1586
Vol. 55, No. 11
using a TOSO CCPD system equipped with a Tskgel (ODS-120T) column.
Anal. Calcd for C48H64N6O6·1/2H2O: C, 69.45; H, 7.89; N, 10.12. Found: C,
Infrared (IR) spectra were recorded with a JASCO A100 spectrometer. 69.11; H, 7.84; N, 9.99.
The pH was measured by TOA pH instrument (Model HM-7E). 1H- and
Spectral Data for Bis(L-Tyr–D-Phe)-N,N-dodecane-1,12-diamine (6b)
Compound 6b was prepared in a similar manner to 6a and obtained as a col-
13C-NMR spectra were obtained using a JEOL a-500 or GX270 NMR spec-
trometers and NMR samples were dissolved in DMSO-d6/CDCl3 (ratio: orless solid in 69.0% yield; mp 135—137 °C; from (ether/MeOH ), Rf (B):
0.5 ml/0.2 ml) with TMS as an internal reference. FAB mass spectral data 0.08; IR (KBr) cmꢃ1 3300 (m), 2920 (m), 1650 (s); 1H-NMR (CDCl3/
were obtained on a JEOL JMS-HX110 spectrometer, and relevant data were DMSO-d6ꢀ0.5/0.2 ml, a-500) d 1.20—1.25 (m, 8H, –(CH2)– ꢂ4), 1.39—
tabulated as m/z. Elemental analyses were performed by the Laboratory 1.42 (m, 2H, –(CH2)–), 2.59 (dd, Jꢀ7.63, 14.03 Hz, 1H, bH1 in Tyr2), 2.81
Center of Elemental Analysis, University of Ehime (Ehime, Japan).
(dd, Jꢀ5.50, 14.03 Hz, 1H, bH2 in Tyr2), 2.83 (dd, Jꢀ8.24, 13.43 Hz, 1H,
Synthesis of Bis(peptides)-N,N-dodecane-1,12-diamine The double- bH2 in Phe1). 3.09—3.24 (m, 3H, –CH2NHCO– and bH1 in Phe1), 4.01 (dd,
stranded peptides, 6a—e and 7a, b used in this study were prepared accord- Jꢀ5.50, 7.63 Hz, 1H, aH2 in Tyr2), 4.59 (ddd, Jꢀ4.88, 8.24, 8.54 Hz, 1H,
ing to the procedures described in our previous papers.4,5)
Bis(L-Tyr(OBzl))-N,N-dodecane-1,12-diamine (3c) Boc–L-Tyr(OBzl)OH
aH1 in Phe1), 6.67 (d, Jꢀ8.55 Hz, 2H, Tyr2), 6.81 (d, Jꢀ8.24 Hz, 2H, Tyr),
7.15—7.26 (m, 5H, Phe1), 7.81 (t, Jꢀ5.50 Hz, 1H, –NHaCO–), and 8.73 (d,
Jꢀ8.54 Hz, 1H, –NHbCO–); 13C-NMR (CDCl3/DMSO-d6ꢀ0.5/0.2 ml) ppm
26.67, 29.05, 29.20, 29.26, 29.28, 38.37, 38.09, 39.22, 54.31, 54.81, 115.55,
124.77, 126.46, 128.13, 129.31, 130.46, 137.55, 156.67, 168.39, and 170.54.
(3.13 g, 8.44 mmol) and CDI (1.64 g, 10.12 mmol) were dissolved in dry
CHCl3 (35 ml). The mixture was stirred for 1.0 h, and to this was added
1,12-diaminododecane (0.82 g, 4.1 mmol) at 4 °C. The solution was stirred
overnight. The solvent was evaporated to dryness. After the addition of FAB-HR-MS (NBA) Calcd for C48H65N6O6 (MꢁHꢁ) m/z: 821.4967. Found:
aqueous MeOH, the precipitate was collected, washed with 5% citric acid, 821.4958.
