Oxyfold: A simple and efficient solid-supported reagent for disulfide bond formation

Article abstract of DOI:10.1002/asia.201100202

The synthesis and use of novel polymer-supported reagents for disulfide bond formation is described. This family of supported reagents consists of a series of oxidized methionines grafted onto a solid support. Their cost and the simplicity of their preparation through N-carboxyanhydride polymerization on beads make them reactants of choice for the formation of disulfide bridges in peptides. Copyright

Full text of DOI:10.1002/asia.201100202

FULL PAPERS
DOI: 10.1002/asia.201100202
Oxyfold: A Simple and Efficient Solid-Supported Reagent for Disulfide Bond
Formation
Pascal Verdiꢀ,[a] Luisa Ronga,[b] Michꢁle Cristau,[a, b] Muriel Amblard,[a] Sonia Cantel,[a]
Christine Enjalbal,[a] Karine Puget,[b] Jean Martinez,*[a] and Gilles Subra*[a]
Abstract: The synthesis and use of novel polymer-supported reagents for disulfide
bond formation is described. This family of supported reagents consists of a series
of oxidized methionines grafted onto a solid support. Their cost and the simplicity
of their preparation through N-carboxyanhydride polymerization on beads make
them reactants of choice for the formation of disulfide bridges in peptides.
Keywords: amino acids · methio-
nine oxidation peptides
supported reagents
·
·
·
Introduction
antagonist activity and improving selectivity or enzymatic
stability. Formation of disulfide bonds is probably one of the
most challenging steps to achieve, regarding the formation
of unwanted byproducts (i.e. oxidation of Met, Trp, and Tyr
residues) and oligomerization. To minimize the latter phe-
nomenon, compounds to be cyclized are highly diluted in
aqueous buffer or organic solution and require time-con-
suming removal of solvent at the end of the intramolecular
reaction. This is particularly problematic for parallel synthe-
sis. Dimethyl sulfoxide (DMSO) is one of the oxidizing reac-
tants most commonly used for the disulfide bond formation.
This solvent has the advantage of being water-soluble. One
of its major drawbacks resides in its elimination from the re-
action medium, which requires evaporation under strong
vacuum or repeated lyophilizations. Furthermore, the di-
methyl sulfide generated during the reaction is volatile and
toxic.
On the other hand, the use of supported reactants is fast
expanding in organic chemistry, in particular, since the auto-
mation and parallelization of organic synthesis. Supported
reactants are particularly advantageous for promoting intra-
molecular reactions. In fact, they are described to cause a
“pseudodilution” phenomenon, which makes it possible to
minimize oligomerization and to use smaller amounts of sol-
vents. The use of supported oxidation reactants has subse-
quently been considered for the conventional synthesis of
cyclic peptides, but also for the automated synthesis of
chemical libraries of peptides bearing disulfide bridges.
They exhibit numerous advantages:
The synthesis of peptides today is highly automated, elonga-
tion of the peptide chain being generally carried out on
solid support, even at the industrial scale.[1] However, some
treatment steps are carried out in solution, after cleavage of
the peptide chain from the solid support and purification.
This is the common procedure used for the formation of di-
sulfide bridges. Many marketed peptide drugs possess one
or several disulfide bridges, that is, Vasopressin (antidiuret-
ic),[2] Octreotide (growth hormone inhibitor),[3] Lanreotide
(antiplatelet),[4] Ziconotide (anestesic),[5] and Oxytocin
(labor inducer).[6,7] Disulfide bridges greatly contribute to
establish the tertiary bioactive structure of peptides and ana-
logues. Moreover, during SAR studies or pharmacological
optimization of peptides and pseudopeptides, disulfide cycli-
zation between cysteine residues is a common strategy to in-
troduce topological constraints, thus modulating agonist or
[a] Dr. P. Verdiꢀ,+ Dr. M. Cristau, Dr. M. Amblard, Dr. S. Cantel,
Prof. C. Enjalbal, Prof. J. Martinez, Dr. G. Subra
Institut des Biomolꢀcules Max Mousseron IBMM
UMR CNRS 5247
15 avenue Charles Flahault 34000 Montpellier (France)
Fax : (+33)4-67548654
E-mail: gilles.subra@univ-montp1.fr
[b] Dr. L. Ronga,+ Dr. M. Cristau, Dr. K. Puget
Genepep, Domaine des Pins, 48 Impasse des Eglantiers
34980 Saint Clꢀment de Riviꢁre (France)
[+] Equal contribution.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201100202. It contains ESI+ mass
spectrometry data of peptides, calculation of methionine oxide units,
and MALDI-TOF MS spectra of methionine oligomers.
