Enhanced Microwave Assisted Method for
On-Bead Disulfide Bond Formation. Synthesis
of –Conotoxin MII
Athanassios S. Galanis1,2, Fernando Albericio2, Morten Grøtli1
Department of Chemistry, Medicinal Chemistry, University of Gothenburg, SE-41296
Göteborg, Sweden1; Institute for Research in Biomedicine, Barcelona Science Park,
08028 Barcelona, Spain, and Department of Organic Chemistry, University of
Barcelona, 08028 Barcelona, Spain2
Supporting Information
Abbreviations…….………………………………………………………………
S2
Experimental Procedures
Linear precursors synthesis using classical SPPS.................................................
S3
Linear precursors synthesis using microwave assisted heating..........................
S4
DMSO oxidation in solution (method 1).............................................................
S5
Iodine oxidation in solution (method 1)..............................................................
S5
Air oxidation on-bead (method 2a &b).............................................................
S5
Displacement cyclization (method 2a &b)……………………………………..
S6
Iodine oxidation on-bead (method 2a)..................................................................
S8
Iodine oxidation in solution (method 2b)............................................................
S9
Analytical data of synthesized peptides….............................................................
S10
HPLC chromatograms of synthesized peptides….................................................
S12
Mass spectra of synthesized peptides…................................................................. S14
S1
ABBREVIATIONS
Abbreviations used for amino acids and the designations of peptides follow the rules
of the IUPAC-IUB Commission of Biochemical Nomenclature in J. Biol. Chem.
1982, 247, 977983. The following additional abbreviations are used: αCtxMII,
alpha-Conotoxin MII; nAChRs, nicotinic acetylcholine receptor; DTNP, 2,2’dithiobis(5-nitropyridine); Npys; 3-nitro-2-pyridylthio; RAM, rink amide linker; TG,
TentaGel resin; PS, polystyrene resin; HOBt, 1-hydroxybenzotriazole; DIC, N,N’diisopropylcarbodiimide,
TBTU;
1-[bis(dimethylamino)methylene]-1H-
benzotriazolium tetrafluoroborate 3-oxide; DIEA, N,N-diisopropylethylamine; TFA,
trifluoracetic acid; DMF, N,N-dimethylformamide; DCM, dichloromethane; NMP, 1Methyl-2-pyrrolidinone; DMSO, dimethylsulfoxide; Et3N, triethylamine; m.w.,
microwave; MeCN, acetonitrile; ESI-MS, electrospray mass spectrometry; RP-HPLC,
reverse phase - high performance liquid chromatography; Trt, trityl; tBu, tert-Butyl.
Amino acid symbols denote L-configuration. All reported solvent ratios are expressed
as v/v.
S2
Experimental Procedures
Synthesis of the linear precursors by classical SPPS.
Rink amide pre-attached Tenta Gel (500 mg, loading: 0.24 mmol/g) or Polystyrene
(280 mg, loading: 0.43 mmol/g) resins were placed in a 3 mL-polypropylene syringe
fitted with a polyethylene filter disc. The resin was then washed with DMF (3x).
