Small molecule diselenide additives for in vitro oxidative protein folding

Article abstract of DOI:10.1039/c5cc10451c

The in vitro oxidative folding of disulfide-rich proteins can be challenging. Here we show a new class of small molecule diselenides, which can be easily prepared from inexpensive starting materials, used to enhance oxidative protein folding. These compounds were tested on a model protein, bovine pancreatic trypsin inhibitor. Two of the tested diselenides showed considerable improvement over glutathione and were on par with the previously described selenoglutathione.

Full text of DOI:10.1039/c5cc10451c

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Small Molecule Diselenide Additives for In Vitro Oxidative Protein
Folding
Post Sai Reddya and Norman Metanisa*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
The in vitro oxidative folding of disulfide-rich proteins can be
challenging. Here we show new class of small molecule
a
diselenides, which can be easily prepared from inexpensive
starting materials, used to enhance oxidative protein folding.
These compounds were tested on
a model protein, bovine
pancreatic trypsin inhibitor. Two of the tested diselenides showed
considerable improvement over glutathione and were on par with
the previously described selenoglutathione.
The in vitro refolding of disulfide-rich proteins can be a
challenging task,1,
2
although this process is generally
spontaneous because all the information required for correct
folding of a protein into its native state is found in the amino
acid sequence.3 However, as the number of disulfide bonds
increases, proteins rich with cysteine (Cys) residues may
exhibit slow folding and low yields of native protein due to
Figure 1. a. Structures of reduced and oxidized glutathione (X = S; GSH and GSSG) and
selenoglutathione, (X= Se; GSeH and GSeSeG), respectively; b. Structures of small
molecule diselenides 1-3 tested as additives for in vitro oxidative protein folding.
multiple
possible
intermediates,
including
trapped
RNase A and bovine pancreatic trypsin inhibitor (BPTI),
although it provided lower yield of native protein than
GSeSeG.8 Later Beld et al. tested other commercially available
small molecule diselenides and compared them to their
disulfide counterpart as additives for oxidative folding in
vivo.12 All diselenides showed better oxidative folding
enhancement than their disulfide analogs. In particular,
selenocystamine showed the best in vivo activity,12 perhaps
due to its smaller size compared to aromatic diselenides tested
and its enhanced solubility, hence was chosen for further
studies. More recently, selenocystamine improved the
accumulation of disulfide containing proteins in chloroplasts of
eukaryotic green algae.13
intermediates. Trapped intermediates are long-lived species
highly stabilized by disulfide bonds, in which reduced cysteine
residues are buried in the protein interior and rendered
inaccessible for oxidation. To facilitate oxidative refolding,
small compound additives are frequently used, typically, a
redox buffer composed of reduced and oxidized glutathione
(GSH and GSSG, respectively, Figure 1a) or other thiol-
containing additives.4-6
Previous studies showed that the oxidative refolding of
disulfide-rich proteins in the presence of selenoglutathione
(Figure 1a),
a selenocysteine (Sec)-containing analog of
glutathione, performs better than glutathione in this respect
for many proteins, both in terms of the rate of refolding and
the yield of native state proteins.7-11 Furthermore,
selenocystamine, a commercially available diselenide, was
shown to enhance the in vitro oxidative folding of two proteins
Selenium is
a
particularly effective electrophile and
nucleophile in nucleophilic exchange reactions,14, 15 due to its
greater polarizability in comparison to sulfur and the lower pKa
for selenol side-chain of Sec compared to thiol of Cys (5.2
versus 8.5, respectively).16 Owing to the rapid oxidation of
selenols by atmospheric oxygen, catalytic amounts of
diselenides can be used for protein refolding under aerobic
conditions.8, 12 It was also suggested that folding enhancement
observed with diselenides is due to enhanced thiol-disulfide
exchange reactions, which can rescue trapped intermediates.11
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1. MsCl, Et3N,
a.
Furthermore, Sec has been used as an intramolecular catalyst
for oxidative protein refolding through Cys-to-Sec substitutions
into the protein sequence.17-24 Strategic Sec substitution into
disulfide-rich polypeptides has proven to be advantageous in
facilitating this process and providing higher yields of native
structures.21-24 Recently, we reported that Sec-substitution
into BPTI could be exploited as a tool to facilitate the folding
process and to steer folding in predictive and more productive
routes.21, 23, 24
CH2Cl2, 0 oC, 2 h
OH
O
Ref. 31
O
O
2. Na2Se2 prepared
TFA/MeOH (1:1)
HO
1
O
O
Se
OH
)
rt, 1 h, quantitative
2
crude yield
from Se, N2H4.H2O,
NaOH, DMF, rt, 3-4 h,
76% (for 2 steps)
OH
O
4
5
L-malic acid
b.
