Microwave-mediated reduction of disulfide bridges with supported (tris(2-carboxyethyl)phosphine) as resin-bound reducing agent

Article abstract of DOI:10.1021/co300104k

We report on the synthesis and use of a new supported reagent consisting in tris(2-carboxyethyl)phosphine (TCEP) immobilized on hydrophilic PEG based resin beads. Used in conjunction with a 5 min microwave (MW) irradiation, supported TCEP reduced disulfide bridges in free thiols in peptides having two or more cysteine residues. Separation of reaction products from reducing agent was easily performed by simple filtration.

Full text of DOI:10.1021/co300104k

Letter
pubs.acs.org/acscombsci
Microwave-Mediated Reduction of Disulfide Bridges with Supported
(Tris(2-carboxyethyl)phosphine) as Resin-Bound Reducing Agent
Guillaume Miralles,† Pascal Verdie,† Karine Puget,‡ Amelie Maurras,‡ Jean Martinez,† and Gilles Subra†,*
́
́
†Institut des Biomolec
́
ules Max Mousseron IBMM, UMR 5247, Universite
Charles Flahault 34000 Montpellier, France
‡Genepep SA, Les Coteaux Saint Roch, 12 Rue du Fer a
́
Montpellier 1, Universite
́
Montpellier 2, CNRS, 15 avenue
̀
Cheval, 34430 St Jean de Vedas, France
́
S
* Supporting Information
ABSTRACT: We report on the synthesis and use of a new
supported reagent consisting in tris(2-carboxyethyl)phosphine
(TCEP) immobilized on hydrophilic PEG based resin beads.
Used in conjunction with a 5 min microwave (MW)
irradiation, “supported TCEP” reduced disulfide bridges in
free thiols in peptides having two or more cysteine residues.
Separation of reaction products from reducing agent was easily performed by simple filtration.
KEYWORDS: disulfide bond, cysteine, supported reagent, reduction, thiol, TCEP
ris(2-carboxyethyl)phosphine (TCEP) is a very useful
efficient and very elegant methodology has been recently
Treducing agent used in biochemistry and chemistry developed for the Fmoc/tBu solid phase synthesis of C-
terminus peptide thioester precursors.15 C-terminal bis(2-
laboratories. Odorless, water-soluble, and stable in a large
range of pH,1 it is the reactant of choice to denaturate proteins,
sulfanylethyl)amido peptides are cleaved from the support
and purified as stable cyclic disulfides. Reduction using TCEP
opens the disulfide bridge, initiating the shift between amide
and thioester suitable for disulfide exchange and NCL.16
In this case, TCEP removal is achieved by a preparative
HPLC. To simplify the workup and speed of the treatments,
the whole synthetic process using solid supported reagents
constitutes an attractive alternative. Actually, only TCEP
immobilized on cross-linked agarose gel has been described.
However its use is limited to aqueous solutions. Moreover, the
agarose gel loading, given by supplier datasheet, is quite low (8
μmol/mL) and is not convenient in the context of TCEP
applications involving gram- or even milligram-scale samples.
As an example, 125 μL of gel are required to reduce 1 mg of a
peptide of MW 1000 g mol−1. At least, the reduction speed
decreases quickly with the sample concentration: 15 min are
required to reduce protein samples at a concentration of 0.1
mg/mL but required 1 h at 1 mg/mL.17
In this context, we first designed a high loaded supported
reducing agent consisting in TCEP immobilized on hydrophilic
cross-linked PEG resin beads ChemMatrix (CM). Then, we
used this new resin, both in aqueous or organic solvents, to
quickly reduce disulfide bonds under short microwave
irradiation.
Cross-linked polymer resin beads are useful auxiliaries both
used for the synthesis of supported reagents or as solid supports
for organic synthesis including peptide synthesis. The nature of
polymer matrix is as important as the solvent used for a given
breaking the tertiary structure established by disulfide bonds
between cysteine prior to SDS-PAGE analysis. This reagent is
particularly suitable when free thiol have to be obtained as
reactive moieties for their immobilization onto maleimide or
sulfhydryl functionalized supports2 or for further chemical
labeling.3 Indeed, compared to sulfur-containing agents, such as
ethanedithiol (EDT) or dithiothreitol (DTT), TCEP will not
compete with thiols as a nucleophile in most chemical
modifications of cysteines. Similarly, TCEP is also used to
keep reductive conditions when free thiols are used reagents,
for example, Michael addition on dehydroalanine residues4 or
on maleimide conjugates.5,6
Mild reductive agents such TCEP are also useful in peptide
chemistry for bioconjugation purposes. Cysteines are often
introduced at the C- or N-terminus of a bioactive sequence as a
template for conjugation with a fluorescent probe, or another
bioactive moiety (cell penetrating, homing peptide, DNA, ...).
