Receptor Recognition of δ Opioid Agonists
J. Am. Chem. Soc., Vol. 118, No. 31, 1996 7289
by FAB-MS and were consistent with the amino acid sequence and
structure of the peptides. The analytical results are listed in Table 7.
c-[D-Pen , D-Pen ]enkephalin (DPDPE, 1). The title compound
4-Methylbenzhydrylamine polystyrene resin (0.51 mmol/g) 1% cross-
link with divinylbenzene (Bachem California, Torrance, CA) was used
as a solid support for the syntheses of the title compound 8, and the
following protected amino acids were added in a stepwise fashion to
2
5
was prepared by the methods described above and was found to be
6
,20
R
R
R
R
identical to the compound previously synthesized.
the growing peptide chain: N -Boc-Gly, N -Boc-Val, N -Boc-Val, N -
R
R
Deltorphin I (6). The title compound was prepared by the methods
described above (without the cyclization procedure) and was found to
Boc-L-Asp-â-benzyl ester, N -Boc-Phe, N -Boc-D-Ala, and optically
R
pure N -Boc-(2S, 3R)-TMT. An excess (2 equiv) of protected amino
acids [except for N -Boc-(2S, 3R)-TMT], HOBT, and DIC was used
3
1
R
be identical to the compound previously synthesized.
1
General procedure for synthesizing [TMT ]DPDPE analogues
for the coupling reactions, which were monitored by ninhydrin tests.
1
R
R
20
is illustrated by the preparation of [(2S,3R)-TMT ]DPDPE (3). N -
Boc-S-p-MeBzl-D-Pen-resin (0.74 g, 0.68 mmol/g, 0.5 mmol) was used
as starting material, and the following protected amino acids were added
in a stepwise fashion to the growing peptide chain: N -Boc-Phe, N -
Boc-Gly, N -Boc-D-Pen(S-p-MeBzl), and optically pure N -Boc-(2S,
N -Boc-(2S,3R)-TMT (1.2 equiv) was added to the growing peptide
chain using BOP reagent (1.44 equiv) and DIEA (1.7 equiv) in NMP
for 15 h. The resin was washed and dried, the protecting groups were
removed, and the peptide was cleaved from the resin in a similar fashion
as that for preparation of the DPDPE analogues (see above). The crude
product was dissolved in acetonitrile and 0.1% TFA water solution
mixture (15:85, v/v) and purified on a Vydac 218TP1010 C18 RP-HPLC
R
R
R
R
R
3
R)-TMT. The analytical data of N -Boc-(2S,3R)-TMT are mp 67.5-
1
6
8.3 °C. H-NMR (CDCl
3
, TMS) δ 6.42 (s, br, 2H, 3′, 5′ aromatic-
-H), 3.34 (m, 1H, C -H), 2.40 (s, Ar-CH ),
′), 1.46 (s, 9H, t-Bu), 1.36 (d, J ) 7.3 Hz, 3H, C
). C-NMR (CDCl ) δ 176.2, 155.7, 153.7, 138.7, 129.4, 117.0,
Hs), 4.72-4.79 (m, 1H, C
R
â
3
3
column (25 cm × 1 cm) with linear gradient elution of 15-70% CH -
CN in 0.1 trifluoroacetic acid (aqueous solution) 1% min at a flow
rate of 3 mL/min. The more lipophilic impurities were washed from
2
.16 (s, Ar-CH
3
â
-
1
3
CH
3
3
-
1
1
2
3
3
15.2, 80.5, 56.6, 38.5, 28.3, 21.5, 17.5, 15.6. IR (KBr, cm ): 3375,
975, 1712, 1689, 1609, 1161, 856. CIMS m/e (relative intensity)
the column with 95-100% CH CN in 0.1% TFA for 10 min, and after
3
equilibrium (11 min, 15% CH CN) the column was ready for use again.
3
+
24.20 (M + 1, 0.5), 73.15 (100). HR-CIMS calcd for C17
H
25NO
5
The UV detector was set at 280 nm during the entire purification
process. The major peak was isolated and lyophilized to afford a white
+
23
23.1733; found (M + 1) 323.1747. [R]
D
) +1.55° (c 0.38, CHCl
3
).