Fmoc-Cys(Acm)-OH (129.2mg, 0.36 mmol) for peptides 11 and 12 or FmocCys(Mmt)-OH (221.6 mg, 0.36 mmol) for peptides 21 and 22 were dissolved in ~ 1ml
DMF and HOBt (54 mg, 0.36 mmol) was added. The mixture was cooled down in ice
bath, DIC reagent (59 L, 0.36 mmol) was added and was stirred for 5 min in the ice
bath and then for 5 min more at r.t.. The pre-activated derivatives were added to the
syringe containing the resin in order to react for 1.5 h at r.t. Next, the resin was treated
as follows: filtration to remove the excess of amino acid derivative and the coupling
reagents, with DMF (5x) and DCM (5x). After filtration, Kaiser and chloranil test
indicated the completion of the coupling reaction. In case of not completed reaction
the coupling step was repeated as before. The resin was further washed and swelled
with DMF (5x) and then was treated with 20% piperidine in DMF (1 x 2 min, 1 x 10
min, 1 x 3 min). Those steps were repeated for each amino acid derivative for the
elongation and completion of the linear peptide sequence on resin. Coupling reactions
were performed for 1 to 2 h for each amino acid derivative. The quantities of coupling
reagents were the same as the first coupling. The quantities of derivatives were:
Fmoc-Leu-OH: 127.2 mg; Fmoc-Asn(Trt)-ΟΗ: 214.8 mg; Fmoc-Ser(tBu)-ΟΗ: 138.0
mg; Fmoc-His(Trt)-ΟΗ: 223.1 mg; Fmoc-Glu(tBu)-ΟΗ: 153.2 mg; Fmoc-Val-ΟΗ:
122.2 mg; Fmoc-Pro-ΟΗ: 121.9 mg and Fmoc-Gly-ΟΗ: 107.0 mg. According to
positive Kaiser and chloranil test the couplings that were repeated were for Ser(tBu)13,
His(Trt)12, Glu(tBu)11, Pro6 for all synthesized peptides and Cys(Mmt)3 for peptides
S3
21
and
22
(H-Gly1-Cys2-Cys3-Ser4-Asn5-Pro6-Val7-Cys8-His9-Leu10-Glu11-His12-
Ser13-Asn14-Leu15-Cys16-NH2). The re-coupling reactions were performed with TBTU
(115.6 mg, 0.36 mmol) reagent in addition of DIEA (125 μl, 0.72 mmol), without preactivation, except of Cys(Mmt) that was re-coupled using the HOBt / DIC reagents in
order to restrict racemized mixtures formation. After the coupling of the N-terminus
Gly and its Fmoc- deprotection, aliquots of the on-resin-peptides were swelled with
DCM (3x). These aliquots were cleaved from the solid support and the side chain
protecting groups were removed by treatment with TFA: TIS:DCM = 9.4:0.5:0.1 for
3.5 h at ambient temperature. The cleavage mixture containing the linear peptides was
evaporated under reduced vacuum and the free peptides were precipitated with diethyl
ether, filtered, washed with diethyl ether, diluted with water, and lyophilized. Results
of the synthesis of the linear precursors are presented at Table S1. The overall yield
for all synthesized crude peptides was in a range of 77%-89%.
Synthesis of the linear precursors by microwave assisted synthesis.
All the washing / swelling steps of the resin were carried out to the filtered
equipped syringes as like in classical SPPS. Coupling reactions and Fmocdeprotection were performed after the transfer of the resin from the filtered equipped
syringes to a proper glass vial adapted to the microwave cavity. Vials of 0.1-0.5 ml
and 2-5 ml were used. Coupling reactions were achieved by microwave heating at 60
0
C for 5-7 min. The Fmoc-removal was performed with piperidine mixture by heating
the resin for 2 min at 60 0C. Then, the resin was transferred to the filtered equipped
syringe, the piperidine mixture was filtered, and the resin was transferred again to the
microwave vial and heated for 2 min at 60 0C. One more treatment of the resin with
20% piperidine was performed for 1 min at 60 0C. Results of the synthesis of the
S4
linear precursors are presented at Table S1. The overall yield for all synthesized crude
peptides was in a range of 77%-89%.
Cyclizations.
Experimental procedures for DMSO mediated oxidation in solution (method 1).
The crude linear peptides 11 and 21 (10 mg, 0.0054mmol) were dissolved in 0.5-2.5
ml of DMSO and then 50 ml of 0.1M phosphate buffer pH=7.5-8.0 was added
directly. The mixture was stirred at r.t for 2.5h for the large loop (peptide 21) and 8h
for the small loop (peptide 11). The oxidation was monitoring by HPLC
chromatography and ESI-MS. After the completion of the oxidation the round flask
was freeze-dried with dry-ice and methanol and the monocyclic bis(Acm)
intermediates were lyophilized at least twice due to the phosphate buffer elimination.
Experimental procedures for Iodine mediated oxidation in solution (method 1).
