Simple single-step single-enzyme synthesis of [14C]-GSH

Article abstract of DOI:10.1002/jlcr.1735

The tri-peptide [¹⁴C]-glutathione ([¹⁴C]-GSH) was synthesized in a single step by GSH synthetase catalyzed reaction of L-γ-glutamyl-L-cysteine and [¹⁴C]-glycine. Preparative reverse phase HPLC afforded [¹⁴C]-GSH in 30% yield and 98% purity. Preparation of GSH synthetase from E. coli via recombinant DNA and the interconversion of [¹⁴C]-GSH to the disulfide [¹⁴C]-GSSG for storage are discussed. Copyright

Full text of DOI:10.1002/jlcr.1735

Research Article
Received 17 March 2009,
Revised 7 October 2009,
Accepted 2 November 2009
Published online 2 March 2010 in Wiley Interscience
(
www.interscience.wiley.com) DOI: 10.1002/jlcr.1735
Simple single-step single-enzyme synthesis
of [ C]-GSH
1
4
Ã
Steve A. de Keczer, Tatyana Voronin, Jennifer Yao, Fang-Jie Zhang, and
Mohammad R. Masjedizadeh
1
4
14
The tri-peptide [ C]-glutathione ([ C]-GSH) was synthesized in a single step by GSH synthetase catalyzed reaction of
1
4
14
L-c-glutamyl-L-cysteine and [ C]-glycine. Preparative reverse phase HPLC afforded [ C]-GSH in 30% yield and 98% purity.
Preparation of GSH synthetase from E. coli via recombinant DNA and the interconversion of [ C]-GSH to the disulfide [ C]-
GSSG for storage are discussed.
1
4
14
1
4
14
14
Keywords: [ C]-glutathione; [ C]-GSH; [ C]-glycine; L-g-glutamyl-L-cysteine; GSH synthetase
Introduction
glycine catalyzed by GSH synthetase was first demonstrated by
1
Block and Snoke in 1952 using a [ C]-gly assay. However, this
4
6
1
was not a preparative method to isolate [ C]-GSH and the GSH
4
The tri-peptide glutathione (g-glutamyl-cysteinyl-glycine, GSH)
participates in
a number of cellular functions including synthetase was a part of a complex pigeon liver cell matrix
antioxidant activity, cell signaling, and drug detoxification. containing other enzymes.
GSH concentration has been related to the pathogenesis of
many human diseases including aging, AIDS, cancer, cystic
fibrosis, ischemia, liver and heart disease, metabolic diseases couple the three amino acids: L-glutamate, L-cysteine, and
More recently, enzymatic routes have relied on two enzymes
g-glutamylcysteine synthetase and glutathione synthetase) to
(
6
–10
6–7
(
sickle cell anemia, and stroke. In a preclinical setting, GSH is
diabetes and obesity), neurodegenerative diseases, seizure, glycine.
It is known that these enzymes act sequentially.
Thus, it was expected that in a cell-free reaction, L-g-glutamyl-L-
1
,2
used to evaluate the formation of reactive drug metabolites. cysteine should couple with glycine catalyzed by GSH synthe-
Formation of a GSH adduct is often associated with those tase.
reactive metabolites that can cause covalent binding (CVB) of
drug-related material to cellular proteins and associated adverse Results and discussion
effects. Because the GSH adducts are novel metabolites and not
readily available synthetically, their quantitation with standard All necessary components for a single-step single-enzyme
1
4
LC/MS/MS methods is not possible. Therefore, the standard synthesis of [ C]-GSH are commercial items. An in-house
procedure for quantifying reactive metabolite formation re- preparation of GSH synthetase provided a more active enzyme
1
4
3
mains the C (or H) labeling of the drug and subsequent than the commercial source (Figure 1).
1
measurement of C CVB to protein.
4
3
In Table 1, the first three experiments were designed to
4
35
Masubuchi has reported the use of [ S]-GSH in the compare two versions of the in-house Glutathione Synthetase:
evaluation of non-labeled drug candidates and has reported a GSS-1 (his-tagged, no thrombin cleavable sites) and GSS-2 (his-
good correlation between CVB results using both labeled drug tagged, thrombin cleavable site), with commercial GSS-3. The
3
5
candidates as well as [ S]-labeled GSH. Because of its long-term cocktail (pH 6.5) used was adapted from a cocktail reported by
1
4
8
stability, the use of [ C]-labeled GSH would allow for long-term Murata et al. for a three amino acid two enzyme matrix
1
4
storage of this compound. It could be used as needed in CVB synthesis of [ C]-GSH. The pH was adjusted from ꢀ6.5 to ꢀ7.5
studies thus alleviating the need to individually label molecules for the remaining sets of experiments 4–10 and only the more
of interest and should allow for rapid determination of CVB in a active in-house synthetase GSS-1 was used.
preclinical setting.