R
1
All the N -tert-butyloxycarbonyl (Boc) protected amino acids (2 equiv)
powder; yield 45%. Amino acid analysis result for [(2S,3R)-TMT ]-
R
except for N -Boc-(2S,3R)-TMT were coupled to the growing peptide
DELT I (8): (2S,3R)-TMT 0.95 (1.00), D-Ala 1.03 (1.00), Phe 1.00
chain using diisopropylcarbodiimide (DIC 2.5 equiv) and N-hydroxy-
(1.00), Asp 1.16 (1.00), Val 1.80 (2.00), Gly 1.10 (1.00). The analytical
R
benzotriazole (HOBT, 2.5 equiv) as coupling reagents. N -Boc-(2S,
data are presented in Table 7.
Amino acid analysis result for [(2R,3R)-TMT ]DELT I (9): (2R,3R)-
1
3
R)-TMT (1.2 equiv)2 was added to the growing peptide chain using
1
BOP reagent (1.44 equiv) and DIEA (1.7 equiv) in 1-methyl-2-
pyrolidinone (NMP) as solvent. After coupling of the last amino acid,
the resin was washed with dichloromethane (6 × 30 mL) and methanol
TMT 0.98 (1.00), D-Ala 1.10 (1.00), Phe 1.00 (1.00), Asp 1.10 (1.00),
Val 1.92 (2.00), Gly 1.09 (1.00). Amino acid analysis result for
1
[
(
(
(2R,3S)-TMT ]DELT I (10): (2R, 3S)-TMT 0.86 (1.00), D-Ala 1.10
(4 × 35 mL) and dried by nitrogen gas flow (9 psi) for 10 min. The
1.00), Phe 0.96 (1.00), Asp 1.04 (1.00), Val 1.80 (2.00), Gly 1.09
1.00). The analytical data of [(2S,3S)-TMT ]DELT I was reported
resin was then stored in Vacuo for 24 h. Cleavage of all side-chain
protecting groups and the peptide from the resin was achieved with
liquid HF (approximately 10 mL) and 0.5 g of p-cresol and 0.5 g of
1
20
previously.
Radioligand Binding Assays. Membranes were prepared from
whole brains taken from adult male Sprague-Dawley rats (250-300
g) obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN).
Following decapitation, the brain was removed, dissected, and homog-
enized at 0 °C in 20 volumes of 50 mM Tris-HCl (Sigma, St. Louis,
MO) buffer adjusted to pH 7.4 using a Teflon-glass homogenizer. The
memberane fraction obtained by centrifugation at 48 000g for 15 min
at 4 °C was resuspended in 20 volumes of fresh Tris buffer and
incubated at 25 °C for 30 min to dissociate any receptor bound
endogeneous opioid peptides. The incubated homogenate was centri-
fuged again as described, and the final pellet was resuspended in 20
volumes of fresh Tris-HCl buffer.
3
4
thiocresol, as outlined above followed by cyclization. The crude
product was dissolved in acetonitrile and 0.1% TFA water solution
mixture (15:85, v/v) and purified on a Vydac 218TP1010 C18 RP-HPLC
column (25 cm × 1 cm) with linear gradient elution of 15-75% CH
3
-
CN in 0.1% trifluoroacetic acid (aqueous solution) at a flow rate of 3
mL/min. The more lipophilic impurities were washed from the column
with 95-100% CH
3
CN in 0.1% TFA for 10 min, and after equilibrium
11 min, 15% CH CN) the column was ready for use again. The UV
(
3
detector was set at 280 nm during the entire purification process. The
major peak was isolated and lyophilized to afford a white powder. Yield
1
5
5 mg (16%). Amino acid analysis result for [(2S,3R)-TMT ]DPDPE
(3): (2S, 3R)-TMT 0.95 (1.00), Gly 1.04 (1.00), Phe 1.00 (1.00). The
Radioligand binding inhibition assay samples were done as previ-
analytical data are presented in Table 7.
14
3
2
4
5
35
ously published using cyclo-[ H][D-Pen , p-Cl-Phe , D-Pen ]enkephalin
(
R
The analytical data of N -Boc-(2R,3R)-TMT are mp 139.0-140.0
3
4
3
[ H][p-ClPhe ]]DPDPE, δ) at a concentration of 0.75 nM and [ H]-
1
°
C dec. H-NMR (CDCl
3
, TMS) δ 6.49 (s, br, 2H, 3′, 5′ aromatic-
-H), 3.54 (m, 1H, C -H), 2.36 (s, Ar-CH ),
′), 1.32 (s, 9H, t-Bu), 1.38 (d, J ) 7.4 Hz, 3H, C
). C-NMR (CDCl ) δ 186.6, 117.2, 115.5, 80.7, 57.6, 36.3, 28.0,
3
36
D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH , [ H]CTOP, µ) (New
2
Hs), 4.52-4.67 (m, 1H, C
R
â
3
England Nuclear, Boston, MA) at a concentration of 0.5 nM as the
radioligands.