1) The formation of disulfide bridges is an intramolecular
reaction, these supported reactants avoid having to work
under very dilute conditions, thereby minimizing the
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Chem. Asian J. 2011, 6, 2382 – 2389

volume of solvent and allowing miniaturization and use
of robot wells with a volume that is often small.
2) The supported reactants allow the disulfide bridges in
peptides and proteins to be obtained under very mild
and simple conditions without the use of toxic, foul-
smelling, or volatile oxidants.
3) The supported oxidation reactants can be easily eliminat-
ed and do not require any lyophilization or evaporation,
which greatly facilitates automation.
Results and Discussion
In this study, a new family of supported reagents for the for-
mation of disulfide bonds is presented. These supported re-
agents consist of a series of oxidized methionines (sulfoxide
or sulfone derivatives) grafted onto a solid support used to
mimic DMSO in solution.[10,11] Methionine residues can be
introduced easily by using a stepwise solid-phase stepwise
synthesis (SPPS) method or, more interestingly, by oligome-
rization of N-carboxyanhydride (NCA) derivatives initiated
by amino groups on solid support.
4) The supported oxidation reactants can be recycled and
reoxidized after use.
Very few supported oxidation reactants, which are effec-
tive for the formation of disulfide bridges, have been de-
scribed to date.
Supported Methionine Sulfoxide as the Oxidizing Reagent
As a proof of concept, we first demonstrated that methio-
nine sulfoxide could promote oxidation. Fluorenylmethoxy-
carbonyl (Fmoc)-protected methionine was anchored on dif-
ferent matrices including amino poly(ethyleneglycol)polya-
crylamide (PEGA; 0.29 mmolgÀ1), amino poly(ethylenegly-
col)polystyrene (PEG-PS; 0.24 mmolgÀ1), and aminomethyl
polystyrene (0.81 mmolgÀ1) by using standard benzotriazole-
1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophos-
phate (BOP)/diisopropylethylamine (DIEA) activation.
After Fmoc deprotection and acetylation, oxidation was per-
formed by using 35% oxygen peroxide for 48 h, yielding
resins 4a–c, respectively (Scheme 1).[12]
2-(Methylsulfinyl)acetic acid, fixed on polyethylene
glycol-polystyrene (PEG-PS), is a good DMSO substitute
for the synthesis of ketones by the Swern oxidation reaction,
but has not been used successfully for the formation of di-
sulfide bridges. Only the Ellman reagent[8] (5,5’-dithiobis(2-
nitrobenzoic) acid or DTNB), developed initially for meas-
uring the concentration of free thiols, has been adapted for
the formation of disulfide bridges and is commercially avail-
able.[9] It remains, however, expensive to manufacture.
We proposed an oxidation mechanism involving methio-
nine S-oxide (Scheme 2) similar to the known mechanism of
disulfide formation by DMSO, which is not dependent on
Scheme 1. Preparation of Ac-Met[O]-resins.
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G. Subra et al.
