Phenylhydrazide as an Enzyme-Labile Protecting Group
(0.36 mmol, 71%) of 12 as a yellowish oil: 1H NMR (CDCl3,
400 MHz) 0.88 (t, J ) 7.0 Hz, 3H), 1.2-1.3 (m, 24H), 1.42 (s,
9H), 1.62-1.69 (m, 2H), 2.76 (t, J ) 7.6 Hz, 2 H), 3.33-3.36
(m, 2H), 3.75-3.79 (m, 2H) 4.65-4.72 (m, 2H), 5.13 (bs, 1H),
6.02 (bd, J ) 4.3 Hz, 1H), 6.80-6.91 (m, 2H), 7.04-7.07 (m,
1H), 7.19-7.24 (m, 2H), 8.63 (bs, 1H); 13C NMR (100 MHz)
14.0 (+), 22.6 (-), 25.6 (-), 28.2 (+, 3C), 28.9-29.6 (all -, 10C),
30.0, 31.9, 44.0, 45.6 (all -), 55.0(+), 80.9 (o), 113.7 (+, 2C),
121.2 (+), 129.1 (+, 2C), 141.6, 148.1, 169.5, 170.3, 201.4 (all
o); HPLC-ESI-MS (C4) tR ) 32.35 min, purity 86%; M )
629.3 [M + Na]+, 573.3 [M + Na - tBu]+, M calcd for
173.0 (all o); HPLC-ESI-MS (C4) tR ) 10.01 min, purity 81%,
M ) 493.1 [M - H]-, M calcd for C19H35N4O7S2 495.1947, found
495.1957.
En zym a tic Deblock in g of Boc-Gly-Leu -NHNHP h 21.
Deprotection of 0.53 mmol of 21 (200 mg) is carried out in a
mixture of 210 mL of phosphate buffer (100 mM, pH 7.0) and
9 mL of MeCN with 70 mg of tyrosinase under oxygen
bubbling. Workup B after 2 days yields 142 mg of 36 (0.49
mmol, 93%) as colorless crystals and 10 mg of 21 (0.027 mmol,
1
5%): mp 137-140 °C; H NMR (CDCl3 + MeOD, 400 MHz)
0.88-0.92 (m, 6H) 1.41 (s, 9H), 1.52-1.68 (m, 3H), 3.66-3.82
(m, 2H), 4.46-4.52 (m, 1H); 13C NMR (100 MHz) 21.3, 22.5,
24.6 (all +), 27.9 (+, 3C), 40.7, 43.4 (both -), 50.4 (+), 80.0
(o), 156.3, 170.0, 174.6 (all o); HPLC-ESI-MS (C4) tR ) 9.85
min, purity 83%, M ) 287.1 [M - H]-, 575.1 [2M - H]-, M
calcd for C13H24N2O5Na 311.1583, found 311.1591.
C
32H54N4O5SNa 629.3713, found 629.3693.
En zym a tic Deblock in g of S-P a lm itoyl-N-ter t-bu tylox-
yca r b on ylglycyl-L-cyst ein e P h en ylh yd r a zid e 12. The
deprotection of 0.116 mmol of 12 (70 mg) was carried out in
30 mL of phosphate buffer and 4 mL of MeCN with 30 mg of
tyrosinase under bubbling of oxygen for 7 d. Workup A yielded
45 mg (0.087 mmol, 75%) of Boc-Gly-Cys(Pal)-OH as a yel-
lowish solid and 17 mg (0.028 mmol, 24%) of 12: mp 137-
139 °C; 1H NMR (CDCl3, 400 MHz) 0.81 (m, 3H), 1.1-1.3 (m,
24H), 1.37 (s, 9H), 1.48-1.56 (m, 2H), 2.21 (t, J ) 7.6 Hz, 2H),
3.30-3.39 (m, 2H), 3.58-3.80 (m, 2H), 4.20-4.36 (m, 1H);
HPLC-ESI-MS (C4) tR ) 29.92 min, purity 66%, M ) 515.3
[M - H]-, 1031.4 [2M - H]-, M calcd for C26H48N2O6SNa
539.3131, found 539.3108.