The cysteine side chain was also used to graft peptides and
proteins on micro or nanoscale objects, such as polymers,7
liposomes,8 gold nanoparticles,9 and glass microarrays.10
Covalent linkage can be achieved via thiol nucleophilic
substitution on a halogenated derivative,11 1,4-Michael
addition12 or hetero-disulfide bond formation by reacting
with an activated disulfide (e.g., NPys).13
Thiol-free reductive conditions afforded by TCEP are of high
interest for native chemical ligation (NCL). An unprotected
peptide fragment bearing a N-terminus cysteine may react
chemoselectively with a C-terminus thioester peptide yielding
an amide bond after a S−N acyl transfer.14 NCL is still
extensively used and developed, and remains the method of
choice for the chemical synthesis of proteins. In addition, an
Received: August 31, 2012
Revised: January 14, 2013
Published: February 25, 2013
© 2013 American Chemical Society
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ACS Combinatorial Science
Letter
Scheme 1. (A) Synthesis of TCEP-ChemMatrix-Supported Reducing Agent and (B) TCEP-CM-Mediated Reduction
Mechanism
Figure 1. FT-IR spectra of ( A) starting ChemMatrix resin, (B) supported-TCEP resin 1, and (C) TCEP bound ChemMatrix 2 resin after reduction
of disulfide bridge.
reaction.18 Because most of reactive sites are buried inside the
resin bead, swelling of beads is essential for optimal penetration
of reagents within the cross-linked matrix.19 Aminomethyl
ChemMatrix was chosen for supported TCEP preparation. In
contrast to resins traditionally used for the synthesis of peptides
and heterocycles, this hydrophilic cross-linked resin is
exclusively composed of PEG. It has been used successfully
for the synthesis of long and difficult peptides20 yielding
significant improvement of crudes. Its excellent swelling
properties, both in water or in aprotic polar solvents, such as
dimethyl formamide (DMF) and N-methyl pyrrolidone
(NMP), makes ChemMatrix a matrix of choice for the
applications foreseen for supported TCEP reducing agent.
TCEP-ChemMatrix Preparation and Characterization.
TCEP-CM 1 was prepared straightforward by activating one
carboxylic function of TCEP with one equivalent of HBTU in
the presence of DIEA. HBTU was introduced dropwise on
TCEP solution along with 1 equiv of DIEA to activate only one
carboxylic function, thus minimizing the possibility of cross-
linking TCEP on aminomethyl ChemMatrix (AM-CM).
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Table 1. Reduction of Disulfide Bridges with Supported TCEP-CM: (A) Starting Oxidized Peptides 3−9, (B) Reduced Peptides
3′−6′, and (C) Reduced Longer Peptides 7′−9′
A
a
a
disulfide compound
RT (min)
purity (%)
MW calcd
672.3
[M + H]+ m/z found
3
4
5
0.93
0.98
1.07
76
90
75
673.3
H-LCAGPCL-NH2
H-MCAGMCL-NH2
724.3
955.4
725.3
956.5
679.3
H-PFCNAFTGC-NH2
(Boc-Tyr-NH-C2H4-S)2
6
7
1.59
2.63
93
83
678.3
d
2269.1
2270.2
pGlu-CRRLCYKQRCVTYCRGR-NH2 (gomesin)
H-KWCFRVCYRGICYRRCR-OH (tachyplesin acid)
d
8
9
2.72
3.07
91
95
2263.1
3865.8
2264.0
d
3866.6
H-KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW-OH (mesenthericin)
B
a
a
reduced
compound
time required for complete conversion (μwave/
RT
purity
(%)
MW
calcd
c
solvent
RT)
(min)
ESI+ [M + H]+ m/z found
3′
4′
5′
6′
6′
6′
6′
EtOH/H2O/AcOH (6/3/1)
EtOH/H2O/AcOH (6/3/1)
EtOH/H2O/AcOH (6/3/1)
DMF/H2O/AcOH (6/3/1)
THF/H2O/AcOH (6/3/1)
EtOH/H2O/AcOH (6/3/1)
H2O
5 min/60 min
5 min/b
0.93
0.97
1.06
1.23
1.23
1.23
1.23
72
85
70
92
90
92
90
674.3
726.3
957.4
340.2
340.2
340.2
340.2
675.3
727.3
958.6
341.2
341.2
341.2
341.2
5 min/60 min
30 s/20 min
30 s/20 min
30 s/20 min
5 min/60 min
C
c
reduced compound
solvent
time required for complete conversion (μwave/RT)
MW calcd
MALDI-MS [M + H]+ m/z found
7′
8′
9′
EtOH/H2O/AcOH (6/3/1)
EtOH/H2O/AcOH (6/3/1)
EtOH/H2O/AcOH (6/3/1)
5 min/b
5 min/b
5 min/b
2273.1
2267.1
3867.8
2274.0
2268.0
3868.9
a
RT and purity % were calculated by peak integration at 214 nm during LC analyses. Compounds 3−6 and longer peptides 7−9 were analyzed in
b
c
different conditions (see Supporting Information). Complete conversion was not observed at RT after one hour. 6′ corresponds to the monomer
d
Boc-Tyr-NH-(CH2)2-SH. m/z of the molecular ion was determined by MALDI-TOF mass spectrometry.