OH
Na2Se2 prepared
from Se, N2H4.H2O,
O
O
Ref. 33, 34
TFA/MeOH (1:1)
HO
OH
2
O
O
OMs
Se
)
rt, 1 h, quantitative
crude yield
NaOH, DMF, rt, 3-4 h,
40% (for 2 steps)
2
Glycerol
6
7
c.
1. HONSu, DCC, THF,
0 oC, 1 h
2. NaBH4, H2O, 0 oC,
HS
(Boc)2O, 1 N NaOH,
S
S
2 h at 0 o
C
35 % HCHO, H2
O
OH
O
OH
OH
Small
seleno-compounds
containing
diselenide
or
H2
N
N
N
then 3 h at rt, 73%
rt, 5 h, 96%
H
Boc
O
30 min, 69% (crude yield)
O
S
selenosulfide bonds have been reported. Seleno-analogs of
lipoic acid have been previously synthesized;25 however, their
use in protein folding was never demonstrated. In addition,
Tomoda and coworkers reported the synthesis of
diselenothreitol (DSTox),26 an analog of the well-studied
additive for protein folding, oxidized dithiothreitol (DTTox).
However, DST was found to exhibit much lower redox
potential than DTT and was therefore suggested to be a poor
additive for protein folding.26 Recently, Raines and coworkers
9
L-Cysteine
8
1. MsCl, Et3N, CH2Cl2,
oC-rt, 1-2 h
2. Ethanolic Na2Se2,
S
TFA/CH2Cl2 (1:1)
rt, 30 min, 91%
0
Se
)
2
3
OH
N
N
Boc
EtOH, 0 oC-rt, overnight
Boc
11
10
36% (for 2 steps)
Scheme 1. Chemical synthesis of diselenides 1, 2 and 3.
trapped intermediates, where free cysteines are usually
inaccessible to oxidant. Third, we wanted to explore the effect
showed that an analog of reduced dithiothreitol DTTred
,
of changing the net charge of the diselenides
and amino polar groups in ensure enhanced solubility in
buffer, and their net charge is altered from negative in
selenoglutathione to neutral in and , and positively charged
in . Changing the net charge can be useful for certain
1-3. The hydroxy
1-3
dithiobutylamine (DTBA), in which an amino group is present,
was a superior reductant for certain proteins over DTTred, due
the lower pKa values of its thiols.27 Later, Raines and coworkers
reported the synthesis of a seleno-DTBA analog and its use in
protein reduction but did not extend the study to oxidative
protein refolding.28
1
2
3
proteins, as recently described for the reduction of proteins by
superior reductant DTBA and seleno-DTBA.27, 28
Our results indicate that
2 and 3 enhance the refolding of BPTI
These studies show that small molecule diselenides may
become useful additives for in vivo and/or in vitro protein
refolding in academic laboratories and pharmaceutical
industries, by enhancing oxidative protein refolding and
increasing the recovery of native protein structures, for
example, from inclusion bodies.
in the same order as with selenoglutathione and are almost
10-fold better than glutathione.
The diselenides
1-3 were prepared from commercially
available starting materials; L-malic acid, glycerol, and L-
cysteine, respectively. L-malic acid (Scheme 1a) was converted
to acetonide protected alcohol
Then was mesylated, followed by treatment with Na2Se2
(generated from Se, N2H4
H2O, and NaOH in DMF)32 giving
diselenide in 76% yield. Acetonide deprotection was
achieved by TFA, providing diselenide in quantitative crude
4
via a known procedure.31
The most studied seleno-additive is selenoglutathione (GSeH),
which, like glutathione (Figure 1a), is a tripeptide with a
negative net charge at neutral pH (−2 for GSeH vs. −1 for GSH).
