Oxidative folding of lysozyme with aromatic dithiols, and aliphatic and aromatic monothiols

Article abstract of DOI:10.1016/j.bmc.2011.11.049

In vitro protein folding of disulfide containing proteins is aided by the addition of a redox buffer, which is composed of a small molecule disulfide and/or a small molecule thiol. In this study, we examined redox buffers containing asymmetric dithiols 1-5, which possess an aromatic and aliphatic thiol, and symmetric dithiols 6 and 7, which possess two aromatic thiols, for their ability to fold reduced lysozyme at pH 7.0 and 8.0. Most in vivo protein folding catalysts are dithiols. When compared to glutathione and glutathione disulfide, the standard redox buffer, dithiols 1-5 improved the protein folding rates but not the yields. However, dithiols 6 and 7, and the corresponding monothiol 8 increased the folding rates 8-17 times and improved the yields 15-42% at 1 mg/mL lysozyme. Moreover, aromatic dithiol 6 increased the in vitro folding yield as compared to the corresponding aromatic monothiol 8. Therefore, aromatic dithiols should be useful for protein folding, especially at high protein concentrations.

Full text of DOI:10.1016/j.bmc.2011.11.049

Bioorganic & Medicinal Chemistry 20 (2012) 1020–1028
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Oxidative folding of lysozyme with aromatic dithiols, and aliphatic
and aromatic monothiols
⇑
Amar S. Patel, Watson J. Lees
Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th St., Miami, FL 33199, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In vitro protein folding of disulfide containing proteins is aided by the addition of a redox buffer, which is
composed of a small molecule disulfide and/or a small molecule thiol. In this study, we examined redox
buffers containing asymmetric dithiols 1–5, which possess an aromatic and aliphatic thiol, and symmet-
ric dithiols 6 and 7, which possess two aromatic thiols, for their ability to fold reduced lysozyme at pH 7.0
and 8.0. Most in vivo protein folding catalysts are dithiols. When compared to glutathione and glutathi-
one disulfide, the standard redox buffer, dithiols 1–5 improved the protein folding rates but not the
yields. However, dithiols 6 and 7, and the corresponding monothiol 8 increased the folding rates 8–17
times and improved the yields 15–42% at 1 mg/mL lysozyme. Moreover, aromatic dithiol 6 increased
the in vitro folding yield as compared to the corresponding aromatic monothiol 8. Therefore, aromatic
dithiols should be useful for protein folding, especially at high protein concentrations.
Received 28 September 2011
Revised 16 November 2011
Accepted 19 November 2011
Available online 1 December 2011
Keywords:
Protein folding
Lysozyme
Aromatic thiol
Redox buffer
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
3%) of the aliphatic thiol (pKa approx. 9) is in the reactive thiolate
form. The mechanism of disulfide formation involves the nucleo-
Most pharmaceutically relevant proteins and many extracellu-
lar proteins contain disulfide bonds.1,2 When disulfide-containing
proteins are overexpressed in bacteria, inclusion bodies, which
are insoluble protein aggregates, are commonly produced.3 To ob-
tain protein in its biologically active form, it is necessary to resolu-
bilize the protein and then fold it in vitro. For in vitro protein
folding, a significant challenge is the rapid formation of the correct
disulfide bonds between cysteine residues.4–6
To enhance the in vitro folding rate of disulfide-containing pro-
teins, protein catalysts and a limited number of small molecule thi-
ols and selenols have been used.7–14 Protein catalysts, for example,
protein disulfide isomerase (PDI), which is used in vivo, can in-
crease in vitro protein folding rates significantly. However, the
use of PDI for in vitro folding is impractical because of its high cost
and low catalytic activity.8 Therefore, small molecule aliphatic thi-
ols, such as glutathione (GSH) (pKa = 8.7), cysteine (pKa = 8.7), b-
mercaptoethanol (pKa = 9.6), and dithiothreitol (DTT) (pKa = 9.2)
are usually employed for in vitro protein folding.9,12 A small mole-
cule thiol is generally used in combination with a small molecule
disulfide to form a redox buffer, for example oxidized glutathione
(GSSG) and reduced glutathione (GSH). Small molecule thiols are
less expensive than PDI and can easily be removed from the native
protein. However, folding rates with small molecule thiols are
slow, especially at pH values below 7.5, as very little (less than
philic attack of a thiolate anion (R1Sꢀ) on a disulfide bond
(R2SSR3), displacing one sulfur (R3S) of the disulfide bond and
forming a new disulfide bond (R1SSR2), Scheme 1. R3Sꢀ acts as
the leaving group, and R2S acts as the center sulfur.9 Recently, more
efficient thiol based redox buffers containing aliphatic dithiols,
synthetic peptides, or aromatic monothiols have been em-
ployed.10,15–19
Aliphatic dithiols, for example, ( )-trans-1,2-bis(2-mercapto-
acetamido)cyclohexane (BMC), and synthetic peptides with two
cysteine residues in close proximity, for example, Cys-Gly-Cys
(CGC), can significantly increase the overall yield of folded pro-
tein.6 Raines et al. illustrated that BMC increased the folding yield
of native ribonuclease A (RNase A) twofold relative to its monothiol
analog N-methylmercaptoacetamide (NMA).10 The increase in
yield was proposed to be due to the presence of a second thiol
group in BMC versus a single thiol group in NMA.10 Furthermore,
Raines et al. showed that addition of BMC to Saccharomyces cerevi-
siae growth medium increased the heterologous production of
Schizosaccharomyces pombe acid phosphatase.10 Raines et al. also
demonstrated that the dithiol containing peptide CGC could
⇑
Corresponding author. Tel.: +1 305 348 3993; fax: +1 305 348 3772.
E-mail address: leeswj@fiu.edu (W.J. Lees).