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Scheme 2. Proposed mechanism for intramolecular disulfide formation mediated by supported methionine S-oxide.
pH values between three and eight.[13,14] This mechanism re-
quires protonation of sulfoxide to yield an unstable adduct
that is rapidly consumed by a thiolate ion to give the disul-
fide. The protonation favored by acidic pH is counterbal-
anced by the decrease in thiolate formation, leading to an
overall pH-independent oxidation. On the contrary, it has
been demonstrated that Ellmanꢃs mediated oxidation is
more efficient at high pH values (pHꢀ6) due to the stabili-
zation of the thiolate nitrobenzoic leaving group formed
during disulfide exchange.[15]
(600 rpm) for 24 hours and aliquots of 40 mL were diluted to
200 mL with a CH3CN/H2O (50:50) solution and then fil-
tered. They were immediately subjected to LCMS analyses.
The percentage of oxidized peptide was calculated as a func-
tion of the areas of the chromatographic peaks obtained at
214 nm (excluding the DMSO peak but taking into account
the possible presence of byproducts) and reported as a func-
tion of time (Figure 1).
In a first instance, the heptapeptide 6 of sequence H-
LCAGPCL-NH2 was chosen as a model for studying disul-
fide bond formation. It was synthesized on solid support,
cleaved from the solid support, purified, and finally oxidized
with different reagents (Table 1). It was dissolved in phos-
Table 1. Oxidation of peptide 6 (H-LCAGPCL-NH2).
Entry Oxidant
Calculated
Resin
Equivalent relat-
resin loading weight
ed to peptide 6
[mmolgÀ1
0.27
]
[mg]
1
2
4a AcMet[O]-
PEGA
4b AcMet[O]-
PEG-PS
111
5
5
0.26
115
Figure 1. Oxidation of peptide 6 (H-LCAGPCL-NH2). ꢄ: 10% DMSO;
~
&
^
: resin 4a (PEGA); : resin 4b (PEG-PS); : resin 4c (PS).
3
4
4c AcMet[O]PS
0.7
n.a.
43
5
471
DMSO 10%[a]
n.a.[b]
[a] The DMSO solution was prepared with phosphate buffer, pH 7.5.
[b] 200 mL, 221 mg, 2.83 mmol. n.a.=not applicable.
The best results were obtained with resin 4a. Oxidation
promoted by resin 4b (PEG-PS matrix) was slightly slower.
As expected, PS resin 4c was much less efficient, probably
due to the poor swelling properties of hydrophobic polystyr-
ene beads in aqueous media, but yielded similar results to
those obtained with DMSO-mediated oxidation. After
48 hours, peptide 6 was completely oxidized whatever sup-
ported reactant (4a–c) was used. Interestingly, no dimer was
observed when resins 4a–c were used. One explanation
could be proposed to explain such results. An unstable sulf-
oxide adduct was formed that underwent a rapid attack by
the sulfhydryl group, followed by a subsequent dehydrata-
tion, thereby resulting in an almost impossible on-resin di-
merization (Scheme 2). According to the first set of experi-
ments, PEGA matrix was selected for the continuation of
the study.
phate buffer (pH 7.5) to give a 3 mm solution (2.0 mgmLÀ1).
Phosphate buffer is commonly used to dissolve peptides[16]
at physiological pH. A concentration of 3 mm was slightly
higher than what was already described (0.7–2 mm)[15] for
mediated disulfide bond formation by other supported re-
agents, such as supported Ellmanꢃs reagent. Two milliliters
of the peptide solution was distributed in Bohdan miniblok
syringes containing different supported oxidation reagent
resins 4a–c (Table 1, entries 1–3). Supported oxidation reac-
tants were used at five equivalents relative to the amount of
peptide to be oxidized. Finally, peptide 6 was dissolved in
phosphate buffer containing 10% of DMSO. All the experi-
ments were carried out in the presence of air at room tem-
perature. The reactors were placed on an orbital shaker
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A Solid-Supported Reagent for Disulfide Bond Formation
Table 2. Comparison of oxidation of peptides 6–11 with DMSO and resin 4a.
lution was used (3–11%). This
side reaction was not detected
in samples oxidized with resin
4a, even after 24 hours. No
undesired oxidation was de-
tected in peptide 7 containing
tryptophan or in peptide 8
containing methionine. Lastly,
to check the putative influ-
ence of the polymer matrix on
oxidation, a solution of pep-
tide 9 was stirred with or with-
out 110 mg of acetylated
amino-PEGA resin (bearing
no methionine sulfoxide).
