The Journal of Organic Chemistry
ARTICLE
acid, HOBT = 1-hydroxybenzotriazole, EDCI = N-ethyl-N0-(3-dimethy-
laminopropyl)carbodiimide hydrochloride, HBTU = O-(benzotriazol-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, DIPEA = N,N0-
diisopropylethylamine, DBU = diazabicyclo[5.4.0]undec-7-ene.
General Procedure 1 for the Synthesis of Fmoc Protected
Peptide tert-Butyl Esters. To a stirred solution of Fmoc-protected
amino acid (1 mmol), glycine tert-butyl ester hydrochloride (1 mmol),
and HOBT (1.2 mmol) in dry methylene chloride (10 mL) was added
EDCI (1.2 mmol) followed by DIPEA (3 mmol) at 0 °C. After 30 min of
stirring at 0 °C, the reaction mixture was warmed to room temperature
and stirred for 2 h. The solvents were removed, and the product was
purified by column chromatography eluting with 50% ethyl acetate in
hexanes.
2H), 7.65 (d, J = 7.5 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.26 (t, J = 7.0 Hz,
2H), 4.38ꢀ4.34 (m, 4H), 4.20ꢀ4.19 (m, 3H); 13C NMR (125.6 MHz,
CD3OD) δ 201.4, 170.5, 157.9, 144.1, 141.4, 127.7, 127.1, 125.1,
119.8, 67.1, 51.3, 48.8, 48.4, 47.4, 47.2, 46.3. ESI-HRMS calcd for
C19H18N2O4SNa [M + Na]+, 393.0885; found, 393.0891.
General Procedure 3: Fmoc-SPPS of the Peptidyl Thioe-
sters on the Aminomethyl Polystyrene Resin
Derivatization of Aminomethyl Polystyrene Resin with
Fmoc-AA-Gly-OH Thioamides. In a 10-mL glass reaction vessel,
aminomethyl polystyrene resin (244 mg, 0.1 mmol) was swelled
in DMF (4 mL) for 30 min, after which the solvent was removed
by filtration. To a stirred solution of Fmoc-protected thioamide
(0.4 mmol), HOBT (54 mg, 0.4 mmol), and HBTU (152 mg,
0.4 mmol) in dry DMF (3 mL) was added DIPEA (70 μL, 0.4 mmol)
at room temperature. After stirring for 4 min, the so-formed solution of
activated Fmoc-protected thioamide was added to the peptide synthesis
vessel with additional DMF (1 mL). The resulting mixture was shaken for
2 h before the solvent was decanted, and the resin was washed thoroughly
using DMF (3 ꢁ 2 mL) and dichloromethane (3 ꢁ 2 mL).
Fmoc-D-Phe-Gly-OtBu (5). Prepared by the general procedure 1
with a yield of 1.15 g (89%), [R]22D +16.1 (c 1, CHCl3); IR (CHCl3)
1740, 1705, 1660 cmꢀ1; 1H NMR (500 MHz) δ 7.76 (d, J = 8.0 Hz, 2H),
7.53 (dd, J = 8.0, 10.5 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 8.33ꢀ7.22
(m, 7H), 6.32 (s, 1H), 5.37 (d, J = 5.5 Hz, 1H), 4.50ꢀ4.33 (m, 3H), 4.19
(t, J = 7.0 Hz, 1H), 3.96ꢀ3.82 (m, 2H), 3.12 (s, 2H), 1.46 (s, 9H); 13C
NMR (125.6 MHz) δ 172.0, 168.7, 156.2, 144.0, 141.5, 136.6, 129.5,
129.0, 128.0, 127.4, 127.3, 125.3, 120.2, 82.7, 67.3, 56.3, 47.3, 42.3, 38.7,
28.3. ESI-HRMS calcd for C30H32N2O5Na [M + Na]+, 523.2209; found,
523.2217.
