Mycothiol disulfide reductase: Solid phase synthesis and evaluation of alternative substrate analogues

Article abstract of DOI:10.1039/b716380k

A solid phase synthesis of des-myo-inositol mycothiol disulfide and its alpha-configured methyl- and benzyl-glycoside derivatives has been developed. Kinetic characterisation of these compounds has demonstrated their viability as alternative substrates for use in mycothiol disulfide reductase enzyme assays. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b716380k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Mycothiol disulfide reductase: solid phase synthesis and evaluation of
alternative substrate analogues
Matthew J. G. Stewart,a Vishnu Karthik Jothivasan,a Andrew S. Rowan,a
Jennifer Waggb and Chris J. Hamilton*a,b
Received 24th October 2007, Accepted 19th November 2007
First published as an Advance Article on the web 7th December 2007
DOI: 10.1039/b716380k
A solid phase synthesis of des-myo-inositol mycothiol disulfide and its alpha-configured methyl- and
benzyl-glycoside derivatives has been developed. Kinetic characterisation of these compounds has
demonstrated their viability as alternative substrates for use in mycothiol disulfide reductase enzyme
assays.
mycothiol disulfide reductase (Mtr) (also referred to as my-
cothione reductase) helps to maintain an intracellular reducing
Background
environment by reducing MSSM back to MSH (Fig. 1).3–5 MSH
deficient bacteria show a significant increase in sensitivity to
oxidative stress6,7 making the mycothiol redox pathway a potential
therapeutic target in M. tuberculosis.
Glutathione (GSH) is the principle antioxidant thiol found in most
eukaryotes and Gram negative bacteria. Most Actinomycetes
(eg. Mycobacterium tuberculosis, Streptomyces coelicolor) lack
GSH and instead utilise the cysteinyl pseudo-disaccharide my-
cothiol (MSH) (1-D-myo-inosityl-2(-N-acetyl-L-cysteinyl) amino-
2-deoxy-a-D-glucopyranoside) as their principal low molecular
weight thiol (Fig. 1).1,2 Like GSH, MSH is believed to play
a key role in the inactivation of potentially damaging radicals
and reactive oxygen species and is oxidised to the symmetrical
mycothiol disulfide (MSSM), in the process. NADPH-dependent
Mtr inhibitor studies are severely restricted by the scarcity of
MSH, which is difficult to prepare in sufficient quantity. Small
quantities of MSH are routinely obtained by whole cell synthesis in
typical yields of 1 mg per litre of M. smegmatis cell culture,8,9 while
chemical syntheses are convoluted.10–13 Interestingly, the inositol
portion of MSH is not always essential for substrate recognition
by certain MSH-processing enzymes. The truncated substrate des-
myo-inositol mycothiol disulfide 4 is a proven alternative substrate
for Mtr3 and the mycothiol-S-bimane analogue 7 (where inositol
is replaced with S-cyclohexyl)14,15 is recognised as an alternative
substrate for mycothiol-S-conjugate amidase.16 Such simplified
analogues are more synthetically accessible than MSH and have
potential as alternative substrates to facilitate inhibitor studies of
Mtr, and (possibly) other MSH-dependent enzymes.
Routine access to sufficient quantities of 4 and/or other
simplified mycothiol disulfide analogues as alternative substrates
for Mtr inhibition assays is clearly desirable. Whilst solution
phase syntheses of 18 and its symmetrical disulfide 43 have
previously been reported, a solid phase approach would enable
the preparation of parallel libraries of MSH analogues. Herein
we report a solid phase method for the synthesis of 1 and the
alpha-configured methyl- and benzyl-glycosides (2 and 3). Their
subsequent oxidation to symmetrical disulfides 4–6 and evaluation
as alternative Mtr substrates is also described.
Results and discussion
The methyl- and benzyl-glycosides of glucosamine (1017 and 11)
were prepared by hydrazinolysis of their N-acetyl precursors 818
Fig. 1 Mycothiol, the mycothiol disulfide reductase pathway and my-
cothiol analogues.