After 24 hours, no significant
Peptide Sequence
Oxidant
4 h
24 h
% oxidized (% dimer) % oxidized (% dimer)
resin 4a
57
40
15
3
57
48
47
44
100
90
78
100
20
80
60
100
91
100
97 (3)
100
89 (11)
6
H-LCAGPCL-NH2
DMSO 10%[a]
resin 4a
7
H-WCAGNCL-NH2
H-MCAGMCL-NH2
DMSO 10%[a]
resin 4a
8
DMSO 10%[a]
Crustacean Cardioactive Peptide resin 4a
9
H-PFCNAFTGC-NH2
Vasopressin
H-CYFQNCPRG-NH2
Brain Binding Peptide
H-CLSSRLDAC-NH2
DMSO 10%[a]
resin 4a
10
11
DMSO 10%[a]
resin 4a
97 (3)
100
90 (10)
DMSO 10%[a]
[a] The DMSO solution was prepared with phosphate buffer, pH 7.5.
Parallel Oxidation Experiments
difference could be found in the disulfide bond formation
(60 and 62% respectively).
Five other peptides 7–11 (Table 2) were synthesized by
Fmoc SPPS and cleaved from the support by using appropri-
ate trifluoroacetic acid (TFA)/scavengers treatment. Model
peptides were chosen with a variable number of residues in
between the cysteine residues ranging from three to seven.
Cysteine residues were positioned at the C terminus, N ter-
minus, and within the peptide sequence. These peptides
were purified by preparative LCMS by using the mono-
charged ion to trigger fraction collection.[17] Even when the
crude purity after cleavage was satisfactory (>90% deter-
mined by HPLC), we chose to use pure peptides to be able
to detect potential small amounts of undesirable side prod-
ucts (dimers, oxidized side chains) during disulfide bridge
formations. Crustacean Cardioactive Peptide (CCAP),[18]
Vasopressin,[2] and Brain Binding Peptide[19] (BBP) were se-
lected as models. Moreover, to verify the effects of oxyfold
resins on methionine or tryptophan residues, peptides 7 and
8, analogues of model peptide 6, were prepared. Indeed, oxi-
dation of tryptophan and methionine has already been re-
ported during disulfide bond formation.[20] It is worth noting
that the retention time of the oxidized form of these methio-
nine and tryptophan-containing peptides was different
enough from the reduced form in our LCMS analysis condi-
tions. Purified peptides were dissolved in phosphate buffer
pH 7.5 (2 mL, 3 mm) and were shaken for 24 hours in
Bohdan miniblock syringes with five equivalents of resin 4a
(Ac-Met[O]-PEGA, 111 mg). In parallel, the same peptides
were dissolved in 10% DMSO as a control. An aliquot of
each solution was submitted to LCMS analysis after 4 hours
of stirring. Results are present-
Increasing the Number of Methionine Sulfoxide
To reduce the volume of resin required for disulfide bond
formation, we increased the number of methionine S-oxide
motifs on the resin by preparing a polymethionine grafted
onto the resin. We hypothesized that the oxidation rate was
only dependent on the number of methionine S-oxide resi-
dues on the support. To that purpose, the protocol used for
preparation of resins 4 was used, but couplings, washings,
and deprotection steps a to c (Scheme 1) were repeated sev-
eral times to yield resins 5 after H2O2 (35%) treatment. The
initial loading of amino PEGA resin used for the syntheses
of resins of type 5 was 0.40 mmolgÀ1.
