3 and workup as described above afforded 21 (264 mg, 0.20
mmol, 77%). FTIR (ATR): 3360, 1744, 1218 cm-1. 1H NMR
(400 MHz, CDCl3): δ 7.66 (s, 1H), 7.54 (s, 1H), 7.31–7.11
(m, 18H), 6.93–6.91 (m, 2H), 5.78–5.75 (m, 1H), 5.68 (d, J )
7.2 Hz, 1H), 5.58 (d, J ) 9.0 Hz, 1H), 5.36 (dd, J ) 6.6 Hz,
2H), 5.25–5.21 (m, 1H), 4.88–4.79 (m, 3H), 4.71 (d, J ) 6.1
Hz, 1H), 4.57–4.39 (m, 6H), 4.27 (dd, J ) 12.5 Hz, 1H),
4.09–4.06 (m, 1H), 4.02–3.92 (m, 2H), 3.89 (ddd, J ) 9.9, 4.3,
1.5 Hz, 1H), 3.80–3.74 (m, 2H), 3.73–3.62 (m, 3H), 3.66 (s,
3H), 3.28–3.08 (m, 4H), 2.01 (s, 3H), 1.97 (s, 3H), 1.96 (s,
3H), 1.80 (s, 3H), 1.36 (s, 9H). 13C NMR (75 MHz, CDCl3): δ
171.1, 171.1, 170.6, 170.0, 169.4, 169.3, 155.7, 143.6, 143.3,
138.3, 137.9, 137.9, 137.2, 128.6, 128.5, 128.5, 128.3, 128.1,
128.0, 127.9, 127.9, 127.8, 122.0, 121.1, 87.6, 85.8, 85.6, 81.0,
80.3, 78.0, 77.4, 75.9, 75.3, 75.2, 74.9, 73.6, 72.7, 70.7, 68.6,
67.8, 61.6, 53.7, 52.7, 51.9, 28.4, 27.9, 27.8, 20.7, 20.7, 20.6,
20.2. HRMS (ESI): m/z calculated for C64H77N8O19 (M + H)+
1261.5305, found 1261.5354.
J ) 11.2 Hz, 1 H), 4.85 (d, J ) 10.8 Hz, 1 H), 4.66–4.47 (m,
6 H), 4.39–4.25 (m, 2 H), 3.95 (t, J ) 9.2 Hz, 1 H), 3.84–3.61
(m, 8 H), 3.21 (dt, J ) 25.0, 12.5 Hz, 1 H), 2.84–2.67 (m, 1
H), 2.20–2.10 (m, 2 H), 2.00–1.86 (m, 1H). 13C NMR (75 MHz,
CDCl3) δ 170.7, 155.9, 145.8, 138.5, 138.0, 137.9, 136.1, 129.6,
128.4, 128.3, 128.3, 128.1, 128.0, 127.9, 127.7, 127.6, 127.5,
127.4, 121.8, 86.9, 81.7, 79.2, 78.1, 75.5, 75.0, 74.5, 74.0, 73.3,
69.1, 67.6, 52.8, 52.4, 52.1, 37.9, 30.9, 28.3. HRMS (ESI): m/z
calculated for C51H54N4NaO9 (M + Na)+ 889.3789, found
889.3815.
(2S,4S)-N-Cbz-4-(4-[ꢀ-D-Glc(Bn)4])-[1,2,3]triazol-1-
yl}pipecolic Acid Methyl Ester (34). Preparation according
to general procedure A afforded 34 (42 mg, 0.049 mmol, 60%)
as a white amorphous solid. Rf (1/1 EtOAc/heptane) ) 0.36.