Its oxidized dimeric form, GSeSeG, contains an intermolecular
diselenide bond instead of a disulfide. The preparation of
selenoglutathione requires multi-step synthesis from
4
·
5
5
1
yield. The product was purified via C18 preparative reversed-
1
phase (RP) HPLC and confirmed by H-,13C- and 77Se-NMR and
30
protected Sec, which also requires multi-step synthesis,29,
ESI-MS (see SI).
followed by peptide synthesis, which also uses a non-standard
protected glutamate.7 In contrast, smaller molecule
In the same manner, glycerol (Scheme 1b) was converted to its
34
acetonide protected mesylate
Mesylate
6
by a known procedure.33,
32
was treated with Na2Se2 to give diselenide 7 in
diselenides (e.g. 1-3 in Figure 1b) can be readily prepared from
6
commercially available and cheap starting materials (Scheme
1). These kind of smaller diselenides with different
functionalities and with different net charge may turn to be
advantageous in protein refolding, one good example is
selenocystamine vide supra.12
40% yield. Acetonide
same manner as with
7
deprotection was performed in the
, giving diselenide in quantitative
5
2
crude yield. The product was purified by preparative RP-HPLC
and confirmed by 1H-,13C- and 77Se-NMR and ESI-MS (see SI).
Finally, thiazolidine (Thz)
excellent yield (96%) from a solution of L-cysteine and 35%
HCHO in water.35, 36 Treatment of
with (Boc)2O and 1N NaOH
afforded Boc-Thz-carboxylic acid ), which is also
commercially available, in 73% yield. Compound was
8 (Scheme 1c) was obtained in
Here we designed a straightforward synthesis of the diselenide
molecules 1-3 (Scheme 1), and study the efficacy in promoting
oxidative folding of model protein, BPTI.
8
(
9
Our aims in synthesizing diselenides 1-3 were threefold. First,
9
we wanted to synthesize simpler, less costly alternatives to
selenoglutathione. Second, we wanted to reduce the size of
these additives relative to selenoglutathione (Figure 1), in
order to afford better penetration into the protein interior of
converted to its –ONSu activated ester using N-
hydroxysuccinimide (HONSu) and dicyclohexylcarbodiimide
(DCC) in THF at 0 oC, and subsequent reduction37 with aqueous
o
NaBH4 at 0 C gave alcohol 10 in 69% crude yield. The crude
alcohol was treated with MsCl and Et3N in CH2Cl2 to give the
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corresponding mesylate 10a, which was treated directly with
38
in situ generated ethanolic Na2Se2 to afford diselenide 11 in
36% total yield. Diselenide 11 was subjected to Boc-
deprotection with 50% TFA in CH2Cl2 at room temperature to
give diselenide 3 as a TFA salt in 91% crude yield, which was
purified by preparative RP-HPLC and confirmed by 1H-,13C- and
77Se-NMR and ESI-MS (see SI).
To elucidate the efficacy of diselenides 1-3 in promoting
oxidative protein folding, we chose BPTI as our model
system.39-41 The oxidative renaturation of BPTI (Figure 2) was
monitored based on the method of Weissman and Kim.41
Figure 3. Oxidative folding of BPTI using a. GSSG; b. diselenide 1; c. 2; and d. 3. All
experiments were carried in parallel; the oxidant (final concentration 150 µM) was
added to reduced BPTI (final concentration 30 µM) in Tris-HCl buffer at pH 8.7 . At time
points, samples were taken and quenched.11 & is impurity co-eluted with N’, $ is N*
eluted earlier than anticipated.
However, in contrast to GSSG, the folding with
within 200 min, while folding with GSSG was not complete
even after 20 h (Figure 3b). BPTI folding with diselenides and
was substantially faster than with GSSG (Figure 3c-d). The
initial oxidation of reduced protein by and were very rapid
and comparable to that observed with GSeSeG
only 1 min, the two trapped intermediates N’ and N*
dominate.11 Diselenides
and continue to enhance the
folding throughout the folding process, resembling the kinetics
of GSeSeG (Figure 4). Although the structures of and look
similar, an additional methylene group in diselenide , in
comparison to seem to cause some effects on folding rates,
1 was complete
2
3
2
3
Figure 2. Folding mechanism of BPTI.41 The initial oxidation of the reduced (R) form is
fast and affords many intermediates containing one disulfide bond, which rearrange
(dotted arrows) to give [30-51] and [5-55] intermediates. Qualitative estimates of the
relative rates of the other steps are indicated. Oxidation of the single disulfide
intermediates give [14-38, 30-51], N’, and [5-55, 14-38] N*, respectively. The
conversion of these trapped intermediates is rate-limiting and goes through unfolding
11
,
where after
2
3
1
2
ꢁꢂ
and isomerization to give [5-55, 30-51] ꢀ , which is readily oxidized to the native (N)
1
ꢁꢂ
state.
