Large-Scale Preparation of N-Butanoyl- l -glutathione (C4-GSH)

Article abstract of DOI:10.1021/acs.oprd.9b00120

A novel two-step synthesis of N-butanoyl-l-glutathione (C4-GSH) was developed involving the chemical modification of the commercially available starting material l-glutathione (GSH). This process not only has the advantage of selective acylation of the GSH amino group without the use of previous solid-phase organic synthesis and/or protecting group chemistry but also highlights the use of inexpensive reagents such as butyric anhydride and sodium methoxide as well as environmentally acceptable solvents such as water and methanol. This chromatography- and salt-free synthesis of C4-GSH is cost-effective, safe, efficient, and easy to scale up.

Full text of DOI:10.1021/acs.oprd.9b00120

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Communication
Large-Scale Preparation of N-Butanoyl-L-glutathione (C4-GSH)
Francesca Bartoccini, Michele Mari, Michele Retini, Alessandra Fraternale, and Giovanni Piersanti
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.9b00120 • Publication Date (Web): 01 Aug 2019
Downloaded from pubs.acs.org on August 1, 2019
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Large-Scale Preparation of
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N‑Butanoyl‑L‑glutathione (C4-GSH)
Francesca Bartoccinia,b, Michele Maria,b, Michele Retinia,b,
Alessandra Fraternalea, Giovanni Piersantia,b,*
aDepartment of Biomolecular Sciences, University of Urbino Carlo
Bo. P.zza Rinascimento 6, 61029 Urbino, PU, Italy
bGluos s.r.l., Piazza Brancaleoni, 1, 61049 Urbania (Italy)
*e-mail: giovanni.piersanti@uniurb.it
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TABLE OF CONTENTS
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O
CO2H
O
CO2H
O
H
H
two steps
N
N
N
N
CO2H
H2N
N
CO2H
H
H
H
O
O
SH
SH
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C4-GSH
GSH
Advantages: Practical, Efficient, Mild, Selective, Safe
Challenges: Scalability, Sustainability, Chemoselectivity
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ABSTRACT: A novel two-step synthesis of N-butanoyl-L-glutathione
(C4-GSH) has been developed involving the chemical modification of
the commercially available starting material L-glutathione (GSH).
This process not only has the advantage of selective acylation of
the GSH amino group without the use of previous solid-phase organic
synthesis and/or protecting group chemistry, but it also
highlights the use of inexpensive reagents such as butyric
anhydride and sodium methoxide as well as environmentally
acceptable solvents such as water and methanol. This
chromatography- and salt-free synthesis of C4-GSH is cost-
effective, safe, efficient, and easy to scale-up.
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KEYWORDS: short peptides; large scale; glutathione; methanolysis;
water
1. INTRODUCTION
The synthesis of novel glutathione (GSH) derivatives and
analogues have attracted an increasing interest due to their
potential therapeutic effects and pharmacological properties in
the treatment of a wide range of diseases.1 Indeed, native reduced
GSH (γ-L-glutamyl-L-cysteinyl-glycine), a thiol group-containing
pseudopeptide, serves as an important intracellular water-soluble
nonenzymatic antioxidant and detoxificant.2 However, the use of
GSH as a therapeutic agent is limited due to its poor stability
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and low bioavailability; therefore, researchers have explored new
potential approaches to maintain, restore, or increase cellular
GSH levels by providing a source of thiols (i.e. cysteine) for GSH
synthesis.3 In contrast to most peptide linkages that are formed
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between an -amino group and an adjacent carboxyl group, the
linkage between the glutamic acid residue and the cysteine residue
of GSH is from the -carboxyl group of glutamic acid. This unique
structure causes GSH to exist as a zwitterion in aqueous solution,
thus making it difficult to permeate cell membranes (Figure 1).
