Rate enhancement of the oxidative folding of lysozyme by the use of aromatic thiol containing redox buffers

Article abstract of DOI:10.1016/j.bmc.2007.11.047

Almost all therapeutic proteins and most extracellular proteins contain disulfide bonds. The production of these proteins in bacteria or in vitro is challenging due to the need to form the correctly matched disulfide bonds during folding. One important pa

Full text of DOI:10.1016/j.bmc.2007.11.047

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2579–2590
Rate enhancement of the oxidative folding of lysozyme by the use
of aromatic thiol containing redox buffers
Minakshi C. Gurbhele-Tupkar, Lissette R. Perez, Yenia Silva and Watson J. Lees*
Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA
Received 26 September 2007; revised 13 November 2007; accepted 16 November 2007
Available online 22 November 2007
Abstract—Almost all therapeutic proteins and most extracellular proteins contain disulfide bonds. The production of these proteins
in bacteria or in vitro is challenging due to the need to form the correctly matched disulfide bonds during folding. One important
parameter for efficient in vitro folding is the composition of the redox buffer, a mixture of a small molecule thiol and small molecule
disulfide. The effects of different redox buffers on protein folding, however, have received limited attention. The oxidative folding of
denatured reduced lysozyme was followed in the presence of redox buffers containing varying concentrations of five different aro-
matic thiols or the traditional aliphatic thiol glutathione (GSH). Aromatic thiols eliminated the lag phase at low disulfide concen-
trations, increased the folding rate constant up to 11-fold, and improved the yield of active protein relative to GSH. The yield of
active protein was similar for four of the five aromatic thiols and for glutathione at pH 7 as well as for glutathione at pH 8.2. At pH
6 the positively charged aromatic thiol provided a higher yield than the negatively charged thiols.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
ence of biological catalysts such as protein disulfide
isomerase or molecular chaperones can have a dramatic
effect on folding but these tend to be expensive and have
low catalytic activity.7 Folding aids, which are proposed
to alter the interactions between or within proteins, re-
tard protein aggregation/precipitation. Common folding
aids include urea, guanidine hydrochloride, arginine,
and glycerol.5,8–14 These aids, however, with limited
exception,10,11 increase the yield of active protein but de-
crease the in vitro folding rate constants of model disul-
fide containing proteins such as lysozyme. Folding aids
also tend to be protein specific and thus have to be opti-
mized for each protein.2
The folding of proteins in vitro has served as a method
of producing therapeutic proteins. When proteins, espe-
cially those containing disulfides, are overexpressed in
bacteria they can aggregate/precipitate inside the cell
forming inclusion bodies.1–5 These inclusion bodies,
which contain inactive protein, are then isolated and res-
olubilized using a denaturant such as guanidine hydro-
chloride or urea. The resulting denatured reduced
protein is then folded in vitro. The in vitro folding step
is usually the lowest yielding. Many therapeutic pro-
teins, almost all of which contain disulfide bonds, are
produced using a variation of this procedure. For disul-
fide containing proteins the common model systems are
bovine pancreatic trypsin inhibitor (BPTI), ribonuclease
A (RNase A), and lysozyme.
The composition of the redox buffer is very important
for the successful folding of disulfide containing pro-
teins. The redox buffer is usually composed of a mixture
of a small molecule disulfide and/or a small molecule
thiol. The traditional mixture contains glutathione
disulfide and glutathione, although other small molecule
aliphatic thiols and their corresponding disulfides have
been used, for example, mercaptoethanol, thioglycolic
acid, cysteine, cystamine, and dithiothreitol (DTT).1,2,15
The redox buffer both oxidizes protein thiols to form
protein disulfide bonds and helps rearrange mismatched
disulfide bonds to form native disulfide bonds. With
redox buffers containing small molecule disulfides and/
or thiols, the oxidation and rearrangement occurs via
Several methods have been developed to improve the
in vitro folding of disulfide containing proteins. The
key parameters for the folding of proteins are the pH
of the solution, the composition of the redox buffer,
and the presence of folding aids.6 In addition, the pres-
Keywords: Protein folding; Lysozyme; Aromatic thiol; Redox buffer.
*
Corresponding author. Tel.: +1 305 348 3993; fax: +1 305 348
3772; e-mail: leeswj@fiu.edu
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.047

2580
M. C. Gurbhele-Tupkar et al. / Bioorg. Med. Chem. 16 (2008) 2579–2590
the thiol-disulfide interchange reaction. Mechanistically,
redox buffers and folding aids are thus different as folding
aids affect interactions within or between proteins and re-
dox buffers enhance the thiol-disulfide interchange
reactions.
hanced (reactions E–H). Aromatic thiolates are better
leaving groups than aliphatic thiolates, such as glutathi-
one, due to their lower thiol pKa values. In addition,
aromatic thiolates are better nucleophiles than aliphatic
thiolates with similar thiol pKa values.21 Only intramo-
lecular reactions between a protein thiol and protein
disulfide (reaction A) and reactions that are rate limited
by protein conformational changes will be unaffected by
switching to an aromatic thiol from glutathione.22 As a
result of these factors aromatic thiols increase the oxida-
tive folding rate of RNase A by up to 25-fold relative to
glutathione depending on the pH.7,22–25 In order to
examine the generality of the rate enhancement, we
decided to examine the folding of lysozyme.
Several new small molecule thiol redox buffers have re-
cently been introduced. These compounds can be subdi-
vided into two classes; dithiols and aromatic thiols.16
Redox buffers that contain a small molecule aliphatic
dithiol or peptide dithiol increase the yield of active pro-
tein but do not increase the rate constant of protein fold-
ing more than 3-fold relative to glutathione.16–20
Dithiols are proposed to improve protein folding by
shortening the lifetime of kinetically stable mixed disul-
fides formed between the protein and the small molecule
thiol. With dithiols the mixed disulfide can be displaced
intramolecularly by the second thiol on the dithiol in-
stead of intermolecularly, as is the case for monothiols.
Intramolecular displacement is proposed to be more ra-
pid than intermolecular displacement.
The folding of denatured reduced lysozyme into its ac-
tive form has been extensively studied under a wide vari-
ety of conditions and its folding pathway is relatively
well characterized.10,14,26–31 However, unlike RNase A,
the yield of active protein is sensitive to the conditions
used. At relatively low protein concentrations, 0.2 mg/
mL, with low concentrations of folding aids, the yield
of active protein can be 20%.10 At higher concentrations
of folding aids the yield increases substantially,
approaching 100% under certain circumstances.10
Therefore, lysozyme is an ideal protein for testing fold-
ing aids and protein folding technology. Although redox
buffers are known to affect protein folding, only a lim-
ited number of studies have investigated the effects of
different redox buffers on folding and they have focused
Aromatic thiols can increase the rate of almost all of the
thiol-disulfide interchange reactions that occur during
protein folding under standard conditions, relative to
aliphatic thiols such as glutathione, Scheme 1. Any reac-
tion in which the aromatic thiolate, the deprotonated
thiol form, acts as a leaving group will be enhanced
(reactions B, C, F, and H) and any reaction in which
the aromatic thiolate acts as a nucleophile may be en-
Reactions of Protein Thiolate
R
S
+
R
S
S
S
S
S
S
RSSR
A
C
B
D
S
S
S
S
R
S
S S
S
S
R
S
+
R
S
SH
S
R
S
S
S
S
HS S
HS S
HS S
Reactions of Small Molecule Thiolate (RS-)
R S
RSSR
R
R
S
RSSR
HS
R
S
+
HS
E
HS
HS
F
+
S
S S
S
S
R
S
+
R
S
+
R
S
+
RSSR
+
R
R
S
HS S
R
HS
HS
G
H
S
S
HS S
HS S
HS S
Scheme 1. Thiol-disulfide interchange reactions that occur during protein folding with a small molecule thiol (RSH) and a small molecule disulfide
(RSSR).

