Preparation of N-tBoc l-glutathione dimethyl and di-tert-butyl esters: Versatile synthetic building blocks

Article abstract of DOI:10.1016/j.bmc.2006.10.022

The title l-glutathione derivatives, containing acid- and base-labile esters, respectively, were obtained in good overall yields. N-^tBoc l-glutathione dimethyl ester was prepared via Fischer esterification of l-glutathione disulfide (GSSG) using HCl in dry methanol, protection of the amine with ^tBoc₂O, and tributylphosphine cleavage of the disulfide in wet isopropanol. Alternatively, Fischer esterification and ^tBoc-protection of l-glutathione (GSH) also furnished N-^tBoc glutathione dimethyl ester accompanied by a small amount of S-^tBoc that was removed chromatographically. The di-tert-butyl ester was obtained by S-palmitoylation of GSH in TFA as solvent, N-^tBoc-protection, esterification using ^tBuOH mediated by diisopropylcarbodiimide/copper(I) chloride, and saponification of the thioester. These l-glutathione derivatives are versatile synthetic building blocks for the preparation of S-glutathione adducts.

Full text of DOI:10.1016/j.bmc.2006.10.022

Bioorganic & Medicinal Chemistry 15 (2007) 1062–1066
Preparation of N-tBoc L-glutathione dimethyl and
di-tert-butyl esters: Versatile synthetic building blocks
J. R. Falck,a,* Bhavani Sangrasa and Jorge H. Capdevilab
aDepartments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038, USA
bDepartments of Medicine and Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
Received 16 September 2006; accepted 11 October 2006
Available online 13 October 2006
Abstract—The title L-glutathione derivatives, containing acid- and base-labile esters, respectively, were obtained in good overall
yields. N-tBoc L-glutathione dimethyl ester was prepared via Fischer esterification of L-glutathione disulfide (GSSG) using HCl
in dry methanol, protection of the amine with tBoc2O, and tributylphosphine cleavage of the disulfide in wet isopropanol. Alterna-
tively, Fischer esterification and tBoc-protection of L-glutathione (GSH) also furnished N-tBoc glutathione dimethyl ester
accompanied by a small amount of S-tBoc that was removed chromatographically. The di-tert-butyl ester was obtained by S-palm-
itoylation of GSH in TFA as solvent, N-tBoc-protection, esterification using tBuOH mediated by diisopropylcarbodiimide/copper(I)
chloride, and saponification of the thioester. These L-glutathione derivatives are versatile synthetic building blocks for the prepara-
tion of S-glutathione adducts.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
The tripeptide L-glutathione (L-c-glutamyl-L-cysteinyl-
glycine; GSH) (4) is a common constituent in most ani-
mal cells1 and many bacteria.2 It participates in a variety
of critical physiologic functions, inter alia, signal trans-
duction,3 immunity,4 maintenance of cellular osmolali-
ty,5 defense against reactive oxygen species (ROS) and
free radicals,6 and protein folding.7 Of particular inter-
est is the bioactivation8 or inactivation of xenobiotics,
drugs, and endogenous substrates by GSH-S-conjuga-
tion mediated by a widely distributed family of
GSH-S-transferases.9 The latter category of substrates
includes metabolites of the cyclooxygenase, lipoxyge-
nase, and cytochrome P450 branches of the arachido-
nate cascade.10 As part of our longstanding interest in
the structure elucidation and total synthesis of GSH-S-
conjugates of eicosanoids,11 we required N-protected
L-glutathione derivatives bearing orthogonally protect-
ed esters as synthetic intermediates. Herein, we report
reliable, multi-gram preparations of the base-labile
building block N-tBoc L-glutathione dimethyl ester12
(3) and its acid-labile di-tert-butyl ester analog (9).
The synthesis of 3 began by dissolving commercial
L-glutathione disulfide (1) in MeOH and saturating with
dry HCl gas (Scheme 1).13 After several days, all
volatiles were removed in vacuo and, typically, the crude
tetramethyl ester dihydrochloride salt13 was directly
t
N-carbamoylated using Boc-anhydride in the presence
of NaHCO3 to give 2.14 Disulfide cleavage15 using
n-Bu3P proceeded smoothly and furnished N-tBoc L-glu-
tathione dimethyl ester12 (3) as a white, crystalline solid
in 54% overall yield.
