S1
Supporting Information for
Accelerating chemoselective peptide bond formation using bis(2-selenylethyl)amido peptide selenoester surrogates
Laurent Raibaut,‡a Marine Cargoët,‡a Nathalie Ollivier,a Yun Min Chang,a Hervé Drobecq,a Emmanuelle Boll,a Rémi Desmet,a Jean-Christophe M. Monbaliu,*b Oleg Melnyk*a
a UMR CNRS 8161 CNRS, Université de Lille, Institut Pasteur de Lille, 1 rue du Pr Calmette, 59021 Lille Cedex, France.
b Center for Integrated Technology and Organic Synthesis, Department of Chemistry, Building B6a, Room 3/16a, University of Liège, Sart-Tilman, B-4000 Liège, Belgium.
‡ Laurent Raibaut and Marine Cargoët contributed equally to this work
Corresponding authors:
Dr Oleg Melnyk, E-mail : [email protected] site: http://olegmelnyk.cnrs.fr
Phone: +33 (0)3 20 87 12 14; ORCIDCent Nat de la Recherche Scientifique (CNRS)Institut de Biologie de Lille1 rue du Pr Calmette, CS 50447, 59021 Lille cedex, France
Dr Jean-Christophe M. Monbaliu, PhD.Center for Integrated Technology and Organic Synthesis - CiTOS University of Liège, Department of Chemistry, Building B6a, Room 3/16aQuartier Agora, Allée du six Aout, 13B-4000 Liège (Sart Tilman), Belgium
t +32 (0) 4 366 35 10 - [email protected] www.citos.ulg.ac.be
Electronic Supplementary Material (ESI) for Chemical Science.This journal is © The Royal Society of Chemistry 2016
S2
Table of Contents1. General Methods ..............................................................................................................................3
Reagents and solvents...................................................................................................................................3Analyses ...........................................................................................................................................................3HPLC purification............................................................................................................................................4
2. Peptide synthesis.............................................................................................................................4General procedure for automated peptide synthesis ...............................................................................42.1 Synthesis of SEAoff peptide segments (9a-c, 18, 19) ...........................................................................42.2 Synthesis of MPA thioesters 12a,b ........................................................................................................72.3 Synthesis of peptide 13d and Cys peptides 14, 21, 22 .......................................................................8
3. Synthesis of diselenide 7 & triselenide 8 ...................................................................................11
4. Synthesis of SeEAoff peptide segments .....................................................................................184.1 Protocol for the synthesis of SeEAoff peptides 10a-c using triselenide 8......................................18
4.1.1 Optimization of the exchange reaction (Fig. 1). Synthesis of SeEAoff peptide 10a starting from SEAoff peptide 9a using selenide compounds 7 or 8. ....................................................................................184.1.2 Synthesis of SeEAoff peptide 10a starting from peptide 9a .................................................................184.1.3 Synthesis of SeEAoff peptide 10a by using peptide thioester 12a as starting material ...................204.1.4 Synthesis of SeEAoff peptide 10b............................................................................................................214.1.5 Synthesis of SeEAoff peptide 10c ............................................................................................................22
4.2 Protocol for the synthesis of SeEAoff peptide 10d by using peptide acid 13d as starting material ...........................................................................................................................................................234.3 Protocol for the synthesis of SeEAoff peptide 10a by exchange using diselenide 7 ....................23
4.3.1 Protocol for the synthesis of SeEAoff peptides using diselenide 7 + Se(s) .........................................23
5. Kinetic measurements (Fig. 2) .....................................................................................................255.2 General procedure for kinetic measurements....................................................................................255.3 Synthesis of peptide 15a by reaction of SEAoff peptide 10a with Cys peptide 14 ........................25
6. SeEA/SEA kinetically Controlled Ligation..................................................................................266.1 Synthesis of peptide 23d .......................................................................................................................266.2 Total synthesis of K1 domain of human hepatocyte growth factor (23c, Fig. 5) ..........................27
7. Synthesis of SeEA peptide 31 ......................................................................................................307.1 One-pot synthesis of MPA peptide thioester 26 (one-pot process I) .............................................307.2 One-pot synthesis of SeEA peptide 30................................................................................................31
7.2.1 Synthesis of AcA-MPA .............................................................................................................................317.2.2 Synthesis of peptide 29 ............................................................................................................................347.2.3 Preparation of SeEAoff peptide 30 (one-pot process II) .......................................................................37
7.3 One-pot assembly of peptide 31 (one-pot process III) ..................................................................................39Step 1....................................................................................................................................................................39Step 2....................................................................................................................................................................39
7.4 One-pot synthesis of NK1-B (one-pot process IV) .........................................................................................41
8. Computational analysis.................................................................................................................43
References...........................................................................................................................................43
S3
1. General Methods
Reagents and solvents2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium fluorophosphate (HBTU) and N-Fmoc protected
amino acids were obtained from Iris Biotech GmbH. Side-chain protecting groups used for the amino acids
were Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-
OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc- Trp(Boc)-OH,
Fmoc-Tyr(tBu)-OH, Fmoc-Cys(StBu)-OH or Fmoc-Cys(Trt)-OH. Synthesis of bis(2-sulfanylethyl)aminotrityl
polystyrene (SEA PS) resin was carried out as described elsewhere.1 Rink-PEG-PS resin (NovaSyn TGR)
and Wang resin (100-200 Mesh) were obtained from Novabiochem. 4-mercaptophenylacetic acid (MPAA),
3-mercaptopropionic acid (MPA), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), metallic selenium
and sodium borohydride (NaBH4) were purchased from Sigma-Aldrich. All other reagents were purchased
from Acros Organics or Merck and were of the purest grade available.
Peptide synthesis grade N,N-dimethylformamide (DMF), dichloromethane (CH2Cl2), diethylether (Et2O),
acetonitrile (CH3CN), heptane, LC–MS-grade acetonitrile (CH3CN, 0.1% TFA), LC–MS-grade water (H2O,
0.1% TFA), N,N-diisopropylethylamine (DIEA), acetic anhydride (Ac2O) were purchased from Biosolve and
Fisher-Chemical. Trifluoroacetic acid (TFA) was obtained from Biosolve. Water was purified with a Milli-Q
Ultra Pure Water Purification System.