M. C. Gurbhele-Tupkar et al. / Bioorg. Med. Chem. 16 (2008) 2579–2590
2581
exclusively on the yield of active protein.15,32–34 With the
standard redox buffer, glutathione and glutathione
disulfide, the optimal conditions have traditionally been
reported as a ratio of [GSH]/[GSSG] and the results
have varied from 10:1 to 1:1.27,30,32,35
(DTE) had been removed by gel permeation chromatog-
raphy,36 was rapidly diluted 100-fold into renaturation
buffer containing 0.1 M buffer (Bis–Tris propane for
pH 7.0 or Bis–Tris for pH 6.0), 1 mM EDTA, 0.5 M
guanidine hydrochloride (GdnHCl), and redox buffer.37
The EDTA was added to prevent metal catalyzed air
oxidation of the thiols.32 The common additive GdnHCl
was included to minimize protein aggregation during
folding.8,37 The final concentration of lysozyme was
0.1 mg/mL or 7 lM. Aliquots were removed from the
folding reaction at specific times and the enzymatic
activity determined by adding the aliquots to a suspen-
sion of M. lysodeikticus.32,37,38 The decrease in light
scattering was then monitored for 2 min at 450 nm.
The change in light scattered per minute is proportional
to enzymatic activity.
Herein, the standard redox buffer for the folding of
denatured/reduced lysozyme, glutathione and glutathi-
one disulfide, is optimized at five different glutathione
disulfide concentrations. The protein folding rates ob-
tained with the optimized standard redox buffer are then
compared with those obtained using redox buffers con-
taining one of five different aromatic thiols. A systematic
study of the effects of a series of redox buffers on the
folding of lysozyme has not been undertaken previously,
even though redox buffers and pH have large effects on
protein folding.6 Aromatic thiols are also known to sig-
nificantly enhance the folding of scrambled RNase A
under a variety of conditions.24 The data obtained with
lysozyme are discussed with respect to the results ob-
tained with RNase A, and general rules for the folding
of disulfide containing proteins efficiently in vitro with
aromatic thiols are presented.
2.1. Folding with glutathione
The folding of reduced lysozyme was performed in re-
dox buffers composed of various concentrations of
GSH and GSSG at pH 7. Five GSSG concentrations
were selected to encompass the range of optimal values
previously reported, 0.06, 0.2, 0.5, 2.0, and 5.0 mM. At
each GSSG concentration 1, 2, 4, 7, and 10 mM GSH
were examined, Figure 1. Lower concentrations of
GSH were examined at 0.06 and 0.2 mM GSSG and
higher concentrations of GSH were examined at 5 mM
GSSG.
2. Results
The folding of reduced lysozyme was undertaken in the
presence of the traditional redox buffer, glutathione and
glutathione disulfide, and redox buffers containing five
different aromatic thiols. Initially, the concentrations
of glutathione and glutathione disulfide were optimized
for protein folding. The glutathione was then replaced
with an aromatic thiol and again the thiol concentration
was varied. The effect of pH on folding was also
investigated.
The optimum concentration of GSH at each GSSG con-
centration was determined either by inspection or by fit-
ting the curve to an exponential function. At 0.06 and
0.2 mM GSSG a noticeable lag occurred before the
appearance of significant enzymatic activity, as indi-
cated in Figure 1 for 0.2 mM GSSG. This lag period
made fitting the folding curve to a single exponential
function inappropriate.30,37,39 The optimum GSH con-
centration was thus determined by inspection. At 0.5,
2, and 5 mM GSSG the lag period had diminished and
The folding of reduced lysozyme was followed using the
recovery of enzymatic activity. Denatured reduced lyso-
zyme (10 mg/mL), from which the dithioerythreitol
90
90
[GSH]
[GSH]
0.2 mM GSSG
2.0 mM GSSG
1 mM
2 mM
4 mM
7 mM
10 mM
1 mM
2 mM
4 mM
7 mM
10 mM
60
30
0
60
30
0
0
100
200
300
0
100
200
300
time (min)
time (min)
Figure 1. Recovery of enzymatic activity during the folding of denatured reduced lysozyme (7 lM) in the presence of 0.2 or 2 mM GSSG and various
GSH concentrations. Assays were performed at pH 7.0 and 25 °C in the presence of 1 mM EDTA and 0.5 M GdnHCl. The curves for 2 mM GSSG
were fit to an exponential, y = A(1 ꢀ eꢀkt), where A is maximal activity, k is the folding rate constant, and t is time.

2582
M. C. Gurbhele-Tupkar et al. / Bioorg. Med. Chem. 16 (2008) 2579–2590
Table 1. Folding of reduced lysozyme at optimum conditions using a
redox buffer of GSH and GSSG at pH 7
2.2. Folding with aromatic thiols
A series of aromatic thiols was selected to improve the
folding rate and yield of lysozyme, Scheme 2. The thiols
were chosen for their solubility in buffer containing
0.5 M GdnHCl at pH 6–8 and to provide a range of thiol
pKa values. Thiols 1, 3, and 4 were used previously to fold
RNase A but thiols 2 and 5 have not been used previously
for protein folding experiments. p-Mercaptobenzyl alco-
hol previously used with RNase A was not adequately
soluble in 0.5 M GdnHCl at pH 6. Aromatic thiols 2
and 5 were selected because at pH 7 they would be soluble
in water, the former would be negatively charged and the
latter would be positively charged. Furthermore, their
thiol pKa values were predicted to differ substantially.
GSSG Optimum
Maximal
Time to 90%
GSH/GSSG
ratio
concd GSH concda % activityb of maximal
(mM) (mM)
activityc (min)
d
d
0.06
0.2
0.5
2.0
5.0
1
17:1
5:1
1
60
60
65
65
190
180
150
180
2
4:1
3.5: 1
2:1
7
10
a For 0.06 and 0.2 mM GSSG, the optimum concentration was
determined by inspection. For 0.5 mM GSSG and higher, the opti-
mum concentration was determined by the value of Ak.
b Maximal activity was estimated from the activity at long time points,
between 240 and 300 min. The average standard deviation of three
independent measurements performed on separate days was 15%.
c Due to significant differences in lag periods, the time to 90% com-
pletion was used, vide supra. The average standard deviation of three
independent measurements performed on separate days was 25 min.
d Maximal activity was not reached within 300 min.
Aromatic thiols 1, 3–5 were purchased or prepared as
previously reported.23,40,41 Aromatic thiol 2 was synthe-
sized in five steps from p-mercaptotoluene, Scheme 3.
The brominated intermediate is also used in the prepara-
tion of the quaternary ammonium salt. The thiol pro-
tecting group was changed midway through the
synthesis to prevent benzoic acid precipitating out of
solution along with the product in the final step.
the folding curve was fit to a single exponential. The
exponential function used was y = A(1 ꢀ eꢀkt) where A
is the maximal enzymatic activity achieved, k is the
apparent folding rate constant, and t is time. The con-
centration of GSH that provided the greatest value of
The folding of reduced lysozyme was undertaken in the
presence of various concentrations of each aromatic
thiol and 0.2 mM GSSG. The concentration of
0.2 mM GSSG was selected for several reasons. Lyso-
zyme folds rapidly in the presence of 0.2 mM GSSG
and 1 mM GSH. Our previous results obtained with
RNase A used 0.2 mM GSSG. Also, we expected the
lag phase to disappear in the presence of 0.2 mM GSSG
and an aromatic thiol, due to the greater reactivity of
aromatic thiols and disulfides, formed by the rapid reac-
tion of GSSG with the aromatic thiol before the lyso-
zyme is added.21,24 The equilibrium concentrations can
be calculated using a set of simultaneous equations.21,24
All the aromatic thiols were assayed at 1, 2, 4, 6, and
8 mM. Aromatic thiols 2, 4, and 5, which were more sol-
uble, were assayed at 8, 10, 15, and 20 mM. The folding
curves were again fit to an exponential function,
y = A(1 ꢀ eꢀkt) where A is the maximal enzymatic activ-
ity achieved, k is the apparent folding rate constant, and
t is time, Figures 2 and 3. Even though the folding of
lysozyme is complex with many different reactions, a
simple exponential fit appears to fit the data quite well.
A
optimal, Table 1. Other GSH concentrations did, how-
k, the initial rate of protein folding, was defined as
*
ever, provide A k values similar to those obtained at
*
the optimum concentration. The following were all with-
in 20% of the optimal: 0.5 mM GSSG, 1–4 mM GSH;
2.0 mM GSSG, 4–7 mM GSH; 5.0 mM GSSG, 4–
10 mM GSH. The optimum concentrations also exhib-
ited the highest maximal activities or within 5% of the
highest for a given GSSG concentration. The folding
experiment was then repeated two additional times at
the optimum GSH concentrations, Table 1.
R =
R
1 -CH2COOH; pKa = 6.6
2 -CH2PO3H; pKa = 6.6
3 -COOH, pKa = 5.95
4 -SO3H; pKa = 5.7
SH
5 -CH2N+(CH3)3; pKa = 5.5
The value of A k corresponds to the initial rate of pro-
*
tein folding. In each case, with the exception of thiol 2,
Scheme 2. Aromatic thiols.
O
Cl
O
O
O
EtO
EtO
EtO
EtO
HO
HO
P
P
Br
P
1) NaOEt/EtOH
2)Ac2O
P(OEt)3
105 ˚C
HCl
hν
NBS
Δ
NEt3/toluene
6
7
8
9
2
S
S
SH
S
S
SH
O
O
O
O
Ph
Ph
Ph
Scheme 3. Preparation of aromatic thiol 2.

