than morphine [23]. The duration of the antinociceptive effect of i.t. [Dmt1 ]DALDA was four times longer than that of morphine at equipotent doses [20,23] ∗
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0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2003.07.004
and, unlike morphine, it did not induce respiratory depression at a dose of 30 × ED50 [20]. Surprisingly, this compound also produced a potent antinociceptive effect (36 times more potent than morphine in the mouse tail-flick test) after subcutaneous (s.c.) administration and this effect was again of long duration (12 h at a dose of 5 × ED50 ), whereas an equipotent dose of morphine was only effective for 3 h [23]. This result indicated that [Dmt1 ]DALDA is capable of crossing the blood–brain barrier (BBB) quite effectively. To examine whether [Dmt1 ]DALDA might be taken up into cells and might cross biological barriers via a transcellular pathway, it is of interest to develop a fluorescent analogue for confocal laser scanning microscopy (CLSM) and flow cytometry studies. Fluorescent analogues of peptides or small proteins are best prepared by incorporation of relatively small fluorophores, such as the 1-dimethylaminonaphthalene-5-sulfonyl (dns = dansyl) group [9,14] or the anthraniloyl (atn) group [21]. The presence of a small fluorophore is unlikely to change the overall structural characteristics of the peptide to an extent that would greatly interfere with biological activity, whereas the attachment of larger fluorophores to relatively
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2.2. Synthesis of fluorescent α,β-diaminopropionic acid derivatives
OH O S O
X=
H N
H2N O
O N H
H N
O
N NH2
(dns)
O
O C
NH
X
HN
H2N
NH2 HN
(atn)
Fig. 1. Structural formulas of [Dmt1 ,Dap(dns)4 ]DALDA and [Dmt1 ,Dap (atn)4 ]DALDA.
small peptides may produce a substantial decrease in receptor binding affinity. Thus, incorporation of relatively large Bodipy and Alexa fluorophores into dermorphin and deltorphin-1 resulted in a significant reduction in and ␦ receptor binding affinity, respectively [1,5], and Alexa derivatives of the smaller opioid tetrapeptides TIPP and endomorphin-1 displayed receptor binding affinities that were 2–3 orders of magnitude lower than those of their respective parent peptides [1]. In the present paper, we describe the syntheses, opioid activity profiles, and fluorescence parameters of two fluorescent [Dmt1 ]DALDA analogues containing either -dansyl-l-␣,-diaminopropionic acid [Dap(dns)] or -anthraniloyl-l-␣,-diaminopropionic acid [Dap(atn)] [21] in place of the Lys4 residue (Fig. 1).
2. Materials and methods 2.1. Analytical methods Precoated plates (silica gel 60 F254 , 250 m; Merck Darmstadt, Germany) were used for ascending TLC in the following solvent systems (all v/v): (I) n-BuOH/AcOH/H2 O (4:1:1); (II) n-BuOH/pyridine/AcOH/H2 O (15:10:3:12); (III) CHCl3 /MeOH/AcOH (85:10:5). Preparative reversedphase HPLC was performed on a Vydac 218-TP1022 column (22 mm × 250 mm) with a linear gradient of 5–22% (first 20 min) and 22–28% (next 20 min) acetonitrile in 0.1% TFA at a flow rate of 13 ml/min. Analytical reversedphase HPLC was performed on a Vydac 218-TP54 column (5 mm × 250 mm) with a linear gradient of 5–30% acetonitrile in 0.1% TFA over 30 min at a flow rate of 1.5 ml/min. The same column was also used for the determination of the capacity factors K under the same conditions. Proton magnetic resonance spectra were recorded at 25 ◦ C on a Varian VXR-400S spectrometer using tetramethylsilane as internal standard. Molecular weights of compounds were determined by FAB mass spectrometry on an MS-50 HMTCTA mass spectrometer interfaced to a DS-90 data system (Dr. M. Evans, Department of Chemistry, University of Montreal).
2.2.1. N␣ -Boc-N -dansyl-l-␣,-diaminopropionic acid (Boc-Dap(dns)-OH) The synthesis of this derivative was analogous to that reported for the preparation of N␣ -benzyloxycarbonyl-Nε dansyllysine [19]. Yield: 92%. TLC Rf 0.85 (I), 0.82 (II), 0.52 (III); 1 H NMR (400 MHz, DMSO-d6 ) δ 8.55–8.50 (d, 1H), 8.38–8.32 (d, 1H), 8.18–8.10 (m, 2H), 7.75–7.62 (m, 2H), 7.38–7.35 (d, 1H), 6.98–6.95 (d, 1H), 4.10–4.02 (m, 1H), 3.55–3.50 (m, 2H), 3.20–3.08 (m, 1H), 2.95–2.90 (s, 6H), 1.42–1.38 (s, 9H); FAB-MS m/e 438. 2.2.2. N␣ -Boc-N -(N -Fmoc)-anthraniloyl-l-␣,diaminopropionic acid (Boc-Dap(atn[Fmoc])-OH) To a solution of N␣ -Boc-N -anthraniloyl-l-␣,-diaminopropionic acid [21] (300 mg, 0.928 nM) in dioxan (7 ml) aq. NaHCO3 (10%) (5 ml) was added under stirring, followed by addition of Fmoc-Cl (238 mg, 1.02 mM) in dioxan (2 ml) at 0 ◦ C. The reaction mixture was stirred overnight at r.t. and, after addition of H2 O (100 ml), was extracted with ethyl ether to remove the excess of Fmoc-Cl. The aqueous layer was acidified to pH 4.0 with aq. KHSO4 (5%) and extracted with ethyl acetate. The ethyl acetate extract was washed with aq. NaCl (saturated), dried over MgSO4 , filtered and evaporated to yield solid N␣ -Boc-N -(N -Fmoc)-anthraniloyl-l␣,-diaminopropionic acid (361 mg, 72%). TLC Rf 0.94 (I), 0.87 (II), 0.68 (III); 1 H NMR (400 MHz, DMSO-d6 ) δ 11.04–11.02 (s, 1H), 9.00–8.92 (s, 1H), 8.18–8.10 (s, 1H), 8.00–7.95 (d, 2H), 7.78–7.65 (d, 3H), 7.55–6.98 (m, 6H), 7.20–7.12 (m, 1H), 4.58–4.38 (m, 3H), 4.10–4.05 (m, 1H), 3.65–3.60 (s, 2H), 1.45–1.38 (s, 9H); FAB-MS m/e 546. 2.2.3. N␣ -Acetyl-N -dansyl-␣,-diaminopropionic acid amide (Ac-Dap(dns)-NH2 ) This compound was prepared by coupling Boc-Dap(dns)OH to a p-methylbenzhydrylamine resin, removal of the Boc group with 50% (v/v) TFA in CH2 Cl2 , acetylation with acetic anhydride and cleavage from the resin by HF/anisole treatment. TLC Rf 0.45 (I), 0.83 (II), 0.31 (III); 1 H NMR (400 MHz, DMSO-d6 ) δ 8.55–8.50 (d, 1H), 8.37–8.32 (d, 1H), 8.00–7.95 (m, 1H), 7.85–7.80 (d, 1H), 7.70–7.62 (m, 2H), 7.38–7.32 (d, 2H), 7.20 (s, 1H), 4.30–4.25 (m, 1H), 3.20–3.15 (m, 1H), 3.05–2.95 (m, 1H), 2.92–2.88 (s, 6H), 1.80–1.78 (s, 3H); FAB-MS m/e 379. 2.2.4. N␣ -Acetyl-N -anthraniloyl-␣,-diaminopropionic acid amide (Ac-Dap(atn)-NH2 ) This compound was prepared in the same manner as Ac-Dap(dns)-NH2 with Boc-Dap(atn[Fmoc])-OH as starting product. Prior to HF cleavage the Fmoc group was removed with 30% (v/v) piperidine in DMF. TLC Rf 0.38 (I), 0.77 (II), 0.17 (III); 1 H NMR (400 MHz, DMSO-d6 ) δ 8.30–8.25 (m, 1H), 8.05–8.00 (d, 1H), 7.55–7.45 (m, 2H), 7.30–7.15 (m, 2H), 6.88–6.83 (d, 1H), 6.72–6.68 (m, 1H), 4.48–4.42