2
for example through steric hindrance, although diselenide
provided quantitative final yield of folded protein.
1-3
The folding reactions were carried out in degassed buffer
under aerobic conditions. Oxidized GSSG or diselenides 1-3
(final concentration 150 µM) was added to reduced BPTI (30
µM) in Tris buffer (100 mM Tris-HCl, 200 mM KCl, 1 mM EDTA,
pH 8.7). At determined time points, 80 µL aliquots were
removed, quenched with HCl (30 µL of 2 M), and injected into
a Waters ACQUITY UPLC using Waters HSS T3 column (1.8 µm,
2.1 x 100 mm), heated at 40 oC, and eluted with a 10:90 to
26.5:73.5 gradient (buffer A = 0.1% TFA in acetonitrile : buffer
B = 0.1% TFA in water) over 5 min, increasing to 40:60 (A : B)
over 19 min. The chromatograms were monitored at 214 nm.
For kinetic analysis, the sum of all peaks of the protein states
was assigned as 100%. The peak assignment for native BPTI (N)
was confirmed by co-injection with an authentic standard, and
the peaks of the intermediates N’ and N* that accumulate
during folding were assigned according to their mass and
previous literature.11, 21, 23, 41 Under these conditions, oxidative
refolding of BPTI in the presence of GSSG was still not
complete after 20 h (Figure 3a).21, 23
Figure 4. Folding of BPTI with GSSG, diselenides 1-3, and GSeSeG; the data points for
In the initial stages of BPTI renaturation, diselenide
1 exhibited
GSeSeG were taken from Ref 11.
a folding behaviour slower than that with GSSG up to 20 min,
where N* was the dominate intermediate (Figure 3b).
Since oxidative folding is performed under aerobic conditions,
and because selenols are quite sensitive to air oxidation, the
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concentration of the diselenide oxidant is not altered 17. K. H. Gowd, V. Yarotskyy, K. S. Elmslie, J. J. Skalicky, B. M.
12
throughout the folding process.8,
We believe that these
Olivera and G. Bulaj, Biochemistry, 2010, 49, 2741-2752.
small molecule diselenides (and perhaps others) may become 18. T. S. Han, M. M. Zhang, K. H. Gowd, A. Walewska, D.
useful additives for in vitro and/or in vivo protein refolding in
Yoshikami, B. M. Olivera and G. Bulaj, ACS Med. Chem. Lett.,
2010, , 140-144.
academia and biopharmaceutical industries.
1
19. M. Muttenthaler, S. T. Nevin, A. A. Grishin, S. T. Ngo, P. T.
Choy, N. L. Daly, S. H. Hu, C. J. Armishaw, C. I. A. Wang, R. J.
Lewis, J. L. Martin, P. G. Noakes, D. J. Craik, D. J. Adams and
P. F. Alewood, J. Am. Chem. Soc., 2010, 132, 3514-3522.
20. A. D. de Araujo, B. Callaghan, S. T. Nevin, N. L. Daly, D. J.
Craik, M. Moretta, G. Hopping, M. J. Christie, D. J. Adams and
P. F. Alewood, Angew. Chem. Int. Ed., 2011, 50, 6527-6529.
Conclusions
A new class of small molecule diselenides was prepared from
commercially available and cheap starting materials. Our
results indicate that these synthetic diselenides are superior to
glutathione, which is typically used as additive for in vitro
oxidative folding. Two of the diselenides demonstrated folding
capabilities similar to that of selenoglutathione, which was
previously shown to enhance the oxidative folding of many
proteins, different in size, number of disulfide bonds and
folding mechanisms. This class of small molecule diselenides
may demonstrate even better folding capabilities with other
proteins, for example those exhibiting a different folding
mechanism than BPTI.
21. N. Metanis and D. Hilvert, Angew. Chem. Int. Ed., 2012, 51
,
5585-5588.
22. A. M. Steiner, K. J. Woycechowsky, B. M. Olivera and G.
Bulaj, Angew. Chem. Int. Ed., 2012, 51, 5580-5584.
23. N. Metanis and D. Hilvert, Chem. Sci., 2015, 6, 322-325.
24. N. Metanis and D. Hilvert, Curr. Opin. Chem. Biol., 2014, 22
27-34.
,
25. K. E. Reed, T. W. Morris and J. E. Cronan, Proc. Natl. Acad.
Sci. USA, 1994, 91, 3720-3724.
26. M. Iwaoka, T. Takahashi and S. Tomoda, Heteroatom Chem.,
2001, 12, 293-299.