M. C. Gurbhele-Tupkar et al. / Bioorg. Med. Chem. 16 (2008) 2579–2590
2583
90
60
30
0
90
2
5
60
[2]
[5]
30
8 mM
8 mM
10 mM
15 mM
20 mM
10 mM
15 mM
20 mM
0
0
50
100
150
0
50
100
150
time (min)
time (min)
Figure 2. Recovery of enzymatic activity during the folding of denatured reduced lysozyme (7 lM) at pH 7 in the presence of various concentrations
of aromatic thiol 2 or 5. Assays were performed at 25 °C in the presence of 0.2 mM GSSG, 1 mM EDTA, and 0.5 M GdnHCl. The 0 min time point
was taken immediately after mixing the denatured reduced protein with the refolding mixture. The curves were fit to an exponential function
y = A(1 ꢀ eꢀkt), where A is maximal activity, k is the folding rate constant, and t is time.
100
80
60
40
20
0
100
80
60
40
20
0
0.15
0.1
0.05
0
0.15
0.1
0.05
0
pH 7
pH 6
0
5
10
15
20
25
0
5
10
15
20
25
[2] (mM)
[2] (mM)
100
80
60
40
20
0
100
80
60
40
20
0
0.15
0.1
0.05
0
0.15
0.1
0.05
0
pH 7
pH 6
0
5
10
15
20
25
0
5
10
15
20
25
[5] (mM)
[5] (mM)
Figure 3. A or maximum activity (circles) and k (squares) values as a function of thiol concentration. Values were normalized to those in Tables 2 and
3, and the error is twice the curve fitting error for each point. At high thiol concentrations (20 mM 5, 15 mM 2 at pH 7, and 20 mM 5, 8 mM 2 at pH
6) the approximate experimental error was 10% in A and 15% in k.
the greatest A k value was obtained at the highest thiol
*
concentration examined. For thiol 2, the greatest A
for each aromatic thiol, Table 2. The standard devia-
tions were approximately 10% in A, 15% in k, and
k
value occurred at 15 mM. The folding experiments were
*
20% in A k. Similar errors were obtained with
ribonuclease A.24 The results with lysozyme were also
*
then repeated multiple times at the best concentrations

2584
M. C. Gurbhele-Tupkar et al. / Bioorg. Med. Chem. 16 (2008) 2579–2590
Table 2. Refolding kinetics of lysozyme with various aromatic thiols
(ArSH) and glutathione at pH 7.0 and glutathione at pH 8.2
90
60
30
0
ArSH pKa Thiol
concd (mM) activity, A
Maximal % k (minꢀ1
)
pH 7a
1
2
3
4
5
6.6
6.6
8
15
8
45 5 (7)
67 5 (7)
70 3 (5)
62 5 (6)
72 6 (6)
64 (2)
0.125 0.014 (0.021)
0.113 0.013 (0.019)
0.064 0.005 (0.008)
0.105 0.009 (0.014)
0.053 0.006 (0.007)
0.014 (0.002)
5.95
5.70 20
5.5
GSHb 8.7
20
7
pH 8.2c
70 (10)
GSH 8.7
2
0.095 (0.011)
a The error was determined from at least seven assays with each assay
being performed independently on separate days. The error is the
standard deviation of the mean at 95% confidence using the Student’s
t test, =ts/n0.5. The error in parentheses is the standard deviation. The
GSSG concentration was 0.2 mM unless specified otherwise.
b The GSH concentration was 7 mM and the GSSG concentration was
2 mM and is based on three independent measurements. The lag
period under these conditions is small.
0
20
40
60
time (min)
Figure 5. The folding of denatured reduced lysozyme (7 lM) in the
presence of 50 mM thiol 5 and 0.2 mM GSSG. Assays were performed
at pH 7.0 and 25 °C in the presence of 1 mM EDTA and 0.5 M
GdnHCl.
c The GSH concentration was 2 mM and the GSSG concentration was
0.4 mM and is based on five independent measurements.
examine what happens at much higher concentrations
of 5, the folding of lysozyme in the presence of 50 and
100 mM of 5 and 0.2 mM GSSG was investigated,
Figure 5. The folding rates and maximum activity were
0.122 minꢀ1 and 78%, and 0.153 minꢀ1 and 80%, respec-
tively, for 50 and 100 mM of 5. The results are the aver-
age of two experiments performed on separate days. To
ensure that the enhanced rate was due to the presence of
the thiol group and not due to the high concentration of
a quaternary ammonium salt, a control experiment was
carried out. Folding was performed with 1 mM GSH
and 0.2 mM GSSG in the presence of 50 and 100 mM
benzyltrimethyl ammonium bromide, the non-thiol con-
taining analog of 5. The control experiment showed a
slight decrease in the folding rate and activity as the con-
centration of benzyltrimethyl ammonium bromide was
increased from 0 to 50 to 100 mM; from 2 to 4 h the
activity decreased by 10% and 20%, respectively.
independent of the disulfide used with the aromatic thi-
ols, the commercially available GSSG or aromatic disul-
fide (ArSSAr), as is the case with ribonuclease A.22 For
thiols 1 and 5, the 0.2 mM GSSG was replaced with
0.2 mM ArSSAr. The improved folding rates in the pres-
ence of aromatic thiols relative to glutathione can be
seen in Figure 4.
Aromatic thiol 5 maintains a high maximal activity, A,
but has a low protein folding rate constant. However,
it is also clear from Figure 2 that as the concentration
of 5 is increased the folding rate constant increases. To
90
2
GSH
60
The folding of reduced lysozyme at pH 6 in the presence
of the aromatic thiols and 0.2 mM GSSG was also
examined. Aromatic thiols 1–4 were assayed at 1, 2, 4,
6, and 8 mM. The aromatic thiols 2, 4, and 5 were then
assayed at 8, 10, 15, and 20 mM. Aromatic thiol 5 was
only assayed at high concentrations as the best concen-
tration was expected to be greater than 8 mM. The opti-
mum concentrations were 8 mM for 1–3 and 15 mM for
4, Figure 6. The best concentration for 5 was again the
highest concentration tested, 20 mM. As is the case at
pH 7, we expect the optimal concentration for 5 to be
higher still, Figure 5. The folding experiments were then
repeated several times at the best concentrations for
each aromatic thiol, Table 3.
90
30
60
30
2
0
0
8
16
24
0
0
100
time (min)
200
300
Figure 4. The folding of denatured reduced lysozyme (7 lM) in the
presence of 15 mM aromatic thiol (circles) and 1 mM GSH
2
(diamonds). The insert corresponds to the early time points for
aromatic thiol 2. Assays were performed at pH 7.0 and 25 °C in the
presence of 0.2 mM GSSG, 1 mM EDTA, and 0.5 M GdnHCl. The
0 min time point was taken immediately after mixing the reduced
denatured protein with the refolding mixture. The insert was fit to an
exponential function y = B + A(1 ꢀ eꢀkt), see text.
3. Discussion
The optimum glutathione and glutathione disulfide con-
centrations for the folding of lysozyme were reinvesti-