◦
and 919 at 120 C (Scheme 1a). The strategy for the solid phase
reactions was to immobilise the cysteine motif onto a polystyrene
resin via an S-trityl linkage as this would also serve to protect
the thiol during the amide coupling reaction. Supported CysNAc
15 was prepared by loading CysNAc 12 onto polystyrene trityl
chloride resin using standard procedures (Scheme 1b).20 Immo-
bilised N-Fmoc cysteine 16 and its pentafluorophenyl ester 17
aSchool of Chemistry and Chemical Engineering, David Keir Building,
Stranmillis Road, Queen’s University Belfast, Belfast, United Kingdom BT9
5AG
bSchool of Chemical Sciences and Pharmacy, University of East Anglia, Nor-
wich, Norfolk, United Kingdom NR4 7TJ. E-mail: c.hamilton@uea.ac.uk
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by base-catalysed enolisation of the 5(4H)-oxazalone) interme-
diate, which itself forms via rearrangement of the pre-activated
carboxylate intermediate. Usually the carboxylate is pre-mixed
with the activating agent for five minutes prior to addition to the
amine nucleophile. With cysteine, racemisation can be minimised
by avoiding carboxylate pre-activation, using a weaker and/or
more sterically hindered base and reducing the solvent polarity.21
A similar strategy was therefore explored for the solid phase syn-
thesis of 3 using the phosphonium coupling reagent PyBOP. This
involved pre-mixing the solvated aminosugar 11 with the cysteine-
derivatised resin 15 prior to the addition of the activating reagents
(ie. PyBOP, HOBt and base). When 2,6-collidine was used as
a base, NMR analysis of the cleaved product revealed a 4 : 1
epimeric mixture of 3. This was evident in the carbon NMR
spectra where several of the carbons were represented by two
closely spaced signals. Of particular note was the presence of two
distinct anomeric carbon signals at 96.35 and 96.25 ppm for the
major and minor epimers, respectively. In the proton NMR spectra
two doublets were observed for the anomeric proton of the major
and minor epimers at 4.84 and 4.79 ppm, respectively. Two closely
overlapping singlets for the N-acetate methyl protons (1.91 ppm)
were also observed. Since the anomeric proton signals shouldered
the NMR solvent peak (in D2O) and the acetate signals were too
close together to allow the epimer ratio to be determined by their
integration, epimer ratios were determined by integration of the
anomeric carbon signals from an inverse gated coupling 13C NMR
spectra of epimeric 3. When 2,6-collidine was replaced with 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) the degree of epimerisation
was reduced (7 : 2) and switching to 2,6-di-tert-butylpyridine
reduced this even further to give a 9 : 1 ratio of epimeric 3.
However, when 2,6-di-tert-butylpyridine was used as the base
for coupling 11 with immobilised CysNFmoc 16 no detectable
level of epimerisation was evident (presumably because the Fmoc
carbamate moiety further disfavours oxazolone formation). These
conditions were therefore employed for the preparation and
purification of all three MSH analogues 1–3 (Scheme 1c). After
coupling the amino sugar onto 16 the Fmoc protecting group was
removed (20% piperidine) and the free amine was acetylated by
treatment with acetic anhydride (1.1 equivalents). The product
thiol was then cleaved from the resin by treatment with 40%
trifluoroacetic acid (TFA). Sometimes, when the acetic anhydride
was used in a small excess some additional O-acylation of the
cleaved product was evident (by NMR). This was easily removed
by treating the cleaved product with a catalytic amount of sodium
methoxide under Zemplen conditions prior to purification. Under
these conditions it was possible to obtain 2 and 3 in about 80%
isolated yields (with respect to immobilised CysNFmoc 16).
The DMF : CH2Cl2 solvent ratios used in the amide bond-
forming steps for compounds 1–3 were dictated by the solubility
of the respective aminosugars. The benzyl glycoside 11 was the
most readily soluble (20% DMF) followed by the methyl glycoside
10 (25% DMF). Glucosamine was only sparingly soluble in DMF,
which may explain why this reaction failed to go to completion, as
evident from the significant amount of unreacted CysNAc 12 in
the cleaved product mixture (by NMR). It is worth noting that the
varying amounts of DMF used in all of the above procedures did
not appear to compromise the epimeric purity of the products.