Scheme 1. Mechanism for thiol/disulfide interchange reaction.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.049

A. S. Patel, W. J. Lees / Bioorg. Med. Chem. 20 (2012) 1020–1028
1021
increase the in vitro folding yield of ribonuclease A threefold rela-
tive to the standard thiol glutathione.20 The increase in yield with
dithiols was proposed to be due to an ‘escape’ mechanism, Figure
1.10,21 When dithiols or monothiols react with a protein disulfide
bond, a mixed disulfide bond is produced. With dithiols, the second
thiol group acts as a clock and provides a set amount of time for the
protein to rearrange. If the protein does not rearrange, the second
thiol of the dithiol makes an intramolecular attack and breaks the
mixed disulfide bond. In the case of monothiols, there is no second
thiol to act as a clock, and the mixed disulfide can become kineti-
cally trapped, and thus the overall yield of native protein de-
creases. Although BMC and synthetic peptides increased the
yield, they did not change the folding rate constant significantly.10
In our previous studies, we have shown that aromatic monothi-
ols (pKa 5.5–6.6) significantly increased the folding rate and to a
lesser extent the overall yield of native protein compared to stan-
dard aliphatic thiols (pKa 8.5–10). With aromatic monothiols, the
folding rates of RNase A were 10–23 times faster at pH 6.0, 7–12
times faster at pH 7.0, and 5–8 times faster at pH 7.70 than with
glutathione.11,17,22 Additionally, it was also demonstrated that at
a high protein concentration (1 mg/mL of lysozyme), aromatic
monothiols increased the folding rate up to 11 times and improved
the yield up to 40% at pH 7.0, and the rate increased up to 7 times
with an up to 25% improvement in yield at pH 8.0 in comparison
with glutathione.19 Aromatic thiols improve the folding rate be-
cause they are better leaving groups and more nucleophilic at pH
7.0 than aliphatic thiols, Scheme 1.23
Besides varying the redox buffer, dilution of the sample can be
used to improve the in vitro folding of disulfide-containing pro-
teins. Generally, protein folding yields decrease with increasing
protein concentration.24–26 Therefore, folding at low protein
concentrations is advantageous (e.g., <0.1 mg/mL), however, large
volumes of refolding solution are needed to perform the refolding
process, making it impractical after a point depending on the scale.
Figure 1. (a) Thiol/disulfide interchange reaction with dithiols, such as PDI, showing escape mechanism.8,10 (b) Thiol/disulfide interchange reaction with monothiols showing
kinetic trap, as a mixed disulfide between monothiol and protein substrate.

1022
A. S. Patel, W. J. Lees / Bioorg. Med. Chem. 20 (2012) 1020–1028
Another efficient strategy to increase the folding yield is the
fore, a polyethylene glycol group was used as the side chain to im-
prove water solubility.
addition of folding aids. The most commonly used folding aids
are guanidine hydrochloride, urea, arginine, and glycerol.27–33
Other folding aids include detergent and surfactants, polyethylene
glycol, molecular chaperones, artificial chaperones, and several
simple compounds, such as acetone, acetamide, sarcosine, and
thiourea.30,34–39 Folding aids are proposed to increase the refolding
yield by interfering with the intermolecular hydrophobic interac-
tions that lead to protein aggregation during in vitro protein fold-
ing. Aggregation causes reduced overall yields. However, folding
aids generally decrease the rate of protein folding.29–31
Based on the advantageous properties of aromatic monothiols
and aliphatic dithiols, a set of new aromatic dithiols (1–7) was de-
signed to examine their effects on protein folding rates and yields.
Two series of aromatic dithiols were synthesized, and the ability of
each dithiol to enhance the folding of lysozyme was determined.
The first series of aromatic dithiols (1–5) consisted of one thiol
group on an aromatic ring and another thiol group on a polyethyl-
ene glycol aliphatic chain, which was either ortho or para to the
aromatic thiol. The second series of aromatic dithiols (6 and 7) con-
sisted of a thiol group on each of two aromatic rings connected by a
hydrocarbon chain containing quaternary ammonium groups. Aro-
matic dithiols 6 and 7 were designed on the basis of the previously
synthesized quaternary ammonium aromatic monothiol 8.18,19
Three aliphatic thiols (9, 10, and the standard thiol glutathione)
were used as controls.
2.1.1. Synthesis of para- and ortho-substituted aromatic
ethylene glycol dithiols (1–5)
The first step in the synthesis of para-substituted aromatic di-,
tri-, and tetraethylene glycol dithiols (1–3) involved the treatment
of the disulfide of 4-mercaptobenzoic acid (11) with oxalyl chlo-
ride (13) and DMF in THF to produce acid chloride 14, Scheme 2.
In the same pot, the acid chloride 14 was treated with di-, tri-, or
tetraethylene glycol disulfide (16, 17, or 18) to form the corre-
sponding polymer (19, 20, or 21). Di-, tri-, and tetraethylene glycol
disulfides (16–18) were synthesized by vigorously stirring their
respective thiols in water under air for a few days with catalytic
base.40,41 The polymer (19, 20, or 21) was then reduced with DTT
in the presence of Et3N to give the corresponding para-substituted
aromatic di-, tri-, or tetraethylene glycol dithiol (1, 2, or 3), respec-
tively. The ortho-substituted aromatic tri- and tetraethylene glycol
dithiols (4 and 5) were synthesized in the same manner as the para
analogs, starting from readily available dithiosalicylic acid (12).
Aromatic monothiol 10 was also synthesized in the same manner
using benzoyl chloride as the starting material.