Activated TCEP was coupled to free amino groups of AM-CM
(Scheme 1A).
using microwave-assisted Fmoc SPPS19 on various solid
supports (see Supporting Information). They were cleaved
from the support by TFA cocktail treatment, and precipitated
in diethyl ether. Peptides were dissolved in water/acetonitrile
(1/1 v/v) and freeze-dried. Compounds 3′−6′ and 9′ (with
two cysteine residues in their sequences) were dissolved in
H2O/DMSO (9/1 v/v) at a 3 mM concentration and
vigorously stirred for 12h to yield cyclic oxidized peptides.
On the other hand, peptides 7′ and 8′ having 4 cysteine
residues were dissolved at 30 μM concentration in ammonium
acetate buffer (0.1 M, pH 8) and stirred vigorously for 48 h. pH
was then lowered to 4 by addition of acetic acid and the media
was finally desalted by RP chromatography. All oxidized
peptides were purified straightforwardly by preparative RP-
HPLC. They were analyzed by LC/MS (see Table 1A and
Supporting Information for spectra and chromatograms).
(BocTyr-NH-C2H4-S-)2 dimer 6 was also chosen as linear
model. It was prepared by coupling Boc-Tyr-OH with
cystamine using BOP/DIPEA activation. After extraction with
ethyl acetate and solubilization with ethanol, 6 was precipitated
by addition of water (yield 84%, purity 93%). It was analyzed
by LC/MS (see Table 1 and Supporting Information for
spectra and chromatograms).
The loading of 1 (0.42 mmol/g) was determined by
phosphorus elemental analysis (see Supporting Information).
Resin 1 was also characterized by FT-IR and compared with
starting CM (Figure 1). Characteristic vibration bands of
carbonyl bonds appeared at 1646 and 1725 cm−1 corresponding
to amide linkage and carboxylic acids. They confirmed the
linkage of TCEP on the polymer matrix. Resin 1 was stored at
room temperature with no particular precaution and was
proved to be stable to oxidation. Indeed, FT-IR was recorded
after 3 and 10 weeks and no difference was observed compared
to the freshly prepared TCEP supported polymer. In particular,
no difference in the baseline was observed at 3400 cm−1,
indicating no phosphine oxide (PO) was present.
Cyclic peptides 3, 4, and 5 (Table 1) containing two cysteine
residues linked via a disulfide bond were selected as model for
TCEP-CM reduction. It is worth noting that the free thiol
linear analogues of the model peptides (3′, 4′, 5′) could be
separated using RP-HPLC (3 min, gradient water/acetonitrile
containing 0.1% TFA on RP C18 column) allowing a
straightforward monitoring of the reduction experiment
courses. Three longer peptides of biological relevance were
also prepared, including 18-mer gomesin21 7, 17-mer
tachyplesin acid22 8, and 37-mer mesentericin23 9. It is worth
noting that peptides 7−8 display two disulfide bridges in their
sequences. Peptides 3′, 4′, 5′, and 7′were synthesized on Rink
amide PS resin and 8′ and 9′ were synthesized on 2-
chlorochlorotrityl PS resin. All the syntheses were performed
Reduction of all peptides was carried out in EtOH/H2O/
AcOH mixture at 0.1 mg/mL which correspond to very
common concentration of peptide samples for analytical or
proteomic studies (i.e., trypsin digestion). Ethanol was used
essentially to ensure a good solubility of deprotected peptides
while acidic water solution was required to quantitatively
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ACS Combinatorial Science
Letter
generate free thiol groups from the thiolate intermediate and
the supported P−S species. Other organic solvents and water
were also used with compound 6 (Table 1). TCEP-CM resin 1
was added to peptide solutions (20 eq. excess). Reactions were
performed at room temperature or under microwave
irradiation. TCEP-CM 1 was removed by a simple filtration
step. For compounds 3−6, the completeness of the reaction
was checked by LC/MS. Single ion recording chromatograms
of oxidized compounds confirmed the complete disappearance
of starting material within 5 min using microwave irradiation.