GSH displays four “free” functional groups (one thiol, one terminal
amino group, and two carboxyl groups, with pKa values of 8.7, 9.2,
2.1, and 3.5, respectively), of which the thiol group is a key
functional element and also the most reactive one, capable of
generating glutathione disulfide (GSSG).4 Therefore, the unique
structural arrays, obstacles to be overcome and interesting
biological activities displayed by GSH make it a particularly
appealing target for derivatization efforts. In addition, mild and
efficient methods to synthesize N-acylglutathiones are desired.
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CO2
O
CO2
O
S
H
H
N
N
H3N
N
CO2H
H3N
N
H
CO2H
H
O
O
SH
1
GSH,
O
R
S-Acyl derivatives of GSH (Ref. 5)
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CO2R
O
O
CO2H
O
H
N
H
N
H2N
N
CO2R
R
N
N
CO2H
H
H
H
O
O
SH
Ester derivatives of GSH (Ref. 6)
SH
N-Acyl derivatives of GSH (Ref. 7)
3
R = n-Pr, C4-GSH,
Figure 1. Structures of glutathione and glutathione derivatives.
2. RESULTS AND DISCUSSION
Currently, the synthesis of GSH derivatives requires clever
manipulation
of
protecting
groups.
However,
a
protection/deprotection event introduces at least two steps into
a sequence, incurring costs from additional reagents and waste
disposal, and generally leads to a reduced overall yield. The
chemoselective functionalization of the highly reactive cysteine
residue5 and the (one or both) terminal carboxyl group6 has been
particularly successful, whereas the more challenging modification
on the α-amino group of the glutamic acid residue has only become
a research focus recently (Figure 1).7 Published methods to
synthesize nonamphoteric derivatives of N-acyl glutathiones have
utilized the following approaches: (a) N-acylation of GSSG with an
activated ester, followed by reduction;7a (b) use of a trityl group
to protect the thiol group of GSH, then N-acylation followed by
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trityl group deprotection;7b (c) use of 4-nitrobenzoyl to protect
the thiol group of GSH, then N-acylation with 1-acyl-1H-
benzotriazole followed by deprotection using pyrrolidine in dry
THF–methanol for 4 h;7c or (d) solid‐phase peptide synthesis using
the appropriate fluorenylmethyloxycarbonyl amino acid and
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carboxylic
acid
under
basic
conditions
with
hydroxybenzotriazole/N,N-diisopropylethylamine activation in
anhydrous N-methylpyrrolidone.7d However, these methods have
various drawbacks and limitations for large-scale applications
because of the limited amount of accessible starting
materials/reagents and sometimes problems with purification from
inorganic compounds. Furthermore, no methods for the
chemoselective acylation of the free amino group of GSH without
involving the thiol group have been reported simply because the
anionic thiol group is more nucleophilic than the amino group.8
Among the different N-acyl glutathione derivatives, N-
butanoyl glutathione (3) (also known as C4-GSH or GSH-C4) has
gained considerable interest lately due to its interesting
antioxidant, antiviral, anti-inflammatory, anti-influenza, and
immunomodulatory activities as well as its very low toxicity.9
Despite these promising features, C4-GSH is still not used in
massive biomedical applications/industries because of its high
price and low-scale production.9d Herein, we report a new and
sustainable two-step synthesis of C4-GSH (3) from commercially
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available GSH (1) using butyric anhydride at room temperature (23
°C) in weakly basic aqueous solution, followed by selective
thioester methanolysis of the S,N-diacylated product, as
illustrated in Scheme 1. Pleasingly, this method did not produce
any side reactions, and column chromatography purification can be
avoided. The possibilities of the chemical modification of
commercially available, low-cost, natural product 1 in combination
with a reduced number of reaction steps, easy product isolation,
and high yields are the most important advantages of the proposed
synthetic route to 3.
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Scheme 1. Synthetic Route for C4-GSH (3)
Butyric anhydride
(2.05 equiv.)
Na2CO3 (1 M)
rt, 4 h
NaOMe
(2 equiv.)