2.1.2. Folding of lysozyme (0.1 mg/mL) with para- and ortho-
dithiols (1–5)
Asymmetric para- and ortho-substituted aromatic dithiols (2–5)
were then examined for their ability to fold reduced lysozyme
(0.1 mg/mL) at pH 7.0 and 8.0, at 25 °C. Lysozyme was selected
as this enzyme has been extensively studied, and its folding path-
way has been well characterized.18,42–46 The folding was carried
out in the presence of various concentrations of aromatic dithiols
(2–5) in combination with either 0.2 or 2.0 mM GSSG. The GSSG
concentrations were chosen to match those of previous experi-
ments.11,16–18 Folding experiments were then compared directly
with 7 mM GSH and 2 mM GSSG (standard conditions), as at these
concentrations the best combination of folding rate and yield for
glutathione was obtained.18
The folding of reduced lysozyme was followed using the recov-
ery of enzymatic activity. Reduced lysozyme (10 mg/mL) was di-
luted 100-fold into renaturation buffer, containing 0.1 M buffer
(bis tris propane–HCl for pH 7.0 or Tris–HCl for pH 8.0), 1.0 mM
EDTA, 0.5 M guanidine hydrochloride (Gdn HCl), and a redox buf-
fer.47,48 EDTA and the folding aid Gdn HCl minimize metal cata-
lyzed air oxidation of thiols and protein aggregation during
folding, respectively.27,46,47 The redox buffer was composed of
GSH or dithiol, and GSSG. At specific times, aliquots were removed
from the folding reaction, and the enzymatic activity was deter-
mined by adding the aliquots to a suspension of Micrococcus lys-
odeikticus.46,47,49 The change in light scattering per minute was
proportional to enzymatic activity. The folding of lysozyme in the
presence of aromatic dithiols (2–5) was compared by fitting enzy-
matic activity versus time to
a single exponential function,
y = A(1 ꢀ eꢀkt), where A is the maximal enzymatic activity, k is
the apparent folding rate constant, and t is time. Although folding
is a complex process a single exponential function has historically
been used, as it fits the data reasonably well in many cases.
A general decrease in the overall yield of native protein with
increasing concentration of aromatic dithiols (2–5) was observed
in the presence of 0.2 mM GSSG, Table 1 and Figure 2. The para-
substituted dithiols, 2 and 3, showed a several fold rate enhance-
ment over GSH (standard conditions) at pH 7.0 but not at pH 8.0
while the ortho-substituted dithiols, 4 and 5, showed little or no
rate enhancement. Similar trends were also observed with 2 mM
GSSG, except that at pH 7.0 dithiols 4 and 5 showed a general in-
crease in yield with increasing dithiol concentration, although
the yield was always at least less than that obtained with GSH
2. Results and discussion
2.1. para- and ortho-Substituted aromatic ethylene glycol
dithiols (1–5)
We initially designed asymmetric small molecule dithiols (1–5)
to mimic PDI, which has two thiol groups, one thiol group more
reactive than the other. In addition, upon oxidation PDI forms a
14-membered ring disulfide. In these small molecule dithiols, the
more reactive thiol group was modeled as an aromatic thiol and
the less reactive thiol group was modeled as an aliphatic thiol; aro-
matic thiols are more reactive than aliphatic thiols at physiological
pH.23 Additionally, the compounds should be water soluble from
pH 6 to 9, the pH range used to fold most proteins in vitro. There-

A. S. Patel, W. J. Lees / Bioorg. Med. Chem. 20 (2012) 1020–1028
1023
Scheme 2. Synthesis of para- and ortho-substituted aromatic ethylene glycol dithiols (1–5).
Table 1
The effect of the stability of the cyclic disulfide and the pres-
ence of an aromatic group were further investigated by examin-
Folding of lysozyme (0.1 mg/mL) with various concentrations of dithiols (1–5), and
monothiols (9–11) in the presence of 0.2 mM GSSG
ing compounds 1, 9, and 10. Compound
1 should be less
Compoundsa
(mM)a
A (%)
k (minꢀ1
)
reducing than 2 and 3 since upon oxidation 1 will form a less sta-
ble cyclic disulfide than 2 and 3.50 Less reducing environments
tend to make it easier to form protein disulfide bonds. However,
dithiol 1 at pH 8.0 showed similar trends as the other para- and
ortho-dithiols 2–5, Table 1. Next, the role of the thiol present on
the aliphatic chain was investigated using monothiols 9 and 10 at
pH 8.0. Monothiol 9 showed not only a decrease in the yield but
also a decrease in the rate compared to the standard conditions,
Table 1. Folding results with monothiol 10, unlike aromatic dithi-
ols, initially showed an increase in yield with increasing concen-
tration followed by a decrease, reaching a maximum at 2 mM,
and the maximum was within 3% that of GSH, Figure 2. Thus,
monothiol 10 showed similar behavior as the standard thiol, glu-
tathione. The results indicated that the attachment of an aromatic
group to an aliphatic thiol decreases the yield of folded protein at
higher thiol concentrations.
pH 7.0
2
0.5
3
0.5
3
0.5
2a
0.5
3
57
23
39
36
55
19
85
2
0.025
0.020
0.038
0.045
0.013
0.001
0.008
0.010
0.010
3
4
5
7 mM GSH/2 mM GSSG
87
pH 8.0
1
0.5
3
0.5
3
0.5
3
53
9
0.072
0.080
0.076
0.10
2
63
16
73
43
58
62
76
27
68
2
3
0.087
0.072
0.041
0.009
0.061
0.023
0.028
0.012
0.027
0.024
0.05
4
0.5
2a
2.2. Aromatic dithiols 6 and 7 with a quaternary ammonium
salt containing linker
5
0.5
3
9
0.25a
1a
A series of symmetrical aromatic dithiols, 6 and 7, having a thiol
group on each of two aromatic rings was prepared. The new series
does not contain an aliphatic thiol, as was the case with the initial
series of dithiols (1–5). Aromatic dithiols 6 and 7 were designed on
the basis of a previously synthesized quaternary ammonium aro-
matic monothiol 8 (thiol pKa = 5.5), which increased the folding
rate and yield of reduced lysozyme.18,19 The criteria for selecting
dithiols 6 and 7 were water solubility, the presence of only aro-
matic thiols, and para-substitution. Additionally, the low thiol
pKa values of 6 and 7 should allow for higher dithiol concentrations
without making the solution too reducing; reduction potential is
dependant upon the concentration of the neutral RSH form. Form-
ing disulfide bonds becomes slower if the solution is too reducing
while higher concentrations of the reactive thiolate leads to faster
reactions, which may correspond to faster folding.18,19 Further-
more, aromatic dithiols 6 and 7 can be compared with aromatic
10
0.5
3
72
68
87
7 mM GSH/2 mM GSSG
a
Solubilities of dithiol 4 at pH 7.0 and monothiol 9 at pH 8.0 were 4 and 2 mM,
respectively.