CCAP (peptide 9) was dissolved in 3 mm (2 mL, 6 mmol
per sample) phosphate buffer at pH 7.5 and oxidized with
different resins (Table 3, entries 1–7) for 72 hours. Equiva-
lents of oxidant were calculated by taking into account the
calculated resin loading and the number of methionine S-
oxide units. Resin 4a was used at 0.1, one, and five equiva-
lents and resins 5a–c were used at five equivalents of oxi-
dant relative to peptide 9. Aliquots of 40 mL were subjected
to LCMS analysis. Results are presented in Figure 2. No sig-
nificant difference could be pointed out while using differ-
ent polymethionine S-oxide chain lengths as far as the same
overall quantity of oxidant (i.e. equivalents of methionine
oxide related to the peptide to be oxidized) was used.
Indeed, when using Ac-[Met(O)]n-PEGA resins with n=
ed in Table 2 and highlighted
Table 3. Oxidation of Peptide 9 (H-PFCNAFTGC-NH2).
that cyclization by the disulfide
bond is highly sequence-de-
pendent. Resin 4a was more
efficient than DMSO to pro-
mote disulfide bond formation
in all cases. Moreover, dimeri-
zation was observed in peptide
10 and in a larger amount in
peptide 11 when DMSO in so-
Entry Oxidant
Calculated resin loading
Resin weight
[mg]
Equivalent(s) related to
peptide 9
[mmolgÀ1
]
1
2
3
4
5
6
7
5a Ac
5b AcAHCTNUGTRENNNUG
G
0.33
0.31
0.28
0.28
0.27
0.27
0.27
30.3
19.3
15.3
46
2.2
22
5
5
5
15
0.1
1
5c Ac
5c Ac
U
4a AcMet[O]PEGA
4a AcMet[O]PEGA
4a AcMet[O]PEGA
110
5
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G. Subra et al.
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using longer polymethionine S-oxide peptide chains (i.e.
resin 5c), the amount of supported reagent could be re-
duced to roughly 10 mg per mL of free thiol peptide solu-
tion. This is particularly convenient for parallel oxidation
experiments in automated synthesizers with small well vol-
umes.
Resins 5 bearing oligomers of methionine S-oxide resi-
dues gave interesting results and allowed us to decrease the
amount of resin used relative to resin 4a. However their
preparation required repeated cycles of Fmoc SPPS-con-
suming protected amino acids, coupling reagents, and labor
time. With the purpose to find less expensive solid supports,
we explored a different strategy based on random short me-
thionine polymers, obtained by on-support polymerization
of methionine NCA.
*
Figure 2. Oxidation of peptide 9 (CCAP) : resin 4a (0.1 equiv); +: resin
~
&
^
4a (1 equiv); : resin 4a (5 equiv); : resin 5a (5 equiv); : resin 5b
(5 equiv); ꢄ: resin 5c (5 equiv); <*?>: resin 5c (15 equiv). See also the
Supporting Information S18 for enlarged figure.
NCA of methionine was prepared from H-Met-OH and
bis(trichloromethyl)carbonate (BTC).[21] Met-NCA 12
(Scheme 3) was poured into a suspension of amino PEGA
resin (0.4 mmolgÀ1) in anhydrous THF. Depending on the
number of equivalents used and on the reaction time, the
length of the polymer could be modulated. Under optimized
conditions, a 50-fold excess of Met-NCA relative to free
amino groups of PEGA resin was stirred during one hour in
anhydrous THF and under an argon atmosphere to yield
resin 13b. In parallel, the same reaction was carried out on
Rink amide PEGA resin (0.36 mmolgÀ1)[22] to yield 13a.
The polymer matrix of 13a was the same as 13b (PEGA).
Beads of 13a were functionalized with Rink amide linker
that enabled the release of methionine oligomers upon TFA
1,3,5, or 7 methionines per peptide (Table 3, entries 1–3 and
7) with five equivalents in all experiments, oxidation
reached completion after 26 hours. These data validated our
hypothesis that oxidation was closely linked to the overall
number of methionine molecules present on the solid sup-
port and not on the way they were distributed within the
polymer matrix. When only 0.1 or one equivalent of resin
4a was used, oxidation was much slower, thus yielding 48
and 61% oxidized CCAP, respectively, after 26 hours stir-
ring.
