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

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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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Organic Process Research & Development

Large-Scale Preparation of 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: [email protected]

ACS Paragon Plus Environment

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Organic Process Research & Development 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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TABLE OF CONTENTS O

CO 2H N H

H N O

C4-GSH

O N H SH

CO 2H

two steps CO 2H

H N

H 2N O

O N H SH

CO 2H

GSH

Advantages: Practical, Efficient, Mild, Selective, Safe Challenges: Scalability, Sustainability, Chemoselectivity


2

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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 highlights anhydride acceptable

and/or the and

protecting

use

sodium

solvents

chromatography-

of

and

group

inexpensive methoxide such

as

salt-free

as

chemistry, reagents well

such

as

water

and

synthesis

of

but

it

as

also

butyric

environmentally methanol. C4-GSH

is

This cost-

effective, safe, efficient, and easy to scale-up.

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


3


Page 4 of 20

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 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.


4

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CO2

H N

H 3N

O

CO2

N H SH

O

CO2H

H 3N O

GSH, 1

CO2R

H N

H 2N

H N

O N H

CO2H

S

O R S-Acyl derivatives of GSH (Ref. 5) O

O

O

N H SH

CO2R

Ester derivatives of GSH (Ref. 6)

R

CO2H N H

H N O

O N H SH

CO2H

N-Acyl derivatives of GSH (Ref. 7) R = n-Pr, C4-GSH, 3

Figure 1. Structures of glutathione and glutathione derivatives.

2. RESULTS AND DISCUSSION Currently,

the

manipulation

synthesis of

of

GSH

protecting

derivatives groups.

requires

clever

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


5


Page 6 of 20

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

carboxylic

fluorenylmethyloxycarbonyl

acid

under

basic

amino

conditions

hydroxybenzotriazole/N,N-diisopropylethylamine N-methylpyrrolidone.7d

anhydrous

acid

However,

and with

activation

these

methods

in 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

antioxidant,

interest

antiviral,

lately

due

anti-inflammatory,

to

its

interesting

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


6

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available GSH (1) using butyric anhydride at room temperature (23 °C)

in

weakly

thioester

basic

aqueous

methanolysis

of

solution, the

followed

by

S,N-diacylated

selective

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. Scheme 1. Synthetic Route for C4-GSH (3)

CO2H H 2N

H N

O

N H O SH L-GSH reduced, 1 (0.4 M)

CO2H

Butyric anhydride (2.05 equiv.) Na2CO3 (1 M) rt, 4 h then 6 M HCl

O

CO2H N H

O

N H

O

C(4+4)-GSH, 2 (1 M)

OH

O

H N

CO2H

S

NaOMe (2 equiv.) MeOH rt, 1 h

O

CO2H N H

O

O

H N O

C4-GSH, 3

O N H SH

CO2H

OMe

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


7


Page 8 of 20

esters (Table 1, 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

(Table 1,

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 >99 %, Table 1, 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 Sbutylated GSH under such basic pH conditions. The “classical” Sto 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


8

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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 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

CO2H

H N

H 2N O

1

O N H SH

Acyl donor CO2H Base, Solvent Temperature time

O n-Pr

CO2H N H

O

H N

N H

O 2

CO2H CO2H

n-Pr

N H

O

S O

O

H N

+ H N 2

4

CO2H

S O

n-Pr


9


Entr y

Acyl (equiv)

Donor

Base

Solvent (M)

Page 10 of 20

Temp.

Tim e (h)

Conv. b

Ratio of 1/2/4 c

Yie ld of 2 (%)d

n-PrCOOH (1) 1

-

H2O (0.5 M)

rt

20

100

0/1/3

-

H2O (0.01)

rt

3

20

1/3/7

-

rt

2

0

-

-

(n-PrCO)2O (1) Na2CO3 (0.1 M) 2

n-PrCOCl (2) NaHCO3 (0.1 M)

3

n-PrCOCl (2)

Et3N (4 equiv)

H2O/CH3CN 0.1 M)

4

n-PrCOFe (2)

-

H2O/CH3CN (20:1)

rt

16

80

1/5/1

-

5

(n-PrCO)O2 (2)

Et3N (4)

H2O/CH3CN 0.1 M)

rt

20

100

0/10/ 1

-

6

n-PrCO2CH2CF3 (2.1)

Na2CO3 (1 M)

H2O (0.4 M)

rt

60

0

1/0/0

-

7

n-PrCO2CH=CH2 (2.1)