5% NaHCO3, and aqueous MeOH, and dried in vacuo. The resulting crude
product, bis(Boc–L-Tyr(OBzl))-N,N-dodecane-1,12-diamide (2c), a colorless
Spectral Data for Bis(L-Phe–L-Tyr-N,N-dodecane-1,12-diamine (6c)
Compound 6c was prepared in a similar manner to 6a and obtained as a col-
solid, was obtained in 94.0% yield. The crude product (3.5 g) was purified orless solid in 75.0% yield; mp 181—184 °C; from (ether/MeOH), Rf (B):
by chromatography on silica gel column (40 g) and eluted in CHCl3 and 2%
0.11; IR (KBr) cmꢃ1 3309 (m), 2853 (m), 1648 (s), 1628 (s); 1H-NMR
MeOH/CHCl3 (stepwise elution); mp 194—196 °C (from MeCN); Rf (A): (CDCl3/DMSO-d6ꢀ0.5/0.2 ml, a-500) d 1.20—1.28 (m, 8H, –(CH2)– ꢂ4),
0.87. HR-FAB-MS (nitrobenzylalchohol, NBA) Calcd for C54H75N4O8
1.37—1.43 (m, 1H, –(CH2)–), 2.70 (dd, Jꢀ8.55, 13.73 Hz, 1H, bH2), 2.83
(dd, Jꢀ7.93, 13.73 Hz, 1H, bH1), 2.92 (dd, Jꢀ5.80, 13.74 Hz, 1H, bH1),
(MꢁHꢁ) m/z: 907.5587. Found: 907.5571.
To compound 2c (4.57 g, 5.04 mmol) was added TFA (26 ml). The mix- 3.05—3.12 (m, 3H, –CH2NHCO– and bH2), 3.70 (dd, Jꢀ4.88, 8.55 Hz, 1H,
ture was stirred for 1 h in an ice bath. After a 5% NaHCO3 and 1 mol/l aH2), 4.53 (ddd, Jꢀ6.41, 7.93, 8.55 Hz, 1H, aH1), 6.69 (d, Jꢀ8.55 Hz, 2H,
NaOH workup and the mixture was stored at ice bath, solid 3c was collected, Tyr), 6.97 (d, Jꢀ8.55 Hz, 2H, Tyr), 7.19—7.21 (m, 2H, Phe), 7.22—7.46 (m,
washed with water, dried in vacuo, and was obtained in 72.1% yield. Com-
3H, Phe), 7.46 (t, Jꢀ5.50 Hz, 1H, –NHaCO–), and 8.19 (d, Jꢀ8.55 Hz, 1H,
pound 3c was purified by silica gel column chromatography eluted with –NHbCO–); 13C-NMR (CDCl3/DMSO-d6ꢀ0.5/0.2 ml) ppm 26.66, 29.03,
CHCl3, 3% MeOH/CHCl3 (stepwise elution); mp 144—146 °C (from
MeCN); Rf (B): 0.45. 1H-NMR (CDCl3/DMSO-d6ꢀ0.5/0.2 ml) d 1.20—
1.28 (m, 8H, –(CH2)– ꢂ4), 1.42—1.50 (m, 2H, –(CH2)– in spacer), 2.66
29.18, 29.26, 29.28, 37.53, 39.06, 39.43, 54.38, 55.42, 115.13, 126.63,
127.41, 128.37, 129.32, 130.17, 136.90, 155.99, 170.73, and 171.84. FAB-
HR-MS (NBA) Calcd for C48H65N6O6 (MꢁHꢁ) m/z: 821.4965. Found:
(dd, Jꢀ8.25, 13.73 Hz, 1H, bH in Tyr), 3.04 (dd, Jꢀ4.57, 13.73 Hz, 1H, bH 821.4971.
in Tyr), 3.12—3.18 (m, 2H, –CH2NHCO– in spacer), 3.47 (dd Jꢀ4.57,
Spectral Data for Bis(L-Tyr–L-Ile)-N,N-dodecane-1,12-diamine (6e)
8.25 Hz, 1H, aH in Tyr), 5.04 (s, 2H, –O–CH2–C6H5), 6.89 (d, Jꢀ8.55 Hz, Compound 5e (81.5% yield) prepared in a similar manner to 5a was purified
2H, Tyr), 7.13 (d, Jꢀ8.55 Hz, 2H, Tyr), 7.29—7.42 (m, 5H, benzyl), and 7.49
by silica gel column chromatography, eluted with CHCl3 and 3% MeOH/
CHCl3 (stepwise elution); mp 199—201 °C (from MeOH); Rf (B): 0.49.