2
.30 (s, Ar-CH
3
â
-
13
CH
3
3
-1
Binding data were analyzed by nonlinear least-square regression
analysis program named Inplot 4.03 (GraphPad, San Diego, CA).
Statistical comparisons between one and two site fits were made using
2
1.8, 16.6, 14.8. IR (KBr, cm ) 3353, 2977, 2936, 1712, 1610, 1592,
1
C
1
486, 1454, 1393, 1367, 1302, 1161, 1030, 860. HR-EIMS calcd for
+
23
17
H
25NO
5
323.1733; found (M + 1) 323.1730. [R]
).
The analytical data of N -Boc-(2R,3S)-TMT are mp 65.5-66.0
D
) +23.3° (c
3
7
F-ration test using a p value of 0.05 as the cut-off for significance.
.05, CHCl
3
R
Data best fitted by a one site model were reanalyzed using the logistic
equation.38 Data obtained from independent measurements are pre-
sented as the arithmetic mean ( SEM.
1
°
4
C. H-NMR (CDCl
.70-5.10 (m, 1H, C
3
, TMS) δ 6.42 (s, br, 2H, 3′, 5′ aromatic-Hs),
-H), 3.33 (m, 1H, C -H), 2.41 (s, Ar-CH ), 2.16
′), 1.54 (s, 9H, t-Bu), 1.36 (d, J ) 6.84 Hz, 3H, C -CH ).
C-NMR (CDCl ) δ 175.2, 155.6, 153.7, 138.2, 129.4, 117.0, 80.4,
6.6, 38.6, 28.3, 27.7, 21.5, 15.6, 14.2. CIMS m/e (relative intensity)
R
â
3
In Vitro Bioassay Methods. Electrically induced smooth muscle
contractions from mouse vas deferens (MVD) and guinea pig ileum
(
s, Ar-CH
3
â
3
1
3
3
39
(GPI) longitudinal muscle-myenteric plexus were used for bioassays.
5
3
1
+
-1
Tissues came from male ICR mice weighing 25-30 g and from male
Hartley guinea pigs weighing 150-400 g. The tissues were tied to
24.20 (M + 1, 8), 268.20 (100). IR (KBr, cm ): 3329, 2967, 1683,
592, 1515, 1450, 1369, 1260, 1142, 1048, 883, 855, 800, 669. HR-
+
CIMS: calcd for C17
H
25NO
5
323.1733; found (M + 1) 323.1757.
(35) Vaughn, L. K.; Knapp, R. J.; Toth, G.; Wan, Y. P.; Hruby, V. J.;
2
3
[R]
D
) -1.38° (c 0.44, CHCl
3
).
Yamamura, H. I. Life Sci. 1989, 45, 1001-1008.
1
Amino acid analysis result for [(2R,3R)-TMT ]DPDPE (4): (2R, 3R)-
(36) Hawkins, K. N.; Knapp, R. J.; Lui, G. K.; Gulya, K.; Kazmierski,
W.; Wan, Y.-P.; Pelton, J. T.; Hruby, V. J.; Yamamura, H. I. J. Pharmacol.
Exp. Ther. 1989, 248, 73-80.
TMT 0.90 (1.00), Gly 1.12 (1.00), Phe 1.00 (1.00). Amino acid analysis
1
result for [(2R,3S)-TMT ]DPDPE (5): (2R,3S)-TMT 0.93 (1.00), Gly
1
(37) Munson, P. J.; Rodbard, D. Anal. Biochem. 1980, 107, 220-239.
1
.15 (1.00), Phe 1.00 (1.00). The analytical data for (2S,3S)-TMT
(
38) De Lean, A.; Munson, P. J.; Rodbard, D. Am. J. Physiol. 1978,
1
20
and [(2S,3S)-TMT ]DPDPE were reported in our previous study.
2
35, E97-E102.
1
General procedure for synthesizing [TMT ]deltorphin I ana-
logues is illustrated by the preparation of [(2S,3R)-TMT ]DELT I (8).
(39) Shook, J. E.; Pelton, J. T.; Wire, W. S.; Herning, L. D.; Hruby, V.
1
J.; Burks, T.F. J. Pharmacol. Exp. Ther. 1987, 240, 772-777.