Pseudoprolines as Removable Turn Inducers
brine, dried over Na2SO4, and filtered, the solvent was
evaporated, and the crude compound was purified by flash
chromatography to give the desired coupled peptide.
were washed with brine and dried over Na2SO4, the solvent
was evaporated, and the residue was purified by flash chro-
matography (silica gel: 95:5 f 80:20 EtOAc/MeOH) to give
the di-deprotected hexapeptide 25 (81 mg, 47% yield) as a
colorless solid: mp 118-120 °C; [R]D ) -34 (c 1.6, MeOH); 1H
NMR (CDCl3/200 MHz) δ 0.91-1.06 (m, 18H), 1.21 (d, J 6.3,
3H), 1.29 (d, J 6.4, 3H), 1.43 (d, J 6.4, 3H), 1.58 (s, 3H), 1.68
(s, 3H), 1.93 (m, 1H), 2.13 (m, 2H), 3.40-3.50 (m, 3H), 3.60
(d, J 6.1, 1H), 3.86 (app t, J 5.8, 1H), 3.93-4.46 (m, 6H), 6.86
(br s, 1H), 6.95 (d, J 9.2, 1H, NH), 7.68 (d, J 7.3, 1H), 8.15 (d,
J 6.0, 1H), 8.47 (d, J 8.8, 1H) [minor conformer (<10%) also
observed]; ESI-MS (positive mode) m/z ) 641 ([MH]+, 30), 663
([MNa]+, 52), 1303 ([2M + Na]+, 100); HR-MS m/z ) 663.3665
[MNa]+, 663.3687 calcd for C30H52N6O9Na.
Cyclo[-Val-Thr-Val-Thr-Val-Thr-] (1) (Method 1). The
pseudoproline-derivatized hexapeptide 24 (183 mg, 0.25 mmol)
was treated with 4 M HCl in dioxane (1 mL) at 0 °C, allowed
to warm to rt, and then stirred at rt for 3 d. The reaction
mixture was diluted with water (15 mL) and extracted several
times with EtOAc. The combined organic fractions were
washed with brine, dried over Na2SO4, and filtered, the solvent
was evaporated, and the residue was purified by flash chro-
matography [silica gel: 80:18:2 CHCl3-MeOH-NH3(aq)] to
give two fractions A (Rf 0.6) and B (Rf 0.5).
General Method for TBS Protection. Under anhydrous
conditions, TBS-Cl (2 equiv per hydroxyl) and imidazole (4
equiv per hydroxyl) were added to a solution of cyclic peptide
in DMF (1 mL/100 mg substrate) and the mixture stirred at
rt for 36 h. The mixture was partitioned between EtOAc and
brine, and then the aqueous layer was extracted with EtOAc
(2×). The combined organic phases were dried (Na2SO4), and
then the solvent was removed under reduced pressure. The
residue was purified by flash chromatography to give the
desired TBS-protected products.
Cyclo[-Val-Thr(ΨMe.Mepro)-Val-Thr(ΨMe.Mepro)-Val-Thr-
(ΨMe.Mepro)-] (24). Under anhydrous conditions, 1.5 equiv of
FDPP (154 mg, 0.40 mmol) was added to a solution of the
linear hexapeptide 7a (197 mg, 0.26 mmol) in acetonitrile (54
mL, C ) 0.005 M), followed by the addition of 3 equiv of DIPEA
(0.14 mL, 0.80 mmol). The reaction mixture was then stirred
at rt overnight, after which the solvent was removed by rotary
evaporation and the residue purified by flash chromatography
(silica gel: 1:1 f 2:1 ethyl acetate/hexane) to give the desired
cyclized hexapeptide 24 (174 mg, 91% yield) as a colorless
solid: mp 234-236 °C; [R]D ) -65 (c 1.6, CHCl3). 1H NMR
(CD3CN/200 MHz) δ 0.83 (d, J 6.8, 9H), 0.92 (d, J 6.8, 9H),
1.31 (d, J 6.2, 9H), 1.47 (s, 9H), 1.50 (s, 9H), 2.06 (dseptet, J
5.9, 6.8, 3H), 4.05 (d, J 5.9, 3H), 4.23 (dq, J 5.9, 6.2, 3H), 4.33
(dd, J 5.9, 6.6, 3H), 6.47 (d, J 6.6 Hz, 3H); 13C NMR (CD3CN/
75 MHz) δ 16.8, 17.9(0), 17.9(1), 24.9, 25.8, 30.3, 57.2, 66.9,
75.0, 96.2, 167.3, 168.0; ESI-MS (positive mode) m/z ) 663 (32),
721 ([MH]+, 42), 743 ([MNa]+, 100); HR-ESI-MS m/z )
743.4318 [MNa]+, 743.4314, calcd for C36H60N6O9Na. Anal.
Calcd for C36H60N6O9‚H2O: C, 58.52; H, 8.46; N, 11.37.
Found: C, 58.37; H,8.44; N, 11.24.
Concentration of fraction A gave the di-deprotected hexapep-
tide 25 (50 mg, 33% yield), which was identical in all respects
to the material obtained above.
