T. Kato et al.
Bioorganic & Medicinal Chemistry 38 (2021) 116111
for the next reaction without purification. A solution of crude mono-
carboxylic acid (7.7 g, 31.2 mmol), diphenylphosphoryl azide (DPPA;
7.4 mL, 34.3 mmol), and Et3N (4.8 mL, 34.3 mmol) in benzene (100 mL)
was refluxed for 30 min. BnOH (3.6 mL, 34.3 mmol) was then added to
the solution, which was then refluxed for 22 h. After removing the sol-
vent, the residue was diluted with EtOAc, washed with 1 M aqueous HCl,
saturated aqueous NaHCO3, and brine before drying over Na2SO4.
Removal of the solvent gave an oily residue, which was purified through
column chromatography on silica gel (20% EtOAc in n-hexane) to give 6
(8.5 g, 78%) as a colorless oil: IR (neat) 3344, 2950, 2147, 1751, 1524,
5.2.9. Methyl 1-(benzyloxycarbonyl)amino-4-(tert-butoxycarbonyl)
aminocyclohexane-1-carboxylate (10)
A mixture of 9 (5.2 g, 15.9 mmol), PPh3 (16.7 g, 63.5 mmol), and
H2O (1.2 mL, 63.5 mmol) in THF (100 mL) was stirred at 60 ◦C for 17 h.
The solution was then evaporated to remove the THF, acidified with
saturated aqueous KHSO4, and washed with Et2O. The aqueous solution
was alkalized to pH 11 with saturated aqueous NaHCO3. Di-tert-butyl
dicarbonate (4.0 mL, 17.5 mmol) in CHCl3 (50 mL) was added to the
stirred alkali solution, which was then refluxed for 5 h, extracted with
CHCl3, and dried over Na2SO4. Removal of the solvent gave a residue,
which was purified through column chromatography on silica gel (33%
EtOAc in n-hexane) to give 10 (4.6 g, 71%) as colorless crystals: IR (KBr)
3349, 2951, 1701, 1528, 1454, 1367, 1302, 1269, 1241, 1169, 1065,
740, 698 cmꢀ 1; 1H NMR (400 MHz, CDCl3): δ 7.34–7.31 (m, 5H), 5.49 (s,
1H), 5.09 (s, 2H), 4.14 (m, 1H), 3.71 (s, 3H), 2.66 (m, 1H), 2.26–2.25
(m, 1H), 2.09 (m, 3H), 1.89 (m, 1H); 13C NMR (400 MHz, CDCl3): δ
174.0, 155.4, 136.2, 128.5, 128.1, 128.0, 66.8, 64.8, 61.6, 52.8, 42.8,
36.0, 31.1; EI-HRMS m/z [M]+ calcd for C21H30N2O6: 406.2104, found:
406.2102.
1456, 1207, 1044, 916, 739 cmꢀ 1
;
1H NMR (400 MHz, CDCl3): δ
7.27–7.38 (m, 5H), 5.11 & 5.13 (br s, 1H), 5.08 (s, 2H), 4.65 & 4.66 (s,
2H), 3.53–3.78 (m, 1H + 3H), 3.35 & 3.36 (s, 3H), 2.13–2.28 (m, 2H),
1.84–1.93 (m, 3H), 1.73–1.79 (m, 1H), 1.62–1.70 (m, 1H), 1.47–1.57
(m, 1H); 13C NMR (100 MHz, CDCl3): δ 174.4, 174.4, 155.5, 155.3,
136.2, 128.5, 128.4, 128.1, 128.1, 128.0, 94.7, 94.6, 73.8, 70.4, 66.7,
58.8, 58.3, 55.2, 55.2, 52.4, 52.3, 30.6, 27.8, 27.5, 26.1; EI-HRMS: m/z
[M]+ calcd for: C18H25NO6 351.1682, found: 351.1683.