Analyses The reactions were monitored by analytical LC–MS (Waters 2695 LC/ZQ 2000 quadripole) on an reverse
phase column XBridge BEH300 C18 (3.5 m, 300 Å, 4.6 × 150 mm) unless otherwise stated at 30 °C using
a linear gradient of 0-100% of eluent B in eluent A over 30 min at a flow rate of 1 mL/min (eluent A = 0.1%
TFA in H2O; eluent B = 0.1% TFA in CH3CN/H2O: 4/1 by vol). The column eluate was monitored by UV at
215 nm and by evaporative light scattering (ELS, Waters 2424). The peptide masses were measured by
on-line LC–MS: Ionization mode: ES+, m/z range 350–2040, capillary voltage 3 kV, cone voltage 30 V,
extractor voltage 3 V, RF lens 0.2 V, source temperature 120 °C, dessolvation temperature 350 °C.
Samples were prepared using 10 L aliquots of the reaction mixtures. The aliquots were quenched by
adding 90 L of 1% aqueous TFA, extracted with Et2O to remove MPAA or MPA before analysis.
MALDI-TOF mass spectra were recorded with a BrukerAutoflex Speed using alpha-cyano-4-
hydroxycinnaminic acid as matrix. The observed m/z corresponded to the monoisotopic ions, unless
otherwise stated.
1H and 13C NMR spectra were recorded on a Bruker Advance-300 spectrometer operating at 300 MHz and
75 MHz respectively. The spectra are reported as parts per million (ppm) down field shift using
tetramethylsilane or dimethylselenide as an internal reference. The data are reported as chemical shift (δ),
multiplicity, relative integral, coupling constant (J Hz) and assignment where possible.
The determination of optical purity of the C-terminal amino acid was done by chiral GC-MS following total
acid hydrolysis in deuterated aqueous acid (C.A.T. GmbH & Co. Chromatographie und Analysentechnik
S4
KG, Heerweg 10, D-72070 Tübingen, Germany).2
HPLC purificationPreparative reverse phase HPLC of crude peptides were performed with an Autopurification prep HPLC–
MS Waters system using a reverse phase column XBridge ODB prep C-18 (5 m, 300 Å, 19 × 100 mm)
and appropriate gradient of increasing concentration of eluent B in eluent A (flow rate of 25 mL/min). The
fractions containing the purified target peptide were identified on-line using MS (ZQ 2000 quadripole).
Selected fractions were then combined and lyophilized.
2. Peptide synthesis
General procedure for automated peptide synthesisPeptide elongation was performed using standard Fmoc/tert-butyl chemistry on an automated peptide
synthesizer (0.2 mmol scale). Couplings were performed using 5-fold molar excess of each Fmoc-L- amino
acid, 4.5-fold molar excess of HBTU, and 10-fold molar excess of DIEA. A capping step was performed
after each coupling with Ac2O/DIEA in DMF. At the end of the synthesis, the resin was washed with CH2Cl2,
diethylether (2 × 2 min) and dried in vacuo.
2.1 Synthesis of SEAoff peptide segments (9a-c, 18, 19) Peptide elongation was performed on SEA PS resin (0.2 mmol, 0.16 mmol/g) using standard Fmoc/tert-
butyl chemistry on an automated peptide synthesizer. Typical procedures for the synthesis of SEAoff
peptide segments were described in previous papers.1, 3 For a detailed protocol see the protocol article 4.
The analytical HPLC and MS analyses of the purified synthetic SEAoff peptides segments (9a-c, 18, 19) are
shown in Figure S1.
Yields for the HPLC purified SEAoff peptides (9a-c, 18, 19):
-Peptide 9a: 50 mg (35% yield, 0.1 mmol scale), MALDI-TOF calcd. for [M+H]+: 1080.6, observed mass: 1080.5 (monoisotopic).
-Peptide 9b: 56 mg (39% yield, 0.1 mmol scale), MALDI-TOF calcd. for [M+H]+: 1108.6, observed mass: 1108.5 (monoisotopic).
-Peptide 19: 35 mg (25% yield, 0.1 mmol scale), MALDI-TOF calcd. for [M+H]+: 1054.4, observed mass: 1054.3 (monoisotopic).
-Peptide 9c: 53 mg (30% yield, 50 mol scale), MALDI-TOF calcd. for [M+H]+: 2784.5, observed mass: 2784.1 (monoisotopic).
-Peptide 18: 47 mg (23% yield, 50 mol scale), MALDI-TOF calcd. for [M+H]+: 3541.6, observed mass: 3541.9 (monoisotopic).
S5
ILKEPVHGA-SEAoff (9a)C18
Time0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
LSU
0.000
200.000
400.000
600.000
800.000
1000.000
1200.000
1400.000
1600.000
1080.5
0
500
1000
1500
Inten
s. [a.
u.]
1060 1070 1080 1090 1100 1110 1120m/z
1063.0 1071.8 1080.6 1089.4 1098.2 1107.00
100
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
1080.6 Theoreticalisotopic prof ile
Time (min)
m /z m /z
ILKEPVHGV-SEAoff (9b)
S6
IRNC(StBu)IIGKGRSYKGTVSITKSGIK-SEAoff (9c)C18
Time0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
LSU
0.000
50.000
100.000
150.000
200.000
250.000
300.000
2784.1
0
200
400
600
800
1000
1200
Inte
ns. [
a.u.
]
2760 2770 2780 2790 2800 2810m/z
2759.0 2770.4 2781.8 2793.2 2804.6 2816.00
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
2784.5
Theoreticalisotopic prof ile
Time (min)
m /z m /z
C(StBu)QPWSSMIPHEHSFLPSSYRGKDLQENY-SEAoff (18)
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
AU
0.0
1.0e-1
2.0e-1
3.0e-1
4.0e-1
5.0e-1
6.0e-1
7.0e-1
8.0e-1
9.0e-1
1.0
1.1
D-F3K1-NH2 purif 2: Diode Array Range: 1.25913.42
1.80
3515.0 3529.6 3544.2 3558.8 3573.4 3588.00
102030405060708090
100
% In
tens
ity
V o y a g e r S pe c # 1[ B P = 3 54 3 .8 , 4 6 14 ]
3541.9
1901 2321 2741 3161 3581 40010
102030405060708090
100
% In
tens
ity
V o y a g e r S pe c # 1[ B P = 3 54 3 .8 , 4 6 14 ]
3541.9
3530 3536 3542 3548 3554 35600
10
20
30
40
50
60
70
80
90
100
3541.6
Time (min)
m /z m /z
Theoreticalisotopic prof ile
C(StBu)HHLEPGG-SEAoff (19)
Time4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00
LSU
0.000
100.000
200.000
300.000
400.000
500.000
600.000
700.000
800.000
LR65 F2 Click purete (2) ELSD SignalRange: 96911.57
1054.369
1076.295
1092.264
0
500
1000
1500
2000
2500
Inte
ns. [
a.u.