Alternatively, Fischer esterification of 4 as conducted
above and subsequent N-carbamoylation of the resul-
tant dimethyl ester hydrochloride salt16 5 led to 3
(Scheme 2).17 The somewhat superior yield of the route
in Scheme 2 (64% overall) versus that in Scheme 1 (54%
overall) is counterbalanced by the need to remove
chromatographically a small amount of N,S-di-tBoc
dimethyl ester by-product.
Due in large part to its poor solubility in even polar
organic solvents, for example, DMF, DMSO, dioxane,
and THF, the conversion of 4 to its di-tert-butyl ester
proved problematic. Little or no esterification was noted
using a variety of chemical and enzymatic procedures:
isobutylene/H2SO4 or CH3SO3H in dioxane,18 tBuOAc/
Keywords: Amino acids and derivatives; Thioesters; Protecting groups;
Peptides.
*
Corresponding author. Tel.: +1 214 648 2406; fax: +1 214 648
6455; e-mail: j.falck@UTSouthwestern.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.022

J. R. Falck et al. / Bioorg. Med. Chem. 15 (2007) 1062–1066
1063
Scheme 1. Reagents and conditions: (i) HCl, MeOH, 0 ꢁC, 80 h; (ii) tBoc2O, NaHCO3, THF/H2O, 23 ꢁC, 29 h, 75% from 1; (iii) Bu3P, n-PrOH/H2O
(2:1), 23 ꢁC, 4 h, 72%.
t
Scheme 2. Reagents and conditions: (i) HCl, MeOH, 0 ꢁC, 80 h, 91%; (ii) Boc2O, NaHCO3, THF/H2O, 23 ꢁC, 15 h, 70%.
HClO4,19 tBoc2O/DMAP/tBuOH,20 tBuBr/K2CO3/
(PhCH2)Et3NCl in DMF or THF,21 tBuOC(O)F/Et3N/
3. Conclusions
t
DMAP in BuOH under reflux,22 Me2NC(OtBu)2H,23
Herein, we provide convenient and efficient preparations
of the dimethyl and di-tert-butyl esters of N-tBoc L-glu-
tathione. Since these synthetic building blocks can be
orthogonally deprotected under basic and acidic condi-
tions, respectively, we anticipate they will find utility in
the preparation of S-glutathione conjugates, peptide
synthesis, and library development.
tBuOH/EDCI/DMAP,24 BuOH/CDI/DBU,25 transeste-
t
t
rification of 5 with BuOH/H2SO4, BuOH/amano AK
t
t
lipase, and BuOH/pig liver esterase.26 To improve its
solubility characteristics as well as obviate the inherent
nucleophilicity of the thiol, 4 was dissolved in a mini-
mum of TFA and S-acylated with acid chlorides of vary-
ing chain lengths.27,28 The palmitoyl thioester
6
displayed satisfactory behavior and could be isolated
as the solid trifluoroacetate salt (Scheme 3). No N-palm-
itoylation under the acidic conditions was noted.29
Sequential N-carbamoylation and esterification of 6
4. Experimental
4.1. General procedures
t
with BuOH afforded N-Boc 7 and N-Boc di-tert-butyl
ester 8, respectively. The latter esterification, however,
could only be effected in satisfactory yield using N,N0-
diisopropylcarbodiimide in the presence of catalytic
CuCl according to Zhu et al.30 Methanolysis31 of 8 with
NaOMe/MeOH gave rise to 9 without event.
1H and 13C spectra were recorded in CDCl3 unless
otherwise specified using tetramethylsilane as internal
reference. The Michigan State University Mass
Spectroscopy Facility provided high-resolution mass
spectra. All reactions were maintained under an argon
t
Scheme 3. Reagents and conditions: (i) H3C(CH2)14C(O)Cl, F3CCO2H, 23 ꢁC, 20 min, then 40 ꢁC, 40 min, 91%; (ii) O(CO2 Bu)2, NaHCO3, THF/
H2O, 23 ꢁC, 15 h, 64%; (iii) tBuOH, (tPrHN@)2C, CuCl, 23 ꢁC, 12 h, then add 7 in CH2Cl2, 40 ꢁC, 48 h, 70%; (iv) NaOMe, MeOH, 1 h, 23 ꢁC, 71%.