(t, Jꢀ5.90 Hz, 1H, –NHaCO–); 13C-NMR (CDCl3/DMSO-d6ꢀ0.5/0.2 ml)
ppm 26.72, 29.08, 29.31, 29.33, 38.83, 40.21, 56.30, 69.69, 114.68, 127.34, Anal. Calcd for C52H84N6O10: C, 65.51; H, 8.88; N, 8.82. Found: C, 65.21;
127.77, 128.39, 130.22, 130.34, 136.97, 157.31, and 174.17. FAB-HR-MS H, 9.00; N, 8.72. Compound 6e was prepared in a similar manner to 6a and
(NBA) Calcd for C44H59N4O4 (MꢁHꢁ) m/z: 706.4530: Found 707.4552.
Bis(L-Tyr–L-Phe)-N,N-dodecane-1,12-diamine (6a) CDI (1.66 g, 10.2
mmol) was added to a solution of Boc–L-Tyr(OBzl)OH (3.16 g, 8.53 mmol)
in dry CHCl3 (35 ml). After stirring for 1.0 h at room temperature, com-
obtained as a colorless solid in 83.0% yield: mp 190—193 °C (from
MeCN/MeOH); Rf (B): 0.10; IR (KBr) cmꢃ1 3298 (m), 2931 (m), 1678 (s),
1643 (s); 1H-NMR (CDCl3): d 0.85—0.89 (m, 6H, bMe and dMe, Ile),
1.07—1.15 (m, 1H, gH in Ile), 1.20—1.30 (m, 8H, –(CH2)n– in spacer),
pound (3a)4) (2.0 g, 4.06 mmol) was added, then the solution was stirred 1.44—1.50 (m, 3H, gH(Ile) and –CH2–), 1.78—1.82 (m, 1H, bH in Ile),
overnight. Compound bis(Boc–L-Tyr(OBzl)–L-Phe)-N,N-dodecane-1,12-di-
2.92 (dd, Jꢀ7.63, 14.3 Hz, 1H, bH in Tyr), 3.08—3.19 (m, 3H, b proton in
amide (4a), a colorless solid, was prepared in a similar manner to that for Tyr and –CH2NHCO–), 4.11 (m, 1H, aH2 in Tyr), 4.22 (m, 1H, aH1 in Ile),
2c and obtained in 97.0% yield. The crude product (4.5 g) was purified by
chromatography on silica gel column (43 g) and eluted in CHCl3 and 3%
6.73 (d, Jꢀ8.2 Hz, 2H, Tyr), 7.05 (d, Jꢀ8.5 Hz, 2H, Tyr), 7.52 (t, Jꢀ5.50 Hz,
1H, –NHaCO–), 8.56 (d, Jꢀ8.85 Hz,1H, –NHbCO–); 13C-NMR (CDCl3/
MeOH/CHCl3 (stepwise elution); mp 199—200 °C (from MeOH); Rf (A): DMSO-d6ꢀ0.5/0.2 ml) ppm 11.19, 15.37, 24.59, 26.71, 29.01, 29.21, 29.24,
0.65; FAB-MS (nitrobenzyl alcohol) m/z 1022 (MꢁH)ꢁ. To a solution of 4a
(2.50 g, 2.08 mmol) in methanol (100 ml) was added 5% Pd/C (0.50 g). The
29.28, 36.47, 37.02, 38.99, 54.12, 58.10, 115.59, 124.76, 130.49, 156.71,
168.14, and 170.55. FAB-HR-MS (NBA) Calcd for C42H69N6O6 (MꢁHꢁ)
mixture was shaken under H2. After the reaction was completed, the catalyst m/z: 753.5285. Found: 753.5260.