The resultant monocyclic bis(Acm) intermediates (~10 mg, ~0.0054 mmol) were
dissolved in 12.5 ml solution mixture of HOAc-H2O (4:1) containing 14 mg of I2
(0.055 mmol; ~10 equivalents). The reaction mixture was stirred for 30 min for
peptide 21 (small loop formation) and 50 min for peptide 11 (large loop formation).
Then, the reaction was quenched by addition of 20 ml of Η2O and the iodine was
extracted 7 times with 35 ml of CCl4. The aqueous phase contained the bicyclic native
αCtxMII was lyophilized. Obtained: 7.1 mg (0.0041 mmol) of crude peptide 11 (yield
77%, purity 84% by HPLC chromatography) and 6.5 mg (0.0038 mmol) of crude
peptide 21 (yield 71%, purity 88% by HPLC chromatography). Best observed yields
are indicated, using the linear peptides released from the TentaGel resin that were
synthesized by the microwave assistance heating procedure.
Experimental procedures for air oxidation on solid support.
S5
Fmoc and side chain protected peptides were oxidized by Et3N method on-bead as
follows. 21 mg of peptide 11 (0.0024 mmol of peptide anchored on resin, calculated
by Fmoc-absorption method) and 32 mg of peptide 21 (0.0037 mmol of peptide
anchored on resin, calculated by Fmoc-absorption method) bounded on RAMTentaGel resin were treated with 1.5 ml of a mixture of TFA: DCM: TIS (1: 94: 5) for
20 min in order to remove Mmt groups. The resin washed with DCM (5x) and DMF
(5x). Then 0.05 Μ of Et3N (15 μl) in 2 ml NMP was added to the reaction syringe at
r.t. The reaction mixture was treated by gently bubble air flow. After 5 h (peptide 11)
and 36 h (peptide 22) the resin washed by DCM (3x), DMF (3x), MeOH(2x) and
NMP(3x). The peptides were treated with 20% piperidine in DMF for the Fmocdeprotection and cleaved from the resin as described above. Obtained: 3.3 mg (0.0017
mmol) of crude peptide 11 (yield 73%, purity 67% by HPLC chromatography) and
1.1 mg (0.0006 mmol) of crude peptide 21 (yield 16%, purity >5% -not clear- by
HPLC chromatograph). Best observed yields are indicated, using the peptides 12 and
22 still anchored on the TentaGel resin that were synthesized by the microwave
assistance heating procedure.
Experimental procedures for the displacement cyclization method on solid support.
(method 2a & b).
Fmoc-Nα-protected and side chains protected bound on resin peptides 12 and 22
(80mg, 0.0093 mmol of peptide anchored on resin, calculated by Fmoc-absorption
method) were put on filtered equipped syringes and the resin was swelled by DCM
(3x) and DMF (2x). Then, 2 ml of 30 % β-mercaptoethanol in DMF was added to the
reaction syringe and was mild stirred for ~3 h in r.t., in order to remove the StBugroup. When microwave heating was applied, the resin was transferred for each
S6
reaction step to a microwave vial (2-5 ml, Biotage). In order to remove tBu-group
using microwave assisted heating the sample was irradiated for 20 min (60 0C). The
reducing agent was filtered and the resin was washed in the filtered equipped syringe
by DCM (5x), DMF (5x) and DCM (5x). At the next step the free thiol group that was
released from the StBu-group, was re-protected with Npys-group by treatment of the
resin with 30 mg of bis(5-nitro-2-pyridyl) disulfide (0.096mmol, ~10eq) (DTNP,
Sigma Aldrich; Buchs; Germany) in 2 ml of DCM. Reaction time in r.t temperature
was 1 h instead of 7 min when microwave heating (60 0C) was applied. The second
thiol group was liberated from the Mmt by treatment of the resin with 2 ml of 1%
TFA in the presence of 5% triisopropylsilane (TIS) as scavenger in DCM, in order to
react with the Npys activated Cys. When the reaction was taking place in r.t., 20 min
were essential for the Mmt removal, while one irradiation step of 7 min was sufficient
applying the microwave heating method. In order to complete the disulfide bond
formation, the resin was washed from the above TFA mixture, and the reaction was
further progress in DCM. The reaction of the free thiol with the Npys-activated Cys
was monitored by measuring the absorbance at 385 nm of 5-nitropyridine-2-thione