1064
J. R. Falck et al. / Bioorg. Med. Chem. 15 (2007) 1062–1066
23
D
atmosphere. Anhydrous solvents were freshly distilled
from sodium benzophenone ketyl, except for CH2Cl2,
which was distilled from CaH2. Extracts were dried over
anhydrous Na2SO4 and filtered prior to removal of all
volatiles under reduced pressure.
10% MeOH/CH2Cl2, Rf ꢀ 0.25; ½aꢁ ꢂ 26 (c 1.1, EtOH);
1H NMR (400 MHz, CD3OD) d 4.58 (t, 1H, J = 6.4 Hz),
4.01 (d, 1H, J = 5.5 Hz), 3.78 (s, 3H), 3.76 (s, 3H), 3.62
(t, 1H, J = 6.8 Hz), 3.32–3.37 (m, 1H), 2.95 (dd, 1H,
J = 6.7, 14.2 Hz), 2.87 (dd, 1H, J = 7.2, 13.9 Hz), 2.43–
2.51 (m, 2H), 2.06–2.15 (m, 1H), 1.93–2.04 (m, 2H);
13C NMR (100 MHz, CD3OD) d 176.3, 175.3, 173.0,
171.7, 57.0, 54.7, 52.9, 52.81, 42.0, 32.9, 30.7, 27.1.
4.2. Chemistry
4.2.1. Bis-N-tBoc L-glutathione tetramethyl ester disulfide
(2). Dry HCl gas was bubbled through a 0 ꢁC solution of
L-glutathione disulfide (1) (6.0 g, 9.8 mmol) in anhy-
drous MeOH (500 mL) until 48 g of HCl was absorbed.
Stirring was continued at 0 ꢁC for 80 h, then all volatiles
were removed in vacuo. The residue was further dried
using a mechanical vacuum pump for 24 h to give the
corresponding tetramethyl ester dihydrochloride salt as
a colorless gum that was used directly in the next step.
4.2.4. N-tBoc L-glutathione dimethyl ester (3) from 5. A
solution of
5 (2.2 g, 6.6 mmol), NaHCO3 (1.2 g,
14.3 mmol, 2.2 equiv), and tBoc2O (1.4 g, 6.6 mmol,
1.0 equiv) in THF/H2O (1:2.4, 25 mL) was stirred at
room temperature. After 15 h, the solution was extract-
ed with EtOAc (4· 50 mL). The combined extracts were
concentrated in vacuo and the residue was purified by
SiO2 column chromatography to give 3 (1.8 g, 70%) as
described above and the by-product S-tBoc, N-tBoc
dimethyl ester (0.39 g, 11%) as a white solid, mp
A solution of the above tetramethyl ester salt (6.0 g),
NaHCO3 (3.3 g, 39.2 mmol, 4 equiv), and tBoc2O
(5.16 g, 23 mmol, 2.4 equiv) in THF/H2O (2.4:1,
150 mL) was stirred at room temperature. After 29 h,
the pH was adjusted to 3 with concd HCl, and the solution
was extracted with EtOAc (5· 10 mL). The combined ex-
tracts were concentrated in vacuo and the residue purified
by SiO2 column chromatography to give 2 (4.0 g, 75%) as
91 ꢁC; lit.17 mp 92 ꢁC. TLC of S-tBoc, N-tBoc dimethyl
23
ester: 5% MeOH/CH2Cl2, Rf ꢀ 0.25; ½aꢁ ꢂ 32 (c 1.0,
D
23
D
EtOH); lit.17 ½aꢁ ꢂ 37:2 (c 1.0, EtOH); 1H NMR
(400 MHz) d 7.29 (t, 1H, J = 5.2 Hz), 6.93 (d, 1H,
J = 7.2 Hz), 5.44 (d, 1H, J = 7.9 Hz), 4.63–4.69 (m,
1H), 4.25 (d, 1H, J = 4.7 Hz), 4.02 (dd, 1H, J = 5.7,
17.9 Hz), 3.95 (dd, 1H, J = 4.7, 18 Hz), 3.69 (s, 3H),
3.68 (s, 3H), 3.22 (dd, 1H, J = 4.8, 15 Hz), 3.13 (dd,
1H, J = 7.0, 14.6 Hz), 2.29 (t, 2H, J = 7.2 Hz), 2.08–
2.14 (m, 1H), 1.92–2.00 (m, 1H), 1.43 (s, 9H), 1.38 (s,
9H); 13C NMR (100 MHz) d 173.0, 172.7, 170.5,
170.2, 169.9, 155.8, 85.8, 80.2, 53.7, 53.1, 53.0, 52.6,
52.5, 41.4, 32.3, 32.2, 28.4, 28.2.
