Novel S-Xanthenyl Protecting Groups for Cysteine
J . Org. Chem., Vol. 62, No. 12, 1997 3847
solution. After extraction with EtOAc (3 × 20 mL), the
combined organic layer was washed with H2O (3 × 20 mL)
and brine (1 × 20 mL) and dried (Na2SO4). Evaporation of
organic solvent gave a white solid which was purified further
by silica gel chromatography. Yield: 0.14 g (81%). Meth od
B (p r efer r ed on la bor a tor y-sca le). 9H-Xanthen-9-ol10 (1.45
g, 7.3 mmol) was added in five portions to a solution of Fmoc-
Cys-OH (6) (2.52 g, 7.3 mmol) and TFA (0.56 mL, 7.3 mmol)
in CH2Cl2 (100 mL). The yellowish reaction solution was
stirred at 25 °C for 30 min under N2 atmosphere, and then
petroleum ether (100 mL) was added. After 3 h, the resultant
white precipitate was collected by filtration, washed with a
mixture of CH2Cl2-petroleum ether (1:1; 3 × 15 mL), and dried
in vacuo over P2O5. Yield: 3.54 g (90%); mp 70-72 °C; Rf
concentrated to give a red solid, which was dried in vacuo over
P2O5 and then dissolved in a solution of TFA (5.2 mL, 68 mmol)
and cysteine (0.77 g, 6.3 mmol) in DME (150 mL). The
reaction solution was stirred at 25 °C for 30 min, diluted with
H2O (100 mL), neutralized to pH 7 by addition of saturated
aqueous Na2CO3 solution, concentrated partially under re-
duced presure to remove DME, and then treated with 10%
citric acid to ∼ pH 6. The resultant white precipitate was
collected by filtration, washed with H2O (3 × 20 mL) and Et2O
(3 × 10 mL), and dried in vacuo over P2O5. Yield: 2.03 g (97%);
mp 150 °C dec; Rf [MeOH-H2O (4:1)] 0.73; 1H NMR (CD3OD)
δ 7.09-7.56 (m, 5H), 6.68-6.78 (m, 2H), 5.43 (s, 1H), 3.81 and
3.80 (adjacent s, equal height, due to diastereomers, 3H), 3.49
(dd, J ) 4.2 and 8.1 Hz, 1H), 2.92-2.99 (m, 1H), 2.77-2.86
(m, 1H). Anal. Calcd for C17H17O4NS (331.39): C, 61.62; H,
5.17; N: 4.23; S, 9.67. Found: C, 60.99; H, 5.05; N, 3.66; S,
9.12.
1
[CHCl3-MeOH (10:1)] 0.46; H NMR (CD3SOCD3) δ 7.86 (d,
J ) 7.8 Hz, 2H), 7.74 (d, J ) 8.4 Hz, 1H), 7.70 (d, J ) 7.5 Hz,
2H), 7.10-7.47 (m, 12H), 5.48 (s, 1H), 4.19-4.30 (m, 3H),
4.02-4.09 (m, 1H), 2.82 (dd, J ) 4.5 and 13.2, 1H), 2.64 (dd,
Nr-(9-F lu or en ylm eth yloxyca r bon yl)cystein e (6). TFA
(25 mL, 0.32 mol) was added to a solution of bis(Fmoc)cystine
(5.54 g, 8.1 mmol) in MeOH (500 mL). Zinc powder (5.32 g,
81.4 mmol) was added next in one portion, and the reaction
mixture was stirred at 25 °C for 30 min [complete reduction
was confirmed by TLC: CHCl3-MeOH-HOAc ) 30:1:0.1, Rf
) 0.53 for Fmoc-cysteine; starting bis(Fmoc)cystine does not
migrate under these TLC conditions]. The mixture was then
diluted with H2O (100 mL) and then partially concentrated
under reduced pressure to remove MeOH. The resultant white
solid was collected by filtration, washed with H2O (until the
filtrate was nonacidic to pH paper), and then dried in vacuo
over P2O5. Yield: 5.01 g (90%); mp 96-98 °C; 1H NMR
(CD3COCD3) δ 7.87 (d, J ) 7.5 Hz, 2H), 7.73 (d, J ) 6.3 Hz,
2H), 7.33-7.42 (m, 4H), 6.82 (d, J ) 6.3 Hz, NH), 4.48 (broad,
1H), 4.37 (d, J ) 6.3 Hz, 2H), 4.27 (t, J ) 6.3 Hz, 1H), 2.95-
3.06 (m, 2H). Anal. Calcd for C18H17NO4S (343.40): C, 62.96;
H, 4.99; N, 4.08; S, 9.34. Found: C, 63.07; H, 5.15; N, 3.89; S,
9.11.