M. C. Gurbhele-Tupkar et al. / Bioorg. Med. Chem. 16 (2008) 2579–2590
2585
90
60
30
0
90
2
5
60
[2]
[5]
30
8 mM
8 mM
10 mM
15 mM
20 mM
10 mM
15 mM
20 mM
0
0
50
100
150
200
0
50
100
150
200
time (min)
time (min)
Figure 6. Recovery of enzymatic activity during the folding of denatured reduced lysozyme (7 lM) at pH 6 in the presence of various concentrations
of aromatic thiol 2 or 5. Assays were performed at 25 °C in the presence of 0.2 mM GSSG, 1 mM EDTA, and 0.5 M GdnHCl. The 0 min time point
was taken immediately after mixing the denatured reduced protein with the refolding mixture. The curves were fit to an exponential function
y = A(1 ꢀ eꢀkt), where A is maximal activity, k is the folding rate constant, and t is time.
et al.42–44 and is close to the 10:1 to 5:1 ratio generally
Table 3. Refolding kinetics of lysozyme with various aromatic thiols
(ArSH) at pH 6
recommended for protein folding.5,45 On the other hand,
the [GSH]2/[GSSG] ratio increased with increasing
[GSSG] concentration,45 5–20 mM. The absolute value
of [GSH]2/[GSSG] is similar to that obtained for RNase
A, 5–20 mM,22,42 and to what is generally recommended
for protein folding, 5–30 mM.5,45 In summary, the redox
buffer has a broad optimum but the [GSH]/[GSSG] ratio
should be selected with consideration of the GSSG con-
centration. It is probable that there is no fixed optimal
ratio of [GSH]/[GSSG].
ArSH pKa
Thiol concd Maximal
(mM)
k (minꢀ1
)
% activity, Aa
1
2
3
4
5
6.6
6.6
5.95
5.7
5.5
8
8
16 (1)
67 (19)
60 (3)
50 (7)
85 (4)
0.092 (0.006)
0.019 (0.0014)
0.061 (0.005)
0.089 (0.015)
0.054 (0.006)
8
15
20
a The error was determined from at least four assays with each assay
being performed on separate days. The error is the standard devia-
tion. The solution contained 0.2 mM GSSG, 1 mM EDTA, and
0.5 M GdnHCl.
The maximal activity, A, achieved at each glutathione
disulfide concentration and optimal glutathione concen-
tration varied only within experimental error, Table 1. A
previous report demonstrated that at higher GSSG con-
centrations (2–5 mM), the folding rate showed a limited
correlation with yield, increasing the GSSG concentra-
tions at pH 8 resulted in slightly slower folding rates
and higher yields.27 The report provided the relative rate
constants for partitioning between productive folding to
native protein, k2, and non-productive folding, k3, in-
stead of maximal activity, A, but the two are mathemat-
ically interchangeable.27,35,46,47 At significantly below
optimal GSH concentration, a lower maximal activity
and lower folding rate is observed, Figure 1, with
2 mM GSSG.
gated. The optimum [GSH]/[GSSG] ratio and GSSG
concentration have been reported previously to be 10:1
and 0.5 mM GSSG, 5:1 and 0.4 mM GSSG, and be-
tween 2:1 and 1:1 and 4 mM GSSG, respec-
tively.27,30,32,35 Given the range of optimum ratios and
concentrations, the optimal conditions for the folding
of denatured reduced lysozyme were reexamined. For
GSSG concentrations of 0.2, 0.5, 2, and 5 mM at opti-
mal GSH concentration, the maximal activity and the
time taken to achieve 90% of the maximal activity were
similar, Table 1. The major difference between the
GSSG concentrations was that the lag period decreased
as the GSSG concentration increased, Figure 1,
suggesting that the lag period may result from the slow
oxidation of protein thiols at low GSSG concentra-
tions.30,37,39 Due to the decrease in the lag period, the
time taken to achieve half-maximal activity was
decreased at higher GSSG concentrations. Optimal fold-
ing might be expected to occur at a constant [GSH]/
[GSSG] ratio or at a constant redox potential, constant
[GSH]2/[GSSG] ratio. The results, including those in
Figure 1, suggest that neither option is valid, but not
conclusively, as the optimal conditions are quite broad.
The [GSH]/[GSSG] ratio decreased with increasing
[GSSG] as has been reported for RNase A by Gilbert
The folding of lysozyme in the presence of a series of
aromatic thiols was investigated and shown to vary with
the thiol pKa value and the thiol concentration. Initially,
the thiol concentration was varied up to 8 or 20 mM
depending on the solubility of the thiol, Table 2. Since
no lag period was observed, probably due to the greater
reactivity of aromatic disulfides relative to GSSG, the
folding curves were fit to an exponential function.21
The aromatic disulfide is produced by the rapid equili-
bration of aromatic thiol and GSSG before the lyso-
zyme is added, the concentrations of which can be
calculated using a series of simultaneous equations.21,24