that was released. When no absorbance was detected at this wavelength the reaction
was stopped. When this extra step was applied in r.t., 20 min were sufficient for no
detection of the characteristic absorbance of Npys-group for the small loop formation
(peptide 12). At the other hand, more than three days were essential for the
completion of the large loop formation (peptide 22). In contrast, when microwave
heating pulses (5 min each) were applied to the bound on resin-peptide, only 1 x 5
min and 6 x 5 min at 60 0C, were sufficient for no-detection of Npys released
absorbance, for peptides 12 and 22, respectively. In between these pulses the resin
was transfer to a filtered equipped syringe, the DCM was filtered and the reaction vial
S7
was filled up again with the resin and freshly DCM solvent. After the end of this
reaction half of the peptides bound on resin quantity were kept for the next cyclization
step on the resin (method 2a). The rest bound on resin peptides 12 and 22 (40 mg
each) were treated by 20% piperidine in order to remove the Fmoc-group and were
cleaved from the resin, as described above, in order to perform the next cyclization
step in solution (method 2b). Obtained from 40 mg of bound on resin peptides after
the cleavage: 7.5 mg of crude peptide 12 (0.0040 mmol; M.W.: 1853.74; yield 88%,
purity 81% by HPLC chromatography) and 6.4 mg of crude peptide 22 (0.0034 mmol;
M.W.: 1853.74; yield 76%, purity 67% by HPLC chromatograph). Best observed
yields are indicated, by the microwave heating method using the peptides 12 and 22
still anchored on the TentaGel resin that were synthesized by the microwave
assistance heating procedure.
Experimental procedures for Iodine mediated oxidation in solid phase (method 2a).
The bound on resin monocyclic bis(Acm) intermediates (40mg for each peptide 12
and 22, 0.00465 mmol of peptide anchored on resin, calculated by Fmoc-absorption
method) were prepared for the second cyclization step by swelling the resin with
DMF (3x). Then, 2 ml of DMF containing 17 mg of I2 (0.07 mmol; ~15 equivalents)
were added to the filtrated equipped syringe. The reaction mixture was gently stirred
for 60 to 90 min for both peptides in r.t. Then, the resin was washed thoroughly with
DCM (5x), CCl4 (5x) and DMF (10x). The bound on resin peptides were treated with
piperidine solution to remove the Fmoc-group and were cleaved for the resin, as
described above. Obtained: 1.3 mg of crude peptide 21 (0.0007 mmol; M.W.:
1709.68; yield 17%, purity 33% by HPLC chromatography) and 0.9 mg of crude
peptide 22 (0.0005 mmol; M.W.: 1709.68; yield 11%, purity 38% by HPLC
S8
chromatography). Best observed yields are indicated, using the monocyclic
intermediates produced from the first microwave assisted displacement cyclization
method, from the TentaGel-resin bound peptides that were synthesized by the
microwave assistance heating procedure.
Experimental procedures for Iodine mediated oxidation in solution (method 2b).
After Fmoc- removal and cleavage from the resin the monocyclic bis(Acm)
intermediates - 7.5 mg of crude peptide 12 (0.0040 mmol) and 6.4 mg of crude
peptide 22 (0.0034 mmol) were dissolved in 12.5 ml solution mixture of HOAc-H2O
(4:1) containing 10 mg and 8.5 mg of I2, respectively (0.04 mmol for peptide 12 and
0.034 mmol for peptide 22; 10 equivalents). The reaction mixture was stirred for 30
min for peptide 12 and 50 min for peptide 22. Then, the reaction was quenched by
addition of 20 ml of Η2O and the iodine was extracted 7 times with 35 ml of CCl4.
The aqueous phase included the bicyclic native αCtxMII was lyophilized. Obtained:
5.3 mg of crude peptide 12 (0.0031 mmol, yield 67%, purity 79% by HPLC
chromatography) and 5.1 mg of peptide 22 (0.0030 mmol, yield 65%, purity 65% by
HPLC chromatography). Best observed yields are indicated, using monocyclic
intermediates produced from the first microwave assisted displacement cyclization
method, cleaved from the TentaGel-resin bound peptides that were synthesized by the
microwave assistance heating procedure.