a white solid, mp 135 ꢁC. TLC: 10% MeOH/CH2Cl2,
23
Rf ꢀ 0.35; ½aꢁ þ 30:5 (c 2.25, CHCl3); 1H NMR
D
(400 MHz) d 8.46–8.58 (m, 1H), 6.72 (d, 1H, J =
8.6 Hz), 5.51–5.56 (m, 1H), 5.32 (d, 1H, J = 8.2 Hz),
4.34–4.44 (m, 1H), 4.15 (dd, 1H, J = 5.8, 18 Hz), 4.09
(dd, 1H, J = 4.9, 18 Hz), 3.76 (s, 3H), 3.74 (s, 3H), 3.06
(dd, 1H, J = 4, 15 Hz), 2.84–2.97 (m, 1H), 2.32–2.44 (m,
2H), 2.14–2.26 (m, 1H), 1.95–2.20 (m, 1H), 1.43 (s, 9H);
13C NMR (100 MHz) d 173.2, 172.8, 171.2, 170.1,
155.9, 80.2, 53.2, 53.1, 52.5, 46.1, 41.5, 32.4, 28.5.
4.2.5. S-Palmitoyl L-glutathione trifluoroacetate (6). Pal-
mitoyl chloride (2.4 mL, 7.8 mmol, 2.4 equiv) was added
dropwise to a solution of L-glutathione (4) (1.0 g,
3.25 mmol) in trifluoroacetic acid (11 mL) under an
argon atmosphere. After stirring at room temperature
for 20 min and at 40 ꢁC for 30 min, the reaction was
quenched by the addition of water (0.25 mL, 14 mmol,
4.3 equiv) and the reaction mixture was stirred for an
additional 1 h at 40 ꢁC. The trifluoroacetic acid was
removed in vacuo, ethyl acetate (20 mL) was added,
the mixture was cooled to 10 ꢁC, and the precipitated
trifluoroacetate salt of 6 (1.6 g, 91%) was collected by fil-
4.2.2. N-tBoc L-glutathione dimethyl ester (3) from 2.
Bu3P (500 lL, 617 mg, 3.05 mmol) was added to a solu-
tion of disulfide 2 (1.6 g, 1.84 mmol) in n-PrOH/H2O
(2:1, 6 mL, degassed with argon) under argon atmo-
sphere. After stirring for 4 h, the n-PrOH was removed
and the aqueous solution was extracted with CH2Cl2
(4· 40 mL). The combined extracts were washed with
water, dried, and concentrated. The residue was purified
by SiO2 column chromatography to give 3 (1.16 g, 72%)
tration, mp 179–181 ꢁC. TLC: MeOH/H2O (2:1),
23
as a white, crystalline solid, mp 94 ꢁC. TLC: 10%
Rf ꢀ 0.67; ½aꢁ ꢂ 15 (c 0.66, EtOH/DMSO (2:1)); 1H
D
23
MeOH/CH2Cl2, Rf ꢀ 0.38; ½aꢁ ꢂ 6:8 (c 1.3, CHCl3);
NMR (400 MHz, CD3SOCD3) d 8.24–8.37 (m, 1H),
4.36–4.45 (m, 1H), 3.60–3.86 (m, 1H), 3.24–3.38 (m,
1H), 2.92–2.99 (m, 1H), 2.84–2.97 (m, 1H), 2.25–2.56
(m, 2H), 2.16 (t, 1H, J = 7.3 Hz), 1.10–1.38 (m, 18H),
0.83 (t, 3H, J = 6.8 Hz); ESMS m/z 546 (M++1); HRMS
(FAB-CI, NBA) Calcd for C26H47N3O7S [M+H]+
546.3214, found 546.3213.