For evaluation as Mtr substrates compounds 1–3 needed to first
be oxidised to their symmetrical disulfides 4–6, ideally without
Scheme 1 Reagents and conditions: (i) NH2NH2·H2O, 120 ◦C; (ii) TFA–
DCM–Et3SiH (40 : 60 : 3); (iii) polystyrene tritylchloride, DIPEA,
CH2Cl2–DMF; (iv) AcOH, trifluoroethanol, CH2Cl2; (v) MeOH, DIPEA,
CH2Cl2; (vi) 11, PyBOP, HOBt, base, DMF–CH2Cl2; (vii) 10, 11 or glu-
cosamine (2 eq.), PyBOP, HOBt, 2,6-di-tert-butylpyridine, DMF–CH2Cl2;
(viii) DMF–piperidine (8 : 2); (ix) Ac2O (1.1 eq.), pyridine (1.1 eq.), DMF;
(x) NH4HCO3 (30 mM), shaking 24–60 h.
were prepared by acidic detritylation of their protected precursors
13–14 followed by removal of the trifluoroacetic acid (in vacuo)
and direct loading onto the resin without further purification.
Solid phase coupling reactions were initially studied between
immobilised CysNAc and aminosugar 11 (Scheme 1b). The use
of HBTU or HATU and excess 11 failed to provide reaction
product 3 (following TFA cleavage from resin). Presumably these
aminium-based peptide coupling reagents react with the excess of
aminosugar 11 to form guanidine byproducts instead. Reactions
of pentafluorophenyl ester 17 with four equivalents of 11 were
extremely sluggish and only trace quantities of 3 were isolated
after the reaction had been agitated for four hours.
Racemisation of the cysteine side chain has previously been
encountered when forming the cysteine-aminosugar amide bond
in MSH, particularly when carbodiimides such as EDCI or DCC
were used as the coupling reagent.13 Racemisation is often caused
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over-oxidation or the need for product purification. In our hands,
oxidation using hemin adsorbed onto Celite22 was not satisfactory
as the oxidised product was heavily contaminated with hemin,
which leached from the Celite solid phase when washing out
the product. Treatment of the thiols with one equivalent of
iodine in 50% aqueous acetonitrile23 efficiently oxidised them to
their disulfides, but required treatment with multiple batches of
activated charcoal to remove any unconsumed iodine.24 Thiols
can be selectively oxidised to their symmetrical disulfides (without
the risk of over-oxidation) under mildly basic conditions. In this
manner, disulfides 4–6 were cleanly prepared by shaking a solution
of thiols 1–3 in 30 mM ammonium hydrogen carbonate in an
open sample vial until the oxidation was complete (Scheme 1c).
Removal of the water and volatile salts by freeze-drying then
provided the clean disulfides 4–6 in quantitative yield. Using this
procedure, thiols 1 and 2 were completely oxidised within 24 h.
Benzyl glycoside 3 was less readily soluble and kept precipitating
out of solution and was periodically redissolved by warming the
solution with a heat gun. This reaction took much longer to reach
completion (60 h). It is worth noting that the epimer-free disulfide
6 gives a single anomeric carbon resonance whereas oxidation of
an epimeric mixture of 3 gives a mixture of three diastereomeric
disulfides, and therefore exhibits three closely spaced anomeric
carbon resonances.
The substrate properties of 4–6 and authentic MSSM with
recombinant hexahistidine-tagged M. tuberculosis Mtr4 were de-
termined (Table 1). In our hands, the substrate properties for 45 and
MSSM5,25 are comparable to those previously reported. Compared
to MSSM, removal of the inositol motif (ie. compound 4) gives
a four-fold increase in Km but with no apparent effect on the
steady state turnover rate (kcat). The methyl glycoside 5 is just
a subtle elaboration of the structure 4, which results in only a
small increase in Km and a 3-fold reduction in kcat. Replacement
of the polyhydroxylated inositol motif of MSSM with a planar,
non-polar, benzyl group 6 results in only a 4-fold increase in Km
and a 2-fold reduction in kcat. The inositol ring of MSSM (as
drawn in Fig. 1) has an apolar patch on the top face. Perhaps the
planar face of the aromatic ring in 6 is able to replace some of the
binding interactions that might occur between Mtr and the inositol
hydrophobic patch in MSSM. If the benzyl group does occupy
the inositol binding pocket it seems plausible that myo-inositol
would be a much stronger inhibitor of Mtr using 6 as a substrate
than with the des-myo-inositol derivative 4. The inhibitory effects
of myo-inositol (25 mM) on the Mtr catalysed turnover of 4, 6,
and MSSM (at their respective Km concentrations) were therefore
studied (Fig. 2). Under these conditions myo-inositol displayed a
small, but comparable level of inhibition (15%) when MSSM or
its benzyl derivative 6 were the substrates. When compound 4 was
the substrate, the level of inhibition was significantly less (3%).