2586
M. C. Gurbhele-Tupkar et al. / Bioorg. Med. Chem. 16 (2008) 2579–2590
Replacing GSSG with aromatic disulfide in the presence
of aromatic thiols does not affect the folding of lysozyme
within experimental error as was shown previously for
RNase A. GSSG is more practical as it is commercially
available. For thiols with high pKa values (2), increasing
the thiol concentration above 10 mM did not signifi-
cantly change the folding rate constant; for thiols with
low pKa values (5), increasing the thiol concentration
above 10 mM significantly increased the folding rate,
Figure 2. Ultimately, thiols with lower pKa values
achieved higher or equivalent folding rates if the thiol
concentration was increased substantially, Figure 5.
and a fixed [RSH]2/[RSSR] ratio. For RNase A at pH
6 and 7 the optimal concentration of thiol in the RSH
form was the same for glutathione and the aromatic thi-
ols, 1–2 mM.24 Overall, the concentration of thiol in the
RSH form is important for folding lysozyme.
Several factors affect the folding rates of disulfide con-
taining proteins with aromatic thiols including the con-
centration of thiol in the neutral RSH form, the
concentration of the thiolate anion, and the reactivity
of the thiolate anion.23,24 For a specific aromatic thiol
the relative concentrations of thiol in the neutral RSH
form and in the thiolate form vary only with the pH
as the thiol pKa is fixed. On the other hand, with a series
of aromatic thiols with different thiol pKa values the rel-
ative concentrations will vary with the thiol pKa values
at a fixed pH. With lysozyme and RNase A, the concen-
tration of thiol in the neutral RSH form affects the equi-
librium between the small molecule thiol and the protein
disulfide, Scheme 4. If the concentration of RSH in the
neutral form is too high, then the environment is too
reducing and the formation of disulfide bonds is diffi-
cult. If the concentration of RSH in the neutral form
is too low, then the environment is too oxidizing and
the formation of disulfide bonds is facile but their rear-
rangement is slow, as there are no free protein thiols. At
low pH, where most of the thiols are in the neutral form,
the equilibrium between aromatic thiols and glutathione
is close to unity.23,24 Thus, the concentration of thiol in
the neutral RSH form affects the equilibria that occur
during protein folding. Partially as a result of these fac-
tors both glutathione and aromatic thiols have optimal
concentrations beyond which the addition of more thiol
leads only to poorer folding, Figures 1 and 6. The con-
centration of thiol in the thiolate form is also important,
as thiolate is the reactive species in the thiol-disulfide
interchange reaction, Scheme 1; the greater the thiolate
concentration the faster the reaction. The reactivity of
the thiolate is linked to the thiol pKa; the lower the aro-
matic thiol pKa, the less reactive the thiolate, although
aromatic thiolates are more reactive than aliphatic thiol-
ates if the pKa of the conjugate acids (thiols) are the
same.21 With RNase A, optimal conditions occurred
when the concentration of thiol in the neutral RSH form
was 1–2 mM.23,24 For lysozyme the value was 4–6 mM.
However, the absolute rate under optimal conditions
was determined by the thiolate concentration and its
reactivity, which varied with the thiol pKa value. With
lysozyme at pH 6 under optimal conditions, compound
2 (pKa = 6.6) has a much smaller concentration of thio-
late (6.3 mM neutral RSH and 1.7 mM thiolate) than
compound 4 (pKa = 5.7, 5 mM neutral RSH, 10 mM
thiolate), although the concentration of RSH in the neu-
tral form is similar for both. The greater thiolate con-
Aromatic thiols significantly enhanced the folding rates
of lysozyme relative to glutathione. The enhanced rate
constant is proposed to be due to the enhanced leaving
group ability of aromatic thiols relative to glutathione
and their enhanced nucleophilicity relative to aliphatic
thiols with similar thiol pKa values.21 With lysozyme,
the greatest folding rate constant obtained with glutathi-
one and glutathione disulfide at pH 7 was 0.014 minꢀ1
.
The optimum folding rate for aromatic thiol 2 was 9
times that of glutathione and the folding rate for 5
was 11 times that of glutathione (7 lM lysozyme,
0.5 M guanidine hydrochloride, pH 7, 25 °C). In com-
parison, under very similar conditions (6 lM lysozyme,
1 M urea, pH 7.4, 25 °C) the in vivo catalyst of thiol-
disulfide interchange reactions during protein folding,
protein disulfide isomerase (PDI), enhanced the folding
rate constant of lysozyme approximately 15-fold, 3:1 ra-
tio of lysozyme to PDI, relative to glutathione under the
same conditions.48 In the absence of urea (10 lM lyso-
zyme, pH 7, 37 °C) the folding rate constant was en-
hanced 7-fold by PDI, 2:1 ratio lysozyme to PDI, but
at 1 lM lysozyme the rate enhancement was 60-fold,
2:1 ratio of lysozyme to PDI.49 Even at pH 6, aromatic
thiols can fold lysozyme faster than glutathione does at
pH 7 or at a comparable rate to glutathione at pH 8.2.
Similarly, with RNase A the folding rate constants in
the presence of aromatic thiols under optimal conditions
at pH 7 were 7–12 times those of glutathione and the
rate constants at pH 6 with aromatic thiols were greater
than those of glutathione at pH 7.7.24 In summary, aro-
matic thiols can be used to significantly enhance the
folding rates of lysozyme at a range of pH values.
The optimal or best thiol concentrations for the folding
of lysozyme are similar but not identical for glutathione
and aromatic thiols when the concentration of thiol in
the neutral RSH form is taken into account, as opposed
to the thiolate form (RSꢀ). With 0.2 mM GSSG, the
optimal GSH concentration was 1 mM (pKa = 8.7;
1 mM RSH), the optimal concentration of aromatic
thiol 2 was 15 mM (pKa = 6.6; 4 mM RSH and 11 mM
thiolate), and the optimal concentration of aromatic
thiol 5 was at least 100 mM (pKa = 5.5; 3 mM RSH
and 97 mM thiolate). For lysozyme at pH 6 the optimal
concentration of the aromatic thiols in the RSH form
was 4–6 mM. Thus, the optimal concentrations of aro-
matic thiols in the RSH form were similar at pH 6
and 7, 4–6 mM, but the optimal concentration for gluta-
thione was less at pH 7, 1 mM. A fixed concentration of
RSH also corresponds to a fixed [RSH]/[RSSR] ratio
PSSP + 2RSH
2RS
2PSH + RSSR
Scheme 4. Reaction of small molecule thiol (RSH) with protein
disulfide (PSSP) to form small molecule disulfide (RSSR) and protein
thiol (PSH).