S9
Table S1. Analytical data of oxidized peptides in solution (Method 1).
peptide
11
21
monocyclic - bis(Acm)
M.W.
Purity / tR (min)a
Calc
Exp
87.92% (19.90min)
1853.74b
927.87c
618.91d
n.d.
928.3
619.2
90.66% (18.08min)
1853.74b
927.87c
618.91d
n.d.
928.3
619.2
bicyclic -native peptide
M.W.
Purity / tR (min)a
Calc
Exp
83.77% (20.91min)
1709.68b
855.84c
570.90d
n.d.
855.9
571.1
87.74% (21.07min)
1709.68b
855.84c
570.90d
n.d.
855.7
571.1
RP-HPLC with UV-detection (215nm), Column: Genesis C18, 120 mm  4.6 mm; Jones Chromatography; solvent A = 0.1% TFA (v/v) and solvent B = MeCN containing
0.1% TFA (v/v); linear gradient, 0% to 45% B in 30 min; flow rate, 1 mL/min. b(M+H+); c(M+2H+)/2; d(M+3H+)/3
a
S10
Table S2. Analytical data of monocyclic-bis (Acm) peptides and native bicyclic peptides. The first cyclization achieved by DTNP method on
resin while the second cyclized loop performed either on resin or after the cleavage of peptides (methods 2a and 2b, respectively).
monocyclic - bis(Acm)
bicyclic -native peptide
SS formation by m.w. no-oxidation
method on resin
peptide
M.W.
Purity / tR (min)a
Calc
12
22
Iodine oxidation on
resin
(Method 2a)
Iodine oxidation in
solution
(Method 2b)
Purity / tR (min)a
Purity / tR (min)a
Exp
80.74% (19.88min)
1853.74b
927.87c
618.91d
n.d.
928.0
619.0
67.39% (18.04min)
1853.74b
927.87c
618.91d
n.d.
928.1
619.2
M.W.
Calc
33.23% (20.97min)
37.76% (20.99min)
Exp
79.16% (21.06min)
1709.68b
855.84c
570.90d
n.d.
855.9
571.2
65.33% (20.82min)
1709.68b
855.84c
570.90d
n.d.
855.5
571.2
RP-HPLC with UV-detection (215nm), Column: Genesis C18, 120 mm  4.6 mm; Jones Chromatography; solvent A = 0.1% TFA (v/v) and solvent B = MeCN containing
0.1% TFA (v/v); linear gradient, 0% to 45% B in 30 min; flow rate, 1 mL/min. b(M+H+); c(M+2H+)/2; d(M+3H+)/3
a
S11
Figure S1. Analytical RP-HPLC profile of crude native αCtxMII peptides 12 (A) and
22 (B) following the Method 2a. Both disulfide bonds have been closed while
peptides are still anchored on resin. First SS bond was formed by the non-oxidative
method while second oxidation performed by iodine-mediated method.
S12
Figure S2. Analytical HPLC chromatogram of co-injected samples of native aCtMII
obtained by the stepwise synthesis and cyclization steps of 11, 21 (oxidations in
solution) and 21 and 22 (first cyclization using the displacement method in
combination with microwave assisted heating, second oxidation in solution).
S13
Figure S3. Mass spectra of linear [bis(SH), bis(Acm)]-aCtxMII peptide 11
S14
Figure S4. Mass spectra of linear [SH, S(SButhio), bis(Acm)]-aCtxMII peptide 12
S15
Figure S5. Mass spectra of monocyclic bis(Acm) peptide 11 oxidized in solution
S16
Figure S6. Mass spectra of monocyclic bis(Acm) peptide 12 cyclized by the nonoxidative method on-resin.
S17
Figure S7. Mass spectra of bicyclic native aCtxMII produced by air oxidations
methods in solution of peptide 11.
S18
Figure S8. Mass spectra of bicyclic native aCtxMII produced by method 2b of
peptide 12.
S19