Advantageously, longer peptide sequences 7−9 were analyzed
by MALDI-TOF MS to obtain a sufficient resolution required
to distinguish clearly the isotopic pattern of the oxidized and
reduced species. Thus, as already observed for compounds 3−
6, a 5 min MW irradiation used along with TCEP-CM was
required to observe the total disappearance of the molecular ion
corresponding to oxidized species to the profit of reduced
peptide (see Supporting Information for MALDI-TOF mass
spectra).
Complete reduction of compound 6 was even quicker as it
was obtained in 20 s under microwave irradiation whatever the
solvent used. On the contrary, reductions at room temperature
proceeded slower. For example, RT reduction of compounds 3,
5, and 6 were complete after 1h but reduction of compounds 4,
7, 8, and 9 do not reach completion even after 1 h reaction.
After MW-mediated TCEP-CM reduction, reduced com-
pounds can be isolated by freeze-drying, without any
chromatography step. After reduction, the resin was recovered
and analyzed by FT-IR (Figure 1 C). As expected, a
characteristic phosphine oxide broad band at 3400 cm−1
appeared in the spectra.
Summing-up, we reported the straightforward and simple
synthesis of a new supported reagent TCEP-CM for reduction
of disulfide bridges. Immobilized on a hydrophilic polymer
matrix, TCEP-CM could be used in aqueous media, compatible
with the solubility of unprotected peptides. We showed that it
was also possible to use mixtures of organic solvents as far as
they are miscible with water, broadening the range of TCEP-
CM potential applications. Combined with microwave
irradiation, quantitative reduction occurred in 5 min, even on
the 37-mer mesentericin 9 or on peptides 7 and 8 displaying
four cysteines (gomesin and tachyplesin). Separation of free
thiol-containing compounds from the reducing agent was easily
done by simple filtration. The high loading (0.42 mmol/g) of
this resin is particularly advantageous because TCEP-CM
mediated reductions require a small quantity of solid support
related to the sample volume. TCEP-CM could also be a
particularly attractive additive to protect liquid biological
samples from oxidation.
ChemStation A.10.02 software, using a 50 mm ×3 mm C18
reversed-phase column PHENOMENEX Kinetex column.
Standard conditions were eluent system A (water/acetoni-
trile/MSA 98/2/0.1) and system B (acetonitrile/water/MSA
98/2/0.1). A flow rate of 500 μL/min and a gradient of (0−
100)% B over 5 min were used. Detection and purity
determination by peak area integration was performed at 214
nm.
LC/MS Analyses. Samples were prepared in an acetonitrile/
water mixture (50/50 v/v), containing 0.1% TFA. The LC/MS
system consisted of a Waters Alliance 2695 HPLC, coupled to a
Micromass (Manchester, U.K.) ZQ spectrometer (electrospray
ionization mode, ESI+). All the analyses were carried out using
a Merck Chromolith Speed rod C18, 25 × 4.6 mm reversed-
phase column. A flow rate of 3 mL/min and a gradient of (0−
100)% B over 3 min (or over 15 min) were used. Eluent A:
water/0.1% HCO2H; eluent B: acetonitrile/0.1% HCO2H.
Retention times (RT) are reported in minutes. Positive ion
electrospray mass spectra were acquired at a solvent flow rate of
200 μL/min. Nitrogen was used for both the nebulizing and
drying gas. The data were obtained in a scan mode ranging
from 100 to 1000 m/z in 0.1 s intervals; 10 scans were summed
up to get the final spectrum.
MALDI mass spectra were recorded on an Ultraflex III
TOF/TOF instrument (Bruker Daltonics, Wissenbourg,
France). α-Cyano-4-hydroxycinnamic acid and 2,5-dihydrox-
ybenzoic acid were used as matrix. A mixture of crude reduced
peptide and matrix solution were deposited onto the MALDI
target according to the dried droplet procedure.