(>15% less at all but one concentration). The faster folding rates of
the para-substituted dithiols, 2 and 3, at pH 7.0 relative to the
ortho-substituted dithiols, 4 and 5, are likely due in part to less ste-
ric hinderance with concomitant lower thiol pKa values.16 Aromatic
thiols also generally showed a decreased rate enhancement over
GSH as the pH was increased from 7.0 to 8.0; the percentage of glu-
tathione in the reactive thiolate form increases fivefold when the
pH is increased from 7.0 to 8.0 but for aromatic thiols the increase
is at most twofold.
Figure 2. Folding of lysozyme (0.1 mg/mL) under (d) standard conditions (7 mM GSH/2 mM GSSG) or 0.2 mM GSSG and various concentrations of ortho-substituted aromatic
tetraethylene glycol dithiol 5 at pH 7.0 or triethylene monothiol 10 at pH 8.0: (j) 0.25 mM, (ꢀ) 0.50 mM (.) 1.0 mM ( ) 2.0 mM ( ) 3.0 mM and (N) 10.0 mM.
D

1024
A. S. Patel, W. J. Lees / Bioorg. Med. Chem. 20 (2012) 1020–1028
Scheme 3. Synthesis of aromatic quaternary ammonium salt dithiols 6 and 7.
monothiol 8 to determine the effect of the second thiol group on
the folding rate and yield of a disulfide-containing protein.
lower yields are normally obtained at higher protein concentra-
tions and thus folding aids, for example Gdn HCl, are usually added
at higher concentration.53 Higher concentrations of Gdn HCl, gen-
erally slow down the folding rate and improve the yield.53 Previ-
ously, it was demonstrated by our group that at high lysozyme
concentration (1 mg/mL), quaternary ammonium salt 8 increased
the folding rate and yield significantly, as compared to standard
conditions.19 Initial studies were performed at 10, 20, 40, 70, and
100 mM of aromatic dithiols, 6 and 7, and aromatic monothiol 8
with 0.5, 1, and 2 mM GSSG to find the best concentrations at both
pH 7.0 and 8.0. At pH 7.0 and 8.0, two combinations of aromatic
dithiols, 6 and 7, and GSSG and one combination of aromatic
monothiol 8 and GSSG were selected as they provided the best
yields, Table 2.
2.2.1. Synthesis of aromatic dithiols 6 and 7
The first step in the synthesis of aromatic dithiols 6 and 7 was
the reaction of p-toluene thiol with benzoyl chloride to yield com-
pound 26, Scheme 3. Compound 26 was then treated with N-bro-
mosuccinimide to provide brominated compound 27,51,52 which
was further treated with N,N,N0,N0-tetramethyl-1,3-propane dia-
mine 28 or N,N,N0,N0-tetramethyl-1,6-hexane diamine 29 to form
compounds 30 and 31, respectively. Compounds 30 and 31 were
then hydrolyzed with a 1:1 mixture of concd HBr and water at
130 °C for 90 min to provide aromatic dithiols
6 and 7,
respectively.
At pH 7.0, aromatic dithiol 6 produced the best yield and aro-
matic monothiol 8 provided the best folding rate, Table 2. Compar-
ative folding studies were conducted at pH 7.0 with the best
combinations of aromatic dithiols, aromatic monothiol, and GSH
with GSSG, Figure 4. The data were fit to a single exponential func-
tion, y = A(1 ꢀ eꢀkt), where A is the maximal enzymatic activity, k is
the apparent folding rate constant, and t is time. The results indi-
cated a significant increase in the folding rates and yields for aro-
matic dithiols 6 and 7, and monothiol 8 relative to GSH at pH 7.0.
The folding rates increased from 11 to 17 times and the yields in-
creased by 26–42% relative to GSH. The best folding rate was ob-
served with 40 mM 8, whereas the best yield was observed with
20 mM 6. When comparing aromatic dithiols versus aromatic
monothiols, aromatic monothiol 8 showed a significant increase
in folding rate relative to aromatic dithiols 6 and 7. However, aro-
matic dithiol 6 significantly increased the yield of native lysozyme
relative to aromatic monothiol 8 based on 10 measurements and a
95% confidence interval. The initial folding rates, A ⁄ k, for 20 mM 6
and 40 mM 8 were 24 and 28 times greater, respectfully, than that
of 7 mM GSH/ 2 mM GSSG at a protein concentration of 1 mg/mL.
2.2.2. Folding of lysozyme with aromatic dithiols 6 and 7
Initially, aromatic dithiols 6 and 7, from 1 to 10 mM, along with
2 mM GSSG were utilized for the folding of reduced lysozyme at
0.1 mg/mL concentration and at pH 7.0 and 8.0. Folding experi-
ments were conducted simultaneously with the standard condi-
tion, 7 mM GSH/2 mM GSSG. The folding of lysozyme was
followed using the recovery of enzymatic activity as described
above. Folding rates increased with increasing concentration of
dithiols 6 and 7, Figure 3. In comparison with glutathione, the fold-
ing rates at pH 7.0 with the dithiols were much faster but at pH 8.0
they were similar, with 6 being slightly slower and 7 being slightly
faster. At pH 7.0 and 8.0, aromatic dithiol 7 showed similar yields
of native lysozyme as 7 mM GSH/2 mM GSSG at all concentrations,
however, with aromatic dithiol 6 the folding yield decreased
slightly with increasing concentration.