Solu tion Sta bility/La bility Stu d ies. Fmoc-Cys(Xan)-OH
or Fmoc-Cys(2-Moxan)-OH (∼1 mg) were dissolved in various
milieus, at a concentration of 1-2 mM. Acid cleavages were
quenched with 5% aqueous NaHCO3. The aqueous layers were
extracted with CH2Cl2 (2 × 1 mL). Organic phases were
concentrated by a stream of N2, treated with petroleum ether
(3 × 10 mL) to remove aromatic scavengers, and dissolved in
MeOH (1 mL) for analysis. Possible reactions were monitored
by TLC: CHCl3-MeOH (10:1), Rf ) 0.46 for Fmoc-Cys(Xan)-
OH and Fmoc-Cys(2-Moxan)-OH; Fmoc-cysteine stays near
origin; and HPLC [Vydac C-18 column (5 µm, 4.6 × 250 mm),
linear gradient of 0.1% TFA in CH3CN and 0.1% aqueous TFA
from 2:3 to 4:1 over a period of 35 min, flow rate 1.2 mL/min,
detection at 254 nm], tR ) 29.4 min for Fmoc-Cys(Xan)-OH; tR
) 11.7 min for Fmoc-cysteine; tR ) 26.7 min for 9H-xanthene;
tR ) 24.3 min for Fmoc-Cys(2-Moxan)-OH; tR ) 20.4 min for
2-methoxy-9H-xanthene.
In other experiments, H-Cys(Xan)-OH, H-Cys(2-Moxan)-OH,
or H-Cys(3-Moxan)-OH were dissolved, at a concentration of
3 mM, in piperidine-DMF (1:4). The fate of S-protected
cysteine was monitored by TLC [MeOH-H2O (4:1)] or analyti-
cal HPLC [Vydac C-18 column (5 µm, 4.6 × 250 mm), linear
gradient of 0.1% TFA in CH3CN and 0.1% aqueous TFA from
1:4 to 4:1 over a period of 45 min, flow rate 1.0 mL/min,
detection at 220 nm]. H-Cys(3-Moxan)-OH (tR ) 11.7 min)
decomposed to 3-methoxy-9H-xanthen-9-one (tR ) 21.1 min),
and 3-methoxy-9H-xanthen-9-ol (tR ) 15.6 min) (Figure 1).
Solid -P h a se Sta bility/La bility Stu d ies. Fmoc-Cys(Xan)-
OH and Fmoc-Cys(2-Moxan)-OH were incorporated onto a
PEG-PS resin (loading: 0.21 mmol/g) using DIPCDI/HOBt
(4:4)-mediated coupling. Portions of these resins (15-25 mg
per experiment) were subjected to various S-Xan cleavage
conditions. The filtrates from the cleavages, containing 9H-
xanthene or 2-methoxy-9H-xanthene, were diluted with MeOH
to a final volume of 25 mL, and absorbance was measured at
250 nm [data used for Figure 3]. To determine the stability
of Fmoc-PAL-PEG-PS resin to these same conditions, the
treated resins were washed with DMF (3 × 2 min) and CH2-
Cl2 (3 × 2 min), dried in vacuo, treated further with piperi-
dine-DMF (1:4, 2.0 mL) at 25 °C for 20 min, and washed with
1
J ) 9.9 and 13.2 Hz, 1H); H NMR (CDCl3) δ 7.75-7.77 (m,
2H), 7.56-7.59 (m, 2H), 7.21-7.41 (m, 8H), 7.05-7.10 (m, 4H),
5.30 (s, 1H), 5.19 (d, J ) 8.0 Hz, NH), 4.33-4.40 (m, 3H), 4.20
(m, 1H), 2.75-2.83 (m, 2H). Anal. Calcd for C31H25NO5S
(523.61): C, 71.11; H, 4.81; N, 2.68; S, 6.12. Found: C, 70.89;
H, 4.78; N, 2.52; S, 5.98.
Nr-(9-F lu or en ylm eth yloxyca r bon yl)-S-(2-m eth oxy-9H-
xa n th en -9-yl)-cystein e (2). Meth od A. The procedure was
exactly as method A for compound 1 but starting with H-Cys-
(2-Moxan)-OH (1.00 g, 3.0 mmol). Yield: 1.39 g (83%).