Combined with their synthetic accessibility, any of 4–6 could
be employed as alternative substrates for routine Mtr inhibition
Fig. 2 The effects of myo-inositol on Mtr catalysed substrate turnover:
assays were carried out with substrates 4 (400 lM), 6 (400 lM) and MSSM
(105 lM) in the presence of myo-inositol (25 mM). Percentage enzyme
activities are relative to control experiments (performed separately with
each substrate) in the absence of myo-inositol. All assays were carried out
in duplicate.
assays. We suggest the derivative 6 to be the most favourable
alternative substrate for such applications as the benzyl moiety
is likely to fill more the binding pocket within Mtr that is normally
occupied by the inositol portion(s) of MSSM. Using this substrate
it should be possible to also detect inhibitors which bind to Mtr
but only overlap the inositol binding region of the active site. Such
compounds could potentially be missed if assays were run using
the more truncated substrate analogues 4 and 5.
Conclusions
A convenient procedure for the solid phase synthesis of simplified
mycothiol mimetics has been devloped which enables efficient cou-
pling of the cysteine and aminosugar motifs without racemisation.
The facile preparation of these alternative substrates will facilitate
the screening of Mtr inhibitors. These solid phase synthesis
protocols will also enable the synthesis of parallel libraries of
mycothiol analogues as inhibitors and/or substrates of Mtr and
other mycothiol-dependent enzymes.
Experimental
General
1H-NMR spectra were recorded on a Bruker AV 300 or DRX
500 NMR FT-spectrometer at 300 or 500 MHz, 13C-NMR were
recorded on the same spectrometers at 75 or 125 MHz, respectively.
Chemical shifts (d) are expressed in ppm and coupling constants
(J) are given in Hz. All NMR spectra were taken at 300 K
except where specified. Electrospray mass spectra were recorded
on a VG Quattro Triple Quadrupole Mass Spectrometer. Optical
rotations ([a]D) were obtained with a Perkin-Elmer Model 341
Polarimeter, using the specified solvent and concentration, and
are quoted in units of 10−1deg cm2 g−1. Analytical TLC was
carried out on glass backed Macherly-Nagel SIL G25 UV254
plates and visualised under UV light or by staining with a 9 :
1 mixture of ethanol : sulfuric acid followed by charring with
a heat gun. Agitation of thiol oxidation reactions were carried
out on a Stuart-SF1 flask shaker. Thiols were visualised on TLC
plates by staining with 0.1% (w/v) Ellman’s reagent dissolved
Table 1 Alternative substrate properties with Mtr
Substrate
Km (lM)
kcat (s−1
)
k )
cat/Km (s−1 lM−1
MSSM
113 ( 10)
463 ( 43)
610 ( 82)
438 ( 43)
68.76 ( 2.27)
70.05 ( 5.88)
25.54 ( 0.83)
36.99 ( 1.57)
0.61 ( 0.08)
0.15 ( 0.04)
0.04 ( 0.01)
0.09 ( 0.01)
4
5
6
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in a 10 : 1 solvent mixture of 0.45 M Tris-HCl (pH 8.5) and
ethanol. Thiol titrations were carried out by addition of reaction
samples to a 50 mM solution of Ellman’s reagent (5,5ꢀ-dithiobis-
(2-nitrobenzoic acid)) in Mtr assay buffer in a disposable cuvette
and measuring the absorbance increase at 412 nm. Polystyrene
trityl chloride resin (100–200 mesh) was purchased from Fluka
(cat No. 93005). Resin loadings were determined by combustion
analysis of sulfur. Compounds 8,18 9,19 10,17 1226 and 1327 were
prepared as previously described. Recombinant M. tuberculosis
Mtr (hexahistidine-tagged) was over expressed and purified from
an M. smegmatis mc2155 transformant as previously described.4
Spectrophotometric assays of Mtr were carried out in using a
temperature controlled PerkinElmer UV Lambda 25 spectropho-
tometer. Kinetic data were analysed (by non-linear regression)
using Grafit Version 5 (Erithacus Software Ltd).