]
1030 1040 1050 1060 1070 1080 1090 1100 1110m/z
1039.0 1045.8 1052.6 1059.4 1066.2 1073.00
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
1054.4
Time (min)
m /z m /z
Theoreticalisotopic prof ile
Figure S1. Analytical HPLC profiles (=215 nm) for purified synthetic peptide segments (9a-c, 18, 19) and MALDI-TOF data corresponding to each product.
S7
2.2 Synthesis of MPA thioesters 12a,bTypical procedures for the synthesis of MPA thioester peptides using SEAoff peptides 9a,b were described
in detail elsewhere.4, 5 The analytical HPLC and MS analyses of the purified MPA thioester peptides 12a,b are shown in Figure S2.
Yields for the HPLC purified MPA peptide thioesters
-Peptide 12a: 9.5 mg (68% yield, 10 mol scale), MALDI-TOF calcd. for [M+H]+: 1051.6, observed mass: 1051.5 (monoisotopic).
-Peptide 12b: 7 mg (49% yield, 10 mol scale), MALDI-TOF calcd. for [M+H]+: 1079.6, observed mass: 1079.5 (monoisotopic).
ILKEPVHGA-S(CH2)2COOH (12a)C18
Time0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
LSU
0.000
200.000
400.000
600.000
800.000
1000.000
1200.000
1400.000
1600.000
1032 1041 1050 1059 1068 10770
100
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
1051.6
1051.5
1073.515
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Inten
s. [a.
u.]
1010 1020 1030 1040 1050 1060 1070 1080 1090m/z
Theoreticalisotopic prof ile
Time (min)
m /z m /z
ILKEPVHGV-S(CH2)2COOH (12b)C18
Time0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
LSU
0.000
200.000
400.000
600.000
800.000
1000.000
1200.000
1400.000
1600.000
1079.5
0
500
1000
1500
Intens
. [a.u.]
1065 1070 1075 1080 1085 1090m/z
1061.0 1071.8 1082.6 1093.4 1104.2 1115.00
100
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
1079.6
Theoreticalisotopic prof ile
Time (min)
m /z m /z
Figure S2. Analytical HPLC profiles (=215 nm) for purified synthetic peptide segments 12a,b and MALDI-TOF data corresponding to each product.
S8
2.3 Synthesis of peptide 13d and Cys peptides 14, 21, 22Peptide elongation were performed on Rink-PEG-PS resin (NovaSyn TGR, 0.25 mmol, 0.25 mmol/g) or on
pre-loaded Fmoc-Gly-Wang resin (0.1 mmole, 0.61 mmol/g) using standard Fmoc/tert-butyl chemistry on
an automated peptide synthesizer. The analytical HPLC and MS analyses of the purified peptides 13d, 14, 21 and 22 are shown in Figure S3.
Isolated yield for the HPLC purified peptides 13d, 14, 21, 22:
Peptide 13d: 33 mg (43% yield, 0.1 mmol scale), Maldi-TOF calc. for [M+Na]+: 790.3, observed mass: 790.2 (monoisotopic).
Peptide 14: 89 mg (62% yield, 0.1 mmol scale), MALDI-TOF calcd. for [M+H]+: 1093.6, observed mass: 1093.6 (monoisotopic).
Peptide 21: 134 mg (13% yield, 0.25 mmol scale), MALDI-TOF calcd. for [M+H]+: 3755.6, observed mass: 3755.8 (monoisotopic).
Peptide 22: 77 mg (55% yield, 0.1 mmol scale), MALDI-TOF calcd. for [M+H]+: 1065.5, observed mass: 1065.6 (monoisotopic).
Ac-GFGQGFGG-COOH (13d)
765.0 773.8 782.6 791.4 800.2 809.00
100
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
790.3
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
LSU
0.000
200.000
400.000
600.000
800.000
1000.000
1200.000
1400.000
1600.000Theoretical
isotopic prof ile
743.0 757.2 771.4 785.6 799.8 814.00
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
790.2806.2
Time (min)
m /z m /z
S9
CILKEPVHGV-NH2 (14)C18
Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
AU
-4.0e-1
-2.0e-1
0.0
2.0e-1
4.0e-1
6.0e-1
8.0e-1
1.0
1.2
1090.0 1092.8 1095.6 1098.4 1101.2 1104.00
100
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
1093.6
1093.6
0.0
0.2
0.4
0.6
0.8
4x10
Inte
ns. [
a.u.
]
1080 1085 1090 1095 1100 1105 1110m/z
Time (min)
m /z m /z
Theoreticalisotopic prof ile
CRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEV-NH2 (21)
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
AU
0.0
2.0e-1
4.0e-1
6.0e-1
8.0e-1
1.0
1.2
1.4
1.6
C-F2K1 purif 2: Diode Array Range: 2.03915.62
0.82
3750 3754 3758 3762 3766 37700
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
3755.6
3736.0 3750.2 3764.4 3778.6 3792.8 3807.00
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
Voyager Spec #1[BP = 3757.8, 3025]
3755.8
Time (min)
m /z m /z
Theoreticalisotopic prof ile
S10
CILKEPVHGA-NH2 (22)
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
LSU
0.000
200.000
400.000
600.000
800.000
1000.000
1200.000
1400.000
LR capping + Cys Pol (2) ELSD SignalRange: 20709.86
13.52
1065.496
1060 1070 10801059 1062 1065 1068 1071 10740
10
20
30
40
50
60
70
80
90
100
% In
tensit
y
1065.6
Time (min)
m /z m /z
Theoreticalisotopic prof ile
Figure S3. Analytical HPLC profiles (=215 nm) for the purified synthetic peptide segments 13d, 14, 21 and 22 and MALDI-TOF data corresponding to each product.