Interestingly, increasing the number of equivalents of
resin 5c from 5 to 15 (Table 3, entries 3 and 4, and Figure 2)
did not yield significant improvement in kinetics. When
Scheme 3. Preparation of Ac-polyMet[O] resin 16 by using Met-NCA 12. TIS=triisopropylsilane.
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A Solid-Supported Reagent for Disulfide Bond Formation
treatment. This experiment was designed to evaluate the
degree of polymerization obtained by NCA polymerization
initiated by the amino groups on the solid support. Indeed,
13a was cleaved and after concentration of the TFA cocktail
in vacuo, the mixture of methionine oligomers 14 was ana-
lyzed by MALDI-TOF mass spectrometry. Protonated and
sodated species of H-(Met)x-NH2 peptides, separated by
131 u (u=unified atomic mass unit), were detected. The
length of detected oligomers was comprised between four to
fourteen residues (see Figure S13 in the Supporting Infor-
mation). Resin 13b was treated with acetic anhydride in di-
chloromethane and after washings with H2O2 (35%) for
48 hours yielded resin 16. Elemental analysis performed on
an aliquot of resin 16 indicated 6.2% of sulfur. This value
corresponds to an average length of 6.9 methionine sulfox-
ide residues per amino function of the starting resin.[23]
Oxidation Yield
Along with the efficiency of disulfide bond formation, a
very important aspect was the yield of recovery of the oxi-
dized peptide. When buffers “easy” to lyophilize (i.e. ammo-
nium acetate) are used to solubilize the free sulfhydril pep-
tide, three or four sequential lyophilizations are required to
remove the salts. When needed, oxidized peptides are puri-
fied by RP-HPLC and finally weighted.
Surprisingly, very low yields are reported in the literature
when supported oxidants are used. As an example, oxidation
of reduced somatostatin with the supported Ellmanꢃs re-
agent yielded somatostatin (disulfide) in an overall yield of
30% (without including the purification process).[15] More
recently, urotensin analogues were oxidized with immobi-
lized supported Ellmanꢃs reagent.[9] The oxidation proceed-
ed quickly but low yields of oxidized peptide were recov-
ered (5.4% for urotensin II and 19.2% for the urotensin an-
tagonist). In the elegant pioneering work of Annis et al., the
authors explain that a significant amount of peptide remains
covalently trapped inside the resin beads. This is the result
of the mechanism of oxidation based on disulfide exchange
that involves a “stable” intermediate linked to the resin. In
the case of oxyfold, the proposed mechanism (Scheme 2)
did not proceed through this “stable” disulfide intermediate.
To check this hypothesis, 30 mg (38 mmol) of the crude TFA
salt of peptide 6 (99% purity) were dissolved in phosphate
buffer (10 mL) and acetonitrile (5 mL). Oxyfold resin 16
(113 mg, 5 equiv) was added to the solution and was mixed
for 48 hours. Completion of the disulfide bond was checked
by LCMS analysis (Figure 4).
Five equivalents of resin 16 were used to promote disul-
fide bond formation in peptides 6 and 7. Equivalents were
calculated by taking into account the calculated resin load-
ing (0.22 mmolgÀ1) and the average number of methionine
S-oxide units determined by elemental analysis (6.9). Re-
sults are presented in Figure 3. For both peptides, resin pre-
The resin was filtered, washed three times with water/ace-
tronitrile (10 mL, 1:1 v/v), and the solution was lyophilized.
A white powder (338 mg) corresponding to the mixture of
phosphate salts and oxidized peptide was obtained. The
same experiment was performed without peptide and phos-
phate salt (308 mg) was obtained. The weight difference be-
tween the two experiments (30 mg) indicated that the oxi-
dized peptide was quantitatively recovered. The crude pep-
tide/phosphate salt mixture was then purified by RP-HPLC.