M. C. Gurbhele-Tupkar et al. / Bioorg. Med. Chem. 16 (2008) 2579–2590
2587
centration results in greater folding rates with 4 than 2
under optimal conditions even though the thiolate of 2
is inherently more reactive than that of 4.
4. Conclusions
In conclusion, the folding of lysozyme and ribonuclease
A follows the same general trends, which suggest that
aromatic thiols should enhance the folding rates of
many, if not almost all, disulfide containing proteins un-
der a variety of conditions. The rate enhancement ex-
pected from replacing glutathione with an aromatic
thiol at pH 7 is approximately 10-fold, which increases
as the pH decreases. The optimal aromatic thiol varies
with the pH of the solution. An aromatic thiol with a
thiol pKa approximately one unit lower than the pH is
ideal. The optimal aromatic thiol concentration also
varies with the aromatic thiol, solution pH, and protein
but a concentration of 1–6 mM in the neutral RSH form
appears to be general in the presence of 0.2 mM
disulfide.
The maximal activity or yield of active protein obtained
with aromatic thiols under optimal conditions at pH 6
and 7 varied with the aromatic thiol and in some cases
was greater than that obtained with glutathione at pH
7 or 8.2. Interestingly, the maximal activity did not show
a strong dependence on the concentration of glutathi-
one, Figure 1, unless the glutathione concentration
was changed from optimal by a factor of 3 or more.30
At pH 7 with aromatic thiols, the maximal activity
was also relatively unaffected by the thiol concentration,
although in one case the maximal activity decreased as
the thiol concentration increased, 1. No correlation
was observed between the folding rate constant and
maximal activity.37,47,49 At pH 6, the same general trend
in maximal activity was observed with aromatic thiol 1
giving the lowest maximal activity followed by 4 then
2 and 3, and finally 5. With RNase A the folding yields
under optimal conditions were also relatively indepen-
dent of the aromatic thiol, although thiols 2 and 5 were
not studied. The maximal activity obtained for lysozyme
with positively charged 5 at pH 6 was the highest we ob-
tained at any pH value or with any thiol, Table 3. All the
other thiols will be negatively charged at pH 6 or 7. A
previous report demonstrated that acetylating or carba-
moylating the lysine residues on lysozyme reduced the
net positive charge on the protein and decreased the
folding yield in a systematic manner.15 In addition, the
same report has shown that a positively charged disul-
fide, cystamine, was better at folding lysozyme than a
negatively charged disulfide, dithiodiglycolic acid, at
the same concentration.15 The authors proposed that
the introduction of additional positive charges derived
from the oxidizing agent, as a result of the formation
of mixed disulfides between the protein and the oxidiz-
ing agent, suppressed aggregation during the early
stages of folding. The pI of hen egg white lysozyme is
approximately 11 so the protein has a net positive
charge at both pH 6 and 7. Therefore, we conclude that
the net charge of the thiol may be important for the
folding yield of proteins with aromatic thiols.
5. Experimental
5.1. General information
Hen egg white lysozyme (>23,000 U/mg) was purchased
from Roche Applied Science and used without purifica-
tion. Reduced lysozyme was prepared according to the
procedure of Goldberg et al.50 Biochemicals were pur-
chased from Sigma. The p-mercaptobenzoic acid, p-mer-
captophenylacetic acid, and the reagents used to
synthesize p-mercaptophosphonic acid were purchased
from Aldrich. The p-mercaptobenzenesulfonic acid,23,40
p-mercaptobenzyl trimethyl ammonium bromide,41
4,40-dithiobis(benzeneacetic acid),22 and 4,40-dithi-
obis(benzenesulfonic acid)23,40 were synthesized as de-
scribed previously. The synthetic aromatic thiols and
disulfides were greater than 97% pure by proton
NMR. The 4,40-dithiobis(benzyl trimethyl ammonium
bromide) was synthesized by stirring the corresponding
thiol in water for several months in the dark to produce
a mixture containing 90% disulfide and 10% thiol. 13C
NMR spectra were referenced to 77.00 ppm (CDCl3)
1
and 49.00 ppm (CD3OD). H NMR spectra were refer-
enced to TMS at 0.00 ppm. Elemental analyses were per-
formed by Complete Analysis Inc., Parsippany, NJ.
Summarizing, the folding rate constant of denatured re-
duced lysozyme was increased 10-fold at pH 7 by the
addition of aromatic thiols instead of glutathione to
the redox buffer. The rate enhancement is similar to that
reported for PDI in vitro at approximately 10 lM lyso-
zyme and a 3 or 2:1 ratio of lysozyme to PDI. The use of
aromatic thiols also eliminated the lag period observed
at low glutathione disulfide concentrations. The optimal
concentration of aromatic thiol occurred when the con-
centration of thiol in the neutral RSH form, as opposed
to the thiolate form, was 4–6 mM. The maximal activity
is similar for four of the five aromatic thiols at pH 7 and
for glutathione at pH 7 and 8.2, Table 2. The maximal
activity for the positively charged aromatic thiol at pH
6 and 7 was greater than that of the negatively charged
thiols, glutathione included, suggesting that there is a
charge effect for the folding of lysozyme with aromatic
thiols, Figure 5 and Table 3.
5.2. Enzymatic assays
5.2.1. Preparation of denatured reduced lysozyme.50,51
Lysozyme (20 mg/mL) in 0.1 M Tris–HCl, pH 8.6, con-
taining 6 M GdnHCl and 0.15 M dithioerythreitol was
incubated for 2 h at room temperature. The solution
was then acidified to pH 3 by the addition of 0.1 M acetic
acid and applied to a column packed with SephadexTM G-
25 fine, which had been pre-equilibrated with 0.1 M acetic
acid solution. The fractions containing reduced lysozyme,
as determined by Ellman’s reagent (5,50-dithiol-bis(2-
nitrobenzoic acid)), were collected and added together
in a 50 mL centrifuge tube. The fractions containing
DTE, which eluted from the column after the protein,
were discarded. The concentration of the reduced protein
was determined from its absorbance at 280 nm using a UV
spectrophotometer (e280 nm = 2.37 mL mgꢀ1 cmꢀ1).32,52
The reduced lysozyme displayed no enzymatic activity.

2588
M. C. Gurbhele-Tupkar et al. / Bioorg. Med. Chem. 16 (2008) 2579–2590
The reduced lysozyme was then divided into portions con-
taining 5 mg of protein and each portion was placed in a
15 mL centrifuge tube. Each portion was lyophilized
and stored at ꢀ20 °C.
formed along with the regular experiments. Varying
concentrations of aromatic thiol, 20 lL, in the absence
of lysozyme solution were added to 980 lL of M. lys-
odeikticus solution and the change in light scattering at
450 nm was monitored. To test if the aromatic thiol
was altering the enzymatic activity, a native lysozyme
solution was prepared, 10 mg/mL, and diluted 100-fold
with renaturation buffer containing varying concentra-
tions of aromatic thiol (10 lL of native lysozyme solu-
tion added to 990 lL of specific renaturation buffer).
Then, 20 lL aliquots were assayed with 980 lL of
M. lysodeikticus solution. For comparison, the activity
of lysozyme in the absence of the aromatic thiols was
determined. Native lysozyme was diluted with Bis–Tris
buffer containing no aromatic thiols or oxidized disul-
fides (10 lL of native lysozyme solution and 990 lL of
buffer). Then, 20 lL aliquots were assayed with 980 lL
of M. lysodeikticus solution.
5.2.2. Calibration of assay for native lysozyme. A stock
solution of native lysozyme (1 mg/mL) was prepared by
dissolving the native protein in 0.1 M Bis–Tris propane,
pH 7.0, with 1 mM EDTA. The protein concentration
(mg/mL) was determined spectrophotometrically by mea-
suring the absorbance at 280 nm (e280 nm
=
2.63 mL mgꢀ1 cmꢀ1).32,53 From the stock solution of
lysozyme, solutions varying in concentration from
0.002 mg/mL to 0.075 mg/mL were prepared. The activity
of each solution was then determined using the enzymatic
assay (vide infra). The activity was then plotted versus the
native protein concentration. The rate was found to vary
linearly with respect to protein concentration.
5.2.3. Assay for lysozyme activity.32,37,38 In all experi-
ments the lysozyme activity was measured by mixing
20 lL aliquots of native or renatured lysozyme solution
(0.1 mg/mL) with 0.980 lL of a M. lysodeikticus solu-
tion (0.25 mg/mL) in 66 mM monobasic potassium
phosphate, pH 6.2, equilibrated at 25 °C. The samples
were mixed by repeatedly inverting the cuvette for
15 s. After the solution was mixed, the light scattered
at 450 nm was monitored for 2 min. The slope of the line
was used to determine the lytic activity of lysozyme.
5.3. Synthesis
5.3.1. S-(4-Methylphenyl)benzenecarbothioic acid (6).41,54
p-Toluenethiol (25.83 g, 0.208 mol) and benzoyl chloride
(29.60 g, 0.211 mol) were added to toluene (1 L). The
solution was cooled to 0 °C and triethylamine (27.0 g,
0.267 mol) was added slowly over 1 h. After stirring for
an additional hour, the mixture was filtered and the solid
was washed with toluene. The filtrate was then extracted
withwater (500 mL), dried (MgSO4), filtered, and concen-
trated in vacuo. The crude solid was recrystallized from
ethanol to provide 33.38 g of product, 70% yield.
5.2.4. Refolding of denatured reduced lysozyme.37,50 The
lyophilized reduced lysozyme was dissolved at 10 mg/
mL in 6 M GdnHCl, 0.1 M acetic acid, pH 2.5
(e280 nm = 2.37 mL mgꢀ1 cmꢀ1).32,52 The pH of the pro-
tein solution was then adjusted to the pH of the folding
experiments by addition of 0.1 M base, Bis–Tris pro-
pane for pH 7.0 or Bis–Tris for pH 6.0. The pH of stock
solutions containing the thiol or disulfide was also ad-
justed to the appropriate pH prior to use with base.
Refolding of reduced lysozyme was achieved in a deox-
ygenated renaturation buffer (0.1 M Bis–Tris propane–
HCl, pH 7.0, 1 mM EDTA, 0.5 M GdnHCl, or 0.1 M
Bis–Tris–HCl, pH 6.0, 1 mM EDTA, 0.5 M GdnHCl,
and various concentrations of GSH or aromatic thiol
and GSSG or aromatic disulfide). Ten microliters of re-
duced lysozyme solution (10 mg/mL) was added to
990 lL of renaturation buffer in a 1.5 mL Eppendorf
tube (100-fold dilution). To ensure reproducible mixing
conditions, the renaturation buffer was added to an
Eppendorf tube, the protein was deposited as a droplet
on the wall of the tube above the buffer meniscus, and
mixing was achieved by vigorous agitation with a Vortex
mixer for 15 s. Subsequently, aliquots were removed at
specific times and assayed for enzymatic activity. All
the refolding experiments were conducted at 25 °C. In
general, we did not observe any turbidity after several
hours of folding, consistent with the literature.27 The
exceptions occurred when the aromatic thiol concentra-
tion was close to saturation.
5.3.2. S-(4-Bromomethyl)phenyl benzenecarbothioic acid
(7).41
S-(4-Methylphenyl)benzenecarbothioic
acid
(19.08 g, 84 mmol) and N-bromosuccinimide (15.0 g,
84 mmol) were added to benzene (125 mL) that had
been deoxygenated by bubbling Ar through it for
15 min. The mixture was then irradiated with a 250 W
GE heat lamp, which provided sufficient energy to reflux
the solvent. After refluxing for 30 min, the solution was
cooled to 0 °C, filtered, concentrated in vacuo, and par-
titioned between 600 mL CH2Cl2 and 300 mL H2O. The
water layer was washed with 150 mL CH2Cl2. The com-
bined organics were dried (MgSO4), filtered, and con-
centrated in vacuo. The crude solid was recrystallized
from hexanes to provide 17.21 g of product (approx.
64% yield), as a 24:1 mixture of S-(4-bromo-
methyl)phenyl benzenecarbothioic acid and S-(4-dibro-
momethyl)phenyl benzenecarbothioic acid.
5.3.3. Diethyl (4-benzoylthiophenyl)methylphosphonate
(8).55 A 24:1 mixture of S-(4-bromomethyl)phenyl ben-
zenecarbothioic acid and S-(4-dibromomethyl)phenyl
benzenecarbothioic acid (6.06 g, 19.7 mmol), and tri-
ethylphosphite (8.54 g, 51.5 mmol) were stirred at
105 °C under a flow of Ar for 90 min. The reaction mix-
ture was cooled and the excess triethylphosphite was dis-
tilled off under reduced pressure. The residue was
purified by silica gel flash chromatography (1:1 to 1:8
hexane/ethyl acetate). The resulting solid was then
recrystallized from CH2Cl2/hexanes to provide 5.03 g
5.2.5. Control experiments. To exclude the possibility of
a direct effect of the aromatic thiol on the cell wall of
M. lysodeikticus, a series of control experiments was per-
1
of product, 70% yield. H NMR (CDCl3, 300 MHz) d
8.02 (d, J = 7.2 Hz, 2 H), 7.61 (t, J = 7.2 Hz, 1 H),