S11
3. Synthesis of diselenide 7 & triselenide 8
HN
ClCl
Se22- (major)
H2NSe
SeH2N
Se
SeSe
CF3COO-CF3COO-
Se(s) + NaBH4
+ Se2- + Se32-
EtOH reflux
1) EtOH/NaOH 1 M
6 7 (21%) 8 (12%)
+
2) TFA
Scheme S1. Synthesis of diselenide 7 & triselenide 8.
CAUTION: H2Se is highly toxic. The reaction must be performed in an efficient fume hood with appropriate
protection (glasses, lab coat and gloves).
1-Preparation of sodium diselenide: Absolute ethanol (27 ml) was added dropwise with magnetic stirring
to metallic selenium powder (1.5 g, 19.8 mmol, 1.5 eq) and sodium borohydride (0.5 g, 13.2 mmol) cooled
in an ice bath. After the vigorous exothermic reaction had occurred, the mixture was further stirred and
heated at reflux for 1.5 hr with N2 passing into the liquid in order to expel H2Se. The nitrogen flow containing
H2Se and going out of the reaction vessel is passed through NaOH and NaOCl traps respectively. The
resulting brown/red colored solution was then cooled down at room temperature and used immediately in
the next step.
2- Preparation of compounds 7 and 8: The bis(2-chloroethyl)amine hydrochloride 6 (1.2 g, 6.6 mmol, 0.6
eq) was dissolved in 1 M NaOH/EtOH: 1/1 by volume (5 mL) and then was added dropwise over a period
of 20 min to the above solution of Na2Se2 or Na2Se3. The solution was then stirred for 1 hour under argon
at room temperature. The reaction mixture was diluted with 1 M NaOH (15 mL) and extracted with CH2Cl2
(3 × 20 mL). The organic phase was dried over solid Na2SO4 and evaporated to dryness in vacuo. The
crude product was purified by reversed-phase HPLC using a linear water-acetonitrile gradient to give 495
mg (21%) of 1,2,5-diselenazepane as the trifluoroacetate salt 7 and 80 mg (12%) of 1,2,3,6-
triselenazocane as the trifluoroacetate salt 8 (yellow powders).
The characterization of diselenide 7 can be found elsewhere.6
HR-MS, NMR 1H/13C and HPLC analyses for the purified triselenide 8 are shown below in Figures S4-S12.
The presence of 77Se atoms in compound 8 and the formation of aggregates make the NMR spectra of
compound 8 complex. The same complexity was observed for compound 7 as discussed elsewhere.6
Compound 8 was found to degrade partially into 7 upon storage (see below), but this did not alter the
usefulness of triselenide 8 for accessing SeEA peptides.
S12
Figure S4. HR-MS analysis for purified triselenide compounds 8. Calcd. for [M+H]+: 311.831, observed mass: 311.830 (monoisotopic).
-1.0-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)
0.0E+00
5.0E+07
1.0E+08
1.5E+08
2.0E+08
2.5E+08
3.0E+08
3.5E+08
4.0E+08
4.5E+08
5.0E+08
2.71
2.72
2.72
2.73
2.74
2.88
2.89
2.89
2.90
2.90
3.05
3.06
3.35
3.37
3.40
3.52
3.54
3.55
3.57
3.58
3.61
3.63
3.64
3.66
3.67
3.69
3.72
4.03
4.04
4.05
4.06
4.07
4.08
8.00
9.69
Figure S5. 1H NMR (300 MHz) spectrum for compound 8 (DMF-d7).
S13
-3-2-10123456789101112f1 (ppm)
-1.00E+07
0.00E+00
1.00E+07
2.00E+07
3.00E+07
4.00E+07
5.00E+07
6.00E+07
7.00E+07
8.00E+07
9.00E+07
1.00E+08
1.10E+08
1.20E+08
1.30E+08
1.40E+08
1.50E+08
1.93
1.95
1.97
2.71
2.72
2.72
2.89
3.42
3.43
3.43
3.44
3.44
3.45
3.46
3.55
3.88
3.89
3.90
3.90
3.91
3.92
8.00
9.80
2.53.03.54.0f1 (ppm)
0.0E+00
1.0E+07
2.0E+07
3.0E+07
4.0E+07
5.0E+072.71
2.71
2.72
2.72
2.73
2.88
2.88
2.89
2.90
2.90
3.42
3.43
3.43
3.44
3.44
3.45
3.46
3.55
3.88
3.89
3.90
3.90
3.91
3.92
Figure S6. 1H NMR (300 MHz) spectrum for compound 7 (DMF-d7). See ref 6
S14
2.72.82.93.03.13.23.33.43.53.63.73.83.94.04.14.24.34.44.54.6f1 (ppm)
-2.0E+07
0.0E+00
2.0E+07
4.0E+07
6.0E+07
8.0E+07
1.0E+08
1.2E+08
1.4E+08
1.6E+08
1.8E+08
2.0E+08
2.2E+08
2.4E+08
2.6E+08
2.8E+08
3.0E+082.71
2.72
2.72
2.73
2.74
2.88
2.89
2.89
2.90
2.90
3.05
3.06
3.35
3.37
3.40
3.52
3.54
3.55
3.57
3.58
3.61
3.63
3.64
3.66
3.67
3.69
3.72
4.03
4.04
4.05
4.06
4.07
4.08
Figure S7. 1H NMR (300 MHz, DMF-d7) spectrum for compounds 7 and 8 (superposition of NMR spectra acquired separately, see Figures S5 & S6).
This figure shows that the triselenide compound 8 isolated by HPLC contains minor amounts of the diselenide 7.
S15
-30-20-100102030405060708090100110120130140150160170f1 (ppm)
-5.0E+07
0.0E+00
5.0E+07
1.0E+08
1.5E+08
2.0E+08
2.5E+08
3.0E+08
3.5E+08
4.0E+08
4.5E+08
A (q)159.92
B (q)116.93
15.0
917
.92
23.3
724
.59
25.1
325
.31
25.7
626
.20
28.9
929
.27
29.5
529
.83
30.1
130
.38
30.6
634
.10
34.3
734
.65
34.9
335
.21
35.4
935
.76
40.0
042
.10
43.7
148
.00
48.3
248
.71
48.9
849
.11
49.4
353
.54
55.3
9
111.