Pure oxidized peptide 6 (17 mg) as the TFA salt was ob-
Figure 3. Oxidation of peptide 6 (left) and 7 (right) ꢄ: resin 16 (5 equiv);
^
: resin 5c (5 equiv).
pared by polymerization of Met-NCA followed by oxidation
proved to be at least as efficient as Ac-Met[O]7-PEGA.
Resin 16 was easier to prepare (two synthetic steps) than
resin 5c, which required fifteen SPPS steps. Moreover, it
opened the way for large-scale economic preparation of sup-
ported reactants to promote disulfide bonds in peptides.
Figure 4. LC monitoring oxidation of the crude peptide 6.
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G. Subra et al.
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LCMS analysis
tained, which indicated that the overall yield of disulfide
bond formation, including purification, was 57%, that is,
twice the values reported in the literature.[9,15]
Samples were prepared in an acetonitrile/water (50:50 v/v) mixture con-
taining 0.1% TFA. The LCMS system consisted of a Waters Alliance
2695 HPLC, coupled to a Micromass (Manchester, UK) ZQ spectrometer
(electrospray ionization mode, ESI+). All the analyses were carried out
by using
a Chromolith flash 25ꢄ4.6 mm column. A flow rate of
Conclusions
3 mLminÀ1 and a gradient of 0–100% B over 2.5 min were used. Eluen-
t A: water/0.1% HCO2H; eluent B: acetonitrile/0.1% HCO2H. Positive-
ion electrospray mass spectra were acquired at a solvent flow rate of
200 mLminÀ1. Nitrogen was used for both the nebulizing and as a 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 acquire the final spec-
trum.
In this study, we have demonstrated the efficiency of sup-
ported methionine sulfoxide to promote disulfide bond for-
mation in peptides. The hydrophilic PEGA was the most ef-
ficient tested solid support. Kinetics were dependent on the
quantity of methionine oxide units grafted onto the solid
support. However, it was also demonstrated that the
number of methionine sulfoxide units could be increased by
preparing methionine oligomers, thus enhancing the number
of oxidized methionines per gram of resin and consequently
reducing the quantity of resin to be used in each reaction
vessel (i.e. 10 mgmLÀ1 of Ac-Met[O]7-PEGA). Random oli-
gomerization initiated by amino groups on solid support
could be performed with methionine NCA to yield easily a
supported reagent as efficient as the oligomers prepared by
conventional stepwise peptide synthesis.
LCMS purification
Samples were prepared in an acetonitrile/water (50:50 v/v) mixture, con-
taining 0.1% TFA. The LCMS autopurification system consisted of a
Waters 2525 binary pump, a Waters 2676 injector/fraction collector, cou-
pled to a Waters Micromass ZQ spectrometer (electrospray ionization
mode, ESI+). All the purifications were carried out by using a reverse-
phase XBridge Prep C18 19ꢄ100 mm, 5 mm OBD particle size column. A
flow rate of 20 mLminÀ1 and a gradient of 0–60% B over 20 min were
used. Eluent A: water/0.1% TFA; eluent B: acetonitrile/0.1% TFA. Posi-
tive-ion electrospray mass spectra were acquired at a solvent flow rate of
204 mLminÀ1. Nitrogen was used for both the nebulizing and as a 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 acquire the final spec-
trum. Collection control trigger is set on single protonated and diproto-
nated ion with a MIT (minimum intensity threshold) of 8ꢄ105.
Experimental Section
Preparation of Met NCA (13)
Materials
Methionine (5 g, 33.51 mmol) was dissolved in dry freshly distilled THF
(120 mL) and the mixture was kept under argon in a three-neck round-
bottomed flask flushed with argon. BTC (3.31 g, 11.17 mmol) was added
under argon and the reaction vessel was washed with dry THF (5 mL).
