Fully Enzymatic Peptide Synthesis using C-Terminal tert-Butyl Ester Interconversion
(50 mL) followed by filtration (3ꢂ). The combined filtrates
were concentrated under vacuum and the remaining solid
was redissolved in 100 mL of EtOAc. This solution was
washed with saturated aqueous NaHCO3 solution (75 mL,
2ꢂ), aqueous HCl solution (pH 1, 75 mL, 2ꢂ), brine
(75 mL) and dried (Na2SO4). The organic phase was concen-
trated under vacuum and the resulting crude material was
purified by preparative HPLC giving Cbz-Phe-OMe 2a as a
colourless oil; yield: 0.41 g (93%).
75 MHz): d=21.9, 22.9, 24.6, 27.9, 38.0, 41.5, 47.1, 53.6, 67.0,
82.3, 119.9, 125.0, 125.1, 126.9, 127.0, 127.7, 128.3, 129.5,
136.0, 141.3, 143.8 (2C), 156.0, 170.2, 171.5; FIA-ESI(+)-
TOF-MS: m/z=557.2982, calcd. for C34H41N2O5 [M+H]+.
H-Leu-Phe-O-t-Bu (27): yield: 0.37 g (99%); 1H NMR
(DMSO-d6, 300 MHz): d=8.65 (1H, d, J=7.5 Hz), 7.24–7.20
(5H, m), 4.41 (1H, q, J=7.2 Hz), 3.57–3.52 (1H, m), 2.97
(2H, dd, J=2.4 and 7.8 Hz), 1.73–1.30 (1H, m), 1.54–1.32
(11H, m), 0.88 (6H, dd, J=6.9 Hz); 13C NMR ([D6]DMSO,
75 MHz): d=21.7, 22.8, 23.5, 27.4, 36.7, 41.7, 51.3, 53.9, 80.7,
126.4, 128.1, 129.1, 136.8, 163.3, 170.0, 171.4; FIA-ESI(+)-
TOF-MS: m/z=335.2351, calcd. for C19H31N2O3 [M + H]+:
335.2329.
Typical Procedure for the Hydrolysis of Peptide t-Bu
Esters (Procedure C)
Boc-Phe-Leu-Phe-O-t-Bu (30): yield: 0.62 g (83%);
1H NMR (CDCl3, 300 MHz): d=7.23–7.07 (10H, m), 6.49
(1H, d, J=7.8 Hz), 6.36 (1H, d, J=7.8 Hz), 4.98 (1H, d, J=
7.8 Hz), 4.61 (1H, q, J=7.8 Hz), 4.64–4.57 (2H, m), 2.97
(2H, dd, J=6.3 Hz), 1.56–1.31 (21H, m) 0.79 (6H, dd, J=
6.0 Hz); 13C NMR (CDCl3, 75 MHz): d=22.0, 22.8, 24.5,
27.9, 28.2, 38.1, 41.1, 51.7, 53.7, 55.6, 80.2, 82.2, 126.9, 128.3,
128.6, 129.3, 129.5, 136.1, 136.6, 155.4, 170.2, 170.9, 171.1;
Alcalase-CLEA (0.5 g) was added to a solution of Cbz-Phe-
Leu-Ala-O-t-Bu (6, 540 mg, 1 mmol) in 18 mL of dioxane
and 2 mL of water. The mixture was shaken at 378C with
150 rpm for 36 h. After filtration, the solids were re-sus-
pended in MeOH (50 mL) followed by filtration (3ꢂ). The
combined filtrate were concentrated under vacuum and the
resulting crude acid was purified by preparative HPLC
giving Cbz-Phe-Leu-Ala-OH 23 as a white solid; in 79%
yield: 0.38 g (79%).
FIA-ESI(+)-TOF-MS:
m/z=582.3572,
calcd.
for
C33H48N3O6 [M+H]+: 582.3538.
Cbz-Phe-Leu-Ala-O-t-Bu (16): yield: 0.56 g (82%);
1H NMR (CDCl3, 300 MHz): d=7.24–7.05 (10H, m), 6.89
(1H, d, J=6.9 Hz), 6.82 (1H, d, J=7.2 Hz), 5.64 (1H, d, J=
8.4 Hz), 5.00–4.90 (2H, m), 4.42–4.29 (3H, m), 3.02–2.90
(2H, m), 1.55–1.33 (12H, m), 1.24 (3H, d, J=7.2 Hz), 0.79
(6H, d, J=5.7 Hz); 13C NMR (CDCl3, 75 MHz): d=18.2,
22.1, 22.7, 24.5, 27.9, 38.4, 41.3, 48.6, 51.7, 56.0, 81.8, 126.9,
127.9, 128.0, 128.4, 128.5, 129.3, 136.3, 156.0, 171.0, 171.1,
171.7; FIA-ESI(+)-TOF-MS: m/z=540.3040, calcd. for
C30H42N3O6 [M+H]+: 540.3068.