1211
5.00
118.
8712
2.75
159.
2215
9.69
160.
1616
0.62
162.
0716
2.46
162.
85
100105110115120125130135140145150155160165170175f1 (ppm)
-5.0E+07
0.0E+00
5.0E+07
1.0E+08
1.5E+08
2.0E+08
2.5E+08
3.0E+08
3.5E+08
4.0E+08
4.5E+08
A (q)159.92
B (q)116.93
111.
12
115.
00
118.
87
122.
75
159.
2215
9.69
160.
1616
0.62
162.
0716
2.46
162.
85
Figure S8. 13C NMR (75 MHz) spectrum for HPLC purified compound 8 (DMF-d7). The quartets at 159.92 and 116.93 ppm are for the trifluoroacetate ion.
S16
0123456789101112f2 (ppm)
1
2
3
4
5
6
7
8
9
10
11
f1 (
ppm
)
2.83.03.23.43.63.84.04.2f2 (ppm)
3.0
3.5
4.0
f1 (
ppm
)
Figure S9. 1H-1H COSY spectrum for compound 8 (DMF-d7).
-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f2 (ppm)
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
f1 (
ppm
)
2.53.03.54.0
10
20
30
40
50
60
Figure S10. 1H-13C HSQC spectrum for compound 8 (DMF-d7).
S17
2.22.32.42.52.62.72.82.93.03.13.23.33.43.53.63.73.83.94.04.14.24.34.44.54.64.74.8f1 (ppm)
0.0E+00
5.0E+07
1.0E+08
1.5E+08
2.0E+08
2.5E+08
3.0E+08
3.5E+08
4.0E+08
2.71
2.72
2.72
2.73
2.74
2.88
2.89
2.89
2.90
2.90
3.05
3.06
3.35
3.37
3.40
3.52
3.54
3.55
3.57
3.58
3.61
3.63
3.64
3.66
3.67
3.69
3.72
4.03
4.04
4.05
4.06
4.07
4.08
Figure S11. 1H NMR (300 MHz, DMF-d7) spectrum for compound 8 after 20 h in DMF-d7 at 27°C (blue trace) and superposition with the 1H NMR spectrum of triselenide 8 taken immediately after HPLC purification (red trace, see Fig. S5 & S7).
This figure shows that the triselenide compound 8 isolated by HPLC is partially converted into diselenide 7 in DMF-d7 solution.
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
LSU
0.000
50.000
100.000
150.000
200.000
250.000
300.000
350.000
400.000
450.000
500.000
550.0007.70
6.64 m/z225 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300 305 310 315 320 325 330 335 340 345 350 355
%
0
100DC31 Tri 4 174 (7.826) Cm (172:179) 1: Scan ES+
2.41e6310
232
230228
282
280234 278
277
284
286
Time (min)
m /z m /z
Theoreticalisotopic prof ile
7
8HN
Se
SeSe
H2NSe
SeSe
CF3COO-
H2NSe
Se
CF3COO-
Figure S12. Analytical HPLC profile (=215 nm) of purified triselenide compounds 8 after several days in lyophilized form. Note that triselenide compound 8 degraded partially into diselenide compound 7 during storage. The loss of selenium from polyselenides of the type RSenR (n > 3) is well documented, see ref 7.
S18
4. Synthesis of SeEAoff peptide segments
4.1 Protocol for the synthesis of SeEAoff peptides 10a-c using triselenide 8
4.1.1 Optimization of the exchange reaction (Fig. 1). Synthesis of SeEAoff peptide 10a starting from SEAoff peptide 9a using selenide compounds 7 or 8.
TCEP-HCl (29 mg, 0.1 mmol) was dissolved in 0.2 M pH 4.2 sodium acetate buffer (1 mL). NaOH (5 M)
was then used to adjust the pH to 5.5. Peptide 9a (10 mg, 0.7 mol) and selenide compound 8 (24 mg, 7
mol, 10 eq) or a mixture of 7 (30 mg, 7 µmol, 10 equiv) and metallic selenium (1.6 mg, 3 equiv) were
dissolved in the above solution. The final peptide concentration was 10 mM (pH 5.5). The reactions were
performed at 37°C under nitrogen atmosphere and monitored by LC-MS.
4.1.2 Synthesis of SeEAoff peptide 10a starting from peptide 9a
TCEP-HCl (29 mg, 0.1 mmol) was dissolved in 0.2 M pH 4.2 sodium acetate buffer (1 mL). NaOH (5 M)
was then added to adjust the pH to 4.1.
Peptide 9a (4 mg, 2.8 mol) and selenide compound 8 (12 mg, 28 mol, 10 equiv) were dissolved in the
above solution (312 L, final peptide concentration 9 mM). The reaction mixture was shaken at 37°C under
nitrogen atmosphere and monitored by LC-MS (Figure S13). After 24 h, the mixture was diluted with water-
TFA 1% (2 mL) and purified by reversed-phase HPLC using a linear water-acetonitrile gradient containing
0.05% TFA to give the purified product 10a (2.2 mg, 51% yield).
The determination of the optical purity for the C-terminal amino acid residue within peptide 10a was
performed by chiral GC-MS analysis after acid hydrolysis in deuterated acid. The analysis was done by
C.A.T. GmbH & Co. company (Chromatographie und Analysen technik KG, Heerweg 10, D-72070
Tübingen, Germany). The analysis indicated a D-Ala content of 0.23%.
S19
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
AU
0.0
5.0e-1
1.0
1.5
2.0
2.5
NO
SHSH
NO
SeSe
t1h
t6h
Time (min)
9a
10a
Se=TCEP
Figure S13. LC-MS analysis of the crude exchange reaction after 24 h (9a→10a).
Time0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
LSU
0.000
25.000
50.000
75.000
100.000
125.000
150.000
175.000
200.000
225.000
250.000
275.000
300.000
325.000
10.10
1151.0 1159.4 1167.8 1176.2 1184.6 1193.00
100
0
10
20
30
40
50
60
70
80
90
100
% Int
ensit
y
1176.51174.5
1174.2
0
500
1000
1500
2000
Inte
ns. [
a.u.