5.2.6. Methyl 1-(benzyloxycarbonyl)amino-4-hydroxycyclohexane-1-
carboxylate (7)
5.2.10. 1-(9-Fluorenylmethoxycarbonyl)amino-4-(tert-butoxycarbonyl)
aminocyclohexane-1-carboxylic acid (11)
A mixture of 6 (8.5 g, 24.2 mmol) and ZrCl4 (2.8 g, 12.1 mmol) in
MeOH (100 mL) was refluxed for 13 h. The solution was then evapo-
rated, and the residue was purified through column chromatography on
silica gel (10% MeOH in CHCl3) to give 7 (6.0 g, 80%) as a colorless oil:
A solution of 0.5 M aqueous NaOH (21.4 mL, 10.7 mmol) was added
to a stirred solution of 10 (2.1 g, 5.1 mmol) in THF (10 mL). After stir-
ring at room temperature for 20 h, the solution was acidified with 1 M
aqueous HCl and evaporated to remove the THF. The aqueous solution
was extracted with CHCl3, dried over Na2SO4, and evaporated to give a
crude carboxylic acid (2.0 g, quantitative). A mixture of the crude car-
boxylic acid (2.0 g, 5.1 mmol) and 10% Pd-C (50 mg) in MeOH (30 mL)
was vigorously stirred under a H2 atmosphere at room temperature for
32 h. The Pd-C catalyst was then filtered off, and the filtrate was evap-
orated to give a crude amino acid (1.3 g, quantitative). A mixture of the
crude amino acid (1.3 g, 5.1 mmol) and DIPEA (3.1 mL, 17.7 mmol) in
CH2Cl2 (30 mL) was stirred at room temperature for 30 min, after which
TMS–Cl (1.3 mL, 10.1 mmol) was added, and the solution was refluxed
for 3 h. Fmoc-Cl (1.6 g, 6.1 mmol) was then added to the stirred solution
at 0 ◦C, after which the solution was stirred at room temperature for
21 h. The solution was then diluted with 1% aqueous HCl, extracted with
CH2Cl2, and dried over Na2SO4. Removal of the solvent gave a white
solid, which was purified through column chromatography on silica gel
(0.1% AcOH and 50% EtOAc in n-hexane) to give 11 (1.71 g, 68%) as
colorless crystals: IR (KBr) 3338, 2975, 1726, 1688, 1530, 1452, 1368,
1268, 1168, 1108, 1062, 991, 739 cm-1; 1H NMR (400 MHz, CDCl3): δ
7.73 (d, J = 7.2 Hz, 2H), 7.55–7.57 (m, 2H), 7.36 (d, J = 6.4 Hz, 2H),
7.26–7.30 (m, 2H), 5.71 (br s, 1H), 5.01–5.08 (m, 1H), 4.40 (m, 2H),
1
IR (neat) 3351, 2952, 1717, 1520, 1455, 1234, 1068, 1004 cmꢀ 1; H
NMR (400 MHz, CDCl3): δ 7.27–7.38 (m, 5H), 5.09 & 5.11 (s, 1H), 5.08
(s, 2H), 3.93 (m, 1H), 3.69 (s, 3H), 2.15–2.33 (m, 2H), 1.78–1.90 (m,
3H), 1.42–1.75 (m, 3H); 13C NMR (100 MHz, CDCl3): δ 174.4, 174.4,
155.5, 155.3, 136.2, 128.5, 128.4, 128.1, 128.1, 128.0, 94.7, 94.6, 73.8,
70.4, 66.7, 58.8, 58.3, 55.2, 55.2, 52.4, 52.3, 30.6, 27.8, 27.5, 26.1; EI-
HRMS: m/z [M]+ calcd for: C16H21NO5 307.1420, found: 307.1417.