1
Experiments 4–9 were attempts to simplify the cocktail.
Though [ C]-GSH is a useful tool for CVB studies, it is not a Experiment 4 can be considered to be the control using gly in
4
1
4
commercial item. Methods for the [ C]-labeling of GSH fall into excess (to avoid potential problems related to the separation of
8
two categories: chemical and enzymatic. A reported multi-step g-glutamylcysteine from glutathione). Experiment 5 showed
1
4
14
synthesis of [ C]-GSH from K CN has an overall yield of 1%.
5
Although yields have been significantly improved, all chemical
routes involve a number of protection and deprotection steps Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
and have the possibility of racemization. Hydrolysis and
*
Correspondence to: Steve A. de Keczer, Roche Palo Alto, 3431 Hillview Avenue,
Palo Alto, CA 94304, USA.
purity. The enzymatic coupling of L-g-glutamyl-L-cysteine and E-mail: Steve.de_keczer@roche.com
derivatization have been used for the determination of optical
2
J. Label Compd. Radiopharm 2010, 53 110–112
Copyright r 2010 John Wiley & Sons, Ltd.

S. A. de Keczer et al.
SH
OH
SH
O
O
glutathione
synthetase
O
O
O
C
O
C
H
1
4
1
4
+
H N
2
N
HO
N
HO
H N
N
OH
OH
H
H
Cocktail
H
H
H N
H
H
2
O
2
O
[14C]-Gly
[14C]-GSH
L- -glutamyl-L-cysteine
Figure 1. Single-step single-enzyme synthesis.
Table 1. Comparisons of GSS sources, and cocktail components
Expt.
Glu-cys
Gly
GSS
ATP
MgCl2
K2HPO4
(mmol)
LiKAcP
(mmol)
EDTA
% GSH
Remarks
(
mmol)
(mmol)
(mg)
(mmol)
(mmol)
(mmol)
(h)
1
2
3
4
5
6
7
8
9
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.09
0.02
0.02
0.02
0.025
0.025
0.025
0.025
0.025
0.02
10
10
10
10
10
10
10
10
10
23
0.01
0.01
0.01
0.025
0.0125
0.025
0.025
0.0125
0.0125
0.056
0.02
0.02
0.02
0.025
0.025
0.025
0.025
0.025
0.025
0.113
0.01
0.01
0.01
0.025
0.025
0.025
0.025
0.025
0.025
0.113
0.02
0.02
0.02
0.025
0.025
0
0.025
0
0
0.005
0.005
0.005
0.005
0.005
0.005
0
0.005
0.005
0.023
18 (1)
14 (1)
6 (1)
pH 6.5, GSS-1
pH 6.5, GSS-2
pH 6.5, GSS-3
pH 7.5, GSS-1
pH 7.5, GSS-1
pH 7.5, GSS-1
pH 7.5, GSS-1
pH 7.5, GSS-1
pH 7.5, GSS-1
pH 7.5, GSS-1
23.4 (20)
49.2 (20)
63.5 (20)
11.6 (20)
62.0 (20)
61.7 (20)
a
b
10
0.089
0
30 (15)
a
14
[
C]-gly.
b
Isolated yield; Glu-cys (L-g-glutamyl-L-cysteine), Gly (glycine), GSS (glutathione synthetase), LiKAcP (lithium potassium
acetylphosphate), EDTA (ethylenediaminetetraacetic acid disodium salt), GSH (glutathione).