Boc-Phe-Leu-Phe-Leu-Phe-O-t-Bu (32): yield: 0.49 g
(starting from 0.65 mmol; 89%); 1H NMR (DMSO-d6,
300 MHz): d=8.22 (1H, d, J=7.2 Hz), 7.99–7.86 (3H, m),
7.29–7.11 (15H, m), 6.90 (1H, d, J=8.4 Hz), 4.57–4.48 (1H,
m), 4.29–4.22 (3H, m), 4.06–4.05 (1H, m), 3.02–2.64 (6H,
m), 1.20–1.06 (24H, m), 0.87–0.77 (1H, m); 13C NMR
(CDCl3, 75 MHz): d=21.5, 22.9, 23.9, 27.4, 36.6, 37.0, 41.1,
41.2, 50.5, 50.8, 53.2, 54.0, 55.5, 77.9, 80.4, 125.9, 126.0, 126.3,
127.8, 128.0, 129.0 (2C), 137.0, 137.5, 138.1, 155.0, 170.1,
170.2, 170.4, 171.2, 171.6; FIA-ESI(+)-TOF-MS: m/z=
842.5035, calcd. for C48H68N5O8 [M+H]+: 842.5062.
For-Met-Leu-O-t-Bu (20): yield: 0.40 g (90%); 1H NMR
(CDCl3, 300 MHz): d=8.13 (1H, s), 6.51–6.46 (2H, m), 4.68
(1H, q, J=6.9 Hz), 4.23–4.35 (1H, m), 2.59–2.53 (2H, dd),
2.07–1.87 (5H, m), 1.66–1.34 (12H, m), 0.87 (6H, dd, J=3.9
and 6.0 Hz); 13C NMR (CDCl3, 75 MHz): d=15.0, 22.0, 22.7,
24.9, 28.0, 31.7, 41.5, 50.4, 51.7, 82.1, 160.6, 170.1, 171.4;
Acknowledgements
FIA-ESI(+)-TOF-MS:
m/z=347.2003,
calcd.
for
C16H32N2O4S [M+H]+: 347.1999.
Jessica Rommens is gratefully acknowledged for the synthesis
and subsequent hydrolysis of compound 16. Furthermore, we
thank Math Boesten for his analytical support and Dr. Theo
Sonke for fruitful discussions.
For-Met-Leu-OMe (21): yield: 0.28 g (92%); 1H NMR
(CDCl3, 300 MHz): d=8.10 (1H, s), 7.24–7.18 (2H, m), 4.78
(1H, q, J=7.8 Hz), 4.50–4.43 (1H, m), 3.04 (3H, s), 2.54
(2H, dd, J=7.2 Hz), 2.07–1.61 (5H, m), 1.61–1.48 (3H, m),
0.85 (6H, dd, J=3.3 and 5.7 Hz); 13C NMR (CDCl3,
75 MHz): d=15.0, 21.6, 22.6, 24.7, 29.6, 31.9, 40.7, 50.3, 50.9,
52.2, 161.0, 170.9, 172.8; FIA-ESI(+)-TOF-MS: m/z= References
305.1539, calcd. for C13H25N2O4S [M+H]+: 305.1530.
Fmoc-Leu-OMe (25): yield: 0.44 g (85%); 1H NMR
(CDCl3, 300 MHz): d=7.69 (2H, d, J=7.5 Hz), 7.54–7.51
(2H, m), 7.35–7.18 (5H, m), 5.07 (1H, d, J=8.1 Hz), 4.36–
4.30 (3H, m), 4.16 (1H, t, J=6.9 Hz), 3,67 (2H, s), 1.63–1.47
(3H, m), 0.89–0.87 (6H, dd); 13C NMR (CDCl3, 75 MHz):
d=21.8, 22.8, 24.7, 41.8, 47.2, 52.3, 67.0, 120.0, 125.1, 127.0,
127.7, 141.3, 143.8, 155.9, 173.9; FIA-ESI(+)-TOF-MS:
m/z=368.1855, calcd. for C22H26NO4 [M + H]+: 368.1856.
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1
Fmoc-Leu-Phe-O-t-Bu (26): yield: 0.63 g (88%); H NMR
(CDCl3, 300 MHz): d=7.67 (2H, d, J=7.5 Hz), 7.50 (2H, d,
J=6.9 Hz), 7.33–7.04 (9H, m), 6.38 (1H, d, J=7.2 Hz), 5.16
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m), 4.14–4.10 (2H, m), 2.99 (2H, dd, J=6.0 Hz), 1.50–1.25
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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