]
1160 1165
1170 1175 11801185 1190
m/z
Theoreticalisotopic prof ile
Time (min)
m /z m /z
Figure S14. HPLC (=215 nm) and MALDI-TOF analysis of the purified peptide 10a.
S20
4.1.3 Synthesis of SeEAoff peptide 10a by using peptide thioester 12a as starting material
TCEP-HCl (29 mg, 0.1 mmol) was dissolved in 0.2 M pH 4.2 sodium acetate buffer (1 mL). NaOH (5 M)
was then added to adjust the pH to 5.5.
Peptide 12a (5 mg, 2.8 mol) and selenide compound 8 (15 mg, 28 mol, 10 equiv) were dissolved in the
above solution (398 L, final peptide concentration 9 mM). The reaction mixture was shaken at 37°C under
nitrogen atmosphere and monitored by LC-MS (Figure S15). After 30 h, the mixture was diluted with water-
TFA 1% (2 mL) and purified by reversed-phase HPLC using a linear water-acetonitrile gradient containing
0.05% TFA to give the purified product 10a (2.5 mg, 46% yield).
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
LSU
0.000
500.000
1000.000
1500.000
SRO
NO
SeSe
t3h
t18h
Time (min)
12a
10a
Figure S15. LC-MS analysis of the crude exchange reaction after 18 h (12a→10a).
S21
4.1.4 Synthesis of SeEAoff peptide 10b
Peptide 10b was synthesized on a 6.9 μmol scale starting from peptide 9b by using the same procedure as
described above for the conversion of 9a into 10a. The reaction mixture was shaken at 37°C under
nitrogen atmosphere and monitored by LC-MS (Figure S16). After 72 h, the mixture was diluted with water-
TFA 1% (2 mL) and purified by reversed-phase HPLC using a linear water-acetonitrile gradient containing
0.05% TFA to give the purified product 10b (4.3 mg, 40% yield).
Time0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
LSU
0.000
100.000
200.000
300.000
400.000
500.000
600.000
NO
SHSH
NO
SeSe
t0
t72h
Time (min)
9b
10b
Se=TCEP
Figure S16. LC-MS analysis of the crude exchange reaction after 72 h (9b→10b).
Figure S17. HPLC (=215 nm) and MALDI-TOF analysis of the purified peptide 10b.
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
LSU
0.000
100.000
200.000
300.000
400.000
500.000
600.000
700.000
800.000
1202.275
0
2000
4000
6000
8000
Inte
ns. [
a.u.
]
1192.5 1195.0 1197.5 1200.0 1202.5 1205.0 1207.5 1210.0 1212.5m/z
1183.0 1190.8 1198.6 1206.4 1214.2 1222.00
100
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
1204.51202.5 Theoretical
isotopic prof ile
Time (min)
m /z m /z
S22
4.1.5 Synthesis of SeEAoff peptide 10c
TCEP-HCl (6 mg, 0.02 mmol) was dissolved in 0.2 M pH 4.2 sodium acetate buffer (1 mL). NaOH (5 M)
was then used to adjust the pH to 4.1.
Peptide 9c (5 mg, 1.4 mol) and selenide compound 8 (5.9 mg, 14 mol, 10 eq), were dissolved in the
above solution (698 L, final peptide concentration 2 mM). The reaction mixture was shaken at 37°C under
nitrogen atmosphere and monitored by LC-MS (Figure S18). After 32 h, the mixture was diluted with water-
TFA 1% (2 mL) and purified by reversed-phase HPLC using a linear water-acetonitrile gradient containing
0.05% TFA to give the purified product 10c (1.9 mg, 38% yield).
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
LSU
0.000
250.000
500.000
750.000
1000.000
1250.000
NO
SHSH
NO
SeSe
t0
t32h
Time (min)
9c
10c
Se=TCEP
Figure S18. LC-MS analysis of the crude exchange reaction after 32 h (9c→10c).
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
LSU
0.000
20.000
40.000
60.000
80.000
100.000
120.000
2761 2769 2777 2785 2793 28010
100
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
2792.3 Theoreticalisotopic prof ile2792.9
0
1000
2000
3000
4000
Inte
ns. [
a.u.
]
2785 2790 2795 2800 2805 2810m/z
Time (min)
m /z m /z
Figure S19. HPLC (=215 nm) and MALDI-TOF analysis of the purified peptide 10c.
S23
4.2 Protocol for the synthesis of SeEAoff peptide 10d by using peptide acid 13d as starting material
Diselenide 7 (4.5 mg, 14 mol, 2 eq) was added to a solution of peptide 13d (5 mg, 7 mol) dissolved in
anhydrous DMF (0.6 mL, final peptide concentration 10 mM). (Benzo-triazol-1-yloxy)tri-
pyrrolidinophosphoniumhexafluorophosphate (PyBOP, 6.8 mg, 14 mol, 2 equiv) and DIEA (6.8 L, 0.39
mmol, 6 equiv) were then added to the solution. The mixture was stirred overnight at room temperature
under argon atmosphere and the progress of the reaction was monitored by LC-MS. The solvent was then
evaporated in vacuo, and the crude product was directly purified by reversed-phase HPLC using a linear
water-acetonitrile gradienttogive 2.2 mg (34%) of peptide 10d.
Ac-GFGQGFGG-SeEAoff (10d)
Figure S20. Analytical HPLC profile (=215 nm) and MALDI-TOF data for the purified synthetic peptide segments 10d. MALDI-TOF calc. for [M+Na]+: 1003.2, observed mass: 1003.1 (monoisotopic).
4.3 Protocol for the synthesis of SeEAoff peptide 10a by exchange using diselenide 7
4.3.1 Protocol for the synthesis of SeEAoff peptides using diselenide 7 + Se(s)
TCEP-HCl (28.7 mg, 0.1 mmol), was dissolved in 0.2 M pH 4.2 sodium acetate buffer (1 mL).
Diselenide compound 7 (24.1 mg, 70 µmol, 10 equiv) and metallic selenium (1.6 mg, 3 equiv) were
suspended in the above solution (703 µL). NaOH (5 M) was then added to adjust the pH to 5.5.