1H NMR (400 MHz, CDCl3): δ 7.44 (s, 1 H), 7.34–7.03 (m,
25 H), 5.17–5.14 (m, 2 H), 4.92 (d, J ) 2.8 Hz, 2 H), 4.85 (d,
J ) 10.9 Hz, 1 H), 4.73–4.47 (m, 6 H), 4.38 (d, J ) 11.0 Hz,
1 H), 4.00–3.59 (m, 7 H), 3.43 (s, 2 H), 2.84–2.78 (m, 1 H),
2.42–2.35 (m, 2 H), 2.19–2.11 (m, 1 H). 13C NMR (75 MHz,
CDCl3) δ 170.7, 155.8, 145.4, 138.4, 137.9, 136.0, 129.6, 128.5,
128.3, 128.2, 128.2, 128.1, 128.0, 127.9, 127.8, 127.8, 127.7,
127.6, 127.5, 127.4, 120.9, 86.8, 81.6, 79.3, 78.1, 75.5, 75.0,
74.7, 73.9, 73.3, 69.0, 67.9, 55.0, 54.9, 53.7, 52.7, 40.5, 32.7,
31.8. HRMS (ESI): m/z calculated for C51H54N4NaO9 (M +
Na)+ 889.3789, found 889.3812.
General Procedure B for Chemoenzymatic Peptide Cou-
pling. The N-Cbz-protected amino acid methyl ester (0.39
mmol) was dried by coevaporation with DMF (2 × 5 mL) and
dissolved in tert-amylOH (4.0 mL). After adding a solution of
Phe-NH2 or Gly-NH2 (1.5 mmol) in tert-amylOH (2.0 mL),
the reaction mixture was stirred at 37 °C. Subsequently, the
dried alcalase suspension in tert-amylOH (1 mL) was added,
and the reaction mixture was stirred at 37 °C. Samples were
taken at regular time intervals and analyzed by HPLC. Upon
(virtually) complete conversion, the reaction mixture was
concentrated in Vacuo to remove most of the volatiles. The
residue was taken up in EtOAc (50 mL) and H2O (20 mL), to
which a few drops of 1 N aqueous HCl were added. The
aqueous layer was extracted with EtOAc (3 × 30 mL), and the
combined organic phase was washed with aqueous KHCO3 (1
M, 40 mL), aqueous HCl (1 N, 40 mL), and brine (40 mL),
dried (Na2SO4), and concentrated in Vacuo. Analytically pure
samples were obtained by recrystallization. A new load of
enzyme (dried precipitate from 500 µL of enzyme solution)
was added three times a week. Quenching solution for HPLC
samples: 50% KH2PO4 (0.1 M, pH 5), 50% MeCN.
Cbz-L-T1M(4-[ꢀ-D-Glc(Ac)4])-Gly-NH2(43). Applyinggen-
eral procedure B (with 0.37 mmol 30, and 5 equiv of Gly-
NH2) for chemoenzymatic peptide coupling gave 43 (165 mg;
65%). Rf (EtOAc) ) 0.25. 1H NMR (400 MHz, CDCl3/CD3OD)
δ 7.93 (s, 1 H), 7.33 (m, 5 H), 5.38 (t, J ) 9.4 Hz, 1 H), 5.28
(t, J ) 9.7 Hz, 1 H), 5.17 (t, J ) 9.7 Hz, 1 H), 5.10–5.04 (m,
2 H), 4.90 (dd, J ) 13.8, 4.5 Hz, 1 H), 4.81–4.64 (m, 3 H),
4.28 (dd, J ) 12.4, 4.8 Hz, 1 H), 4.12 (dd, J ) 12.4, 2.1 Hz,
1 H), 3.98 (ddd, J ) 10.0, 4.8, 2.2 Hz, 1 H), 3.92 (d, J ) 17.1
Hz, 1 H), 3.83 (d, J ) 17.1 Hz, 1 H), 2.05 (s, 3 H), 2.03 (s, 3
H), 2.00 (s, 3 H), 1.85 (s, 3 H). 13C NMR (75 MHz, CD3OD)