Given the successes in terms of improved folding rates and to a
lesser extent folding yields at a protein concentration of 0.1 mg/
mL, we also tested protein folding with dithiols, 6 and 7, and
monothiol 8 at a higher protein concentration (1 mg/mL), which
is more challenging and practical. Higher protein concentrations
reduce folding volumes, a significant advantage for larger scales,
and can simplify subsequent protein purification steps. However,
Table 2
Folding of lysozyme (1 mg/mL) with aromatic dithiols 6 and 7, aromatic monothiol 8,
and GSH at pH 7.0 and 8.0a
Redox buffer
A (%)
k (minꢀ1
)
A ⁄ k (%/min)
pH 7.0
10 mM 6/1.0 mM GSSG
20 mM 6/1.0 mM GSSG
10 mM 7/0.5 mM GSSG
10 mM 7/1.0 mM GSSG
40 mM 8/1.0 mM GSSG
7 mM GSH/2.0 mM GSSG
83
86
70
74
82
44
5
0.012 0.001
0.015 0.003
0.013 0.002
0.011 0.001
0.017 0.002
0.001 0.0002
1.0 0.2
1.2 0.3
0.9 0.2
0.8 0.2
1.4 0.2
0.05 0.01
4
5
6
3
2
pH 8.0
20 mM 6/1.0 mM GSSG
40 mM 6/1.0 mM GSSG
10 mM 7/1.0 mM GSSG
20 mM 7/1.0 mM GSSG
40 mM 8/1.0 mM GSSG
7 mM GSH/2.0 mM GSSG
88
5
0.022 0.003
0.023 0.010
0.016 0.002
0.017 0.002
0.018 0.002
0.003 0.001
2.0 0.3
1.9 1.0
1.3 0.2
0.8 0.4
1.5 0.1
0.19 0.02
77 10
78
49 26
81
73
6
Figure 3. Folding of lysozyme (0.1 mg/mL) at pH 7.0 in the presence of 2 mM GSSG
and 0.5 M Gdn HCl. Comparison of the standard conditions (d) 7 mM GSH, with
6
4
various concentrations of 6, (j) 1 mM (ꢀ) 2 mM (.) 3 mM (N) 7 mM, and (D)
10.0 mM.
a
Errors represent the standard deviation.

A. S. Patel, W. J. Lees / Bioorg. Med. Chem. 20 (2012) 1020–1028
1025
Figure 4. Folding of lysozyme (1 mg/mL) in the presence of 1.7 M Gdn HCl at (a) pH 7.0 and (b) pH 8.0, (d) 7 mM GSH/2 mM GSSG (standard conditions), (ꢀ) 10 mM 6/1 mM
GSSG, (.) 20 mM 6/1 mM GSSG, (N) 10 mM 7/0.5 mM GSSG, ( ) 10.0 mM 7/1 mM GSSG, (j) 40 mM 8/1 mM GSSG, ( ) 40 mM 6/1 mM GSSG, and ( ) 20 mM 7/1 mM GSSG.
D
At pH 8.0, aromatic dithiol 6 produced the best yield and folding
rate, (Table 2 and Fig. 4a and b). The folding rates with 20 mM aro-
matic dithiol 6 and 40 mM aromatic monothiol 8 were 8 and 6
times higher, respectively, than that of 7 mM GSH. The yield
improvements over 7 mM GSH were up to 15%. Comparison of
20 mM aromatic dithiol 6 and 40 mM aromatic monothiol 8 at
pH 8.0, revealed that aromatic dithiol 6 was better in terms of rate
and yield based on 6 measurements and a 95% confidence interval.
With 20 mM aromatic dithiol 7, protein precipitation was some-
times observed by the naked eye and correspondingly the yield
was lower and erratic. The initial folding rate, A ⁄ k, with 20 mM
6 was 10 times larger than that with GSH at a protein concentra-
tion of 1 mg/mL.
When the concentrations of lysozyme and the folding aid Gdn
HCl increased, folding rates decreased, as expected, and the yields
with glutathione also decreased. With 10 mM aromatic dithiols 6
and 7 folding rates decreased approximately sixfold while with
GSH they decreased 8 and 15-fold, at pH 7.0 and 8.0, respectively.
The yields with 10 mM aromatic dithiols 6 and 7 were similar or
increased slightly when the protein and salt concentrations were
increased while those with glutathione decreased. When the aro-
matic thiol concentration was kept the same, a change in pH from
7.0 to 8.0 had little effect upon the yields, but this was not the case
with glutathione. The slower folding rate at pH 7.0 with glutathi-
one might allow other processes to become more significant.
1H NMR spectra were referenced to residual monoprotonated sol-
vent at 7.26 ppm (CDCl3), 3.31 ppm (CD3OD), 4.79 ppm (D2O), or
2.50 ppm (DMSO). 13C NMR spectra were referenced to solvent at
77.00 ppm (CDCl3), 49.00 ppm (CD3OD), or 39.52 ppm (DMSO).
High resolution mass spectroscopy (HRMS) was conducted by the
facility at Florida State University, Tallahassee, FL. Solubility of all
compounds (1–5, 9, and 10) was determined by Ellman’s method.56
4.1. Synthesis of di-, tri-, and tetraethylene glycol disulfides
(16–18) (general procedure using 17 as an example)
Triethylamine (0.5 mL, 3.58 mmol) was added to a solution of
2-[2-(2-mercaptoethoxy)ethoxy]ethanol (triethylene glycol thiol)
(3.5 g, 21.08 mmol) in 25 mL of distilled water. The solution was
stirred vigorously for 1 week under air. The reaction mixture was
acidified with conc HCl, and the aqueous layer was then extracted
with CHCl3 (3 ꢁ 25 mL). The organic layers were combined and
washed with brine (2 ꢁ 25 mL), dried over MgSO4, filtered, and re-
duced under vacuum to give 2.93 g of triethylene glycol disulfide
(17) as a yellow oil, 84% yield.
4.1.1. 2,20-(2,20-Disulfanediylbis(ethane-2,1-diyl)bis)oxy))-
diethanol [diethylene glycol disulfide (16)]41
45% Yield, 1H NMR (CDCl3, 400 MHz) d 3.78–3.73 (m, 8H), 3.60
(t, J = 4.4 Hz, 4H), 2.94 (t, J = 6.3 Hz, 4H), 2.61 (t, J = 6.2 Hz, 2H).
4.1.2. 3,6,13,16-Tetraoxa-9,10-dithiaoctadecane-1,18-diol
[triethylene glycol disulfide (17)]41
3. Conclusion
84% Yield, 1H NMR (CDCl3, 400 MHz) d3.75–3.70 (m, 8H), 3.66–
3.63 (m, 8H), 3.59 (t, J = 4.5 Hz, 4H), 3.04 (t, J = 5.5 Hz, 2H) 2.90 (t,
J = 6.6 Hz, 4H); 13C NMR (CDCl3, 100 MHz) d 72.6, 70.4, 70.3, 69.6,
61.7, 38.4.