MeOH
CO2H
O
O
CO2H
O
S
O
CO2H
O
H
H
H
N
then 6 M HCl
rt, 1 h
N
N
H2N
N
CO2H
N
N
CO2H
N
N
CO2H
H
H
H
H
H
O
O
O
SH
SH
O
O
3
C4-GSH,
2
C(4+4)-GSH,
(1 M)
1
L-GSH reduced,
(0.4 M)
O
OMe
OH
Due to the poor solubility of GSH in common organic solvents
and its high solubility in water, we decided to treat GSH with
different acylating agents in weakly basic aqueous solution at
room temperature. Multiple experiments were carried out to screen
for an effective base, the type and the proper molar equivalents
of acyl donor for GSH acylation; the results are presented in
Table 1. All tested acyl donor afforded a mixture of mono- and
double-acylated products 2 and 4 respectively, in scarse to
excellent conversion with the exception of 2,2,2-trifluoroethyl
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esters (Tableꢀ1, entries 1-12). Moderate conversion was observed
using enolesters such as vinyl butanoate, although some reactivity
was observed in combination with sodium carbonate as a base and
water as exclusive solvent (Tableꢀ1, entries 10,11). Butyric
anhydride, available in bulk at reasonable prices, turned out to
be a very suitable donor substrate leading to product 2 with
complete conversion and with high yield. In all cases, a base as
an additive improved the reaction leading to complete consumption
of substrate (up to >99ꢁ%, Tableꢀ1, entries 13-21). The best result
was obtained with treatment of 1 dissolved in 1 M aqueous Na2CO3
solution (final concentration of GSH, 0.4 M) with butyric anhydride
(2.05 equiv.) at room temperature (Table 1, entry 21). In this
case, full conversion with a very high purity was achieved, and
the impurity content was minimal (1%, by HPLC assay and qNMR).
Notably, no chemoselective monobutanoylation at the amino group
was observed under any conditions, probably because the anionic
thiol group is more nucleophilic than the amino group. In addition,
no acyl migration to the amino group was observed from the mono S-
butylated GSH under such basic pH conditions. The “classical” S-
to N-acyl transfer has been reported to proceed efficiently through
five- or six-membered cyclic transition states with optimum at pH
7.0−7.3. However, for larger ring sizes (te n membered ring in
this case), the S-to-N acyl transfer becomes rate-determining step
and ring-size of the transition state becomes a critical factor in
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the successful outcome of the process. As in the case of
intermolecular S-to-N acyl transfer, slower formation of the amide
bond results in increased competition from thioester hydrolysis.
Of the two carboxylic acid groups present in GSH, one forms a
zwitterion with the amino group, but the other is free; therefore,
we used sodium carbonate (2.5 equiv) in order to neutralize it and
to afford the corresponding S,N doubly butylated GSH. The mild
reaction conditions and the stoichiometric ratio of anhydride
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employed prevented the formation of side reactions, such as -
acylamido ketone formation, the Dakin–West reaction10 on the free
amino acid side, and intramolecular dehydration on the N-acyl
glycine side. Acidification with 6 M aqueous HCl was used to adjust
the bulk pH to 3–4, allowing protonation of the sodium butyrate
side product, which was washed out with methyl tert-butyl ether
(MTBE). Next, 6 M aqueous HCl was added until the pH of the mixture
was adjusted to 1–2. The formed precipitate was collected on a
Buchner funnel and washed with H2O and MTBE to afford the desired
pure S,N doubly acylated GSH (2) derivatives as white solids in
excellent yields.
Table 1. Effect of acyl donor and base on the acylation reactiona
O
CO2H
O
S
CO2H
O
S
CO2H
O
H
H
H
N
Acyl donor
Base, Solvent
N
N
+
n-Pr
N
N
H
CO2H
H2N
N
H
CO2H
H2N
N
CO2H
H
H
O
O
O
Temperature
time
SH
1
4
2
O
n-Pr
O
n-Pr
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Ratio Yie
Tim
e
(h)
of
ld
of
Entr Acyl
Donor
Conv.