D
1H NMR (400 MHz) d 6.97 (t, 1H, J = 5.5 Hz), 6.83
(d, 1H, J = 7 Hz), 5.29 (d, 1H, J = 8.5 Hz), 4.68 (ddd,
1H, J = 8.5, 6.0, 4.6 Hz), 4.30–4.40 (m, 1H), 4.08 (dd,
1H, J = 5.8, 18 Hz), 4.00 (dd, 1H, J = 18, 5.2 Hz), 3.76
(s, 3H), 3.75 (s, 3H), 3.14 (ddd, 1H, J = 14, 7.9,
4.6 Hz), 2.74–2.84 (m, 1H), 2.38 (t, 2H, J = 6.5 Hz),
2.20–2.26 (m, 1H), 1.90–1.99 (m, 1H), 1.83 (dd, 1H,
J = 8, 11 Hz), 1.43 (s, 9H); 13C NMR (100 MHz) d
173.1, 172.8, 170.4, 170.2, 86.04, 80.3, 54.1, 52.7, 52.5,
41.4, 32.1, 28.5, 28.3, 26.4.
4.2.6. N-tBoc S-palmitoyl L-glutathione (7). Sodium
bicarbonate (984 mg, 11.72 mmol, 4 equiv) and di-
tert-butyldicarbonate (767 mg, 3.5 mmol, 1.2 equiv)
were added sequentially to a solution of 6 (1.6 g,
2.93 mmol) in THF/H2O (1:2.4, 10 mL) under an ar-
gon atmosphere. After stirring overnight, the pH
was adjusted to 4 with 6 N HCl and the reaction
mixture was extracted with EtOAc (4· 5 mL). The
4.2.3. L-Glutathione dimethyl ester hydrochloride (5).
L-Glutathione (4) (2.0 g, 6.51 mmol) was esterified in
acidified MeOH as described for 2 to give 5 (2.2 g,
91%) as a free flowing, white solid, mp 98 ꢁC. TLC:

J. R. Falck et al. / Bioorg. Med. Chem. 15 (2007) 1062–1066
1065
combined organic extracts were dried, concentrated
in vacuo, and the solid white residue was azeotroped
with anhydrous benzene (20 mL) and dried under
high vacuum for 1 h to yield 7 (1.2 g, 64%) as a free
flowing solid, mp 104 ꢁC, sufficiently pure to be used
1H NMR (400 MHz) d 6.93 (d, 1H, J = 6.4 Hz),
6.80–6.85 (m, 1H), 5.26 (d, 2H, J = 7.9 Hz), 4.64–
4.72 (m, 1H), 4.16–4.28 (m, 1H), 3.96 (dd, 1H,
J = 5.5, 13 Hz), 3.87 (dd, 1H, J = 5.4, 11.7 Hz),
3.06–3.18 (m, 2H), 2.78–2.86 (m, 1H), 2.36 (t, 2H,
J = 7.4 Hz), 2.18–2.26 (m, 1H), 1.82 (dd, 1H,
J = 7.9, 9.8 Hz), 1.47 (s, 18H), 1.45 (s, 9H); 13C
NMR (75 MHz) d 172.6, 171.6, 170.1, 168.7, 156.0,
82.4, 80.1, 54.6, 53.5, 42.2, 32.5, 29.1, 28.4, 28.14,
28.09, 26.7; ESMS m/z 542 (M++23); HRMS
(FAB-CI, NBA) Calcd for C23H41N3O8S [M+H]+
520.2693, found 520.2692.