Several new aromatic dithiols were designed and successfully
prepared to increase the oxidative folding rate and yield of lyso-
zyme. Aromatic dithiols, 6 and 7, and an aromatic monothiol 8 sig-
nificantly increased protein folding rates and yields as compared to
the traditional aliphatic thiol, glutathione, at a high protein con-
centration (1 mg/mL) and at both pH 7.0 and 8.0. The folding rate
enhancements observed with an aromatic monothiol 8 and two
aromatic dithiols, 6 and 7, were between 8–17 times, and yield
improvements of 15–42% at both pH 7.0 and 8.0 were achieved.
Moreover, aromatic dithiol 6 significantly increased the in vitro
folding yield of lysozyme as compared to aromatic monothiol 8
at both pH 7.0 and 8.0 and high protein concentration (1 mg/mL).
Therefore, protein folding with aromatic dithiols should be useful,
especially for larger scales where high protein concentrations are
used.
4.1.3. 3,6,9,16,19,22-Hexaoxa-12,13-dithiatetracosane-1,24-diol
[tetraethylene glycol disulfide (18)]40
91% Yield, 1H NMR (CDCl3, 400 MHz) d 3.72 (t, J = 6.7 Hz, 4H),
3.68–3.63 (m, 20H), 3.60 (t, J = 4.5 Hz, 4H), 2.89 (t, J = 6.7 Hz, 4H),
2.74 (t, J = 5.7 Hz, 2H); 13C NMR (CDCl3, 100 MHz) d 72.4, 70.4,
70.3, 70.1, 69.4, 61.4, 38.2.
4.2. Synthesis of para- and ortho-substituted aromatic ethylene
glycol dithiols (1–5)
4.2.1. Synthesis of para- and ortho-substituted aromatic
ethylene glycol disulfide polymers57 (general procedure
using polymer 20 as an example)
4. Experimental
4,40-Dithiobenzoic acid (11)23,58,59 (0.213 g, 0.696 mmol) was
Hen egg white lysozyme was purchased from Roche Applied
Sciences and used without further purification. Di-, tri-, and tetra-
ethylene glycol thiols, the reduced forms of 16, 17, and 18, respec-
tively, and 8 were synthesized as described previously.51,54,55
dissolved in 4 mL of THF, and then DMF (10 lL) was added. The
solution was cooled to 0 °C, and oxalyl chloride (13) (0.883 g,
6.96 mmol) was slowly added into the solution via syringe. The

1026
A. S. Patel, W. J. Lees / Bioorg. Med. Chem. 20 (2012) 1020–1028
solution was stirred for 3 h and then reduced under vacuum to give
a yellow oil. The yellow oil was dissolved in 6 mL of CH2Cl2 and
then transferred to a flask containing triethylene glycol disulfide
(17) (0.981 g, 2.98 mmol), Et3N (0.77 mL, 5.57 mmol), and 46 mL
of CH2Cl2 at 0 °C. The reaction mixture was stirred at 0 °C for
30 min and gradually warmed to room temperature, and then stir-
ring continued overnight. The reaction mixture was dissolved in
50 mL of CH2Cl2 and then washed with 50 mL of 0.1 N HCl. The or-
ganic layer was then washed with water (2 ꢁ 50 mL), dried
(MgSO4), filtered, and reduced under vacuum to provide 0.81 g of
polymer 20.
3.69–3.57 (m, 6H), 2.66 (dt, J = 8.2, 6.5 Hz, 2H), 1.55 (t, J = 8.2 Hz,
1H); 13C NMR (CDCl3, 100 MHz) d 166.7, 138.3, 132.6, 131.9,
130.9, 126.0, 124.7, 73.0, 70.7, 70.3, 69.2, 64.3, 24.4. HRMS (ESI+)
calcd for C13H18O4S2 (M+Na)+ 325.0544, obsd 325.0536, calcd for
(M+H)+ 303.0724, obsd 303.0718.
4.2.2.5. 2-[2-(2-(2-Mercaptoethoxy)ethoxy)ethoxy]ethyl-2-mer-
captobenzoate [ortho-substituted aromatic tetraethylene glycol
dithiol (5)]. 29% Overall yield, 1H NMR (CDCl3, 400 MHz) d 8.03
(d, J = 7.5 Hz, 1H), 7.31–7.29 (m, 2H), 7.15 (ddd, J = 8.1, 5.6,
3.0 Hz, 1H), 4.70 (s, 1H), 4.47 (t, J = 4.8 Hz, 2H), 3.83 (t, J = 4.8 Hz,
2H), 3.70–3.57 (m, 10H), 2.67 (dt, J = 8.2, 6.5 Hz, 2H), 1.57 (t,
J = 8.2 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d 166.7, 138.7, 132.6,
132.0, 131.0, 126.1, 124.8, 73.0, 70.8, 70.8, 70.7, 70.4, 69.2, 64.4,
24.4. HRMS (ESI+) calcd for C15H22O5S2 (M+Na)+ 369.0806, obsd
369.0788.
4.2.2. Reduction of para- and ortho-substituted aromatic
ethylene glycol disulfide polymer with dithiothreitol (DTT)
(general procedure using the reduction of compound 20
as an example)
DTT (1.032 g, 6.70 mmol) and Et3N (0.5 mL, 3.59 mmol) were
added to a concentrated solution of polymer 20 (0.81 g) in 15 mL
of CH2Cl2. The reaction mixture was stirred (2 h for di- and trieth-
ylene glycol polymer, and 3 h for tetraethylene glycol polymer) un-
der argon, and then the mixture was extracted with 50 mL of ethyl
acetate. The organic layer was washed with water (2 ꢁ 50 mL),
dried over MgSO4, filtered, and concentrated under reduced pres-
sure to give the crude product. The crude product was then puri-
fied by silica gel chromatography using CH2Cl2/hexane (2:1 for
diethylene glycol dithiol, 4:1 for triethylene glycol dithiol, and
6:1 for tetraethylene glycol dithiol) to provide 0.124 g of para-
substituted aromatic triethylene glycol dithiol (2) as a yellow oil,
30% overall yield from compound 11.