Syn th esis of Boc-Gly-Leu -Met-Gly-Met-Gly-NHNHP h
37. A solution of 0.205 mmol of 34 (120 mg) in 10 mL of CH2-
Cl2 was stirred with 2 mL of TFA at 0 °C for 1 h. The solvent
was evaporated, and the crude residue was directly employed
in the subsequent coupling with 0.25 mmol of 36 (95 mg) under
standard conditions as described above. Flash chromatography
(SiO2, 3% MeOH in DCM) yielded 125 mg (0.165 mmol, 81%)
of a yellowish foam: mp 185-187 °C; 1H NMR (CDCl3
+
MeOD, 400 MHz) 0.80-0.91 (m, 6H), 1.41 (s, 9H), 1.44-1.61
(m, 3H), 1.95-2.15 (m, 10H), 2.46-2.58 (m, 4H), 3.58-3.89
(m, 6H), 4.13-4.26 (m, 2H), 4.48-4.54 (m, 1H), 6.76-6.83 (m,
3H), 7.14-7.18 (m, 2H); HPLC-ESI-MS (C4) tR ) 18.57 min,
purity 81%, M ) 755.1 [M + H]+, M ) 777.3 [M + Na]+, M )
655.1 [M - Boc + H]+, M calcd for C33H54N8O8S2Na 777.3404,
found 777.3404.
En zym a tic Deblock in g of Boc-Met-Gly-NHNHP h 22.
Deprotection of 1.66 mmol 22 (657 mg) is performed in a
mixture of 250 mL of phosphate buffer (100 mM, pH 7.0) and
30 mL of MeCN with 290 mg of tyrosinase and with oxygen
bubbling. After 3 days, workup procedure B yielded 305 mg of
32 (1.00 mmol, 60%) and 160 mg of 22 (0.40 mmol, 24%): 1H
NMR (CDCl3 + MeOD, 400 MHz) 1.38 (s, 9H), 1.80-1.85 (m,
1H), 1.95-2.04 (m, 1H), 2.03 (s, 3H) 2.45-2.53 (m, 2H), 3.83-
3.98 (m, 2H), 4.03-4.12 (m, 1H); 13C NMR (100 MHz) 14.78
(+), 27.9 (+, 3C), 29.7, 31.7, 40.7 (all -), 53.1 (+), 80.0 (o),
155.8, 171.1, 172.5 (all o); HPLC-ESI-MS (C4) tR ) 9.25 min,
purity 87%; M ) 305.0 [M - H]-, 611.0 [2M - H]- 231.1, M
calcd for C12H23N2O5S 307.1327, found 307.1339.
En zym a tic Deblock in g of Boc-Gly-Leu -Met-Gly-Met-
Gly-NHNHP h 37. Deprotection of 0.106 mmol 37 (80 mg) was
carried out in a mixture of 210 mL of phosphate buffer (100
mM, pH 7.0) and 9 mL of MeCN with 70 mg of tyrosinase
under oxygen addition. After 15 h, workup B yielded 40 mg of
Boc-Gly-Leu-Met-Gly-Met-Gly-OH (0.060 mmol, 57%) and 18
mg of 37 (0.024 mmol, 22%): 1H NMR (D2O, 400 MHz) 0.72-
0.83 (m, 6H) 1.30 (s, 9H), 1.44-1.56 (m, 3H), 1.85-2.09 (m,
6H), 2.13-2.26 (m, 1H), 2.37-2.54 (m, 2H), 2.59 (bs, 3H),
2.76-2.88 (m, 2H), 3.62-3.74 (m, 2H), 3.78-3.95 (m, 4H),
4.21-4.27 (m, 1H), 4.31-4.46 (m, 2H); HPLC-ESI-MS (C4)
tR ) 14.08 min, purity 55%, M ) 663.1 [M - H]-.
Tr a p p in g Exp er im en ts in th e P r esen ce of Eth yl Acr y-
la te: F or m a tion of 40. A mixture of 0.253 mmol of 22 (100
mg) was treated with 50 mg of tyrosinase in 50 mL of buffer,
3 mL of MeCN, and 1.5 mmol of ethyl acrylate (150 mg, 6
equiv) with oxygen bubbling for 12 h. The pH was adjusted to
8-9 by addition of NaHCO3, and the mixture was extracted
three times with 50 mL of EtOAc. The organic layer was dried
over MgSO4 and concentrated in vacuo, and the crude residue
was purified by flash chromatography (SiO2, DCM/EtOAc 95:
5) yielding 19 mg of 40 (0.098 mmol, 39%). The pH of the
aqueous layer was adjusted to pH 2 and the mixture is
extracted with EtOAc to yield 45 mg 32 (0.147 mmol, 58%):
1H NMR (CDCl3, 400 MHz) 1.26 (t, J ) 7.1 Hz, 3H), 2.78 (bd,
J ) 6 Hz, 1H, exchangeable), 2.95 (dd, J ) 6.9, 13.9 Hz, 1H),
3.10 (dd, J ) 4.5, 13.9 Hz, 1H), 4.20 (q, J ) 7.1 Hz, 2H), 4.41
(bdd, J ) 4.6, 6.7 Hz, 1H), 7.18-7.30 (m, 5H); 13C NMR (100
MHz) 14.39 (+), 40.80 (-), 61.93 (-), 71.46 (+), 127.1 (+), 128.6
(+, 2C), 129.8 (+, 2C), 136.6 (o), 174.4 (o); GC-MS tR ) 7.331
min, purity >99%, M ) 194 [M]+, 176 [M - H2O]+, 91, 65, M
calcd for C11H14O3 194.0943, found 194.0869.