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(m, 1H), 3.50–3.42 (m, 2H), 1.95–1.92 (s, 3H); FAB-MS m/e 265. 2.3. Peptide synthesis Peptides were synthesized by the manual solid-phase technique using Boc protection for the ␣-amino group, except for Dmt which was Fmoc protected. Side chain protection was as follows: tosyl (Arg), Fmoc [Dap(atn)]. 1,3-Diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) were used as coupling agents. Peptides were assembled on a p-methylbenzhydrylamine resin according to a published protocol [18]. After peptide assembly was complete, Fmoc protection was removed with 30% (v/v) piperidine in DMF and peptides were cleaved from the resin by HF/anisole treatment in the usual manner. Crude peptides were purified by preparative reversed-phase HPLC and were found to be at least 98% pure as assessed by TLC and analytical HPLC. 2.3.1. H-Dmt-d-Arg-Phe-Dap(dns)-NH2 TLC Rf 0.30 (I), 0.81 (II); K 1.12; FAB-MS m/e 831. 2.3.2. H-Dmt-d-Arg-Phe-Dap(atn)-NH2 TLC Rf 0.14 (I), 0.77 (II); K 1.15; FAB-MS m/e 717. 2.4. Pharmacological testing in vitro The guinea pig ileum (GPI) [11] and mouse vas deferens (MVD) [7] bioassays were carried out as reported in detail elsewhere [4,15]. A dose–response curve was determined with [Leu5 ]enkephalin as standard for each ileum and vas preparation, and IC50 values of the compounds being tested were normalized according to a published procedure [22]. Ke values for naloxone as antagonist in the GPI assay were determined from the ratio of IC50 values obtained in the presence and absence of a fixed naloxone concentration (5 nM) [8]. Opioid receptor binding studies were performed as described in detail elsewhere [15]. Binding affinities for and ␦ receptors were determined by displacing, respectively, [3 H]DAMGO (Multiple Peptide Systems, San Diego, CA) and [3 H]DSLET (Multiple Peptide Systems) from rat brain membrane binding sites, and opioid receptor binding affinities were measured by displacement of [3 H]U69,593 (Amersham) from guinea pig brain membrane binding sites. Incubations were performed for 2 h at 0 ◦ C with [3 H]DAMGO, [3 H]DSLET, and [3 H]U69,593 at respective concentrations of 0.72, 0.78, and 0.80 nM. IC50 values were determined from log dose–displacement curves, and Ki values were calculated from the obtained IC50 values by means of the equation of Cheng and Prusoff [3], using values of 1.3, 2.6, and 2.9 nM for the dissociation constants of [3 H]DAMGO, [3 H]DSLET, and [3 H]U69,593, respectively.
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2.5. Antinociceptive assay The radiant heat tail-flick assay was used for the antinociceptive test in mice. The light intensity was adjusted such that the baseline latencies ranged between 2.5 and 3.5 s. Compounds were injected i.t. according to a published method [23]. Analgesia was defined as a latency response of greater than two times the baseline latency for an individual animal. To avoid tissue damage, a cut-off of 10 s was used. Groups of 8–10 mice were used for each dose, and each mouse was only used once. The percentage of analgesic responders was calculated, and the quantal dose–response curves analyzed using probit analysis (Pharm Tools Pro; McGary Group, Inc., Elkins Park, PA). Data are presented as ED50 with 95% confidence intervals. 2.6. Fluorescence spectroscopy Fluorescence emission spectra were recorded on a SLM 8000 spectrofluorometer with 2 nm spectral resolution for excitation and emission. Solutions of peptides and reference amino acids in Tris–HCl buffer (pH 6.6) at a concentration of 2 × 10−5 M were used. The excitation wavelength was 350 nm for both fluorophores. Fluorescence quantum yields (ϕ) were determined relative to N-acetyl-l-tryptophanamide (NATA) (ϕNATA = 0.14) as reference. The quantum yield was calculated based on the following equation: ES AR nS 2 ϕS = ϕR ER AS n R where the subscripts S and R refer to the sample and reference compound (NATA), respectively. E is the integrated area under the corrected emission spectrum. A is the optical density of the solution at the excitation wavelength (A < 0.05) and (nS /nR )2 is the correction for the refractive index.
3. Results 3.1. In vitro opioid activity profiles In the receptor binding assays, the dansylated analogue H-Dmt-d-Arg-Phe-Dap(dns)-NH2 displayed subnanomolar receptor binding affinity, high ␦ receptor binding affinity and relatively low receptor binding affinity (Table 1). It thus showed only slight preference for receptors over ␦ receptors, but quite high selectivity for versus receptors. Similarly, the peptide analogue containing the anthraniloyl group, H-Dmt-d-Arg-Phe-Dap(atn)-NH2 , bound with very high affinity to receptors (Ki = 0.508 nM), less tightly to ␦ receptors and with modest affinity to receptors. It showed somewhat higher versus ␦ selectivity (Ki␦ /Ki = 6) than the dansylated peptide and similar versus selectivity. In comparison with the parent peptide [Dmt1 ]DALDA, the two fluorescent analogues displayed only about three- to
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Table 1 Opioid receptor binding affinities of fluorescent [Dmt1 ]DALDA analogues
Compound
Ki [nM]
H-Dmt-d-Arg-Phe-Dap(dns)-NH2 H-Dmt-d-Arg-Phe-Dap(atn)-NH2 H-Dmt-d-Arg-Phe-Lys-NH2 Morphine
0.589 0.508 0.143 1.00
± ± ± ±
Ki [nM]
Ki␦ [nM]
0.023 0.029 0.015 0.04
1.88 3.12 2100 32.6
± ± ± ±
0.19 0.05 310 3.7
35.0 22.7 22.3 217
± ± ± ±
Ki ratio (/␦/)
6.5 4.9 4.3 49
1/3/59 1/6/45 1/14700/156 1/33/217
Data represent the mean ± S.E.M. from three to six independent experiments.