added and agitated for 60 min. The resin was washed with DMF
(2 × 10 cm3) then DCM (3 × 10 cm3). The product was cleaved
from the resin by 3 × 3 min treatments with 6 cm3 portions of
TFA–CH2Cl2–Et3SiH (40 : 60 : 3) and the combined filtrates were
concentrated on a rotary evaporator to give an oil. The product
was triturated with ice cold ether (4 cm3) to give a white precipitate
which was dissolved in water (8 cm3) and washed with cold ether
(2 × 2 cm3). The aqueous layer was then freeze-dried to give 2
as a white lyophilate (42 mg, 84%). [a]D +57.1◦ (c 0.71 in H2O);
20
dH (300 MHz, D2O); 1.91 (3H, s, CH3), 2.73 (1H, dd, J 14.2, 6.3,
CHAHBSH), 2.79 (1H, dd, J 14.2, 6.3, CHAHBSH), 3.24 (3H, s,
OCH3), 3.31 (1H, t, J 9.2, H-4), 3.50–3.66 (3H, m, H-3, H-5, H-6a),
3.72 (1H, dd, J 12.2, 2.3, H-6b), 3.79 (1H, dd, J 10.7, 3.6, H-2), 4.34
(1H, t, J 6.3, CHCH2SH), 4.61 (1H, d, J 3.6, H-1); dC (75 MHz,
D2O); 22.63 (CH3), 26.36 (CH2SH), 56.23 (OCH3), 54.71, 56.73
(C-2, CHCH2SH), 61.53 (C-6), 71.05, 71.88, 72.70 (C-3, C-4, C-5),
2(-N -Acetyl-L-cysteinyl) amino-2-deoxy-a-D-glucopyranoside
(1). Resin 16 (0.122 mmol) was pre-swollen by agitation in DMF
(5 cm3) for 10 min and then filtered. A freshly prepared milky
suspension of D-glucosamine (44 mg, 0.245 mmol) in DMF (5 cm3)
was then added and whilst agitating with nitrogen gas a solution
of HOBt (33 mg, 0.244 mmol), PyBOP (127 mg, 0.245 mmol) and
2,6-di-tert-butylpyridine (46 mg, 0.245 mmol) was added and the
resulting mixture was agitated for 3 h by which time the slurry
had turned into a yellow/brown solution. The resin was washed
successively with 6 cm3 portions of DMF (4 × 3 min) followed
by CH2Cl2 (3 × 3 min). The product was then cleaved from the
resin by 3 × 3 min treatments with 8 cm3 portions of TFA–
CH2Cl2–Et3SiH (40 : 60 : 3) and the resin finally washed with
CH2Cl2 (6 cm3). The combined filtrates were concentrated on a
rotary evaporator to give a yellow/brown oil. The product was
precipitated by the addition of ice cold Et2O (4 cm3). The ethereal
solution was decanted and the precipitate washed with ice cold
Et2O (2 × 2 cm3). The precipitate was then dissolved in water
(8 cm3) and extracted with ice cold Et2O (3 × 2 cm3). The acidic
aqueous layer was then neutralised with 30 mM NH4HCO3 and
passed through a Phenomenex Strata strong anion exchange solid
phase extraction tube (1000 mg) which was then washed with 3
column volumes of water. The combined eluents were freeze dried
to give 1 as a white solid (18 mg, 53%). dH (400 MHz, CD3OD)
2.75–2.91 (2H, m, CH2SH), 3.32–3.50 (1H, m) 3.56–3.88 (5H,
m) 4.49–4.53 (1H, m, CHCH2S), 4.64–4.66 (1H, d, J 8.0, H-1-a)
5.08 (1H, d, J 3.2, H-1-b); dC (100 MHz, CD3OD) 22.78, (CH3),
27.46 (CH2SH), 36.27, 56.21, 57.46, 57.59, 63.00, 73.04, 73.46,
+
=
=
99.03 (C-1), 173.18 (C O), 175.23 (C O); m/z (ESI ) 339.1245
(M+H+. C12H23N2O7S requires 339.1226) 699 (88%, 2M + Na+),
361 (100, M+Na+), 339 (10, M+H+).