M. C. Gurbhele-Tupkar et al. / Bioorg. Med. Chem. 16 (2008) 2579–2590
2589
7.51–7.45 (m, 4H), 7.40 (dd, J = 8.3, 2.2 Hz, 2H), 4.05
(quintet, J = 7.3 Hz, 4H), 3.20 (d, J = 21.8 Hz, 2H),
1.27 (t, J = 7.0 Hz, 6H); 13C NMR (CDCl3, 75 MHz) d
190.1, 136.7, 135.3, 133.8, 133.6 (d, J = 9 Hz), 130.8
(d, J = 6 Hz), 128.9, 127.6, 126.0, 62.4 (d, J = 6 Hz),
33.8 (d, J = 38 Hz), 16.5 (d, J = 5 Hz); Anal. calcd for
C18H21O4PS: C, 59.33; H, 5.81. Found C, 59.54; H, 5.76.
W.J.L. M.T.-G. was supported in part by a Student
Summer Research Award funded by the Biomedical Re-
search Initiative NIH/NIGMS R25 GM061347. L.P.
was supported by an undergraduate fellowship NIH/
NIGMS R25 GM061347. Y.S. was supported by an
undergraduate
GM008771.
fellowship
NIH/NIGMS
T34
5.3.4. Diethyl (4-acetylthiophenyl)methylphosphonate (9).
Sodium hydride (1.57 g of 60 wt%, 39.2 mmol) was
added to ethanol (240 mL) and the fizzing solution
was deoxygenated by bubbling Ar through it for
45 min. Diethyl (4-benzoylthiophenyl)methylphospho-
nate (4.19 g, 11.5 mmol) was then added. After 25 min
the reaction was quenched by the addition of acetic
anhydride (11.02 g, 108 mmol). No thiol groups were
detected by Ellman’s reagent. The solution was concen-
trated in vacuo and the slurry was partitioned between
300 mL CH2Cl2 and 150 mL satd aq sodium bicarbon-
ate. The aqueous layer was washed with 150 mL
CH2Cl2. The combined organics were dried (MgSO4),
filtered, and concentrated in vacuo. The crude material
was purified by flash chromatography (1:1 hexanes/ethyl
acetate to neat ethyl acetate) to provide 3.34 g of an oil,
References and notes
1. De Bernardez Clark, E. Curr. Opin. Biotechnol. 1998, 9,
157.
2. De Bernardez Clark, E. Curr. Opin. Biotechnol. 2001, 12,
202.
3. Lange, C.; Rudolph, R. Protein Folding Handbook 2005, 5,
1245.
4. Lilie, H.; Schwarz, E.; Rudolph, R. Curr. Opin. Biotech-
nol. 1998, 9, 497.
5. Rudolph, R.; Lilie, H. FASEB J. 1996, 10, 49.
6. Willis, M. S.; Hogan, J. K.; Prabhakar, P.; Liu, X.; Tsai,
K.; Wei, Y.; Fox, T. Protein Sci. 2005, 14, 1818.
7. Gough, J. D.; Lees, W. J. Bioorg. Med. Chem. Lett. 2005,
15, 777.
8. Orsini, G.; Goldberg, M. E. J. Biol. Chem. 1978, 253,
3453.
9. Expert-Bezancon, N.; Rabilloud, T.; Vuillard, L.; Gold-
berg, M. E. Biophys. Chem. 2003, 100, 469.
10. Dong, X.-Y.; Huang, Y.; Sun, Y. J. Biotechnol. 2004, 114,
135.
1
96% yield. H NMR (CDCl3, 300 MHz) d 7.36 (s, 4 H),
4.02 (quintet, J = 7.3 Hz, 4H), 3.17 (d, J = 21.8 Hz, 2H),
2.41 (s, 3H), 1.25 (t, J = 7.1 Hz, 6H); 13C NMR (CDCl3,
75 MHz) d 193.9, 134.6, 133.4 (d, J = 9 Hz), 130.7 (d,
J = 6 Hz), 126.5, 62.3 (d, J = 7 Hz), 33.7 (d,
J = 38 Hz), 30.2, 16,4 (d, J = 5 Hz); Anal. calcd for
C13H19O4PS: C, 51.65; H, 6.33. Found C, 51.43; H, 6.29.
11. Maeda, Y.; Yamada, H.; Ueda, T.; Imoto, T. Protein Eng.
1996, 9, 461.
12. Rariy, R. V.; Klibanov, A. M. Proc. Natl. Acad. Sci. 1997,
94, 13520.
5.3.5. (4-Thiophenyl)methylphosphonic acid (2).55 A mix-
ture of diethyl (4-acetylthiophenyl)methylphosphonate
(3.12 g, 10.3 mmol) and concd HCl (500 mL) was heated
to reflux for 5 h. The oil bath was at 130 °C. The reaction
mixture was concentrated in vacuo to approximately
100 mL, warmed, filtered, and cooled to 0 °C. The
mixture was filtered, and the solid washed with water be-
fore being lyophilized to provide 1.75 g of product, 83%
13. Arakawa, T.; Tsumoto, K. Biochem. Biophys. Res. Com-
mun. 2003, 304, 148.
14. Reddy, K. R. C.; Lilie, H.; Rudolph, R.; Lange, C. Protein
Sci. 2005, 14, 929.
15. Maeda, Y.; Ueda, T.; Yamada, H.; Imoto, T. Protein Eng.
1994, 7, 1249.
16. Kersteen, E. A.; Raines, R. T. Antioxid. Redox Signal.
2003, 5, 413.
17. Woycechowsky, K. J.; Wittrup, K. D.; Raines, R. T.
Chem. Biol. 1999, 6, 871.
18. Woycechowsky, K. J.; Raines, R. T. Biochemistry 2003,
42, 5387.
19. Cabrele, C.; Fiori, S.; Pegoraro, S.; Moroder, L. Chem.
Biol. 2002, 9, 731.
20. Cabrele, C.; Cattani-Scholz, A.; Renner, C.; Behrendt, R.;
Oesterhelt, D.; Moroder, L. Eur. J. Org. Chem. 2002,
2144.
21. DeCollo, T. V.; Lees, W. J. J. Org. Chem. 2001, 66, 4244.
22. Gough, J. D.; Williams, R. H., Jr.; Donofrio, A. E.; Lees,
W. J. J. Am. Chem. Soc. 2002, 124, 3885.
23. Gough, J. D.; Gargano, J. M.; Donofrio, A. E.; Lees, W.
J. Biochemistry 2003, 42, 11787.
24. Gough, J. D.; Lees, W. J. J. Biotechnol. 2005, 115, 279.
25. Gough, J. D.; Barrett, E. J.; Silva, Y.; Lees, W. J.
J. Biotechnol. 2006, 125, 39.
1
yield. H NMR (CD3OD, 300 MHz) d 7.23–7.14 (m,
4 H), 3.04 (d, J = 21.6 Hz, 2H); 13C NMR (CD3OD,
75 MHz) d 131.7 (d, J = 6 Hz), 131.5, 131.1 (d,
J = 4 Hz), 130.2 (d, J = 3 Hz), 35.4 (d, J = 35 Hz); Anal.
calcd for C7H9O3PS: C, 41.18; H, 4.44. Found C, 41.10;
H, 4.36.
5.4. Determination of thiol pKa values56,57
The thiol pKa of the aromatic thiols was determined in
buffers with and without 0.5 M GdnHCl. The error is
0.1 U and comparison with literature values for some
of the compounds has been published previously.21
The addition of 0.5 M GdnHCl increased the value by
0.1 for the positively charged aromatic thiol and de-
creased the value by 0.1 for the negatively charged aro-
matic thiols, all within experimental error.
26. Yoshii, H.; Furuta, T.; Yonehara, T.; Ito, D.; Linko,
Y.-Y.; Linko, P. Biosci. Biotechnol. Biochem. 2000, 64,
1159.
27. De Bernardez Clark, E.; Hevehan, D.; Szela, S.; Maachu-
palli-Reddy, J. Biotechnol. Prog. 1998, 14, 47.
28. Ueda, T.; Nagata, M.; Imoto, T. J. Biochem. 2001, 130,
491.
Acknowledgments
29. Yasuda, M.; Murakami, Y.; Sowa, A.; Ogino, H.;
Ishikawa, H. Biotechnol. Prog. 1998, 14, 601.
This work was supported in part by the National Sci-
ence Foundation under Grant No. CHE-0342167 to