Syn th esis of Boc-Met-Gly-Met-Gly-NHNHP h 34. A solu-
tion of 1.06 mmol of 22 (420 mg) in 10 mL of CH2Cl2 was
treated at 0 °C with 2 mL of TFA for 1 h. The solvent was
evaporated, and the crude product was directly employed in
the subsequent coupling with 0.98 mmol of 32 (300 mg) under
standard conditions as described above. Flash chromatography
(SiO2, 3% MeOH in DCM) yielded 243 mg (0.42 mmol, 42%)
1
of a yellowish solid: mp 136-138 °C dec; H NMR (CDCl3 +
MeOD, 400 MHz) 1.41 (s, 9H), 1.75-1.85 (m, 1H), 1.90-2.04
(m, 2H), 2.05 (s, 3H), 2.06 (s, 3H), 2.11-2.24 (m, 1H), 2.41-
2.58 (m, 4H), 3.70-3.77 (m, 1H), 3.86-3.95 (m, 1H), 3.95-
4.07 (m, 2H), 4.12 (dd, J ) 5.5, 8.4 Hz, 1H), 4.46 (dd, J ) 5.4,
8.7 Hz, 1H), 6.72-6.88 (m, 3H), 7.14-7.20 (m, 2H); 13C NMR
(100 MHz) 14.79, 14.83 (both +), 27.9 (+, 3C), 29.8, 29.9, 30.1,
30.9, 41.6, 42.7 (all -), 52.8, 53.8 (both +), 80.2 (o), 113.1 (+,
2C), 120.5 (+), 128.6 (+, 2C), 147.5, 156.2, 169.4, 170.3, 172.4,
173.6 (all o); HPLC-ESI-MS (C4) tR ) 15.37 min, purity 91%,
M ) 583.1 [M - H]-, M Calcd for C25H41N6O6S2 585.2529,
found 585.2529.
En zym a tic Deblock in g of Boc-Met-Gly-Met-Gly-
NHNHP h 34. Deprotection of 0.125 mmol of 34 (73 mg) was
carried out in a mixture of 30 mL of phosphate buffer and 3
mL of MeCN with 40 mg of tyrosinase under oxygen bubbling.
After 24 h, workup B yielded 50 mg of Boc-Met-Gly-Met-Gly-
OH (0.101 mmol, 81%) as a brownish oil and 7 mg of 34 (0.012
mmol, 10%): 1H NMR (CDCl3 + MeOD, 400 MHz) 1.39 (s, 9H),
1.79-1.95 (m, 2H), 1.95-2.02 (m, 1H), 2.04 (s, 3H), 2.05 (s,
3H), 2.06-2.14 (m, 1H), 2.44-2.56 (m, 4H), 3.73-3.80 (m, 1H),
3.89-3.93 (m, 2H), 3.95-4.03 (m, 1H), 4.20 (dd, J ) 5.2, 8.3
Hz, 1H), 4.57 (dd, J ) 5.7, 8.2 Hz, 1H); 13C NMR (100 MHz)
15.00, 15.06 (both +), 28.1 (+, 3C), 29.9, 30.0, 30.9, 31.5, 40.9,
42.7 (all -), 52.1, 53.6 (both +), 80.3, 156.0, 169.6, 171.5, 171.9,
ESR Exp er im en ts. Substrate solution: 100 mM 33 in 100
mM phosphate buffer pH 7.0. Spin trap solution: 200 mM 5,5-
dimethylpyrroline N-oxide (DMPO) in phosphate buffer. Im-
mediately before the measurements, 100 µL of buffer, sub-
strate, and DMPO solutions were added to 5.56 mg of
tyrosinase, and the solution was treated with O2 for 7 min.
For the blank sample, 100 µL of buffer, substrate, and DMPO
solution were treated with O2 for 7 min without tyrosinase.
J . Org. Chem, Vol. 67, No. 20, 2002 6909