4.3. Synthesis of aromatic triethylene glycol monothiol (9)
Compound 9 was prepared in 57% overall yield using the gen-
eral procedures for compounds 1–5 with benzoyl chloride and tri-
ethylene glycol disulfide (17) as starting materials.
1H NMR (CDCl3, 400 MHz) d 8.04 (d, J = 7.0 Hz, 2H), 7.55 (t,
J = 7.4 Hz, 1H), 7.42 (t, J = 7.7 Hz, 2H), 4.47 (t, J = 4.8 Hz, 2H), 3.83
(t, J = 3.7 Hz, 2H), 3.70 (t, J = 4.0 Hz, 2H), 3.65–3.58 (m, 4H), 2.66
(q, J = 6.4 Hz, 2H), 1.55 (t, J = 8.2 Hz, 1H); 13C NMR (CDCl3,
100 MHz) d 166.6, 133.1, 130.2, 129.8, 128.4, 73.0, 70.8, 70.4,
69.4, 64.2, 24.4. HRMS (ESI+) calcd for C13H18O4S (M+Na)+
293.0823, obsd 293.0823.
4.2.2.1. 2-(2-Mercaptoethoxy)ethyl-4-mercaptobenzoate [para-
substituted aromatic diethylene glycol dithiol (1)]. 48% Overall
yield, 1H NMR (CDCl3, 400 MHz) d 7.90 (d, J = 8.4 Hz, 2H), 7.28 (d,
J = 8.4 Hz, 2H), 4.45 (t, J = 4.7 Hz, 2H), 3.79 (t, J = 4.8 Hz, 2H),
3.67–3.62 (m, 2H), 2.70 (dt, J = 8.1, 6.3 Hz, 2H), 1.59 (t, J = 8.2 Hz,
1H); 13C NMR (CDCl3, 100 MHz) d 166.1, 138.7, 130.4, 128.2,
127.1, 72.9, 69.0, 64.1, 24.4. HRMS (ESI+) calcd for C11H14O3S2
(M+Na)+ 281.0282, obsd 281.0289.
4.4. Synthesis of quaternary ammonium salt dithiols (6 and 7)
4.4.1. Synthesis of protected quaternary ammonium salt
dithiols 30 and 3160 (general procedure using 30 as an example)
Benzyl bromide derivative 2751,52 (2.10 g, 6.84 mmol) was
added to a stirred solution of N,N,N0,N0-tetramethyl-1,3-propane
diamine (28) (0.38 g, 2.92 mmol) in 20 mL of acetonitrile. The reac-
tion mixture was stirred overnight, diluted with diethyl ether, and
filtered. The solid was then washed with diethyl ether and dried
yielding 2.07 g of a white powder (30), 95% yield.
4.2.2.2. 2-(2-(2-Mercaptoethoxy)ethoxy)ethyl-4-mercaptoben-
zoate [para-substituted aromatic triethylene glycol dithiol
(2)]. 30% Overall yield, 1H NMR (CDCl3, 400 MHz) d 7.90 (d,
J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 4.46 (t, J = 4.8 Hz, 2H), 3.83
(t, J = 4.8 Hz, 2H), 3.71–3.60 (m, 6H), 2.68 (dt, J = 8.1, 6.4 Hz, 2H),
1.58 (t, J = 8.2 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d 166.1, 138.6,
130.4, 128.2, 127.1, 73.0, 70.7, 70.3, 69.3, 64.1, 24.4. HRMS (ESI+)
calcd for C13H18O4S2 (M+Na)+ 325.0544, obsd 325.0538, calcd for
4.4.1.1. N1,N3-Bis(4-benzoylthio)benzyl)-N1,N1,N3,N3-tetrameth-
ylpropane-1,3-diaminium bromide [propane salt (30)]. 95%
Yield, 1H NMR (DMSO, 400 MHz) d 8.00 (d, J = 7.4 Hz, 4H), 7.78–
7.70 (m, 10H), 7.62 (t, J = 7.8 Hz, 4H), 4.74 (s, 4H), 3.37 (t,
J = 8.0 Hz, 4H), 3.11 (s, 12H), 2.50–2.46 (m, 2H). 13C NMR (DMSO,
100 MHz) d 188.6, 135.7, 135.2, 134.6, 134.0, 129.6, 129.5, 129.3,
127.2, 66.5, 60.1, 49.7, 16.9. HRMS (ESI+) calcd for C35H40Br2N2O2S2
(MꢀBr)+ 663.1714, obsd 663.1685.
C
13H18O4S2 (M+H)+ 303.0724, obsd 303.0721.
4.2.2.3. 2-[2-(2-(2-Mercaptoethoxy)ethoxy)ethoxy]ethyl-4-mer-
captobenzoate [para-substituted aromatic tetraethylene glycol
dithiol (3)]. 34% Overall yield, 1H NMR (CDCl3, 400 MHz) d 7.90
(d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 4.45 (t, J = 4.8 Hz, 2H),
3.82 (t, J = 4.8 Hz, 2H), 3.71–3.58 (m, 10H), 2.68 (dt, J = 8.1,
6.5 Hz, 2H), 1.59 (t, J = 8.2 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d
166.1, 138.6, 130.4, 128.2, 127.2, 73.0, 70.8, 70.8, 70.7, 70.3, 69.3,
64.2, 24.4. HRMS (ESI+) calcd for C15H22O5S2 (M+Na)+ 369.0806,
obsd 369.0807.