Peptide 9a (10 mg, 7 mol) was dissolved in the above solution (703 L, final peptide concentration 10
mM). The reaction mixture was shaken at 37 °C under nitrogen atmosphere and monitored by LC-MS. After
988 994 1000 1006 1012 10180
100
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
1003.2
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
AU
0.0
1.0e-1
2.0e-1
3.0e-1
4.0e-1
5.0e-1
6.0e-1
7.0e-1
8.0e-1
9.0e-1
1.0
1.1
Theoreticalisotopic prof ile
985 997 1009 1021 1033 10450
4.8E+4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
1003.1
1019.1400
1017.1277
1015.14901021.1267
1016.1452
1022.11951027.37191015.3238 1019.3176 1023.1284
Time (min)
m /z m /z
S24
20 h, the mixture was diluted with water-TFA 1 % (2 mL) and purified by reversed-phase HPLC (eluent A =
water containing 0.1% TFA, eluent B=acetonitrile in water 4/1 containing 0.1 % TFA, 50 °C, detection at
215 nm, 6 mL/min, 0 to 10 % eluent B in 10 min, then 10 to 25 % eluent B in 45 min, C18 XBridge column)
to give 5.92 mg of pure product (56 %).
A)
Time (min)0.00 5.00 10.00 15.00 20.00 25.00 30.00
0.0
100.0
200.0
300.0
400.0
500.0
600.0
700.0
800.0
900.0
Intensity, light scattering (AU)
m/z
400 600 800 1000 12000
100A2
587.6
1176.3
A: 1173.11±0.00
B)
Figure S21. A) LC-MS analysis of peptide 10a. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 x 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: [M+2H]2+ m/z calcd. 589.3, obs. 587.6. B) MALDI-TOF analysis and comparison with the theoretical profile.
S25
5. Kinetic measurements (Fig. 2)
5.2 General procedure for kinetic measurementsTable S4: Peptides used for the kinetic study
SEAoff, SeEAoff or thioester peptide
9a H-ILKEPVHGA-SEA
12a H-ILKEPVHGA-S(CH2)2COOH
10a H-ILKEPVHGA-SeEA
9b H-ILKEPVHGV-SEA
12b H-ILKEPVHGV-S(CH2)2COOH
10b H-ILKEPVHGV-SeEA
For SEA peptides 9a,b and thioester peptides 12a,b: TCEP-HCl (29 mg, 0.1 mmol) and MPAA (17 mg, 0.1
mmol) were dissolved in 0.1 M pH 7.2 sodium phosphate buffer (1 mL). NaOH (5 M) was then added to
adjust the pH to 7.1.
For SeEA peptides 10a,b: TCEP-HCl (29 mg, 0.1 mmol), MPAA (17 mg, 0.1 mmol) and Se=TCEP (12 mg,
35 mol) were dissolved in 0.1 M pH 7.2 sodium phosphate buffer (1 mL). NaOH (5 M) was then added to
adjust the pH to 7.1. Se=TCEP is used to inhibit the deselenization of SeEA peptides by TCEP.8
Peptides 9a-b or 10a-b or 12a-b (1 mol) and peptide 14 (1.5 mol, 1.5 equiv) were dissolved in the above
solution (300 L, final peptide concentration 3.5 mM, pH 7.1). The ligations were performed at 37°C under
nitrogen atmosphere and monitored by RP-HPLC. For each time point, 10 μL aliquots were withdrawn,
quenched by adding 90 μL of 1% aqueous TFA and extracted with Et2O to remove MPAA. The samples
were stored at –20°C until analysis.
5.3 Synthesis of peptide 15a by reaction of SEAoff peptide 10a with Cys peptide 14
For the ligation of SeEAoff peptide peptide 10a with Cys peptide 14 on a preparative scale, the reaction was
scaled up to 3.3 mol. In this reaction, TCEP-HCl (29 mg, 0.1 mmol), MPAA (17 mg, 0.1 mmol) and
Se=TCEP (26 mg, 0.08 mmol) were dissolved in 0.1 M pH 7.2 sodium phosphate buffer (1 mL). NaOH (5
M) was then added to adjust the pH to 7.1. Peptide 10a (5 mg, 3.3 mol) and peptides 14 (7 mg, 4.9 mol,
1.5 eq) were dissolved in the above solution (942 L, 3.5 mM final concentration). After completion of the
ligation, the mixture was diluted with water-TFA 5% (2 mL), extracted with Et2O (3 × 500 L) to remove
MPAA and purified by reversed-phase HPLC using a linear water-acetonitrile gradient containing 0.05%
TFA to give the purified ligation product 15a (4.8 mg, 56% yield).
S26
Determination of optical purity of amino acid derivative of peptide ligation product 15a by chiral GC-MS
after acid hydrolysis showed 0.43% of D-Ala C-terminus.
Figure S22. Analytical HPLC profile (=215 nm) and MALDI-TOF data for the purified peptide 15a. Calcd. for [M+H]+: 2038.3, observed mass: 2038.2 (monoisotopic).
.
6. SeEA/SEA kinetically Controlled Ligation
6.1 Synthesis of peptide 23d
TCEP-HCl (58 mg, 0.2 mmol) and MPAA (33 mg, 0.2 mmol) were dissolved in 6 M guanidine-HCl, 0.1 M
pH 7.2 sodium phosphate buffer (1 mL). NaOH (5 M) was then added to adjust the pH to 7.1.
Peptide 10d (5.3 mg, 5.4 mol) and peptide 19 (7.6 mg, 5.4 mol) were dissolved in the above solution
(780 L, final peptide concentration 7 mM) resulting in the reduction of SeEAoff and SEAoff groups and in the
removal of cysteine StBu protecting group. The mixture was stirred at 37 °C under nitrogen atmosphere
and the progress of the ligation was monitored by LC-MS. Figure S23A was obtained after 4 h of reaction.
After 7 h, peptide segment 22 (11 mg, 8.1 μmol, 1.5 eq) was added to the reaction mixture which was
further stirred at 37°C under nitrogen. Figure S23B corresponds to the LC-MS analysis of the crude after 48
h. After 48 h, the reaction mixture was diluted with water (2 mL), acidified with 5% aqueous TFA (2 mL) and
extracted with diethylether to remove the excess of MPAA. The crude product was directly purified by
reversed-phase HPLC using a linear water-acetonitrile gradient containing 0.05% TFA to give the purified
ligation product 23d (4.5 mg, 27% yield) (Figure S24).