NM acknowledges the Israel Science Foundation (ISF; grant
No. 1072/14) for financial support. PSR is supported by PBC
Fellowship, Council for Higher Education, Israel. We thank
Rebecca Notis Dardashti for editing this manuscript.
27. J. C. Lukesh, M. J. Palte and R. T. Raines, J. Am. Chem. Soc.,
2012, 134, 4057-4059.
28. J. C. Lukesh, B. VanVeller and R. T. Raines, Angew. Chem. Int.
Ed., 2013, 52, 12901-12904.
29. R. Quaderer, A. Sewing and D. Hilvert, Helv. Chim. Acta,
2001, 84, 1197-1206.
Notes and references
1. R. Rudolph and H. Lilie, Faseb J., 1996, 10, 49-56.
2. S. Misawa and I. Kumagai, Biopolymers, 1999, 51, 297-307.
3. C. B. Anfinsen, Science, 1973, 181, 223-230.
30. N. Metanis, E. Keinan and P. E. Dawson, J. Am. Chem. Soc.,
2006, 128, 16684-16691.
31. A. Venkanna, E. Sreedhar, B. Siva, K. S. Babu, K. R. Prasad and
J. M. Rao, Tetrahedron: Asymmetry, 2013, 24, 1010-1022.
32. A. H. G. Siebum, W. S. Woo, J. Raap and J. Lugtenburg, Eur. J.
Org. Chem., 2004, 2905-2913.
4. W. J. Lees, Curr. Opin. Chem. Biol., 2008, 12, 740-745.
5. K. J. Woycechowsky, K. D. Wittrup and R. T. Raines,
Chemistry & Biology, 1999, 6, 871-879.
6. J. C. Lukesh, K. A. Andersen, K. K. Wallin and R. T. Raines,
Org. Biomol. Chem., 2014, 12, 8598-8602.
7. J. Beld, K. J. Woycechowsky and D. Hilvert, Biochemistry,
2007, 46, 5382-5390.
33. F. S. Gibson, M. S. Park and H. Rapoport, J. Org. Chem., 1994,
59, 7503-7507.
34. M. Goubert, L. Toupet, M. E. Sinibaldi and I. Canet,
Tetrahedron, 2007, 63, 8255-8266.
8. J. Beld, K. J. Woycechowsky and D. Hilvert, Biochemistry,
2008, 47, 6985-6987.
35. O. B. Sutcliffe, R. C. Storr, T. L. Gilchrist and P. Rafferty, J.
Chem. Soc., Perkin Trans. 1, 2001, 1795-1806.
36. P. S. Reddy, S. Dery and N. Metanis, Angew. Chem. Int. Ed.,
2016, 55, 992-995.
9. J. Beld, K. J. Woycechowsky and D. Hilvert, Biochemistry,
2009, 48, 4662-4662.
10. J. Beld, K. J. Woycechowsky and D. Hilvert, J. Biotechnol.,
2010, 150, 481-489.
37. S. V. Jadhav, A. Bandyopadhyay, S. N. Benke, S. M. Mali and
H. N. Gopi, Org. Biomol. Chem., 2011, 9, 4182-4187.
11. N. Metanis, C. Foletti, J. Beld and D. Hilvert, Isr. J. Chem.,
2011, 51, 953-959.
38. D. L. Klayman and T. S. Griffin, J. Am. Chem. Soc., 1973, 95
,
197-200.
12. J. Beld, K. J. Woycechowsky and D. Hilvert, ACS Chem. Bio.,
39. T. E. Creighton, Prog. Biophys. Mol. Bio., 1978, 33, 231-297.
40. T. E. Creighton and D. P. Goldenberg, J. Mol. Biol., 1984, 179
497-526.
2010,
13. L. S. Ferreira-Camargo, M. Tran, J. Beld, M. D. Burkart and S.
P. Mayfield, AMB Expr., 2015, , 39-49.
5, 177-182.
,
5
41. J. S. Weissman and P. S. Kim, Science, 1991, 253, 1386-1393.
14. D. Steinmann, T. Nauser and W. H. Koppenol, J. Org. Chem.,
2010, 75, 6696-6699.
15. R. J. Hondal, S. M. Marino and V. N. Gladyshev, Antioxidants
& redox signaling, 2013, 18, 1675-1689.
16. R. E. Huber and R. S. Criddle, Arch. Biochem. Biophys., 1967,
122, 164-173.
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