-

Toluene (0.1 M)

reflu x

4

0

1/0/0

-

8

n-PrCO2CH=CH2 (2.1)

NaHCO3 equiv)

(6

THF/H2O (1:1; 0.1 M)

reflu x

4

0

1/0/0

-

9

n-PrCO2CH=CH2 (2.1)

DIPEA equiv)

(0.2

Toluene (0.1 M)

reflu x

4

0

1/0/0

-

10

n-PrCO2CH=CH2 (2.1)

Na2CO3 (1 M)

H2O (0.4 M)

rt

4

100

0/1/2

-

11

n-PrCO2CH=CH2 (2.1)

Na2CO3 (1 M)

H2O (0.4 M)

rt

24

100

decom p

-

12

n-PrCOBtf (2.1)

Et3N (2)

H2O/CH3CN 0.1 M)

rt

2

100

0/1/4

-

13

(n-PrCO)2O (2.05)

K3PO4 equiv)

H2O (0.4 M)

rt

6

100

0/10/ 1

-

14

(n-PrCO)2O (2.05)

K2CO3 (1 M)

H2O (0.4 M)

rt

2

100

0/10/ 1

-

15

(n-PrCO)2O (2.05)

NaOH (5 M)

H2O (1 M)

rt

1

100

decom p

-

16

(n-PrCO)2O (2.2)

H2O (0.01 M)

rt

1

100

0/1/0

68

H2O (0.1 M)

rt

1

100

0/1/0

75

(2.5

(3:1;

(3:1;

(1:10;

Na2CO3 (0.1 M) NaHCO3 (0.1 M) 17

(n-PrCO)2O (2.2)

Na2CO3 (0.5 M)


10

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NaHCO3 (0.5 M) 18

(n-PrCO)2O (2.2)

Na2CO3 (0.5 M)

H2O (0.2 M)

rt

2

100

0/1/0

78

19

(n-PrCO)2O (2.2)

Na2CO3 (1 M)

H2O (0.4 M)

rt

2

100

0/1/0

81

20

(n-PrCO)2O (2.1)

Na2CO3 (1 M)

H2O (0.4 M)

rt

2

100

0/1/0

80

21

(n-PrCO)2O (2.05)

Na2CO3 (1 M)

H2O (0.4 M)

rt

4

100

0/1/0

89

aReaction

conditions: 1 (1 g, 3.25 mmol). bConversion was calculated by peak areas in LC/UV/MS. cRatio 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 f1-(1Hequiv) 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. 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

methoxide,

since

this

reaction

in

the

proceeds

presence rapidly

of

sodium

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)


11


Page 12 of 20

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

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.


12

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4. EXPERIMENTAL SECTION 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 was performed with a polarimeter using a sodium lamp (λ 589 nm, D-line); [α]D20 values are 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)-1oxopropan-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


13


Page 14 of 20

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), 2.001.91 (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,

26.3, 18.9, 12.7, 12.6. Mp: 96 - 98 ° C. [α]20

D

= - 30.0 (c = 0.05,

MeOH). HRMS (ESI) m/z calcd for C18H30N3O8S (M + H)+ 448,1748; Found 448.1751. N2-butyryl-N5-((R)-1-((carboxymethyl)amino)-3-mercapto-1oxopropan-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


14

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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), 1.991.91 (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 D

= - 22 (c = 0.56, MeOH). HRMS (ESI) m/z calcd for C14H24N3O7S (M +

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 *[email protected]. Author Contributions All authors have approved the final version of this manuscript.

ACKNOWLEDGMENTS


15


Page 16 of 20

We are grateful to Professor Mauro Magnani for useful discussions. CONFLICT OF INTEREST F.B., A.F. and G.P., are inventors on patent application submitted by the authors and University of Urbino Carlo Bo.

REFERENCES (1) (a) Ehrlich, K.; Viirlaid, S.; Mahlapuu, R.; Saar, K.; Kullisaar,

T.;

Zilmer,

M.;

Synthesis

and

Properties

Langel, of

Ü.;

Novel

Soomets,

Powerful

U.

Design,

Antioxidants,

Glutathione Analogues. Free Radic. Res. 2007, 41, 779–787. (b) Fraternale, A.; Paoletti, M. F.; Nencioni, A. C., L.; Garaci, E.; Palamara, A. T.; Magnani, M. GSH and Analogs in Antiviral Therapy. Mol. Aspects Med. 2009, 30, 99–110. (c) Fraternale, A.; Brundu, S.;

Magnani,

M.