1
4
that reducing the amount of ATP improved the conversion to and [ C]-GSSG (26%) which had been concentrated to dryness
GSH. ATP was reported to be necessary and has been shown to from 5% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA) at
be the best of several nucleotides tested to catalyze the enzyme ambient temperature in air and then redissolved in water was
9
14
reaction. Experiment
potassium acetyl phosphate (LiKAcP) from the cocktail has a from 2 to 7 with 1% aqueous NH OH. This solution could readily
6 showed that eliminating lithium converted to 97% [ C]-GSSG in 1 h by simply adjusting the pH
4
1
4
14
beneficial effect. Experiment 7 was based on the assumption be converted back to 2% [ C]-GSSG, 94% [ C]-GSH by the
that Na EDTA may not be needed in this cell-free reaction, since addition of DTT to give a 20 mM solution. This treatment may
its use to chelate errant metal ions present in the immobilized afford a simple mode of storage in the [ C]-GSSG oxidized form,
2
1
4
1
4
cell matrix may not be needed. However, the elimination of which readily converts back to the reduced [ C]-GSH. The DTT
7
Na
2
EDTA resulted in a drastic reduction in the conversion of gly can be removed by extraction with EtOAc.
to GSH, thus necessitating the use of Na EDTA in further
Because of the cell-free nature of this synthesis, repeated
sequential addition of reagents to convert gly to GSH was not
since literature sources claim that there is no activity in the necessary, nor was the repeated treatment with DTT to release
2
2 2 4
experiments. No attempt was made to alter MgCl or K HPO
8
absence of divalent cation and potassium ion was needed for GSH from a cell matrix.
9
maximum activity. The final two non-labeled experiments 8–9
compare excess gly vs stoichiometric gly (desired for the labeled
synthesis) while incorporating both beneficial effects, the
Experimental
reduced amount of ATP and the absence of LiKAcP. There was Proton spectra were recorded on a Brucker 300 MHz NMR
good reproducibility, and no apparent advantage to using spectrometer. Mass spectrometric analyses were carried out on
excess gly. Thus, experiment 9 was used as the basis for the Fmoc derivatives (where needed) of the amino acids and peptides
1
4
[
C]-labeled synthesis in experiment 10.
4
using a Finnegan LCQ ion trap mass spectrometer equipped with
1
The [ C]-labeling reaction was conducted at 4.5 ꢁ scale. an electrospray ionization (ESI) source, a Thermo Finnigan
1
[
4
C]-GSH was isolated in 30% yield and 98% purity after Surveyor LC pump, a PDA detector (sheath gas flow 70 units
1
4
treatment with DTT (to convert significant [ C]-GSSG to
N
2
/sweep gas flow 15 units N
C]-GSH) followed by Prep-HPLC purification. The bulk of collision energy 35%), and a Packard Radiomatic 610TR Flow
the activity was recovered as a single peak at the retention time Scintillation Analyzer (column: Atlantis-C18, 4.6ꢁ 150 mm, 5 m; A:
; 1cESi SID= 20V, Normalized
2
1
[
4
11
1
4
14
and MW of [ C]-glycine. A portion of the 98% pure [ C]-GSH 0.1% TFA in H O, B: 0.1% TFA in ACN; hold 1% B for 5 min.
2
Prep-HPLC fraction was concentrated to dryness by ambient Gradient: 1% B to 50% B 5–10min., 50% B 15–20 min. Flow Rate:
temperature rotary evaporation (releasing the vacuum with 1 mL/min, 301C, 210 nm. Specific activity was determined by MS.
nitrogen). Dilution of this product revealed an increase in TLC plates (silica gel 250 microns) were obtained from Analtech
1
[
4
C]-GSSG from 1% before concentration to 10% after concen- and scanned using a Bioscan System 200 Imaging Scanner.
tration. Disulfide formation could be avoided by using lyophiliza- Analytical HPLC was conducted using the following conditions:
1
2
14
tion. Alternatively, a DTT untreated mixture of [ C]-GSH (69%) Hypercarb-C18, 4.6 ꢁ 150 mm; ACN: 0.01% TFA 5:95 go to 90:10
J. Label Compd. Radiopharm 2010, 53 110–112
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

S. A. de Keczer et al.
20–25min; 1 mL/min at 210 nm. Prep-HPLC was performed on A single colony was used to start a 2.5 mL overnight culture in LB
Hypercarb-C18, 21.2ꢁ 150 mm; ACN: 0.01% TFA 5:95; 18mL/min media containing 100 mg/mL carbenicillin and was used as the
at 210 nm. The small molecules were reagent-grade and were innoculum for a 250 mL culture of LB media containing 100 mg/
1
4
used as received from vendors. [ C]-glycine was purchased from mL carbenicillin and induced to express recombinant protein with
Moravek Biochemicals, Inc. (Brea CA), L-g-glutamyl-L-cysteine was 0.1 mM IPTG at an OD600 of 0.6 at 221C overnight. Cells were
purchased from Sigma (St. Louis, MO), and glutathione synthetase harvested and lysed in 20mL buffer A (25mM Tris, pH 7.5, 0.3 M
was purchased from Abnova Corporation (Taiwan) and prepared NaCl, 10% glycerol, 5 mM b-mercaptoethanol) plus 2 tablets of
in-house by the Protein and Molecular Sciences Group.