directly in the next step. TLC: 30% MeOH/EtOAc,
23
D
Rf ꢀ 0.25; ½aꢁ ꢂ 8 (c 1.7, CHCl3); 1H NMR
(400 MHz) d 7.50–8.20 (m, 2H), 6.47 (br s, 1H),
5.65 (br s, 1H), 4.71 (d, 1H, J = 5.8 Hz), 4.13–4.35
(m, 2H), 3.72–3.88 (m, 2H), 3.18–3.36 (m, 2H),
2.55 (t, 2H, J = 7.6 Hz), 2.35 (t, 2H, J = 7.3 Hz),
1.45 (s, 9H), 1.25 (s, 26H), 0.88 (t, 3H,
J = 6.7 Hz); 13C NMR (75 MHz) d 200.1, 179.4,
175.4, 174.2, 172.8, 171.6, 156.2, 85.3, 82.2, 80.6,
53.0, 44.2, 41.6, 34.3, 32.1, 29.9, 29.7, 29.6, 29.5,
29.3, 29.2, 28.5, 27.6, 27.8, 25.0, 22.9, 14.3; ESMS
m/z 668 (M++23); HRMS (FAB-CI, NBA) Calcd
for C31H55N3O9S [M+H]+ 646.3737, found 646.3737.
Acknowledgments
Financial support was provided by the Robert A. Welch
Foundation and NIH (GM31278, DK38226,
GM37922).
4.2.7. N-tBoc S-palmitoyl L-glutathione di-tert-butyl ester
(8). tert-Butanol (2.18 mL, 23 mmol, 10.6 equiv) and
N,N0-diisopropylcarbodiimide (3.2 mL, 20.8 mmol,
9.6 equiv) were stirred overnight in the presence of a
catalytic amount of CuCl (25 mg) under an argon
atmosphere. The resulting O-tert-butyl N,N0-diisopro-
pylisourea solution was diluted with dry dichloro-
methane (10 mL) followed by the addition of 7
(1.4 g, 2.16 mmol) and then the reaction mixture was
heated under reflux. After 2 days, the reaction mixture
was filtered through Celiteꢂ and the filter cake was
washed with dichloromethane (150 mL). The filtrate
was concentrated in vacuo and the residue purified
by column chromatography on silica gel impregnated
with triethylamine (2 mL Et3N/100 g SiO2) using
30% EtOAc/hexanes as eluant to give 8 (1.15 g,
References and notes
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70%) as a thick syrup. TLC: 5% MeOH/CH2Cl2,
23
D
Rf ꢀ 0.66; ½aꢁ ꢂ 12:5 (c 2.06, CHCl3); 1H NMR
(300 MHz) d 6.96 (t, 1H, J = 5.2 Hz), 6.77 (d, 1H,
J = 7.0 Hz), 5.24 (d, 1H, J = 7.6 Hz), 4.60 (dt, 1H,
J = 4.8, 7.9 Hz), 4.16–4.22 (m, 1H), 3.90 (dd, 1H,
J = 5.5, 12.6 Hz), 3.34 (dd, 1H, J = 4.6, 14.3 Hz),
3.25 (dd, 1H, J = 7.94, 14.3 Hz), 2.56 (t, 2H,
J = 7.3 Hz), 2.12–2.38 (m, 2H), 1.63 (t, 2H,
J = 7.1 Hz), 1.46 (s, 9H), 1.45 (s, 9H), 1.43 (s, 9H),
1.24 (br s, 26H), 0.87 (t, 3H, J = 7.0 Hz); 13C NMR
(75 MHz) d 200.5, 172.8, 171.5, 170.1, 168.6, 155.9,
82.3, 80.0, 53.6, 44.1, 42.2, 32.4, 32.0, 30.4, 29.78,
29.77, 29.75, 29.71, 29.54, 29.47, 29.3, 29.1, 28.8,
28.5, 28.2, 28.1, 25.7, 22.8, 14.3; ESMS m/z 780
(M++23); HRMS (FAB-CI, NBA) Calcd for
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4.2.8. N-tBoc L-glutathione di-tert-butyl ester (9). A
freshly prepared 0.1 N solution of NaOMe in MeOH
(14 mL, 1.45 mmol) was added to a stirring solution
of 8 (1.0 g, 1.32 mmol) in methanol (14 mL) under
an argon atmosphere. After 1 h, the reaction mixture
was cooled to 5 ꢁC and acidified to pH 5 using 5%
acetic acid in ether. All volatiles were removed in vac-
uo and the residue was purified by column chroma-
tography using 50% EtOAc/hexanes as eluant to
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