Meth od B. The procedure was exactly as method B for
compound 1 but starting with 2-methoxy-9H-xanthen-9-ol10,12
(1.63 g, 7.1 mmol). Yield. 3.27 g (83%); mp 63-65 °C; Rf
1
[CHCl3-MeOH (10:1)] 0.46; H NMR (CDCl3) δ 7.76 (d, J )
6.0 Hz, 2H), 7.57 (m, 2H), 7.20-7.40 (m, 6H), 7.02-7.08 (m,
3H), 6.91 (dd, J ) 2.5 and 10 Hz, 1H), 6.82 (dd, J ) 2.5 and
8.5 Hz, 1H), 5.29 (s, 1H), 5.23 (d, J ) 8.0 Hz, NH), 4.32-4.41
(m, 3H), 4.20 (m, 1H), 3.75 (s, 3H) 2.76-2.84 (m, 2H). Anal.
Calcd for C32H27NO6S (553.63): C, 69.42; H, 4.92; N, 2.53; S,
5.79. Found: C, 69.22; H, 4.95; N, 2.44; S, 5.54.
S-(9H-Xa n th en -9-yl)cystein e (3). A suspension of cys-
teine (0.61 g, 5.0 mmol) in TFA-1,2-dimethoxyethane (DME)
(1:50; 100 mL) was stirred at 25 °C under N2 until a colorless
clear solution was obtained (several hours). Next 9H-xanthen-
9-ol10 (1.0 g, 5.1 mmol) was added in one portion. The reaction
mixture was stirred at 25 °C for 30 min and then neutralized
to pH 7 by addition of saturated aqueous Na2CO3 solution,
concentrated under reduced pressure, diluted with H2O (50
mL), treated with 10% citric acid to pH ∼ 6, and then stirred
at 25 °C for 1 h. The resultant white solid was collected by
filtration, washed with H2O (3 × 10 mL) and EtOAc (3 × 10
mL), and dried in vacuo over P2O5. Yield: 1.33 g (88%); mp
165 °C (dec.); Rf [MeOH-H2O (4:1)] 0.69; 1H NMR (CD3OD) δ
7.38-7.46 (m, 2H), 7.14-7.21 (m, 2H), 6.98-7.04 (m, 4H), 5.35
(s, 1H), 3.37-3.41 (m, 1H), 2.84 (dd, J ) 4.0 and 14.2 Hz, 1H),
2.72 (dd, J ) 8.1 and 14.2 Hz, 1H). Anal. Calcd for C16H15
-
NO3S (301.36): C, 63.77; H, 5.02; N, 4.65; S, 10.64. Found:
C, 63.80; H, 4.93; N, 4.56; S, 10.47.
S-(2-Meth oxy-9H-xa n th en -9-yl)cystein e (4). The pro-
cedure was exactly as for compound 3 but starting with
2-methoxy-9H-xanthen-9-ol10,12 (3.89 g, 17.1 mmol). Yield:
1
5.20 g (97%); mp 185 °C dec; Rf [MeOH-H2O (4:1)] 0.75; H
NMR (CD3OD) δ 7.50-7.54 (two dd,22 ratio: 1:1, J ) 1.5 and
7.8 Hz, 1H), 7.24-7.31 (m, 1H), 7.02-7.14 (m, 4H), 6.86-6.87
(two dd22, ratio: 1:1, J ) 3.0 and 9.0 Hz, 1H), 5.45 (s, 1H),
3.82 (s, 3H), 3.49-3.54 (m, 1H), 2.80-3.00 (m, 2H). Anal.
Calcd for C17H17NO4S (331.39): C, 61.62; H, 5.17; N,4.23; S,
9.67. Found: C, 61.45; H, 4.93; N, 4.23; S, 9.44.
S-(3-Meth oxy-9H-xa n th en -9-yl)cystein e (5). 3-Methoxy-
9H-xanthen-9-one10,12 (1.50 g, 6.6 mmol) was dissolved in 95%
ethanol (150 mL), following which solid NaOH (1.33 g, 33
mmol) and preactivated Zn dust10,12 (1.72 g, 26 mmol) were
added, each in one portion. The suspension was refluxed for
4 h, cooled to 25 °C, filtered to remove unreacted Zn dust, and
washed with ethanol (3 × 10 mL). The pink filtrate was
(22) Two aromatic protons were resolved because the title Cys
derivative exists as a diastereomeric mixture.