Complete Mini, EDTA-Free protease inhibitor tablets (Roche
Molecular), 25mL benzonase nuclease (Sigma), and 1x Bugbuster
reagent (Novagen). Cell extracts were centrifuged at 3400rpm at
1
[
4
C]-Glutathione
4
1C and the supernatant containing soluble glutathione synthe-
L-g-glu-cys (25.6 mg, 0.090 mmol) in 1.8 mL nitrogen purged
water was combined with the cocktail: 31.4 mg ATP
tase was collected. The his-tagged glutathione synthetase was
purified over a 1 mL NiNTA superflow column (Qiagen) in buffer A
on an AKTA FPLC (GE), washed with buffer A plus 5 mM imidazole
until baseline UV absorbance was reached, and eluted with buffer
A plus 200 mM imidazole. The identity of the recombinant protein
was confirmed by western blot using an a-penta-his monoclonal
(
0.056 mmol), 23.2 mg MgCl2 (0.113 mmol), 19.7 mg K HPO4
2
(
2
0.113 mmol), and 8.6 mg Na EDTA (0.023 mmol). The pH was
adjusted from 3–4 to 7–8 with 20 mL 6 M NaOH, and 2.5 mL of
1
mCi/mL aqueous [ C]-gly was added. The solution was
4
2
sterilized (0.45 m filter, rinsing with 0.5 mL nitrogen bubbled
water), and glutathione synthetase (23 mg in 10 mL buffer) was
added. After stirring under nitrogen for 15 h at 371C, the solution
was cooled to ambient temperature, stirred for X30 min with
antibody (GE). Protein concentration was determined by OD280
ꢂ1
(e = 38578 M /cm) using a NanoDrop spectrophotometer (Ther-
mo Scientific) and the purity was analyzed by SDS-PAGE. The yield
was 5.5 mg of purified glutathione synthetase from 250 mL of E.
coli culture. Proteins were aliquoted and stored at ꢂ801C.
0
9
.50mL 200 mM DTT. Prep HPLC purification afforded 1.5 mCi of
1
8% pure [ C]-GSH having a specific activity of 52.9mCi/mmol
4
(
MS ESI1, m/z 309.9 M1H) with a HPLC retention time of 4.63min
and 3 mCi of labeled material at the retention time (2.97 min) and
Conclusion
1
MW of [ C]-gly (Figure 2).
4
We have developed an efficient one-step method for the cell-
1
free preparation of [ C]-GSH from readily available reagents:
4
Glutathione synthetase
1
[
4
C]-gly and g-glu-cys; and catalyzed by glutathione synthetase
14
in a simplified cocktail. The air sensitive [ C]-GSH can be easily
1
4
Construction of glutathione synthetase expression vector
converted to the more stable oxidized form [ C]-GSSG that can
1
be readily reduced back to [ C]-GSH as needed. Purification was
by preparative HPLC.
4
The recombinant glutathione synthetase expression vector was
PCR amplified from BL21(DE3) E. coli genomic DNA using the
0
following primers: 5 - GGG GAC AAG TTT GTA CAA AAA AGC
0
Acknowledgements
0
AGG CTT AAT GAT CAA GCT CGG CAT C- 3 (forward) and 5 -
GGG GAC CAC TTT GTA CAA GAA AGC TGG GTA TTA CTG CTG
0
We thank Rong Teng and Frida Dobrouskin for analytical
support and Yanzhou Liu for NMR analysis. We also wish to
thank Dr David Shaw for helpful discussions.
CTG TAA ACG- 3 (reverse). The PCR product was gel purified
and cloned into an entry vector (pDONR221) followed by an
expression vector (pDEST17) through recombination using the
Gateway cloning system (Invitrogen). Presence of correct insert
was confirmed through restriction enzyme digestion.
References
Expression and purification of glutathione synthetase
[
[
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Figure 2. [ C]-GSH Radio-HPLC.
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J. Label Compd. Radiopharm 2010, 53 110–112