fourfold lower receptor binding affinity and comparable receptor binding affinity. However, their ␦ receptor binding affinity is much higher and, therefore, they have much lower versus ␦ receptor selectivity than the parent peptide. Both fluorescent analogues have higher receptor binding affinity than morphine but somewhat lower versus ␦ and versus receptor selectivities (Table 1). Due to the replacement of the Lys4 residue in [Dmt1 ] DALDA with Dap(dns) or Dap(atn), both fluorescent analogues carry a lower positive charge (2+) than the parent peptide (3+). As observed in an earlier study, gradual augmentation of the positive charge from 1+ to 3+ in a series of dermorphin-(1–4) tetrapeptide analogues produced an enhancement in receptor affinity and a progressive decrease in ␦ receptor affinity, resulting in increasingly higher receptor selectivity [16]. The decrease in versus ␦ receptor selectivity observed with the two fluorescent analogues as compared to the parent peptide is in agreement with results of the latter study. In agreement with the results of the receptor binding assay, both fluorescent [Dmt1 ]DALDA analogues showed high agonist potency in the GPI assay (Table 2). They are almost as potent as the [Dmt1 ]DALDA parent in this assay and 12-fold more potent than morphine. The agonist effects of [Dmt1 ]DALDA, [Dmt1 ,Dap(dns)4 ]DALDA, and [Dmt1 ,Dap(atn)4 ]DALDA in this assay were naloxonereversible with respective Ke values of 1.17 ± 0.08 nM, 3.76 ± 0.75 nM, and 1.54 ± 0.29 nM. Such low Ke (naloxone) values are typical for receptor interactions in the GPI [6]. As expected on the basis of the ␦ receptor binding assay data, the two fluorescent analogues showed also high agonist potency in the MVD assay, being about eight times more potent than the [Dmt1 ]DALDA parent peptide. The determined IC50 (MVD)/IC50 (GPI) ratios are lower than the Ki␦ /Ki binding constant ratios (Tables 1 and 2) because these potent agonists produce their agonist effect in the
MVD assay not only through interaction with ␦ receptors in this assay system but also with receptors which are present in the vas. 3.2. Antinociceptive activity In the mouse tail-flick assay, [Dmt1 ,Dap(dns)4 ]DALDA and [Dmt1 ,Dap(atn)4 ]DALDA showed very high antinociceptive potency when given i.t., as an ED50 of about 4 pmol/mouse was determined for both of them (Table 3). They were about 250 times more potent than morphine in producing the antinociceptive effect. In agreement with the in vitro opioid activity data, the two fluorescent peptide analogues were only about three times less potent than the [Dmt1 ]DALDA parent in this analgesic test. 3.3. Fluorescence spectroscopic parameters The fluorescence emission spectrum of the dansyl group is known to be strongly dependent on the polarity of its surroundings. While Nε -dansyllysine has an emission maximum at 578 nm that is typical for an entirely aqueous environment, a hypsochromic shift of up to 80 nm is observed for the dansyl fluorescence in lipophilic surroundings [2,10]. The fluorescence emission spectra of [Dmt1 ,Dap(dns)4 ]DALDA and the reference amino acid derivative Ac-Dap(dns)-NH2 both show a maximum at 578 nm (Fig. 2 and Table 4) in Tris–HCl buffer (pH 6.6). This result indicates that the dansyl group of the peptide is located in a completely aqueous environment and does not engage in any significant intramolecular interactions. This is also indicated by the similar fluorescence quantum yields of the dansylated peptide (ϕ = 0.025) and the dansylated Dap derivative (ϕ = 0.030) (Table 4). As in the case of the dansyl group, the fluorescence emission maximum of the anthraniloyl group also depends on
Table 2 Guinea pig ileum (GPI) and mouse vas deferens (MVD) assay of fluorescent [Dmt1 ]DALDA analogues Compound
GPI IC50 [nM]
H-Dmt-d-Arg-Phe-Dap(dns)-NH2 H-Dmt-d-Arg-Phe-Dap(atn)-NH2 H-Dmt-d-Arg-Phe-Lys-NH2 Morphine
2.52 2.46 1.41 29.3
± ± ± ±
0.41 0.36 0.29 2.2
Data represent the mean ± S.E.M. from three to five independent experiments.
MVD IC50 [nM] 3.02 2.84 23.1 155
± ± ± ±
0.49 0.38 2.0 31
MVD/GPI IC50 ratio 1.20 1.15 16.4 5.29
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Table 3 Antinociceptive potencies of fluorescent [Dmt1 ]DALDA analogues in the mouse tail-flick test (i.t. administration) ED50
95% CI
Potency relative to morphine
H-Dmt-d-Arg-Phe-Dap(dns)-NH2 H-Dmt-d-Arg-Phe-Dap(atn)-NH2 H-Dmt-d-Arg-Phe-Lys-NH2 Morphine
4.0 pmol/mouse 3.9 pmol/mouse 1.2 pmol/mouse 1.0 nmol/mouse
2.2–7.3 1.9–22.4 0.6–1.4 0.4–1.8
250 256 833 1
Fluorescence Intensity (a.u.)
Compound
4. Discussion
4 418
3
3
4
2
2 (x10) 1 (x10)
578
1
0 400
500
600
700
Wavelength (nm) Fig. 2. Fluorescence emission spectra of [Dmt1 ,Dap(dns)4 ]DALDA (1), Ac-Dap(dns)-NH2 (2), [Dmt1 ,Dap(atn)4 ]DALDA (3), and Ac-Dap(atn)-NH2 (4) in Tris–HCl buffer, pH 6.6. λexc = 350 nm. Spectra were normalized to the same optical density (0.02) at λ = 350 nm.
the polarity of its environment [21]. The observed emission maximum of the anthraniloyl derivative of Dap at 418 nm (Table 4) is typical for aqueous surroundings. Since [Dmt1 ,Dap(atn)4 ]DALDA in Tris–HCl buffer (pH 6.6) also shows an emission maximum at 418 nm, it is evident that the anthraniloyl group in this peptide is exposed to an aqueous environment, as it is the case with the dansyl group in [Dmt1 ,Dap(dns)4 ]DALDA. The fluorescence quantum yield of [Dmt1 ,Dap(atn)4 ]DALDA is very high (ϕ = 0.358), comparable to that of the reference amino acid derivative Ac-Dap(atn)-NH2 (ϕ = 0.328) and 14 times higher than that of the dansylated peptide ([Dmt1 ,Dap(dns)4 ]DALDA) (Table 4).