RP-preparative HPLC purification was performed on a
Waters HPLC 4000 instrument, equipped with a UV detector
486 and Waters Delta-Pack 40 × 100 mm, 15 Å, 100 μm,
reversed-phase column. Standard conditions were eluent
system A (water/0.1% TFA) and eluent system B (acetoni-
trile/0.1% TFA). A flow rate of 50 mL/min and a gradient of
(10−70)% B over 30 min were used, detection 214 nm.
Solvents used for HPLC and LC/MS were of HPLC grade.
IR spectra in solid state were recorded on a FTS 575C
spectrometer Bio Rad.
ICP AES Mass Spectrometry was performed on ICAP
instrument (ThermoFisherScientific). Twenty mg of TCEP-
CM were mineralized in 6 mL of nitric acid/H2O2 (5/1 v/v)
before ICP-AES introduction. Analyses were subcontracted to
CNRS-service central d’analyses UMR 5280, Chemin du canal
69360 SOLAIZE, France.
Synthesis of Supported TCEP Resin 1. Three hundred
milligrams of 1.14 mmol/g loaded ChemMatrix resin was
swollen in DCM. TCEP·HCl (98 mg, 0.342 mmol) was
dissolved in DMF containing DIPEA (117 μL, 0.684 mmol).
HBTU in DMF was added dropwise (130 mg, 0.342 mmol),
and the mixture was allowed to react for 5 min. Then the
mixture was put onto the resin under mechanical stirring for 24
h. Resin was washed with 3 × 10 mL DMF (3 min), 1 × 10 mL
HCl 1 M (3 min), 1 × 10 mL methanol (3 min), 1 × 10 mL
Et2O (3 min), 2 × 10 mL DCM (3 min) and finally dried under
vacuum. About 320 mg of resin 1 was obtained. The loading
was calculated from data obtained by phosphorus elemental
analysis performed by inductively coupled plasma-atomic
emission spectrometry (ICP-AES). 1.30% (weight) of
phosphorus was detected. The loading of TCEP-CM 1 was
determined as 0.42 mmol/g. (See calculations in Supporting
Information.)
EXPERIMENTAL PROCEDURES
■
Analytical Methods. HPLC analyses were performed on a
Beckman Gold apparatus for compounds 3−6 and 3′−6′ and
on a Agilent 1100serie apparatus for peptides 7−9. Beckman
Gold apparatus is composed of the 126-solvent module, the
168 detector, and the 32 Karat software, using a 50 mm ×3.9
mm a C18 reversed-phase column VWR chromolith column.
Standard conditions were eluent system A (water/0.1% TFA)
and system B (acetonitrile/0.1% TFA). A flow rate of 5 mL/
min and a gradient of (0−100)% B over 3 min were used. The
Agilent 1100 series apparatus is composed of the G137
degasser, the G1376A capillary pump, the μ-WPS G1377A
injection system, the MWD G1365B detector, and the
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General Protocol for Reduction of Disulfide Bonds in
Peptides. Five milligrams of TCEP-supported reagent 1 (2.1
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ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed SPPS protocols, loading calculation, LC chromato-
grams, and mass spectra. This information is available free of
charge via the Internet at http://pubs.acs.org/.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +33 4 11 75 96 06. E-mail: gilles.subra@univ-montp1.
fr.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
ACN, acetonitrile; AcOH, acetic acid; BOP, benzotriazole-1-yl-
oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate;
CM, ChemMatrix; DIPEA, N,N-diisopropylethylamine; DTT,
dithiothreitol; DMF, dimethylformamide; EDT, ethanedithiol;
EtOH, ethanol; Fmoc, fluorenylmethyloxycarbonyl; HBTU, O-
benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phos-
phate; MSA, methanesulfonic acid; MW, microwave; NCL,
native chemical ligation; NMP, N-methyl-2-pyrolidone; PEG,
polyethylene glycol; TCEP, tris(2-carboxyethyl)phosphine; RT,
retention time; RP-HPLC, reverse-phase high-performance
liquid chromatography; SPPS, solid-phase peptide synthesis
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on solid-phase chemistry. J. Comb. Chem. 2000, 3, 9−15.
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and protocols of modern solid-phase peptide synthesis. Mol. Biotechnol.
2006, 33, 239−254.
(20) García-Ramos, Y.; Paradís-Bas, M.; Tulla-Puche, J.; Albericio, F.
ChemMatrix for complex peptides and combinatorial chemistry. J.
Pept. Sci. 2010, 16, 675−678.
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