δ 174.0, 172.3, 171.7, 171.3, 171.0, 145.4, 137.7, 129.5, 129.1,
Cbz-L-T1M(4-[ꢀ-D-Glc(Ac)4])-OMe (30). Preparation ac-
cording to general procedure A afforded 30 (1.7 g, mmol, 96%)
1
as a white solid. Rf (EtOAc) ) 0.74. H NMR (400 MHz,
CDCl3/CD3OD) δ 7.75 (s, 1 H); 7.41–7.29 (m, 5 H); 5.37 (t, J
) 9.4 Hz, 1 H); 5.25 (t, J ) 9.7 Hz, 1 H); 5.18 (dd, J ) 10.0,
9.4 Hz, 1 H); 5.12 (s, 2 H); 4.91–4.71 (m, 4H); 4.28 (dd, J )
12.5, 4.9 Hz, 1 H); 4.13 (dd, J ) 12.5, 2.2 Hz, 1 H); 3.94 (ddd,
J ) 10.1, 4.8, 2.2 Hz, 1 H); 3.79 (s, 3 H); 2.07 (s, 3 H); 2.05
(s, 3 H); 2.02 (s, 3 H); 1.86 (s, 1H). 13C NMR (75 MHz, CDCl3)
δ 170.5, 170.0, 169.7, 169.4, 169.0, 155.6, 144.3, 135.8, 128.5,
128.2, 128.0, 123.2, 76.2, 73.7, 73.1, 71.3, 68.3, 67.3, 62.0,
53.9, 53.1, 50.7, 20.6, 20.5, 20.3. IR (film) V 3356, 3140, 2950,
2885, 2250, 1748, 1532 cm-1. HRMS (ESI) m/z calculated for
C28H34N4NaO13 (M + Na)+ 657.2020, found 657.1996.
Fmoc-L-T1P(4-[ꢀ-D-Glc(Bn)4])-OH (31). Preparation ac-
cording to general procedure A afforded 31 (626 mg, 0.695
mmol, 69%) as a white solid. FTIR (ATR): V 3058, 3032, 2863,
2245, 1718 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.74 (d, J )
7.5 Hz, 2 H), 7.55–7.51 (m, 2 H), 7.39–6.94 (m 24 H),
5.01–4.33 (m, 13 H), 4.18–4.03 (m, 2 H), 3.88–3.81 (m, 5 H),
3.58–3.42 (m, 1 H). HRMS (ESI): m/z calculated for
C54H52N4NaO9 (M + Na)+ 923.3632, found 923.3629.
Fmoc-L-T1B(4-[ꢀ-D-Glc(Bn)4])-OH (32). Preparation ac-
cording to general procedure A afforded 32 (662 mg, 0.702
mmol, 71%) as a white amorphous solid. IR (film) V 3058, 3021,
2924, 2859, 1714 cm-1. 1H NMR (400 MHz, CDCl3): δ 7.75
(d, J ) 7.5 Hz, 2 H), 7.61–7.55 (m, 2 H), 7.45–6.95 (m, 25 H),
5.40 (d, J ) 6.9 Hz, 1 H), 4.92 (dd, J ) 24.0, 11.1 Hz, 2 H),
4.82 (d, J ) 10.8 Hz, 1 H), 4.67 (d, J ) 10.9 Hz, 1 H),
4.59–4.23 (m, 10 H), 4.20 (t, J ) 6.8 Hz, 1 H), 4.01 (t, J ) 9.5
Hz, 1 H), 3.81 (t, J ) 8.7 Hz, 1 H), 3.75–3.53 (m, 4 H),
2.02–1.78 (m, 2 H), 1.77–1.58 (m, 2 H), 1.39–1.22 (m, 1 H),
1.14–0.96 (m, 1 H). HRMS (ESI): m/z calculated for
C57H58N4NaO9 (M + Na)+ 965.4101, found 965.4126.
(2S,4R)-N-Cbz-4-(4-[ꢀ-D-Glc(Bn)4])-[1,2,3]triazol-1-
yl}pipecolic Acid Methyl Ester (33). Preparation according
to general procedure A afforded 33 (39 mg, 0.045 mmol, 49%)
as a white amorphous solid. Rf (1/1 EtOAc/heptane) ) 0.26.
1H NMR (400 MHz, CDCl3): δ 7.45 (s, 1 H), 7.39–6.95 (m,
25 H), 5.24–5.08 (m, 3 H), 4.95 (d, J ) 11.1 Hz, 1 H), 4.91 (d,
510
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Vol. 12, No. 3, 2008 / Organic Process Research & Development