Table 4 Steady-state fluorescence parameters of fluorescent [Dmt1 ]DALDA analogues and fluorescent Dap derivatives in Tris–HCl buffer (pH 6.6, 20 ◦ C) Compound
λem max [nm]
ϕ
H-Dmt-d-Arg-Phe-Dap(dns)-NH2 Ac-Dap(dns)-NH2 H-Dmt-d-Arg-Phe-Dap(atn)-NH2 Ac-Dap(atn)-NH2
578 578 418 418
0.025 0.030 0.358 0.328
In comparison with [Dmt1 ]DALDA, the two fluorescent analogues display similarly high receptor binding affinity, agonist potency in vitro and antinociceptive activity in vivo. Thus, they represent valuable pharmacological tools despite their lower versus ␦ receptor selectivity, as compared to the parent peptide. The retained high opioid activity of these analogues is likely to be due to the compactness of the incorporated fluorophores which do not adversely affect binding to opioid receptors. The fluorescence parameters of the two peptide analogues in aqueous solution were compared to those of the N-acetylated and carboxamidated reference amino acid derivatives N-Ac-Dap(dns)-NH2 and N-Ac-Dap(atn)-NH2 , in which the fluorophore is completely exposed to the water. Both fluorescent peptides showed the same fluorescence emission maxima as their respective reference amino acid derivatives and very similar fluorescence quantum yields, indicating that in both of them the fluorophore is completely exposed to the aqueous environment and is not involved in any intramolecular interactions with peptide moieties that would affect the quantum yield and the location of the fluorescence emission maximum. It is important to point out that [Dmt1 ,Dap(atn)4 ]DALDA has about the same high fluorescence quantum yield (ϕ = 0.358) as the reference amino acid derivative N-Ac-Dap(atn)-NH2 (ϕ = 0.328). This result represents the first demonstration that Dap(atn) may retain a high fluorescence quantum yield upon incorporation into a peptide. Obviously, Dap(atn) is an attractive fluorescent amino acid for incorporation into small peptides because of its high fluorescence quantum yield and small size. In the case of both the dansyl and the anthraniloyl group, a blue-shift in the fluorescence emission maximum and an increase in the fluorescence quantum yield is generally observed upon transfer from a polar environment to a more hydrophobic one [2,10,21]. Therefore, it should be possible to use the two fluorescent [Dmt1 ]DALDA analogues in binding studies with receptors or other biopolymers by monitoring changes in the fluorescence intensity or in the fluorescence emission maximum. Peptides carrying a fluorescent label can also be used in studies of their interaction with cells, including cellular uptake and intracellular distribution, by CLSM. Receptormediated cellular uptake of fluorescent deltorphin-1 and dermorphin analogues has been studied by CLSM [1,5].
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More recently, [Dmt1 ]DALDA has been shown to be internalized into Caco-2 cells by a mechanism which does not involve receptor-mediated endocytosis and this internalization was visualized in a CLSM study using [Dmt1 ,Dap(dns)4 ]DALDA [24]. [Dmt1 ,Dap(atn)4 ]DALDA should be even more suitable for such studies because of its high fluorescence quantum yield. Finally, these fluorescent [Dmt1 ]DALDA analogues can be used in studies examining their tissue distribution and penetration of biological barriers.
Acknowledgments This work was supported by a Multicenter Consortium Grant (PO1-DA08924) from the National Institute on Drug Abuse, NIH, USA. References [1] Arttamangkul S, Alvarez-Maubecin V, Thomas G, Williams GT, Grandy DK. Binding and internalization of fluorescent opioid peptide conjugates in living cells. Mol Pharmacol 2000;58:1570–80. [2] Chen RF. Fluorescence of dansyl amino acids in organic solvents and protein solutions. Arch Biochem Biophys 1967;120:609– 20. [3] Cheng YC, Prusoff WH. Relationship between the inhibition constant (KI ) and the concentration of inhibitor which causes 50 percent inhibition (I50 ) of an enzymatic reaction. Biochem Pharmacol 1973;22:3099–102. [4] DiMaio J, Nguyen TM-D, Lemieux C, Schiller PW. Synthesis and pharmacological characterization in vitro of cyclic enkephalin analogues: effects of conformational constraints on opiate receptor selectivity. J Med Chem 1982;25:1432–8. [5] Gaudriault G, Nouel D, Dal Farra C, Beaudet A, Vincent J-P. Receptor-induced internalization of selective peptidic and ␦ opioid ligands. J Biol Chem 1997;272:2880–8. [6] Gillan MGC, Kosterlitz HW, Magnan J. Unexpected antagonism in the rat vas deferens by benzomorphans which are agonists in other pharmacological tests. Br J Pharmacol 1981;72:13–5. [7] Henderson G, Hughes J, Kosterlitz HW. A new example of a morphine sensitive neuroeffector junction. Br J Pharmacol 1972;46:764–6. [8] Kosterlitz HW, Watt AJ. Kinetic parameters of nicotinic agonists and antagonists with particular reference to N-allylnoroxymorphone (naloxone). Br J Pharmacol 1968;33:266–76. [9] Lipkowski AW, Misicka A, Kosson D, Kosson P, Lachwa-From M, Brodzik-Bienkowska A, et al. Biological properties of a new fluorescent biphalin fragment analogue. Life Sci 2002;70:893–7.
[10] Parker CW, Godt SM, Johnson MC. Fluorescent probes for the study of the antibody-hapten reaction. II. Binding of the 5-dimethylaminonaphthylene-1-sulfonamido group by homologous rabbit antibody. Biochemistry 1967;6:3408–16. [11] Paton WDM. The action of morphine and related substances on contraction and on acetylcholine output of coaxially stimulated guinea pig ileum. Br J Pharmacol 1957;12:119–27. [12] Sasaki Y, Ambo A, Suzuki K. Studies on analgesic oligopeptides. VII. Solid phase synthesis and biological properties of Tyr-d-Arg-Phe-beta-Ala-NH2 and its fluorinated aromatic amino acid derivatives. Chem Pharm Bull 1991;39:2316–8. [13] Sasaki Y, Suto T, Ambo A, Ouchi H, Yamamoto Y. Biological properties of opioid peptides replacing Tyr at position 1 by 2,6-dimethyl-Tyr. Chem Pharm Bull 1999;47:1506–9. [14] Schiller PW. Study of adrenocorticotropic hormone conformation by evaluation of intramolecular resonance energy transfer in Nε -dansyllysine21 -ACTH-(1–24)-tetrakosipeptide. Proc Natl Acad Sci USA 1972;69:975–9. [15] Schiller PW, Lipton A, Horrobin DF, Bodanszky M. Unsulfated C-terminal 7-peptide of cholecystokinin: a new ligand of the opiate receptor. Biochem Biophys Res Commun 1978;85:1332–8. [16] Schiller PW, Nguyen TM-D, Chung NN, Lemieux C. Dermorphin analogues carrying an increased positive net charge in their “message” domain display extremely high -opioid receptor selectivity. J Med Chem 1989;32:698–703. [17] Schiller PW, Weltrowska G, Schmidt R, Nguyen TM-D, Berezowska I, Chen Y, et al. Opioid peptide analogs with novel activity profiles as potential therapeutic agents for use in analgesia. In: Shimonishi Y, editor. Peptide science—present and future. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1999. p. 665–9. [18] Schiller PW, Nguyen TM-D, Berezowska I, Dupuis S, Weltrowska G, Chung NN, et al. Synthesis and in vitro opioid activity profiles of DALDA analogues. Eur J Med Chem 2000;35:895–901. [19] Schwyzer R, Schiller PW. Hormon-Rezeptor-Beziehungen: Synthese und Eigenschaften von Nε -Dansyllysin21 -adrenocorticotropin-(1–24)tetrakosipeptid. Helv Chim Acta 1971;54:897–904. [20] Shimoyama M, Shimoyama N, Zhao G-M, Schiller PW, Szeto HH. Antinociceptive and respiratory effects of intrathecal H-Tyr-d-ArgPhe-Lys-NH2 (DALDA) and [Dmt1 ]DALDA. J Pharmacol Exp Ther 2001;297:364–71. [21] Taki M, Hohsaka T, Murakami H, Taira K, Sisido M. A non-natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe. FEBS Lett 2001;507:35–8. [22] Waterfield AA, Leslie FM, Lord JAH, Ling N, Kosterlitz HW. Opioid activities of fragments of -endorphin and of its leucine65 -analogue. Comparison of the binding properties of methionine- and leucine-enkephalin. Eur J Pharmacol 1979;58:11–8. [23] Zhao G-M, Wu D, Soong Y, Shimoyama M, Berezowska I, Schiller PW, et al. Profound spinal tolerance after repeated exposure to a highly selective -opioid peptide agonist: role of ␦-opioid receptors. J Pharmacol Exp Ther 2002;302:188–96. [24] Zhao K, Luo G, Zhao G-M, Schiller PW, Szeto HH. Transcellular transport of a highly polar 3+ net charge opioid tetrapeptide. J Pharmacol Exp Ther 2003;304:425–32.