4.4.1.2. N1,N6-Bis(4-benzoylthio)benzyl)-N1,N1,N6,N6-tetrameth-
ylhexane-1,6-diaminium bromide [hexane salt (31)]. 99% Yield,
1H NMR (DMSO, 400 MHz) d 7.99 (d, J = 7.3 Hz, 4H), 7.79–7.69 (m,
10H), 7.62 (t, J = 7.9 Hz, 4H), 4.67 (s, 4H), 3.33 (br s, 4H), 3.03 (s,
12H), 2.07 (br s, 4H), 1.38 (br s, 4H). 13C NMR (DMSO, 100 MHz)
d 188.5, 135.6, 135.1, 134.5, 133.8, 129.5, 129.3, 127.1, 65.5, 63.5,
49.3, 25.3, 21.7. HRMS (ESI+) calcd for C38H46Br2N2O2S2 (MꢀBr)+
705.2184, obsd 705.2163.
4.2.2.4. 2-[2-(2-Mercaptoethoxy)ethoxy]ethyl-2-mercaptoben-
zoate [ortho-substituted aromatic triethylene glycol dithiol
(4)]. 32% Overall yield, 1H NMR (CDCl3, 400 MHz) d 8.03 (d,
J = 7.6 Hz, 1H), 7.30–7.28 (m, 2H), 7.13 (ddd, J = 8.1, 5.6, 2.9 Hz,
1H), 4.67 (s, 1H), 4.46 (t, J = 4.8 Hz, 2H), 3.82 (t, J = 4.8 Hz, 2H),
4.4.2. Synthesis of quaternary ammonium salt dithiols 6 and 7
(general procedure using 6 as an example)
The quaternary ammonium salt (30) (1.557 g, 2.09 mmol) dis-
solved in 45 mL of water and 45 mL of HBr (48 wt % in water)
was refluxed under argon. After 90 min, the reaction mixture was

A. S. Patel, W. J. Lees / Bioorg. Med. Chem. 20 (2012) 1020–1028
1027
cooled to 0 °C, filtered, and then washed with EtOAc (3 ꢁ 100 mL).
followed by withdrawing 20
l
L of the resulting solution and then
The aqueous layer was reduced partially and then lyophilized sev-
eral times by dissolving the residue in water to provide 1.042 g of 6
as a white powder, 93% yield.
measuring the enzymatic activity.19,46,47,49
Acknowledgment
This work was supported in part by the National Science Foun-
dation under Grant No. CHE-0342167 to W.J.L.
4.4.2.1. N1,N3-Bis(4-mercaptobenzyl)-N1,N1,N3N3-tetramethyl-
propane-1,3-diaminium bromide (6). 93% Yield, 1H NMR (D2O,
400 MHz) d 7.46 (d, J = 8.4 Hz, 4H), 7.41 (d, J = 8.4 Hz, 4H), 4.52
(s, 4H), 3.37 (t, J = 8.3 Hz, 4H), 3.09 (s, 12H), 2.41 (quintet,
J = 8.3 Hz, 2H). 13C NMR (D2O, 100 MHz) d 135.8, 133.5, 129.0,
123.5, 68.5, 60.1, 49.7, 17.0. HRMS (ESI+) calcd for C21H32Br2N2S2
(MꢀBr)+ 455.1190, obsd 455.1175.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.11.049.
References and notes
4.4.2.2. N1,N6-Bis(4-mercaptobenzyl)-N1,N1,N6N6-tetramethyl-
hexane-1,6-diaminium bromide (7). 51% Yield, 1H NMR (D2O,
400 MHz) d 7.50 (d, J = 7.2 Hz, 4H), 7.45 (d, J = 7.7 Hz, 4H), 4.48
(s, 4H), 3.30 (t, J = 8.0 Hz, 4H), 3.08 (s, 12H), 1.92 (br s, 4H), 1.47
(br s, 4H). 13C NMR (D2O, 100 MHz) d 135.3, 133.4, 128.9, 124.1,
67.4, 63.8, 49.6, 25.2, 22.0. HRMS (ESI+) calcd for C24H38Br2N2S2
(MꢀBr)+ 497.1659, obsd 497.1638.
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1.5 mL-Eppendorf tube, 10
lL of reduced lysozyme (10 mg/mL)
was added to 990 L of renaturation buffer. For reproducible mix-
l
ing conditions, the renaturation buffer was placed in an Eppendorf
tube, the protein was added 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, 20 lL aliquots
were removed at specific times and assayed for enzymatic activ-
ity.18,46,47,49All the refolding experiments were performed at 25 °C.
4.5.2. Refolding of denatured reduced lysozyme (1 mg/mL)
Reduced lysozyme (11 mg/mL) was dissolved in 0.1 M acetic
acid containing 6 M Gdn HCl at pH 2.5. The pH of the protein solu-
tion was then adjusted to that of the folding experiment (pH 7.0 or
8.0) by the addition of 3 M KOH. After adjusting the pH, the con-
centration of the protein solution was determined by UV–vis, and
the final concentration was adjusted to 10 mg/mL with 6 M Gdn
HCl, if necessary (e
280nm = 2.37 mL mgꢀ1 cmꢀ1).25,34,47 Stock solu-
tions of GSH, aromatic monothiol, aromatic dithiols, and GSSG
were prepared in deoxygenated 0.1 M bis tris propane–HCl (for
pH 7.0) or 0.1 M Tris–HCl (for pH 8.0), 1.25 M Gdn HCl, and
1 mM EDTA. From the stock solutions, varying amount of GSH
and GSSG, aromatic monothiol and GSSG, or aromatic dithiols
and GSSG were mixed in 0.1 M bis tris propane–HCl at pH 7.0 or
0.1 M Tris–HCl at pH 8.0, 1.25 M Gdn HCl, and 1 mM EDTA and ad-
justed to the appropriate pH with 1 M KOH, if necessary (renatur-
ation buffer). In a 1.5 mL-Eppendorf tube, 900
lL of renaturation
buffer was mixed with 100 L of reduced lysozyme solution
l
(10 mg/mL) to give a 10-fold dilution and a final concentration of
1 mg/mL protein and 1.7 M Gdn HCl. Subsequently, at specific
intervals, 10
diluted with 90
or 0.1 M Tris–HCl at pH 8.0, 1.25 M Gdn HCl, and 1 mM EDTA)
lL aliquots were withdrawn and each aliquots was
l
L of buffer (0.1 M bis tris propane–HCl at pH 7.0

1028
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