Benzyl 2(-N-acetyl-L-cysteinyl) amino-2-deoxy-a-D-glucopyrano-
side (3). Resin 16 (0.28 mmol) was pre-swollen by agitation in
a 4 : 1 co-solvent mixture of CH2Cl2–DMF (20 cm3) for 10 min
and then filtered. A solution of 11 (148 mg, 0.56 mmol) in 4 : 1
CH2Cl2–DMF (0.5 cm3) was then added followed by a solution of
HOBt (74 mg, 0.56 mmol), PyBOP (288 mg, 0.56 mmol) and 2,6-
di-tert-butylpyridine (124 lL, 0.56 mmol) in 4 : 1 CH2Cl2/DMF
(1 cm3) and the resulting mixture was agitated for 2 h. The resin
was washed successively with 10 cm3 portions of DMF (4 × 4 min),
then agitated in 20% (v/v) of piperidine in DMF (40 cm3). After
washing with DMF (3 × 10 cm3) a solution of acetic anhydride
(30 lL, 0.297 mmol) and pyridine (24 lL, 0.297 mmols) in DMF
10 cm3 was then added and agitated for 60 min. The resin was
washed with DMF (2 × 10 cm3) then DCM (3 × 10 cm3). The
product was cleaved from the resin by 3 × 3 min treatments
with 6 cm3 portions of TFA–CH2Cl2–Et3SiH (40 : 60 : 3) and
the combined filtrates were concentrated in vacuo to give an oil.
The product was treated the NaOMe (45 mg, 0.84 mmol) in MeOH
(5 cm3) for 3 h then stirred with DOWEX H+ (2 g) for 1 h. The
resin was filtered and treated with hot water to obtain any product
that had precipitated out during filtration. The aqueous layer was
then freeze dried to give 3 as a white solid (91 mg, 79%). mp
217 ◦C (decomp); [a]D +89.6; (c 1.0 in H2O); dH (300 MHz,
20
D2O); 1.91 (3H, s, CH3), 2.73 (1H, dd, J 14.1, 6.7 CHAHBSH),
2.80 (1H, dd, J 14.1, 5.7 CHAHBSH), 3.35 (1H, t, J 9.0, H-4),
3.59–3.65 (3H, m, H-3, H-5, H-6A), 3.69 (1H, dd, J 12.0, 2.3
H-6B) 3.79 (1H, dd, J 10.7, 3.7 H-2), 4.33 (1H, dd, J 6.7, 5.7
CHCH2SH), 4.40 (1H, d, J 11.7, OCHAHBPh), 4.60 (1H, d, J 11.7,
OCHAHBPh, 4.84 (1H, d, J 3.7, H-1), 7.29 (5H, m, 5 × ArH); dC
(75 MHz, D2O); 22.06 (CH3), 25.91 (CH2SH), 54.16, 55.94 (C-2,
CHCH2S), 60.78 (C-6), 70.07, 70.40, 71.06, 72.45 (CH2Ph, C-3,
=
76.08, 78.31, 92.86, (C-1-a) 96.98 (C-1-b), 172.94 (C O), 173.78
+
+
=
(C O) m/z (ESI ) 347.0879 (M+Na . C11H20N2O7SNa requires
347.0883).
Methyl 2(-N-acetyl-L-cysteinyl) amino-2-deoxy-a-D-glucopyrano-
side (2). Resin 16 (0.148 mmol) was pre-swollen by agitation in
DMF (10 cm3) for 10 min and then filtered. A solution of 10 (58 mg,
0.297 mmol) in CH2Cl2–DMF (3 : 1) (5 cm3) was then added
followed by a solution of HOBt (41 mg, 0.297 mmol), PyBOP
(154 mg, 0.297 mmol) and 2,6-di-tert-butylpyridine (57 mg,
0.297 mmol) in CH2Cl–DMF (3 : 1) (2 cm3) and the resulting
mixture was agitated for 2 h. The resin was washed successively
with 10 cm3 portions of DMF (4 × 4 min), then agitated in 20%
(v/v) of piperidine in DMF (40 cm3). After washing with DMF
(3 × 10 cm3), a solution of acetic anhydride (30 mg, 0.297 mmol)
and pyridine (24 mg 0.297 mmols) in DMF 10 cm3 was then
C-4, C-5), 96.35 (C-1), 128.73 (CH), 128.91 (CH), 129.09 (CH),
+
=
=
137.20 (C), 172.35 (C O), 174.49 (C O); m/z (ESI ) 437.1355
(M+Na+. C18H26N2O7SNa requires 437.1358).