Highly potent fluorescent analogues of the opioid peptide [Dmt1]DALDA Irena Berezowska a , Nga N. Chung a , Carole Lemieux a , Bogumil Zelent b , Hazel H. Szeto c , Peter W. Schiller a,∗ a
Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 b Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6058, USA c Department of Pharmacology, Joan and Sanford I. Weill Medical College of Cornell University, New York, NY 10021, USA Received 18 April 2003; accepted 3 July 2003
Abstract H-Dmt-d-Arg-Phe-Lys-NH2 (Dmt = 2 ,6 -dimethyltyrosine) ([Dmt1 ]DALDA) is a highly potent and selective opioid peptide agonist capable of producing an antinociceptive effect after systemic administration. Fluorescent analogues of [Dmt1 ]DALDA containing either -dansyl-l-␣,-diaminopropionic acid [Dap(dns)] or -anthraniloyl-l-␣,-diaminopropionic acid [Dap(atn)] in place of Lys4 were synthesized. Both analogues retained subnanomolar opioid receptor binding affinity, very high opioid agonist activity in the guinea pig ileum assay and extraordinarily high antinociceptive activity in the mouse tail-flick test (intrathecal administration). The maxima of the fluorescence emission spectra recorded in Tris–HCl buffer (pH 6.6) indicated a completely aqueous environment of the fluorophore in both peptides. The high fluorescence quantum yield (ϕ = 0.358) of the [Dap(atn)4 ] analogue was particularly remarkable. These fluorescent [Dmt1 ]DALDA analogues represent valuable pharmacological tools for various applications, including studies on the binding to receptors and other biopolymers, cellular uptake and intracellular distribution, and tissue distribution. © 2003 Elsevier Inc. All rights reserved. Keywords: [Dmt1 ]DALDA; Opioid peptides; Fluorescent opioid peptide analogues; Fluorescence spectroscopy; Fluorescence quantum yield; Opioid activity profile in vitro; Antinociception
1. Introduction Structural modification of the N-terminal tetrapeptide segment of dermorphin led to a number of potent and selective opioid agonists, including the tetrapeptides H-Tyr-d-Arg-Phe-Lys-NH2 (DALDA) [16] and H-Tyr-d-Arg-Phe--Ala-NH2 [12]. Replacement of the Tyr1 residue in these tetrapeptides with 2 ,6 -dimethyltyrosine (Dmt) produced a further increase in agonist potency and receptor selectivity. Thus, the tetrapeptides H-Dmt-d-Arg-Phe-Lys-NH2 ([Dmt1 ]DALDA) [17,18] and H-Dmt-d-Arg-Phe--Ala-NH2 [13] are among the most potent and most selective opioid agonists reported to date. Like the DALDA parent, [Dmt1 ]DALDA carries a high positive net charge (3+). In the mouse tail-flick assay, [Dmt1 ]DALDA displayed extraordinary potency (ED50 = 1.2 pmol) when given intrathecally (i.t.), being 833 times more potent than morphine [23]. The duration of the antinociceptive effect of i.t. [Dmt1 ]DALDA was four times longer than that of morphine at equipotent doses [20,23] ∗
Corresponding author. Tel.: +1-514-987-5576; fax: +1-514-987-5513. E-mail address: [email protected] (P.W. Schiller).
0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2003.07.004
and, unlike morphine, it did not induce respiratory depression at a dose of 30 × ED50 [20]. Surprisingly, this compound also produced a potent antinociceptive effect (36 times more potent than morphine in the mouse tail-flick test) after subcutaneous (s.c.) administration and this effect was again of long duration (12 h at a dose of 5 × ED50 ), whereas an equipotent dose of morphine was only effective for 3 h [23]. This result indicated that [Dmt1 ]DALDA is capable of crossing the blood–brain barrier (BBB) quite effectively. To examine whether [Dmt1 ]DALDA might be taken up into cells and might cross biological barriers via a transcellular pathway, it is of interest to develop a fluorescent analogue for confocal laser scanning microscopy (CLSM) and flow cytometry studies. Fluorescent analogues of peptides or small proteins are best prepared by incorporation of relatively small fluorophores, such as the 1-dimethylaminonaphthalene-5-sulfonyl (dns = dansyl) group [9,14] or the anthraniloyl (atn) group [21]. The presence of a small fluorophore is unlikely to change the overall structural characteristics of the peptide to an extent that would greatly interfere with biological activity, whereas the attachment of larger fluorophores to relatively
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2.2. Synthesis of fluorescent α,β-diaminopropionic acid derivatives
OH O S O
X=
H N
H2N O
O N H
H N
O
N NH2
(dns)
O
O C
NH
X
HN
H2N
NH2 HN
(atn)
Fig. 1. Structural formulas of [Dmt1 ,Dap(dns)4 ]DALDA and [Dmt1 ,Dap (atn)4 ]DALDA.
small peptides may produce a substantial decrease in receptor binding affinity. Thus, incorporation of relatively large Bodipy and Alexa fluorophores into dermorphin and deltorphin-1 resulted in a significant reduction in and ␦ receptor binding affinity, respectively [1,5], and Alexa derivatives of the smaller opioid tetrapeptides TIPP and endomorphin-1 displayed receptor binding affinities that were 2–3 orders of magnitude lower than those of their respective parent peptides [1]. In the present paper, we describe the syntheses, opioid activity profiles, and fluorescence parameters of two fluorescent [Dmt1 ]DALDA analogues containing either -dansyl-l-␣,-diaminopropionic acid [Dap(dns)] or -anthraniloyl-l-␣,-diaminopropionic acid [Dap(atn)] [21] in place of the Lys4 residue (Fig. 1).