Base
Solvent (M)
Temp.
b
y
(equiv)
1/2/4
2
c
(%)d
7
8
n-PrCOOH (1)
9
1
2
-
H2O (0.5 M)
H2O (0.01)
rt
rt
20
3
100
20
0/1/3
1/3/7
-
-
(n-PrCO)2O (1)
n-PrCOCl (2)
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13
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Na2CO3 (0.1 M)
NaHCO3 (0.1 M)
H2O/CH3CN
0.1 M)
(3:1;
3
4
5
n-PrCOCl (2)
n-PrCOFe (2)
(n-PrCO)O2 (2)
Et3N (4 equiv)
rt
rt
rt
2
0
-
-
-
-
-
H2O/CH3CN (20:1)
16
20
80
1/5/1
H2O/CH3CN
0.1 M)
(3:1;
0/10/
1
Et3N (4)
100
n-PrCO2CH2CF3
(2.1)
6
7
Na2CO3 (1 M)
H2O (0.4 M)
rt
60
4
0
1/0/0
1/0/0
1/0/0
1/0/0
0/1/2
-
-
-
-
-
-
-
-
-
-
n-PrCO2CH=CH2
(2.1)
reflu
x
-
Toluene (0.1 M)
0
n-PrCO2CH=CH2
(2.1)
NaHCO3
equiv)
(6 THF/H2O (1:1; 0.1 reflu
8
4
0
M)
x
n-PrCO2CH=CH2
(2.1)
DIPEA
equiv)
(0.2
reflu
x
9
Toluene (0.1 M)
4
0
n-PrCO2CH=CH2
(2.1)
10
11
12
13
14
15
Na2CO3 (1 M)
Na2CO3 (1 M)
Et3N (2)
H2O (0.4 M)
H2O (0.4 M)
rt
rt
rt
rt
rt
rt
4
100
100
100
100
100
100
n-PrCO2CH=CH2
(2.1)
decom
p
24
2
H2O/CH3CN
0.1 M)
(1:10;
n-PrCOBtf (2.1)
0/1/4
(n-PrCO)2O
(2.05)
K3PO4
equiv)
(2.5
0/10/
1
H2O (0.4 M)
H2O (0.4 M)
H2O (1 M)
6
(n-PrCO)2O
(2.05)
0/10/
1
K2CO3 (1 M)
NaOH (5 M)
2
(n-PrCO)2O
(2.05)
decom
p
1
Na2CO3 (0.1 M)
NaHCO3 (0.1 M)
16
17
(n-PrCO)2O (2.2)
H2O (0.01 M)
rt
rt
1
1
100
100
0/1/0
0/1/0
68
75
(n-PrCO)2O (2.2) Na2CO3 (0.5 M) H2O (0.1 M)
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NaHCO3 (0.5 M)
4
5
6
7
8
9
18
19
20
(n-PrCO)2O (2.2) Na2CO3 (0.5 M) H2O (0.2 M)
rt
rt
rt
2
2
2
100
100
100
0/1/0
0/1/0
0/1/0
78
81
80
(n-PrCO)2O (2.2) Na2CO3 (1 M)
(n-PrCO)2O (2.1) Na2CO3 (1 M)
H2O (0.4 M)
H2O (0.4 M)
10
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(n-PrCO)2O
Na2CO3 (1 M)
(2.05)
21
H2O (0.4 M)
rt
4
100
0/1/0
89
aReaction conditions: 1 (1 g, 3.25 mmol). Conversion was
b
c
calculated by peak areas in LC/UV/MS. Ratio determined by peak
areas in LC/UV/MS. dIsolated yield. en-PrCOF obtained from n-PrCO2H
(2.2 equiv), cyanuric fluoride (1.1 equiv), and pyridine (2.2
equiv) in CH2Cl2, room temperature, 3 h.
benzo[d][1,2,3]triazol-1-yl)butan-1-one obtained from n-PrCO2H (1
equiv), 1H-benzo[d][1,2,3]triazole (4 equiv), and SOCl2 (1 equiv)
in THF, 2 h. rt, room temperature; All data are the average of two
experiments.
f1-(1H-
The next focus of our study was to find an easy and
sustainable way for selective S-debutanoylation. We expected the
S-acyl groups to be “active esters” and especially susceptible to
dilute alkaline conditions. Therefore, we preferred to remove the
S-butanoyl group by methanolysis in the presence of sodium
methoxide, since this reaction proceeds rapidly and almost
quantitatively without causing -elimination or racemization or
affecting amide bonds or other sensitive parts of the molecule.