Concentration of fraction B gave the desired hexapeptide 1
(85 mg, 56% yield) as a colorless solid: mp 188-190 °C; [R]D
1
) -98 (c 0.9, MeOH); H NMR (MeOD/200 MHz) δ 0.90 (d, J
7.0, 9H), 0.93 (d, J 7.0, 9H), 1.13 (d, J 6.4, 9H), 2.30 (dseptet,
J 5.6, 7.0, 3H), 4.02 (d, J 5.6, 3H), 4.07 (d, J 3.7, 3H), 4.20 (dq,
J 3.7, 6.4, 3H), (NH, OH not observed); 13C NMR (MeOD/75
MHz) δ 19.0, 20.3, 21.2, 31.4, 61.7, 62.3, 68.8, 173.3, 174.5;
ESI-MS (positive mode) m/z ) 601 ([MH]+, 7), 623 ([MNa]+,
20), 1223 ([2M + Na]+, 100); HR-MS m/z ) 623.3380 [MNa]+,
623.3381 calculated for C27H48N6O9Na.
Cyclo[-Val-Thr-Val-Thr(ΨMe.Mepro)-Val-Thr(ΨMe.Mepro)-]
(27). The pseudoproline-derivatized hexapeptide 24 (149 mg,
0.21 mmol) was treated with 4 M HCl in dioxane (1 mL) and
stirred at 0 °C for 1 h after which the reaction mixture was
diluted with water (15 mL) and extracted several times with
EtOAc. The combined organic fractions were washed with
brine and dried over Na2SO4, the solvent was evaporated, and
the residue was purified by flash chromatography (silica gel:
95:5 EtOAc/MeOH) to give the monounprotected hexapeptide
27 (46 mg, 32% yield) as a colorless solid: mp 148-150 °C;
[R]D ) -140 (c 1.7, CHCl3); 1H NMR (CD3CN/200 MHz) δ 0.84
(m, 15H), 1.05 (d, J 6.3, 3H), 1.10 (d, J 6.9, 3H), 1.33 (d, J 6.3,
3H), 1.40 (d, J 6.1, 3H), 1.43 (s, 6H), 1.52 (s, 3H), 1.54 (s, 3H),
2.12 (dseptet, J 3.7, 6.6, 1H), 2.39 (m, 2H), 3.67 (dd, J 4.9, 7.4,
1H), 3.90 (app. t, J 8.4, 1H), 3.98 (m, 1H), 4.03 (m, 1H), 4.07-
4.17 (m, 2H), 4.17-4.25 (m, 2H), 4.35 (br.d, J 4.8, 1H, OH
(disappears on D2O exchange)), 4.49 (dq, J 3.7, J 6.3, 1H), 6.70
(d, J 4.8, 1H), 7.35 (m, 2H), 7.76 (d, J 8.4, 1H); 13C NMR (CD3-
CN/75 MHz) δ 15.1, 17.5, 17.8 (3), 17.8 (4), 18.1, 18.9, 19.0,
20.0, 22.4, 25.1, 25.2, 26.8, 27.3, 27.7, 32.4, 32.4, 57.6, 58.2,
60.7, 65.4, 65.5, 66.4, 75.1, 76.0, 95.8, 96.6, 167.7, 168.8, 169.1,
169.6, 170.4, 171.9 (1 signal obscured or overlapping); ESI-
MS (positive mode) m/z 681 ([MH]+, 100), 703 ([MNa]+, 37);
HR-MS m/z ) 703.3960 [MNa]+, 703.4001 calcd for C33H56N6O9-
Na.
Cyclo[-Val-Thr(TBS)-Val-Thr(TBS)-Val-Thr(TBS)-] (23).
Treatment of 1 (78 mg, 0.13 mmol) according to the general
method for TBS protection gave the cyclic hexapeptide 23 as
a colorless solid (88 mg, 70%) after flash chromatography
(silica gel: 2:1 EtOAc/hexane): mp 170-172 °C; [R]D ) -18
(c 1.2, CHCl3); 1H NMR (CDCl3/200 MHz) δ 0.00 (s, 9H), 0.01
(s, 9H), 0.78 (s, 27 H), 0.87 (d, J 6.8, 9H), 0.91 (d, J 6.8, 9H),
1.03 (d, J 6.3, 9H), 2.38 (m, 3H), 3.73 (dd, J 6.8, 6.8, 3H), 4.12
(dd, J 4.3, 5.6, 3H), 4.46 (dd, J 4.3, 6.3, 1H), 7.18 (d, J 6.8,
3H), 7.35 (d, J 5.6, 3H); 13C NMR (CDCl3/75 MHz) δ -4.5, -4.3,
18.3, 19.5, 19.9, 20.2, 26.2, 29.2, 60.5, 63.3, 67.2, 170.3, 171.3;
ESI-MS (positive mode) m/z ) 966 ([MNa]+, 100); HR-MS m/z
) 965.5927, 965.5970 calcd for C45H90N6O9Si3Na.
Acknowledgment. We thank the Australian Re-
search Council for financial support and for the award
of a Queen Elizabeth II research fellowship to K.A.J.
Supporting Information Available: Experimental de-
tails and characterization data for compounds 1-13, 15-22,
and 28 and details of single-crystal X-ray diffraction structure
determination for 24. Copies of 1H and 13C spectra for
compounds 1, epi-1, 2b, 7a, 8, 9, 12, 13, 15-18, 23-25, and
27 and NOESY spectra for compounds 7a, 12, 16, and 24.
X-ray crystal structure of 24 (CIF). This material is available
Cyclo-[-Val-Thr-Val-Thr-Val-Thr(ΨMe.Mepro)-] (25). A
5% (v/v) TFA solution was added dropwise to a solution of the
pseudoproline-derivatized hexapeptide 24 (194 mg, 0.27 mmol)
in CH2Cl2 (10 mL) at 0 °C. The reaction mixture was stirred
at rt for 3 h and then diluted with water (15 mL) and extracted
several times with EtOAc. The combined organic fractions
JO0484732
J. Org. Chem, Vol. 69, No. 25, 2004 8809