2(-N-Acetyl-L-cysteinyl) amino-2-deoxy-a-D-glucopyranoside
disulfide (4). A solution of 1 (34 mg, 0.105 mmol) dissolved in
30 mM aqueous NH4HCO3 (4 cm3) was shaken for 24 h until
all the thiol had been consumed (by titration with Ellman’s
reagent). The water was removed on a rotary evaporator and
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the solids coevaporated three times with water before finally
dissolving in water and freeze drying to give 4 as a white solid
in quantitative yield. Spectral data was in agreement with that
previously reported. 3
Trityl polystyrene-supported cysteines (15–17): general pro-
cedure. For preparation of 16, Trityl-S-CysNHFmoc (4.0 g,
6.8 mmol) was first detritylated by treatment with TFA–DCM–
Et3SiH (40 : 60 : 3) (20 cm3) for 3 h. The solvents were removed in
vacuo. The crude mixture and DIPEA (1.18 cm3, 6.8 mmol) were
then dissolved in anhydrous CH2Cl2–DMF (1 : 1, 20 cm3) and
added to polystyrene trityl chloride resin (1.7 mmol, pre-swollen
in anhydrous DMF for 20 min). The mixture was agitated with
a bubbling flow of argon for 2 h. The resin was filtered, washed
with CH2Cl2 (20 cm3, 4 × 3 min), treated with 10 cm3 of CH2Cl2–
trifluoroethanol–AcOH (7 : 2 : 1) for 15 min, then washed with
four portions of DCM. The resin was finally treated with a 20 cm3
of CH2Cl2–MeOH–DIPEA (16 : 3 : 1) for 30 min then washed
(CH2Cl2, 4 × 20 cm3) filtered and dried under vacuum (40 ◦C)
overnight. Resin 17, was prepared following detritylation of 14 in
a similar manner and 15 was prepared from CysNAc 12 using the
same loading procedures.
Methyl 2(-N-acetyl-L-cysteinyl) amino-2-deoxy-a-D-glucopyrano-
side disulfide (5). A solution of 2 (26 mg, 0.077 mmol) dissolved
in 30 mM aqueous NH4HCO3 (3 cm3) was shaken for 24 h until all
the thiol had been consumed (by titration with Ellman’s reagent).
The water and some of the volatile salts were removed on a
rotary evaporator before dissolving in water and freeze drying
to give 5 as a white solid (26 mg, quantitative). [a]D +23.5◦ (c
20
0.74, DMSO); dH (300 MHz, DMSO-d6); 1.85 (3H, s, CH3), 2.7
(1H, dd, J 13.4, 9.6, CHAHBSH), 3.07–3.14 (2H, m), 3.22 (3H, s,
OCH3), 3.29–3.35 (1H, m), 3.40–3.48 (2H, m), 3.57–3.63 (2H, m),
4.48 (1H, d, J 3.3, H-1), 4.57 (1H, dd, J 9.6, 4.4, CHCH2S); dC
(75 MHz, DMSO-d6); 22.74 (CH3), 41.08 (CH2SS), 52.20, 54.32,
54.95 (OCH3, C-2, CHCH2SS), 61.05 (C-6), 70.73, 70.88, 72.97
=
=
(C-3, C-4, C-5), 98.17 (C-1), 170.49 (C O), 170.80 (C O); m/z
(ESI+) 697.2038 (M+Na+. C24H42N4O14S2Na requires 697.2037)
697 (55%), 483 (65), 143 (100).
Enzyme assays
Prior to conducting detailed assays, the concentrations of the
stock solutions of all the disulfide substrates (MSSM and 4–6)
were determined by titration against NADPH under the assay
conditions outlined below, but in the presence of a large excess
(25–50 nM) of Mtr. The amount of NADPH oxidised during
complete substrate exhaustion using an extinction coefficient of
6220 M−1 (at 340 nm) was used to calculate the initial substrate
concentration. Stock enzyme concentrations were determined by
absorbance of the active site FAD chromophore at 462 nm with
an extinction coefficient of 11 300 M−1. Standard substrate assays
were carried out in a final volume of 1000 lL in disposable cuvettes
at 30 ◦C containing Mtr (5 nM), HEPES (50 mM), 0.1 mM
EDTA, NADPH (0.14 mM) at pH 7.5 and varying concentrations
of substrates. Substrate stock solutions were made up using co-
solvent mixtures of assay buffer and DMSO; final assay mixtures
contained 2% DMSO. Enzyme activity was monitored by the de-
crease in absorbance at 340 nm due to NADPH oxidation. Initial
rates were measured from the linear region of product formation
and Km and kcat values were determined by weighted non-linear
regression analysis of the hyperbola plot of initial rate against
substrate concentration using the Michaelis Menten equation.