2. Materials and methods 2.1. Analytical methods Precoated plates (silica gel 60 F254 , 250 m; Merck Darmstadt, Germany) were used for ascending TLC in the following solvent systems (all v/v): (I) n-BuOH/AcOH/H2 O (4:1:1); (II) n-BuOH/pyridine/AcOH/H2 O (15:10:3:12); (III) CHCl3 /MeOH/AcOH (85:10:5). Preparative reversedphase HPLC was performed on a Vydac 218-TP1022 column (22 mm × 250 mm) with a linear gradient of 5–22% (first 20 min) and 22–28% (next 20 min) acetonitrile in 0.1% TFA at a flow rate of 13 ml/min. Analytical reversedphase HPLC was performed on a Vydac 218-TP54 column (5 mm × 250 mm) with a linear gradient of 5–30% acetonitrile in 0.1% TFA over 30 min at a flow rate of 1.5 ml/min. The same column was also used for the determination of the capacity factors K under the same conditions. Proton magnetic resonance spectra were recorded at 25 ◦ C on a Varian VXR-400S spectrometer using tetramethylsilane as internal standard. Molecular weights of compounds were determined by FAB mass spectrometry on an MS-50 HMTCTA mass spectrometer interfaced to a DS-90 data system (Dr. M. Evans, Department of Chemistry, University of Montreal).
2.2.1. N␣ -Boc-N -dansyl-l-␣,-diaminopropionic acid (Boc-Dap(dns)-OH) The synthesis of this derivative was analogous to that reported for the preparation of N␣ -benzyloxycarbonyl-Nε dansyllysine [19]. Yield: 92%. TLC Rf 0.85 (I), 0.82 (II), 0.52 (III); 1 H NMR (400 MHz, DMSO-d6 ) δ 8.55–8.50 (d, 1H), 8.38–8.32 (d, 1H), 8.18–8.10 (m, 2H), 7.75–7.62 (m, 2H), 7.38–7.35 (d, 1H), 6.98–6.95 (d, 1H), 4.10–4.02 (m, 1H), 3.55–3.50 (m, 2H), 3.20–3.08 (m, 1H), 2.95–2.90 (s, 6H), 1.42–1.38 (s, 9H); FAB-MS m/e 438. 2.2.2. N␣ -Boc-N -(N -Fmoc)-anthraniloyl-l-␣,diaminopropionic acid (Boc-Dap(atn[Fmoc])-OH) To a solution of N␣ -Boc-N -anthraniloyl-l-␣,-diaminopropionic acid [21] (300 mg, 0.928 nM) in dioxan (7 ml) aq. NaHCO3 (10%) (5 ml) was added under stirring, followed by addition of Fmoc-Cl (238 mg, 1.02 mM) in dioxan (2 ml) at 0 ◦ C. The reaction mixture was stirred overnight at r.t. and, after addition of H2 O (100 ml), was extracted with ethyl ether to remove the excess of Fmoc-Cl. The aqueous layer was acidified to pH 4.0 with aq. KHSO4 (5%) and extracted with ethyl acetate. The ethyl acetate extract was washed with aq. NaCl (saturated), dried over MgSO4 , filtered and evaporated to yield solid N␣ -Boc-N -(N -Fmoc)-anthraniloyl-l␣,-diaminopropionic acid (361 mg, 72%). TLC Rf 0.94 (I), 0.87 (II), 0.68 (III); 1 H NMR (400 MHz, DMSO-d6 ) δ 11.04–11.02 (s, 1H), 9.00–8.92 (s, 1H), 8.18–8.10 (s, 1H), 8.00–7.95 (d, 2H), 7.78–7.65 (d, 3H), 7.55–6.98 (m, 6H), 7.20–7.12 (m, 1H), 4.58–4.38 (m, 3H), 4.10–4.05 (m, 1H), 3.65–3.60 (s, 2H), 1.45–1.38 (s, 9H); FAB-MS m/e 546. 2.2.3. N␣ -Acetyl-N -dansyl-␣,-diaminopropionic acid amide (Ac-Dap(dns)-NH2 ) This compound was prepared by coupling Boc-Dap(dns)OH to a p-methylbenzhydrylamine resin, removal of the Boc group with 50% (v/v) TFA in CH2 Cl2 , acetylation with acetic anhydride and cleavage from the resin by HF/anisole treatment. TLC Rf 0.45 (I), 0.83 (II), 0.31 (III); 1 H NMR (400 MHz, DMSO-d6 ) δ 8.55–8.50 (d, 1H), 8.37–8.32 (d, 1H), 8.00–7.95 (m, 1H), 7.85–7.80 (d, 1H), 7.70–7.62 (m, 2H), 7.38–7.32 (d, 2H), 7.20 (s, 1H), 4.30–4.25 (m, 1H), 3.20–3.15 (m, 1H), 3.05–2.95 (m, 1H), 2.92–2.88 (s, 6H), 1.80–1.78 (s, 3H); FAB-MS m/e 379. 2.2.4. N␣ -Acetyl-N -anthraniloyl-␣,-diaminopropionic acid amide (Ac-Dap(atn)-NH2 ) This compound was prepared in the same manner as Ac-Dap(dns)-NH2 with Boc-Dap(atn[Fmoc])-OH as starting product. Prior to HF cleavage the Fmoc group was removed with 30% (v/v) piperidine in DMF. TLC Rf 0.38 (I), 0.77 (II), 0.17 (III); 1 H NMR (400 MHz, DMSO-d6 ) δ 8.30–8.25 (m, 1H), 8.05–8.00 (d, 1H), 7.55–7.45 (m, 2H), 7.30–7.15 (m, 2H), 6.88–6.83 (d, 1H), 6.72–6.68 (m, 1H), 4.48–4.42
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(m, 1H), 3.50–3.42 (m, 2H), 1.95–1.92 (s, 3H); FAB-MS m/e 265. 2.3. Peptide synthesis Peptides were synthesized by the manual solid-phase technique using Boc protection for the ␣-amino group, except for Dmt which was Fmoc protected. Side chain protection was as follows: tosyl (Arg), Fmoc [Dap(atn)]. 1,3-Diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) were used as coupling agents. Peptides were assembled on a p-methylbenzhydrylamine resin according to a published protocol [18]. After peptide assembly was complete, Fmoc protection was removed with 30% (v/v) piperidine in DMF and peptides were cleaved from the resin by HF/anisole treatment in the usual manner. Crude peptides were purified by preparative reversed-phase HPLC and were found to be at least 98% pure as assessed by TLC and analytical HPLC. 2.3.1. H-Dmt-d-Arg-Phe-Dap(dns)-NH2 TLC Rf 0.30 (I), 0.81 (II); K 1.12; FAB-MS m/e 831. 2.3.2. H-Dmt-d-Arg-Phe-Dap(atn)-NH2 TLC Rf 0.14 (I), 0.77 (II); K 1.15; FAB-MS m/e 717. 2.4. Pharmacological testing in vitro The guinea pig ileum (GPI) [11] and mouse vas deferens (MVD) [7] bioassays were carried out as reported in detail elsewhere [4,15]. A dose–response curve was determined with [Leu5 ]enkephalin as standard for each ileum and vas preparation, and IC50 values of the compounds being tested were normalized according to a published procedure [22]. Ke values for naloxone as antagonist in the GPI assay were determined from the ratio of IC50 values obtained in the presence and absence of a fixed naloxone concentration (5 nM) [8]. Opioid receptor binding studies were performed as described in detail elsewhere [15]. Binding affinities for and ␦ receptors were determined by displacing, respectively, [3 H]DAMGO (Multiple Peptide Systems, San Diego, CA) and [3 H]DSLET (Multiple Peptide Systems) from rat brain membrane binding sites, and opioid receptor binding affinities were measured by displacement of [3 H]U69,593 (Amersham) from guinea pig brain membrane binding sites. Incubations were performed for 2 h at 0 ◦ C with [3 H]DAMGO, [3 H]DSLET, and [3 H]U69,593 at respective concentrations of 0.72, 0.78, and 0.80 nM. IC50 values were determined from log dose–displacement curves, and Ki values were calculated from the obtained IC50 values by means of the equation of Cheng and Prusoff [3], using values of 1.3, 2.6, and 2.9 nM for the dissociation constants of [3 H]DAMGO, [3 H]DSLET, and [3 H]U69,593, respectively.