Glutathione

and

Glutathione

Derivatives

in

Immunotherapy. Biol. Chem. 2017, 398, 261–275. (d) Cacciatore, I.; Baldassarre, L.; Fornasari, E.; Mollica, A.; Pinnen, F. Recent Advances in the Treatment of Neurodegenerative Diseases Based on GSH Delivery Systems. Oxid. Med. Cell. Longev. 2012, 240146. (2) (a) Meister, A.; Anderson, M. E. Glutathione. Ann. Rev. Biochem. 1983, 52, 711-760. (b) Meister, A. Glutathione Metabolism and its Selective Modification. J. Biol. Chem. 1988, 263, 1720517208.

(c)

Hayes,

J

.D.;

McLellan,

L.

I.

Glutathione

and

Glutathione-Dependent Enzymes Represent a Co-ordinately Regulated Defence Against Oxidative Stress. Free Radic. Res. 1999, 31, 273-


16

Page 17 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


300. (d) Garcia-Gimenez, J. L.; Pallardo, F. V. Maintenance of Glutathione Levels and its Importance in Epigenetic Regulation. Front. Pharmacol. 2014, 5, 88. (e) Homma T.; Fujii, J. Application of Glutathione as Anti-Oxidative and Anti-Aging Drugs. Cur. Drug Metab. 2015, 16, 560-571. (3) (a) Cacciatore, I.; Cornacchia, C.; Pinnen, F.; Mollica, A.; Di

Stefano,

A.

Prodrug

Approach

for

Increasing

Cellular

Glutathione Levels. Molecules 2010, 15, 1242-1264. (b) Wu, J. H.; Batist, G. Glutathione and potentials.

BBA-Gen.

Glutathione Analogues; Therapeutic

Subjects

2013,

1830,

3350-3353.

(c)

Giustarini, D.; Galvagni, F; Donne, I. D.; Milzani, A; Severi, F.M.; Santucci, A.; Rossi, R. N-Acetylcysteine ethyl ester as GSH Enhancer in Human Primary Endothelial Cells: A Comparative Study with other Drugs. Free Radical Bio. Med. 2018, 126, 202-209. (4)

Gilbert,

H.

F.

Redox

control

of

enzyme

activities

by

thiol/disulfide exchange. Methods Enzymol. 1984, 107, 330–351. (5) (a) Antoniou, A. I.; Pepe, D. A.; Aiello, D.; Siciliano, C.; Athanassopoulos C. M. Chemoselective Protection of Glutathione in the

Preparation

of

Bioconjugates:

The

Case

of

Trypanothione

Disulfide. J. Org. Chem. 2016, 81, 4353–4358. (b) Zampagni, M.; Wright,

D.;

Cascella,

R.;

D'Adamio,

G.;

Casamenti,

F.;

Evangelisti, E.; Cardona, F.; Goti, A.; Nacmias, B.; Sorbi, S.; Liguri,

G.;

Cecchi

C.

Novel

S-acyl

Gglutathione

Derivatives

Prevent Amyloid Oxidative Stress and Cholinergic Dysfunction in


17


Page 18 of 20

Alzheimer Disease Models. Free Radic. Biol. Med. 2012, 52, 1362– 1371. (c) Wright, D.; Zampagni, M.; Evangelisti, E.; Conti S.; D'Adamio, G.; Goti, A.; Becatti, M.; Fiorillo, C.; Taddei, N.; Cecchi, C.; Liguri, G. Protective Properties of Novel S-Acylglutathione

thioesters

Against

Ultraviolet-induced

Oxidative

Stress. Photochem. Photobiol. 2013, 89, 442-452. (d) Cascella, R.; Evangelisti, E.; Zampagni, M.; Becatti, M. D'Adamio, G.; Goti, A.; Liguri, G.; Fiorillo, C.; Cecchi, C. S-Linolenoyl Glutathione Intake

Extends

Life-span

and

Stress

Resistance

via

Sir-2.1

Upregulation in Caenorhabditis Elegans. Free Rad. Biol. Med. 2014, 73, 127-135. (e) Fu, K.; Wang, Q.-F.; Zhan, F.-X.; Yang, L.; Yang, Q.; Zheng G.-X. An Improved Process for Preparation of S-AcetylL-glutathione. Org. Process Res. Dev. 2015, 19, 590−593. (6) (a) Falck, J. R.; Sangras, B.; Capdevila, J. H. Preparation of