The reaction mixture was stirred under reflux for 3 h while the medium
turned into a limpid solution. The reaction was cooled to room tempera-
ture. Ethyl acetate (100 mL) was added and the organic phase was quick-
ly washed three times with a 0.5% NaHCO3 aqueous solution (100 mL).
The organic layer was dried over anhydrous MgSO4 and the solvent was
removed in vacuo. A clear oil was obtained (5.57 g, 95% yield). NCA
was used without further treatment for polymerization experiments.
1H NMR (300 MHz, CDCl3): d=1.98 (s, 3H; CH3), 2.09 (m, 2H; CH2b),
2.52 (t, 2H; CH2g), 4.4 (t, 1H; CHa), 7.19 ppm (s, 1H; NH); 13C NMR
(75 MHz, CDCl3): d=15.04 (CH3), 29.34 (CH2 g), 29.94 (CH2 b), 56.40
(CH a), 152.88 (CO urethane), 170.20 ppm (CO anhydride).
All the solvents were obtained from Acros and were used without purifi-
cation. Protected amino acids O-(1H-benzotriazol-1-yl)-1,1,3,3-tetrame-
thyluronium hexafluorophosphate (HBTU) were purchased from Iris Bi-
otechn GmbH. Other reagents were purchased from Aldrich and Lancas-
ter.
Peptide synthesis
The peptide chains were elongated by means of a Liberty Microwave
Peptide Synthesizer (CEM Corporation, Matthews, NC), an additional
module of Discover (CEM Corporation, Matthews, NC) that combines
microwave energy at 2450 MHz to SPPS by following the Fmoc/tBu
strategy.[24]
Syntheses were conducted on a 0.1 mmol scale on Fmoc-Rink-amide PS
resin. All coupling reactions were performed with 5 equivalents of amino
acid in N,N-dimethylformamide (DMF; 0.2m), 5 equivalents of HBTU in
DMF (0.5m), and 10 equivalents of N,N-diisopropylethylamine (DIPEA)
in N-methyl-2-pyrrolidone (NMP) solution (2m).
Acknowledgements
Fmoc deprotections were performed with a 20% piperidine DMF solu-
tion.
This work was partly founded by the contract A0907015J - APPI pro-
gram; Osꢀo Innovation and La Rꢀgion Languedoc Roussillon.
Each deprotection and coupling reaction was performed with microwave
energy and nitrogen bubbling. Microwave cycle was characterized by two
deprotection steps; the first one was for 30 s, the second one for 180 s.
All coupling reactions were for 300 s.
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Washing steps were performed between coupling and deprotection steps.
Three washes of DMF (7 mL) were used between steps.
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Cleavage of Rink amide-PS resin[25]
Peptides were cleaved from resin during 2 h by using cleavage cocktail
(15 mL, trifluoroacetic acid/water/triisopropylsilane/EDT 94:2:2:2 v/v/v/
v) per well. After removal of the resin by filtration, cleavage cocktail was
removed in a Genevac EZ-2+ solvent centrifugal evaporator.
Compounds were precipitated three times in diethyl ether, dissolved in
an acetonitrile/water 50:50 solution containing 0.1% TFA, and freeze
dried.
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ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 2382 – 2389

A Solid-Supported Reagent for Disulfide Bond Formation
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[22] Rink amide PEGA resin (0.36 mmolgÀ1) was prepared by coupling
Fmoc-Rink amide linker on amino PEGA resin and then by remov-
al of Fmoc protection by DMF/pip treatment.
[23] <>Calculations are presented in the Supporting Information.
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[12] Under these conditions, sulfoxide was the major compound obtained
over sulfone. This was checked by direct infusion ESIMS analysis of
tri, penta, and hepta methionine oligomers cleaved from Rink
amide PEGA resin, previously treated with hydrogen peroxide.
Spectra are presented in the Supporting Information S16–S18.
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Received: February 25, 2011
Published online: June 7, 2011
Chem. Asian J. 2011, 6, 2382 – 2389
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
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