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2.5. Antinociceptive assay The radiant heat tail-flick assay was used for the antinociceptive test in mice. The light intensity was adjusted such that the baseline latencies ranged between 2.5 and 3.5 s. Compounds were injected i.t. according to a published method [23]. Analgesia was defined as a latency response of greater than two times the baseline latency for an individual animal. To avoid tissue damage, a cut-off of 10 s was used. Groups of 8–10 mice were used for each dose, and each mouse was only used once. The percentage of analgesic responders was calculated, and the quantal dose–response curves analyzed using probit analysis (Pharm Tools Pro; McGary Group, Inc., Elkins Park, PA). Data are presented as ED50 with 95% confidence intervals. 2.6. Fluorescence spectroscopy Fluorescence emission spectra were recorded on a SLM 8000 spectrofluorometer with 2 nm spectral resolution for excitation and emission. Solutions of peptides and reference amino acids in Tris–HCl buffer (pH 6.6) at a concentration of 2 × 10−5 M were used. The excitation wavelength was 350 nm for both fluorophores. Fluorescence quantum yields (ϕ) were determined relative to N-acetyl-l-tryptophanamide (NATA) (ϕNATA = 0.14) as reference. The quantum yield was calculated based on the following equation: ES AR nS 2 ϕS = ϕR ER AS n R where the subscripts S and R refer to the sample and reference compound (NATA), respectively. E is the integrated area under the corrected emission spectrum. A is the optical density of the solution at the excitation wavelength (A < 0.05) and (nS /nR )2 is the correction for the refractive index.
3. Results 3.1. In vitro opioid activity profiles In the receptor binding assays, the dansylated analogue H-Dmt-d-Arg-Phe-Dap(dns)-NH2 displayed subnanomolar receptor binding affinity, high ␦ receptor binding affinity and relatively low receptor binding affinity (Table 1). It thus showed only slight preference for receptors over ␦ receptors, but quite high selectivity for versus receptors. Similarly, the peptide analogue containing the anthraniloyl group, H-Dmt-d-Arg-Phe-Dap(atn)-NH2 , bound with very high affinity to receptors (Ki = 0.508 nM), less tightly to ␦ receptors and with modest affinity to receptors. It showed somewhat higher versus ␦ selectivity (Ki␦ /Ki = 6) than the dansylated peptide and similar versus selectivity. In comparison with the parent peptide [Dmt1 ]DALDA, the two fluorescent analogues displayed only about three- to
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Table 1 Opioid receptor binding affinities of fluorescent [Dmt1 ]DALDA analogues
Compound
Ki [nM]
H-Dmt-d-Arg-Phe-Dap(dns)-NH2 H-Dmt-d-Arg-Phe-Dap(atn)-NH2 H-Dmt-d-Arg-Phe-Lys-NH2 Morphine
0.589 0.508 0.143 1.00
± ± ± ±
Ki [nM]
Ki␦ [nM]
0.023 0.029 0.015 0.04
1.88 3.12 2100 32.6
± ± ± ±
0.19 0.05 310 3.7
35.0 22.7 22.3 217
± ± ± ±
Ki ratio (/␦/)
6.5 4.9 4.3 49
1/3/59 1/6/45 1/14700/156 1/33/217
Data represent the mean ± S.E.M. from three to six independent experiments.
fourfold lower receptor binding affinity and comparable receptor binding affinity. However, their ␦ receptor binding affinity is much higher and, therefore, they have much lower versus ␦ receptor selectivity than the parent peptide. Both fluorescent analogues have higher receptor binding affinity than morphine but somewhat lower versus ␦ and versus receptor selectivities (Table 1). Due to the replacement of the Lys4 residue in [Dmt1 ] DALDA with Dap(dns) or Dap(atn), both fluorescent analogues carry a lower positive charge (2+) than the parent peptide (3+). As observed in an earlier study, gradual augmentation of the positive charge from 1+ to 3+ in a series of dermorphin-(1–4) tetrapeptide analogues produced an enhancement in receptor affinity and a progressive decrease in ␦ receptor affinity, resulting in increasingly higher receptor selectivity [16]. The decrease in versus ␦ receptor selectivity observed with the two fluorescent analogues as compared to the parent peptide is in agreement with results of the latter study. In agreement with the results of the receptor binding assay, both fluorescent [Dmt1 ]DALDA analogues showed high agonist potency in the GPI assay (Table 2). They are almost as potent as the [Dmt1 ]DALDA parent in this assay and 12-fold more potent than morphine. The agonist effects of [Dmt1 ]DALDA, [Dmt1 ,Dap(dns)4 ]DALDA, and [Dmt1 ,Dap(atn)4 ]DALDA in this assay were naloxonereversible with respective Ke values of 1.17 ± 0.08 nM, 3.76 ± 0.75 nM, and 1.54 ± 0.29 nM. Such low Ke (naloxone) values are typical for receptor interactions in the GPI [6]. As expected on the basis of the ␦ receptor binding assay data, the two fluorescent analogues showed also high agonist potency in the MVD assay, being about eight times more potent than the [Dmt1 ]DALDA parent peptide. The determined IC50 (MVD)/IC50 (GPI) ratios are lower than the Ki␦ /Ki binding constant ratios (Tables 1 and 2) because these potent agonists produce their agonist effect in the
MVD assay not only through interaction with ␦ receptors in this assay system but also with receptors which are present in the vas. 3.2. Antinociceptive activity In the mouse tail-flick assay, [Dmt1 ,Dap(dns)4 ]DALDA and [Dmt1 ,Dap(atn)4 ]DALDA showed very high antinociceptive potency when given i.t., as an ED50 of about 4 pmol/mouse was determined for both of them (Table 3). They were about 250 times more potent than morphine in producing the antinociceptive effect. In agreement with the in vitro opioid activity data, the two fluorescent peptide analogues were only about three times less potent than the [Dmt1 ]DALDA parent in this analgesic test. 3.3. Fluorescence spectroscopic parameters The fluorescence emission spectrum of the dansyl group is known to be strongly dependent on the polarity of its surroundings. While Nε -dansyllysine has an emission maximum at 578 nm that is typical for an entirely aqueous environment, a hypsochromic shift of up to 80 nm is observed for the dansyl fluorescence in lipophilic surroundings [2,10]. The fluorescence emission spectra of [Dmt1 ,Dap(dns)4 ]DALDA and the reference amino acid derivative Ac-Dap(dns)-NH2 both show a maximum at 578 nm (Fig. 2 and Table 4) in Tris–HCl buffer (pH 6.6). This result indicates that the dansyl group of the peptide is located in a completely aqueous environment and does not engage in any significant intramolecular interactions. This is also indicated by the similar fluorescence quantum yields of the dansylated peptide (ϕ = 0.025) and the dansylated Dap derivative (ϕ = 0.030) (Table 4). As in the case of the dansyl group, the fluorescence emission maximum of the anthraniloyl group also depends on
Table 2 Guinea pig ileum (GPI) and mouse vas deferens (MVD) assay of fluorescent [Dmt1 ]DALDA analogues Compound
GPI IC50 [nM]
H-Dmt-d-Arg-Phe-Dap(dns)-NH2 H-Dmt-d-Arg-Phe-Dap(atn)-NH2 H-Dmt-d-Arg-Phe-Lys-NH2 Morphine
2.52 2.46 1.41 29.3
± ± ± ±
0.41 0.36 0.29 2.2
Data represent the mean ± S.E.M. from three to five independent experiments.