Benzyl 2(-N-acetyl-L-cysteinyl) amino-2-deoxy-a-D-glucopyrano-
side disulfide (6). A solution of 3 (30 mg, 0.072 mmol) dissolved
in a 1 : 1 mixture of hot 30 mM aqueous NH4HCO3 and
MeOH (3 cm3) and was shaken for 60 h until all the thiol
had been consumed (by titration with Ellman’s reagent). As the
reaction progressed the reactants/products began to crash out of
solution and were periodically re-dissolved by warming with a
heat gun. The methanol was removed on a rotary evaporator and
the remaining aqueous solution was freeze-dried to give 6 as a
white solid in quantitative yield. At 300K, the NMR spectra of
3 was complicated by the detection of multiple conformations.
20
The NMR analyses were therefore carried out at 363K. [a]D
13.7◦ (c 0.51, DMSO; dH (500 MHz, DMSO-d6); 1.84 (3H, s,
CH3), 2.83 (1H, dd, J 13.5, 10.0, CHAHBS), 3.11 (1H, dd, J
13.5, 4.2, CHAHBS), 3.13–3.18 (1H, m), 3.40–3.56 (3H, m), 3.60–
3.69 (2H, m), 4.29 (1H, d, J 12.3, CHAHBPh), 4.59 (1H, d, J
12.3, CHAHBPh), 4.70 (1H, J 3.5, H-1), 4.70–4.75 (m, CHCH2S);
dC (125 MHz, DMSO-d6); 22.32 (CH3), 41.02 (CH2S), 52.70,
54.35 (C-2, CHCH2S), 61.23 (C-6), 68.78, 71.01, 71.13, 72.95
(C-3, C-4, C-5) 96.20 (C-1), 127.51 (CH), 127.67 (CH), 128.24
+
+
=
(CH), 137.66 (C), 170.55 (C O); m/z (ESI ) 849.2767 (M+Na .
C36H50N4O14S2Na requires 849.2663.
Acknowledgements
We would like to acknowledge the following for financial sup-
port: Queen’s University Belfast (M.J.G.S), The Department of
Employment and Learning (A.S.R), Wellcome Trust Vacation
Scholarship (VS/02/EAN/6/CH/TH/FH) (J.W), The Overseas
Research Studentship Awards Scheme (V.K.J) and The Guildford
Bench Methodology Fund. We are also grateful to J. Blanchard
for provision of the plasmid encoding recombinant Mtr and
R.C. Fahey, G. Newton and E. Newton for the provision of
authentic mycothiol disulfide. We also thank the EPSRC Mass
Spectrometry Service Centre, Swansea for invaluable support.
Benzyl 2-amino-2-deoxy-a-D-glucopyranoside (11). Com-
pound 919 (1.5 g, 4.81 mmol) was dissolved in hydrazine hydrate
(9 cm3) and stirred at 120 ◦C for 2 days, then concentrated
in vacuo, co-evaporated twice with toluene, and purified by
chromatography (EtOAc–MeOH 7 : 3 as eluent) to yield 11 as a
white solid (1.02 g, 78%), mp 110–114 C; [a]D +148.1 (c 1.0 in
MeOH); dH (500 MHz, CD3OD) 2.67 (1H, dd, H-2, J 10.1, 3.6),
3.31 (1H, m, H-4), 3.51 (1H, dd, J 10.1, 9.0, H-3), 3.63 (1H, ddd
J 9.8, 5.7, 2.3 H-5), 3.69 (1H, dd, J 11.9, 5.7, H-6b), 3.80 (1H, dd,
J 11.9, 2.3, H-6a), 4.50 (1H, d, J 11.5, PhCHACHB), 4.76 (1H,
d, J 11.5, PhCHACHB), 4.93 (1H,d, J 3.6, H-1), 7.26–7.40 (5H,
m, Ph), dC (125 MHz, CD3OD) 57.03 (C2), 70.42 (CH2Ph), 71.96
(C-4), 74.38 (C-5), 75.78 (C-3), 99.25 (C-1), 128.86 (CH), 129.31
(CH), 129.44 (CH), 138.95 (C); m/z (ESI+) 270.1340 (M+H+.
C13H19NO5 requires 270.1341.
◦
20
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