Fmoc Removal and Iterative Peptide Assembly. A solution
of 20% piperidine in DMF (5 mL) was added to the resin, and the
resulting mixture was shaken for 4 min, after which the solvents were
removedbyfiltration. A further 5mLof 20% piperidine in DMFwasadded
to the resin, which was then shaken for 30 min. The solvent was removed,
and the resin was washed with DMF (2 ꢁ 5 mL), dichloromethane
(2 ꢁ 5 mL), isopropyl alcohol (2 ꢁ 5 mL), and hexane (2 ꢁ 5 mL). All
couplings were carried out by adding a solution of protected amino acid
(0.4 mmol), preactivated with HOBT (0.4 mmol), DIPEA (0.4 mmol),
and HBTU (0.4 mmol) in dry DMF (4.0 mL) as described above, to the
resin followed by shaking at room temperature. After 2 h, the resin was
washed with DMF (2 ꢁ 5 mL), dichloromethane (2 ꢁ 5 mL), and DMF
(2 ꢁ 5 mL). The final amino acid was introduced with Boc protection.
Alkylation of Resin-Bound Peptidyl Thioamides. The resin-
bound peptidyl thioamide was treated with a solution of DBU (150 μL,
1 mmol) and benzyl bromide (107 μL, 0.9 mmol) in DMF (4 mL) and
shaken at room temperature for 2 h. The resin was washed with DMF
(2 ꢁ 5 mL), dichloromethane (2 ꢁ 5 mL), and DMF (2 ꢁ 5 mL) and
dried to provide the resin-bound thioimidate.
Cleavage from the Resin with Aqueous TFA. In the case of
tripeptidyl thioesters (L-Val-L-Ala-D/L-Phe-SBn), the resin-bound thioi-
midate was suspended in a 1:1 mixture of TFA/water (10 mL) and
stirred magnetically for 2 h at room temperature. The resin was filtered
and washed with acetonitrile (2 ꢁ 10 mL). The combined filtrates were
concentrated, and the concentrate was neutralized with a saturated
solution of sodium bicarbonate (40 mL) followed by extraction with
chloroform (3 ꢁ 50 mL). The organic layer was washed with brine and
dried. Evaporation of the solvent afforded the crude peptidyl thioesters,
which were subjected to chromatographic purification eluting with 20%
methanol in chloroform.
Cleavage from the Resin with TFA/Water/Anisole. The
resin-bound thioimidate was suspended in 10 mL of a mixture of
TFA/water/anisole (5.0:4.5:0.5 mL) and stirred magnetically for 2 h
at room temperature. A solution of TFA/DCM/Et3SIH (7.5:2.0:0.5,
10 mL) was added to the reaction mixture, which was then stirred for 4 h.
The resin was filtered off and washed with acetonitrile (2 ꢁ 10 mL). The
combined filtrates were concentrated and the concentrate was taken up
in acetonitrile/water (v/v 1:1, 5 mL) and subjected to RP-HPLC
purification (10% f 100% A in B with a flow rate of 12 mL/min over
40 min and 215 nm UV detection) to afford the peptidyl thioester.
L-Val-L-Ala-L-Phe-SBn (10). Following the general procedure 3
with a yield of 32 mg (72%). 500 MHz 1H NMR shows epimerization
(ratio, L/D = 78.1/21.9). 1H NMR (500 MHz) δ 7.62 (d, J = 7.5 Hz, 1H),
7.32ꢀ7.21 (m, 8H), 7.11ꢀ7.09 (m, 2H), 7.03ꢀ7.00 (m, 1H), 4.97ꢀ
4.93 (m, 1H), 4.49ꢀ4.43 (m, 1H), 4.12 (s, 1H), 3.23 (dd, J = 5.0, 14.0
Fmoc-Gly-Gly-OtBu (6). Prepared by the general procedure 1 with
a yield of 4.23 g (84%), IR (CHCl3) 1739, 1704, 1660 cmꢀ1; 1H NMR
(500 MHz) δ 7.76 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.40 (t, J =
7.5 Hz, 2H), 7.31 (dt, J = 7.0, 0.5 Hz, 2H), 6.65 (s, 1H), 5.73 (t, J = 5.5 Hz,
1H), 4.43 (d, J = 7.0 Hz, 2H), 4.22 (t, J = 7.0 Hz, 1H), 3.95 (t, J = 6.0 Hz,
4H), 1.47 (s, 9H); 13C NMR (125.6 MHz) δ 169.4, 169.1, 156.9, 144.0,
141.5, 128.0, 127.3, 125.3, 120.2, 82.8, 67.5, 47.3, 44.6, 42.2, 28.3. ESI-
HRMS calcd for C23H26N2O5Na [M + Na]+, 433.1739; found, 433.1732.