2590
M. C. Gurbhele-Tupkar et al. / Bioorg. Med. Chem. 16 (2008) 2579–2590
30. van den Berg, B.; Chung, E. W.; Robinson, C. V.;
Dobson, C. M. J. Mol. Biol. 1999, 290, 781.
31. Guez, V.; Roux, P.; Navon, A.; Goldberg, M. E. Protein
Sci. 2002, 11, 1136.
45. Wetlaufer, D. B.; Branca, P. A.; Chen, G. X. Protein Eng.
1987, 1, 141.
46. Kiefhaber, T.; Rudolph, R.; Kohler, H. H.; Buchner, J.
Bio/Technology 1991, 9, 825.
32. Saxena, V. P.; Wetlaufer, D. B. Biochemistry 1970, 9, 5015.
33. Yoshii, H.; Furuta, T.; Yonehara, T.; Ito, D.; Linko, P.
J. Chem. Eng. Jpn. 2001, 34, 211.
34. Raman, B.; Ramakrishna, T.; Rao, C. M. J. Biol. Chem.
1996, 271, 17067.
47. Buswell, A. M.; Middelberg, A. P. J. Biotechnol. Bioeng.
2003, 83, 567.
48. Van den Berg, B.; Chung, E. W.; Robinson, C. V.; Mateo,
P. L.; Dobson, C. M. EMBO J. 1999, 18, 4794.
49. Puig, A.; Gilbert, H. F. J. Biol. Chem. 1994, 269, 7764.
50. Goldberg, M. E.; Rudolph, R.; Jaenicke, R. Biochemistry
1991, 30, 2790.
35. Hevehan, D. L.; De Bernardez Clark, E. Biotechnol.
Bioeng. 1997, 54, 221.
36. Lanckriet, H.; Middelberg, A. P. J. J. Chromatogr. A 2004,
1022, 103.
51. Perraudin, J. P.; Torchia, T. E.; Wetlaufer, D. B. J. Biol.
Chem. 1983, 258, 11834.
37. Roux, P.; Delepierre, M.; Goldberg, M. E.; Chaffotte, A.
F. J. Biol. Chem. 1997, 272, 24843.
38. Jolles, P. Method Enzymol. 1962, 5, 137.
39. Anderson, W. L.; Wetlaufer, D. B. J. Biol. Chem. 1976,
251, 3147.
40. Kawai, H.; Sakamoto, F.; Taguchi, M.; Kitamura, M.;
Sotomura, M.; Tsukamoto, G. Chem. Pharm. Bull. 1991, 39,
1422.
52. Wetlaufer, D. B.; Johnson, E. R.; Clauss, L. M. In
Lysozyme; Osserman, E. F., Canfield, R. E., Beychok, S.,
Eds.; Academic Press: New York and London, 1974; p
269.
53. Sophianopoulos, A. J.; Rhodes, C. K.; Holcomb, D. N.;
Van Holde, K. E. J. Biol. Chem. 1962, 237, 1107.
54. Aitken, R. A.; Drysdale, M. J.; Ryan, B. M. J. Chem. Soc.,
Perkin Trans. 1 1998, 3345.
41. Moss, R. A.; Dix, F. M. J. Org. Chem. 1981, 46, 3029.
42. Lyles, M. M.; Gilbert, H. F. Biochemistry 1991, 30, 613.
43. Konishi, Y.; Ooi, T.; Scheraga, H. A. Biochemistry 1982,
21, 4734.
55. Coogan, M. P.; Harger, M. J. P. J. Chem. Soc., Perkin
Trans. 2 1994, 2101.
56. Benesch, R. E.; Benesch, R. J. Am. Chem. Soc. 1955, 77,
5877.
44. Ahmed, A. K.; Schaffer, S. W.; Wetlaufer, D. B. J. Biol.
Chem. 1975, 250, 8477.
57. Lees, W. J.; Singh, R.; Whitesides, G. M. J. Org. Chem.
1991, 56, 7328.