5.2.7. Methyl 1-(benzyloxycarbonyl)amino-4-(methylsulfonyloxy)
cyclohexane-1-carboxylate (8)
A mixture of 7 (6.0 g, 19.4 mmol), Et3N (5.4 mL, 38.7 mmol), and
methanesulfonyl chloride (MsCl) (3.0 mL, 38.7 mmol) in CH2Cl2
(100 mL) was stirred at room temperature for 24 h. The solution was
then washed with 1 M aqueous HCl, saturated aqueous NaHCO3, and
brine before drying over Na2SO4. Removal of the solvent gave an oily
residue, which was purified through column chromatography on silica
gel (50% EtOAc in n-hexane) to give 7 (6.7 g, 89%) as white crystals: IR
(KBr) 3362, 2961, 1718, 1533, 1352, 1260, 1229, 1173, 975, 910, 756,
701, 529 cmꢀ 1; 1H NMR (400 MHz, CDCl3): δ 7.26–7.39 (m, 5H), 5.09 (s,
2H), 4.95 (s, 1H), 4.93 (s, 1H), 3.70 (s, 3H), 3.03 & 3.01 (s, 3H),
2.22–2.28 (m, 2H), 1.99–2.05 (m, 4H), 1.84–1.87 (m, 2H); 13C NMR
(400 MHz, CDCl3): δ 174.1, 173.9, 155.7, 155.4, 136.1, 128.5, 128.2,
128.1, 128.0, 79.0, 66.8, 58.2, 57.7, 52.5, 38.6, 31.5, 30.0, 27.5, 27.1,
26.5; EI-HRMS: m/z [M]+ calcd for C17H23NO7S: 385.1195, found:
385.1190.
4.18 (m, 1H), 3.46 (m, 1H) 2.19 (m, 2H), 1.89 (m, 4H), 1.43 (s, 9H); 13
C
NMR (400 MHz, CDCl3): δ 178.5, 155.2, 143.7, 141.3, 127.7, 127.1,
125.0, 119.9, 79.4, 66.7, 58.0, 48.3, 47.1, 30.9, 28.4, 27.7; FAB-MS m/z
calcd for C27H32N2NaO6 [M+Na]+: 503.2158, found: 503.2156.
5.3. Synthesis of peptides
5.2.8. Methyl 4-azido-1-(benzyloxycarbonylamino)cyclohexane-1-carbox-
ylate (9)
Peptides were synthesized on a solid support using Fmoc solid-phase
methods with commercially available Rink amide resin and Fmoc-amino
acids. The following describes a representative coupling and depro-
A mixture of 8 (6.7 g, 17.3 mmol), sodium azide (2.3 g, 34.6 mmol),
and 15-crown-5 ether (0.7 mL, 3.5 mmol) in DMF (30 mL) was stirred at
90 ◦C for 16 h. The solution was then evaporated to remove the DMF,
diluted with H2O, extracted with EtOAc, and dried over Na2SO4.
Removal of the solvent gave an oily residue, which was purified through
column chromatography on silica gel (30% EtOAc in n-hexane) to give 9
(5.3 g, 92%) as a colorless oil: IR (neat) 3551, 2952, 2360, 2109, 1737,
1520, 1454, 1288, 1233, 1064, 995, 745, 699 cmꢀ 1; 1H NMR (400 MHz,
CDCl3): δ 7.26–7.38 (m, 5H), 5.11 & 5.08 (s, 1H), 5.09 (s, 2H), 3.67 (s,
3H), 3.35–3.41 (m, 1H), 2.03–2.27 (m, 2H), 1.70–1.93 (m, 4H),
1.49–1.59 (m, 2H); 13C NMR (400 MHz, CDCl3): δ 174.0, 155.4, 136.2,
128.5, 128.1, 128.0, 66.8, 64.8, 61.6, 52.8, 42.8, 36.0, 31.1; EI-HRMS
m/z [M]+ calcd for C16H20N4O4: 332.1485, found: 332.1483.
tection cycle on a 25 μmol scale. Fmoc-NH-SAL resin (55.6 mg; loading:
0.45 mmol/g) was soaked in DMF for 30 min. After removing the DMF,
20% piperidine in DMF was added to the resin for deprotection. After
washing out the piperidine, Fmoc-amino acid or 5(6)-carboxyfluores-
cein (CF) (4 equiv), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)
dimethylamino(morpholino)carbenium hexafluorophosphate (COMU)
(4 equiv), and DIPEA (8 equiv) dissolved in DMF were added to the
coupling reaction. The resin was then suspended in a cleavage cocktail
(TFA: 1.9 mL; H2O: 50 μL; TIS: 50 μL) at room temperature for 90 min.
The TFA solution was evaporated to a small volume, after which cold
diethyl ether was added to the solution to precipitate the peptides. The
7