General Procedure 2 for the Synthesis of Thioamides. A
solution of Fmoc-protected peptidyl tert-butyl ester (4 mmol) and
Lawesson’s reagent (2.4 mmol) in dry toluene (40 mL) was heated to
reflux under nitrogen for 2 h. After cooling to room temperature, the
toluene was removed, and the crude reaction mixture was subjected to
column chromatography to afford the thioamide tert-butyl ester that
then was dissolved in a mixture of TFA/CH2Cl2/Et3SiH (15.0/5.0/
2.0 mL). After 2 h of stirring at room temperature, the solvents were
removed, and the concentrate was washed with 40% ethyl acetate/
hexanes to afford the desired product.
Fmoc-L-thionoPhe-Gly-OH (7). Prepared by the general proce-
dure 2 with a yield of 1.20 g (78%), [R]24D ꢀ12.1 (c 0.75, CH3OH); IR
(CHCl3) 3305, 1720, 1705, 1247 cmꢀ1; 1H NMR (500 MHz, CD3OD)
δ 7.77 (d, J = 7.5 Hz, 2H), 7.56 (t, J = 9.0 Hz, 2H), 7.37 (t, J = 7.5 Hz,
2H), 7.31ꢀ7.19 (m, 7H), 4.76 (dd, J = 5.0, 9.5 Hz, 1H), 4.35ꢀ4.10
(m, 5H), 3.36ꢀ3.31 (m, 1H), 2.94 (dd, J = 10.0, 13.0 Hz, 1H); 13C
NMR (125.6 MHz, CD3OD) δ 205.3, 170.2, 156.8, 144.0, 141.3, 137.7,
129.2, 128.2, 127.6, 127.0, 126.5, 125.2, 125.1, 119.7, 71.2, 66.9, 62.8,
48.7, 47.5, 47.3, 47.1, 46.3, 41.4. ESI-HRMS calcd for C26H24N2O4SNa
[M + Na]+, 483.1354; found, 483.1346.
Fmoc-D-thionoPhe-Gly-OH (8). Prepared by the general proce-
dure 2 with a yield of 1.15 g (76%), [R]24D +10.5 (c 0.5, CH3OH); IR
(CHCl3) 3305, 1720, 1705, 1247 cmꢀ1; 1H NMR (500 MHz, CD3OD)
δ 7.77 (d, J = 7.5 Hz, 2H), 7.56 (t, J = 9.0 Hz, 2H), 7.37 (t, J = 7.5 Hz,
2H), 7.31ꢀ7.19 (m, 7H), 4.76 (dd, J = 5.0, 9.5 Hz, 1H), 4.35ꢀ4.10
(m, 5H), 3.36ꢀ3.31 (m, 1H), 2.94 (dd, J = 10.0, 13.0 Hz, 1H); 13C
NMR (125.6 MHz, CD3OD) δ 205.3, 170.2, 156.8, 144.0, 141.3, 137.7,
129.2, 128.2, 127.6, 127.0, 126.5, 125.2, 125.1, 119.7, 71.2, 66.9, 62.8,
48.7, 47.5, 47.3, 47.1, 46.3, 41.4. ESI-HRMS calcd for C26H24N2O4SNa
[M + Na]+, 483.1354; found, 483.1359.
Fmoc-thionoGly-Gly-OH (9). Prepared by the general procedure
2 with a yield of 2.60 g (72%), IR (CHCl3) 3295, 1725, 1714,
1
1247 cmꢀ1; H NMR (500 MHz, CD3OD) δ 7.75 (d, J = 7.5 Hz,
6522
dx.doi.org/10.1021/jo200497j |J. Org. Chem. 2011, 76, 6518–6524