N-tBoc

L-glutathione

dimethyl

and

di-tert-butyl

esters:

Versatile Synthetic Building Blocks. Bioorg. Med. Chem. 2007, 15, 1062-1066. (b) Alt, C.; Eyer, P. Ring Addition of the Alpha-amino Group of Glutathione increases the reactivity of benzoquinone thioethers. Chem. Res. Toxicol. 1998, 11, 1223-1233. (c) Vogel, E. R.; Jackson, W.; Masterson, D. S. Efficient Esterification of Oxidized L-Glutathione and Other Small Peptides. Molecules 2015, 20, 10487-10495. (7) (a) Shen, B.; Bazin, C.; English, A. M. Rapid High-yield NAcylation

of

Aminothiols:

N-Acetylglutathione

and

N-


18

Page 19 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Acetylhomocysteine and their Thiol pK(a) Values. J. Pept. Sci. 2013, 19, 263-267;. (b) Bernardi, D.; Ba, L. A.; Kirsch, G. First Synthesis of N-Acylated Photoactivatable Analogues of Glutathione Bearing an Aryl Azide Moiety. Synthesis 2007, 1, 140-144. (c) Katritzky, A. R.; Abo-Dya, N. E.; Tala, S. R.; Ghazvini-Zadeh, E. H.; Bajaj, K.; El-Feky, S. A.; Efficient and Selective Syntheses of S-Acyl and N-Acyl Glutathiones. Synlett 2010, 9, 1337–1340. (d) Benatti, U.; Brandi, G.; Garaci, E.; Magnani, M.; Millo, E.; Palamara, A. T.; Rossi, L. Preparation of glutathione derivatives as antiviral agents. Patent WO 2005063795 A1 20050714, 2005. (8) Zaretsky, S.; Rai, V.; Gish, G.; Forbes, M. W.; Kofler, M.; Yu, J. C. Y.; Tan, J.; Hickey, J. L.; Pawson, T.; Yudin, A. K. Org. Biomol. Chem. 2015, 13, 7384 –7388. (9) (a) Palamara, A. T.; Brandi, G.; Rossi, L.; Millo, E.; Benatti, U.; Nencioni, L.; Iuvara, A.; Garaci, E.; Magnani, M. New Synthetic

Glutathione

Derivatives

with

Increased

Antiviral

Activities. Antivi. Chem. Chemoth. 2004, 15, 77–85. (b) Limongi, D.; Baldelli, S.; Checconi, P.; Marcocci, M.; De Chiara, G.; Fraternale, A.; Magnani, M.; Ciriolo, M. R.; Palamara A. T. GSHC4 Acts as Anti-inflammatory Drug in Different Models of Canonical and Cell Autonomous Inflammation Through NFkB Inhibition. Front. Immunol. 2019, 10, 155. (c) Sgarbanti, R.; Nencioni, L.; Amatore, D.;

Coluccio, P.;

Fraternale, A.; Sale,

P.;

Mammola,

C. L.;

Carpino, G.; Gaudio, E.; Magnani, M.; Ciriolo, M. R.; Garaci, E.;


19


Page 20 of 20

Palamara, A. T. Redox Regulation of the Influenza Hemagglutinin Maturation Influenza

Process: Therapy.

A

New

Antiox.

Cell-Mediated Redox

Strategy

for

Anti-

Sign. 2011, 15, 593-606.

(d)

Amatore, D.; Celestino, I.; Brundu, S.; Galluzzi, L.; Coluccio, P.; Checconi, P.; Magnani, M.; Palamara, A.T.; Fraternale, A.; Nencioni L. Glutathione Increase by the n‐Butanoyl Glutathione Derivative

(GSH‐C4)

Inhibits

Viral

Replication

and

Induces

a

Predominant Th1 Immune Profile in Old Mice Infected with Influenza Virus. FASEB BioAdvances. 2019, 1, 296–305. (10) (a) Dakin, D.; West, R. A General Reaction of Amino acids. J. Biol. Chem. 1928, 78, 91–104. (b) Dakin, H. D.; West, R. A General Reaction of Amino acids. II. J. Biol. Chem. 1928, 78, 745– 756. (11) Sheldon R. The E factor 25 Years on: the Rise of Green Chemistry and Sustainability. Green Chem. 2017, 19, 18-43.


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

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