MVD IC50 [nM] 3.02 2.84 23.1 155
± ± ± ±
0.49 0.38 2.0 31
MVD/GPI IC50 ratio 1.20 1.15 16.4 5.29
I. Berezowska et al. / Peptides 24 (2003) 1195–1200
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Table 3 Antinociceptive potencies of fluorescent [Dmt1 ]DALDA analogues in the mouse tail-flick test (i.t. administration) ED50
95% CI
Potency relative to morphine
H-Dmt-d-Arg-Phe-Dap(dns)-NH2 H-Dmt-d-Arg-Phe-Dap(atn)-NH2 H-Dmt-d-Arg-Phe-Lys-NH2 Morphine
4.0 pmol/mouse 3.9 pmol/mouse 1.2 pmol/mouse 1.0 nmol/mouse
2.2–7.3 1.9–22.4 0.6–1.4 0.4–1.8
250 256 833 1
Fluorescence Intensity (a.u.)
Compound
4. Discussion
4 418
3
3
4
2
2 (x10) 1 (x10)
578
1
0 400
500
600
700
Wavelength (nm) Fig. 2. Fluorescence emission spectra of [Dmt1 ,Dap(dns)4 ]DALDA (1), Ac-Dap(dns)-NH2 (2), [Dmt1 ,Dap(atn)4 ]DALDA (3), and Ac-Dap(atn)-NH2 (4) in Tris–HCl buffer, pH 6.6. λexc = 350 nm. Spectra were normalized to the same optical density (0.02) at λ = 350 nm.
the polarity of its environment [21]. The observed emission maximum of the anthraniloyl derivative of Dap at 418 nm (Table 4) is typical for aqueous surroundings. Since [Dmt1 ,Dap(atn)4 ]DALDA in Tris–HCl buffer (pH 6.6) also shows an emission maximum at 418 nm, it is evident that the anthraniloyl group in this peptide is exposed to an aqueous environment, as it is the case with the dansyl group in [Dmt1 ,Dap(dns)4 ]DALDA. The fluorescence quantum yield of [Dmt1 ,Dap(atn)4 ]DALDA is very high (ϕ = 0.358), comparable to that of the reference amino acid derivative Ac-Dap(atn)-NH2 (ϕ = 0.328) and 14 times higher than that of the dansylated peptide ([Dmt1 ,Dap(dns)4 ]DALDA) (Table 4).
Table 4 Steady-state fluorescence parameters of fluorescent [Dmt1 ]DALDA analogues and fluorescent Dap derivatives in Tris–HCl buffer (pH 6.6, 20 ◦ C) Compound
λem max [nm]
ϕ
H-Dmt-d-Arg-Phe-Dap(dns)-NH2 Ac-Dap(dns)-NH2 H-Dmt-d-Arg-Phe-Dap(atn)-NH2 Ac-Dap(atn)-NH2
578 578 418 418
0.025 0.030 0.358 0.328
In comparison with [Dmt1 ]DALDA, the two fluorescent analogues display similarly high receptor binding affinity, agonist potency in vitro and antinociceptive activity in vivo. Thus, they represent valuable pharmacological tools despite their lower versus ␦ receptor selectivity, as compared to the parent peptide. The retained high opioid activity of these analogues is likely to be due to the compactness of the incorporated fluorophores which do not adversely affect binding to opioid receptors. The fluorescence parameters of the two peptide analogues in aqueous solution were compared to those of the N-acetylated and carboxamidated reference amino acid derivatives N-Ac-Dap(dns)-NH2 and N-Ac-Dap(atn)-NH2 , in which the fluorophore is completely exposed to the water. Both fluorescent peptides showed the same fluorescence emission maxima as their respective reference amino acid derivatives and very similar fluorescence quantum yields, indicating that in both of them the fluorophore is completely exposed to the aqueous environment and is not involved in any intramolecular interactions with peptide moieties that would affect the quantum yield and the location of the fluorescence emission maximum. It is important to point out that [Dmt1 ,Dap(atn)4 ]DALDA has about the same high fluorescence quantum yield (ϕ = 0.358) as the reference amino acid derivative N-Ac-Dap(atn)-NH2 (ϕ = 0.328). This result represents the first demonstration that Dap(atn) may retain a high fluorescence quantum yield upon incorporation into a peptide. Obviously, Dap(atn) is an attractive fluorescent amino acid for incorporation into small peptides because of its high fluorescence quantum yield and small size. In the case of both the dansyl and the anthraniloyl group, a blue-shift in the fluorescence emission maximum and an increase in the fluorescence quantum yield is generally observed upon transfer from a polar environment to a more hydrophobic one [2,10,21]. Therefore, it should be possible to use the two fluorescent [Dmt1 ]DALDA analogues in binding studies with receptors or other biopolymers by monitoring changes in the fluorescence intensity or in the fluorescence emission maximum. Peptides carrying a fluorescent label can also be used in studies of their interaction with cells, including cellular uptake and intracellular distribution, by CLSM. Receptormediated cellular uptake of fluorescent deltorphin-1 and dermorphin analogues has been studied by CLSM [1,5].
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More recently, [Dmt1 ]DALDA has been shown to be internalized into Caco-2 cells by a mechanism which does not involve receptor-mediated endocytosis and this internalization was visualized in a CLSM study using [Dmt1 ,Dap(dns)4 ]DALDA [24]. [Dmt1 ,Dap(atn)4 ]DALDA should be even more suitable for such studies because of its high fluorescence quantum yield. Finally, these fluorescent [Dmt1 ]DALDA analogues can be used in studies examining their tissue distribution and penetration of biological barriers.
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