Additionally, the methyl butanoic acid ester byproduct has a low
boiling point (102 °C at atmospheric pressure), so both methyl
butanoate and methanol can be easily removed by evaporation. Thus,
treatment of these doubly acylated GSH derivatives with sodium
methoxide in methanol for 1 h at room temperature, followed by
removal of methanol in vacuo and acidification with Dowex 50WX8(H)
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resin, gave the corresponding N-acyl glutathione in 69% yield. The
cation-exchange resin in the column was activated by the
displacement of sodium into the protonated form with sulfuric acid
prior to the main reaction. The substrate 3 in water was pumped
into the column at a controlled reaction temperature, producing a
continuous stream of the desired compound 3, without requiring an
extraction step. NMR and optical rotation data were consistent
with a sample obtained previously.7d To highlight the synthetic
utility of the present protocol, a Kg-scale process was performed,
and the efficiency of the small-scale reaction was retained upon
scale-up, delivering 3 in comparable yield. The current process
generates about 9.4 kg of waste/kg of C4-GSH (E-factor value, not
including water).11
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3. CONCLUSION
We have successfully developed a new, practical, and convenient
process for the synthesis of enantiomerically pure C4-GSH on kg
scale in 69% overall yield, starting from natural and largely
available GSH. The two-step process was conducted under mild
reaction conditions, and it employed environmentally friendly raw
materials and solvents, without the use of hazardous or toxic
chemicals. In addition, the procedure does not require the use of
expensive reagents or chromatography, and the small amounts of
impurities are easily removed.
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4. EXPERIMENTAL SECTION
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General. All reagents and solvents were purchased from commercial suppliers and used
without further purification. 1H NMR and 13C NMR spectra were recorded on 400 spectrometer,
using D2O as solvent. Chemical shifts ( scale) are reported in parts per million (ppm) relative to
the central peak of the solvent. Coupling constants (J values) are given in hertz (Hz). Melting
points were determined on capillary melting point apparatus and are uncorrected. HPLC analysis
were carried out using column Phenomenex C6-phenyl 150 mm x 4.60 mm x 5mm. See the
Supporting Information (SI) for the gradient elution program for HPLC. Optical rotation analysis
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was performed with a polarimeter using a sodium lamp (λ 589 nm, D-line); [α]D values are
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reported in 10−1 deg cm2 g−1; concentration (c) is in g for 100 mL. HRMS analysis was performed
using a Q-TOF microTM mass spectrometer. The purity of synthesized compound
was confirmed to be ≥98.8% purity by qNMR.
N2-butyryl-N5-((R)-3-(butyrylthio)-1-((carboxymethyl)amino)-1-
oxopropan-2-yl)-L-glutamine (2).