(5a) was removed by filtration and obtained in 86.0% yield. Compound 5a
was purified by silica gel column chromatography and eluted with CHCl3
and 3% MeOH/CHCl3 (stepwise elution); mp 132—135 °C (from MeOH);
UV/Vis Titration The acid-base dissociation constant (pKa) was ob-
tained by the UV/Vis titration method. Compounds 6a and 6e were brought
to a final concentration of 3.65ꢂ10ꢃ5 mol/l, the pH in the solution increased
Rf (B): 0.46. Anal. Calcd for C58H80N6O10·1/2H2O; C, 67.61; H, 7.92; N, from 4.9 to 12.6 at 297 (K) as a function of pH in a 10-mm path length
8.16. Found: C, 67.66; H, 7.94; N, 8.25. quartz cell. Various pH (pHꢀ4.9, 6.1, 6.4, 7.7, 8.2, 8.8, 9.2, 10.4, 11.7, 12.2,
Compound 5a (2.0 g, 2.39 mmol) was dissolved in TFA (15 ml). The col- 12,6) solutions were prepared with 0.1 mol/l acetate, 0.1 mol/l sodium ac-
orless solid 6a was collected in a similar manner to that for 3c. Compound etate, 0.2 mol/l borate-sodium chloride, and 0.2 mol/l sodium hydroxide
6a was purified by silica gel column chromatography eluted with CHCl3, 3%
MeOH/CHCl3, and 8% MeOH/CHCl3 (stepwise elution); mp 178—180 °C
(from MeCN/MeOH); Rf (B): 0.10; IR (KBr) cmꢃ1 3296 (m), 2920 (m),
buffer in 85% MeOH solution.
Computational Chemistry The search for optimized conformations of
double-stranded peptides were done using SPARTAN’04 programs11) run-
1648 (s); 1H-NMR (CDCl3/DMSO-d6ꢀ0.5/0.2 ml) d 1.15—1.24 (m, 8H, ning on a personal computer (Pentium 4 CPU). The lowest energy con-
–(CH2)– ꢂ4), 1.36—1.39 (m, 2H, –(CH2)– in spacer), 2.45 (dd, Jꢀ8.85, former determined with the conformationanl search (Monte-Carlo method)
13.73 Hz, 1H, bH1 in Phe1), 2.89 (dd, Jꢀ4.27, 13.73 Hz, 1H, bH2 in Tyr2), computation at the MMFF94 level was optimized using the restricted
2.93 (dd, Jꢀ7.63, 13.74 Hz, 1H, bH1 in Phe1), 3.00—3.18 (m, 3H, bH2 in
Tyr2 and –CH2NHCO–), 3.49 (dd, Jꢀ4.27, 8.85 Hz, 1H, aH2 in Tyr2), 4.62
Hartree-Fock (RHF) level with a 6-31G(d) basis set.
Drugs and Chemicals Dulbecco’s Modified Eagle Medium (DMEM)
(ddd, Jꢀ7.63, 8.55, 8.85 Hz, 1H, aH1 in Phe1), 6.72 (d, Jꢀ8.55 Hz, 2H, and fetal bovine serum (FBS) were obtained from Dainippon Pharma Co.,
Tyr2), 6.96 (d, Jꢀ8.54 Hz, 2H, Tyr), 7.14—7.19 (m, 3H, Phe1), 7.22—7.26 Ltd. (Tokyo). The cellular toxicity kit (WST-1 or Kit-8) was obtained from
(m, 2H, Phe1), 7.44 (t, Jꢀ5.49 Hz, 1H , –NHaCO–), and 7.98 (d, Jꢀ8.55 Hz, Dojindo Laboratories (Kumamoto, Japan). The small double-stranded modi-
1H, –NHbCO–); 13C-NMR (CDCl3/DMSO-d6ꢀ0.5/0.2 ml) ppm 26.65,
29.05, 29.15, 29.28, 29.30, 38.38, 39.09, 39.72, 53.64, 56.18, 115.39,
fied peptides were synthesized according to previously published meth-
ods.4,5) POD (peroxidase)-conjugated anti-phosphotyrosine monoclonal anti-
126.38, 127.73, 128.06, 129.31, 130.08, 137.14, 156.08, 170.63 and 173.67. body (PY20, M141) was purchased from TAKARA Bio Inc., (Tokyo,