A 12-L, three-neck round-bottomed flask equipped with dropping funnel and
motorized stirrer was charged with 1M solution of aqueous Na2CO3
(8 L) and GSH (1) (1 Kg, 3.26 mol, 0.4 M). Butyric anhydride (1.1 L, 6.68
mol, 2.05 equiv) was added dropwise over 2 hours and stirred at room temperature
for 2 hours (100% conversion by HPLC). The solution was acidified to pH 3.5 with 6N HCl
(around 2.2 L). The aqueous phase was washed with methyl tert-butyl ether (3 x 2.6 L) and then
it was acidified to pH 1.5 with 6 N HCl (around 1 L). The solid obtained was filtered and washed
with water (3 x 3 L) to give N,S-dibutanoyl glutathione 2 (1.3 Kg, 89 %) as
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a white amorphous solid.1H NMR (400 MHz, D2O) δ 4.62 (dd, J1 = 8.0
and J2 = 5.0 Hz, 1H), 4.32 (dd, J1 = 9.0 and J2 = 5.0 Hz, 1H), 3.96
(s, 2H), 3.42 (dd, J1 = 14.5 and J2 = 5.0 Hz, 1H), 3.13 (dd, J1 =
145 and J2 = 8.0 Hz, 1H), 2.59 (t, J = 7.0 Hz, 2H), 2.39 (t, J =
7.5 Hz, 2H), 2.24 (t, J = 7.0 Hz, 2H), 2.19-2.12 (m, 1H), 2.00-
1.91 (m, 1H), 1.65–1.54 (m, 4H), 0.88 (t, J = 7.0, 3H), 0.87 (t,
J = 7.0, 3H).13C NMR (100 MHz, D2O) δ 203.7, 177.3, 175.3, 174.9,
173.0, 172.1, 52.6, 52.1, 52.1, 45.3, 41.2, 37.3, 31.6, 29.9,
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26.3, 18.9, 12.7, 12.6. Mp: 96 - 98 ° C.[α]20 = - 30.0 (c = 0.05,
D
MeOH).HRMS (ESI) m/z calcd for C18H30N3O8S (M + H)+ 448,1748; Found
448.1751.
N2-butyryl-N5-((R)-1-((carboxymethyl)amino)-3-mercapto-1-
oxopropan-2-yl)-L-glutamine (3).
A 6-L, round-bottomed flask equipped with magnetic stirring bar was charged
with N,S-dibutanoyl glutathione (2) (1.3 Kg, 2.9 mol) prepared
above and 3 L of methanol. The solution was cooled to 0 °C and sodium methoxide (312
g, 5.8 mol, 2 equiv) was added portionwise in 1 hour (check HPLC). The solution was
stirred for 20 minutes at room temperature and then the solvent was
evaporated under reduce pressure. The resulting crude was taken up
with H2O (200 mL)and poured into a beaker containing Dowex 50W-X8(H) (4 L water wet
resin) and H2O (1.2 L). The suspension was filtered and the resin was washed with H2O (3 x 1.2
L). The filtrate was concentrated at reduced pressure to a volume
of 2 L; methyl tert-butyl ether (3 L) was added and the mixture
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was vigorously stirred for 1 hour at room temperature. The solid obtained
was filtered and washed with methyl tert-butyl ether (3 x 2 L) to
give 3 (760 g, 69%) as a white amorphous solid. 1H NMR (400 MHz,
D2O) δ 4.52 (dd, J1 = 7.0 and J2 = 5.5 Hz, 1H), 4.25 (dd, J1 = 9.0
and J2 = 5.0 Hz, 1H), 3.89 (s, 2H), 2.92–2.83 (m, 2H), 2.42 (t, J
= 7.5 Hz, 2H), 2.23 (t, J = 7.5 Hz, 2H), 2.19 – 2.12 (m, 2H), 1.99-
1.91 (m, 1H), 1.61-1.52 (m, 2H), 0.87 (t, J = 7.5Hz, 3H). 13C NMR
(100 MHz, D2O) δ 177.2, 176.0, 175.3, 173.8, 172.3, 55.5, 52.6,
41.7, 37.4, 31.6, 26.5, 25.5, 18.9, 12.8. Mp : 176 - 178 ° C. [α]20
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= - 22 (c = 0.56, MeOH). HRMS (ESI) m/z calcd for C14H24N3O7S (M +
D
H)+ 378,1329; Found 378.1317.
ASSOCIATED CONTENT
1H and 13C NMR spectra and HPLC analysis for 2 and 3 were reported.
(PDF)
qNMR of compound 3. (PDF)
AUTHOR INFORMATION
Corresponding Author
*giovanni.piersanti@uniurb.it.
Author Contributions
All authors have approved the final version of this manuscript.
ACKNOWLEDGMENTS
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We are grateful to Professor Mauro Magnani for useful discussions.
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CONFLICT OF INTEREST
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F.B., A.F. and G.P., are inventors on patent application
submitted by the authors and University of Urbino Carlo Bo.
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