Time0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
LSU
0.000
25.000
50.000
75.000
100.000
125.000
150.000
175.000
200.000
225.000
250.000
275.000
2038.3
0
1000
2000
3000
4000
5000
Inten
s. [a.
u.]
2025 2030 2035 2040 2045 2050 2055 2060 2065 2070 2075m/z
2030.0 2033.6 2037.2 2040.8 2044.4 2048.00
100
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sit
y
2038.2
Time (min)
m /z m /z
Theoreticalisotopic prof ile
S27
KCL-t4h
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
LSU
0.000
500.000
1000.000
1500.000
NO
SHSH
Cys
Cys Cys
Time (min)
t4h
t48h
Cys
20d
23d
22
Figure S23. HPLC analysis (=215 nm) of the crude kinetically controlled assembly process leading to the
formation of peptide 23d after 4 h (SeEA ligation step, up) and after 48 h (SEA ligation step, down).
2645.2
0
2000
4000
6000
8000Inte
ns. [a
.u.]
2635 2640 2645 2650 2655 2660 2665m/z
2639.0 2643.8 2648.6 2653.4 2658.2 2663.00
100
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
2645.3
Time (min)
m /z m /z
Theoreticalisotopic prof ile
Time4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
LSU
0.000
25.000
50.000
75.000
100.000
125.000
150.000
175.000
Figure S24. Analytical HPLC profile (=215 nm) and MALDI-TOF data for the purified peptide 23d. Calcd. for [M+H]+: 2645.3, observed mass: 2645.2 (monoisotopic).
6.2 Total synthesis of K1 domain of human hepatocyte growth factor (23c, Fig. 5)
TCEP-HCl (29 mg, 0.1 mmol), MPAA (17 mg, 0.1 mmol) and Se=TCEP (26 mg, 0,08 mmol) were dissolved
in 6 M guanidine-HCl, 0.1 M pH 7.2 sodium phosphate buffer (1 mL). NaOH (5 M) was then added to adjust
the pH to 7.1.
Peptide 10c (5 mg, 1.4 mol) and peptide 18 (7 mg, 1.7 mol, 1.2 eq) were dissolved in the above solution
(348 L, final peptide concentration 4 mM). The mixture was stirred at 37 °C under nitrogen atmosphere
A)
B)
S28
and the progress of the ligation was monitored by LC-MS (see Figure 6). After 30 h, peptide segment 21 (7
mg, 1.7 μmol, 1.2 equiv) was added to the reaction mixture which was further stirred at 37°C under nitrogen
for 72 h. The reaction mixture was then diluted with water (2 mL), acidified with 5% aqueous TFA (2 mL)
and extracted with diethylether to remove the excess of MPAA. The crude product was directly purified by
reversed-phase HPLC using a linear water-acetonitrile gradient containing 0.05% TFA to give K1
polypeptide 23c (3.3 mg, 21% yield) (Figure S25).
S29
a)
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
LSU
0.000
10.000
20.000
30.000
40.000
50.000
60.000
70.000
80.000
90.000
100.000
110.000 9638.6
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Inte
ns. [
a.u.
]
7000 8000 9000 10000 11000 12000 13000 14000 15000m/z
Time (min)
m /z
b)
Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
LSU
0.000
200.000
400.000
600.000
K1 domain HGF-SF from One-pot synthesis
K1 domain HGF-SF from KCL one-pot synthesis
Co-injection
Time (min)
Figure S25. a) Analytical HPLC profile (=215 nm) and MALDI-TOF data for the purified K1 polypeptide 23c. Calc. for [M+H]+: 9639.8, observed mass: 9638.6 (average isotopic composition). b) Comparison between synthetic K1 produced by “one-pot” three-segment ligation (see ref 3) and by the SeEA/SEA KCL method discussed in this paper.
S30
7. Synthesis of SeEA peptide 31
7.1 One-pot synthesis of MPA peptide thioester 26 (one-pot process I)The synthesis of peptides 24 and 25 was performed as described elsewhere.9
6 M Gdn.HCl 0.1 M pH 7.2 sodium phosphate buffer was degassed during 30 min. MPAA was dissolved in
this solution to a final concentration of 200 mM. NaOH (5 M) was then added to adjust the pH to 7.1.
Peptide thioester 24 (13.09 mg, 2.12 µmol) and SEAoff peptide 25 (8.84 mg, 2.12 µmol) were dissolved in
the above solution (650 µL, final peptide concentration 3.3 mM). The mixture was stirred at 37 °C under
nitrogen atmosphere. After 20 h, the exchange of the SEA group by MPA was started by adding a 20 %
MPA solution in water containing 0.2 M TCEP (pH=3.9, 650 µL). After 20 h, the reaction mixture was
diluted with 10 % aqueous acetic acid (22 mL), extracted with Et2O (3 x 15 mL) to remove MPAA and
purified by RP-HPLC (eluent A = water containing 0.1 % TFA, eluent B=acetonitrile in water 4/1 containing
0.1 % TFA, 50 °C, detection at 215 nm, 6 mL/min, 0 to 10 % eluent B in 5 min, then 10 to 40 % eluent B in
60 min, C18 XBridge column) to give 9.84 mg of thioester peptide 26 (46.5 %).
Time (min)
2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50
0.000
100.000
200.000
300.000
400.000
500.000
600.000
Intensity, light scattering (AU)
m/z200 400 600 800 1000 1200 1400 1600 1800 2000
0
100
A: 7579.12±0.61
A11689.93A12
632.62
A13583.99
A10758.78
A9843.09
A8948.53
A71083.8
Intensity (AU)
Figure S26. LC-MS analysis of thioester peptide 26. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 x 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: M calculated (mean) 7580.01, found 7579.12±0.61.
S31
7578.5
3788.1
0
1
2
3
4
4x10
4000 5000 6000 7000 8000 9000 10000 11000 12000m/z
Intensity (AU)
Figure S27. MALDI-TOF analysis of thioester peptide 26. Matrix : 2,5-dihydroxybenzoic acid (DHB), positive mode. [M+H]+ calcd. (mean) 7580.01, found 7578.5.
7.2 One-pot synthesis of SeEA peptide 307.2.1 Synthesis of AcA-MPA
O O
AcA-MPAS
CO2H
N-hydroxysuccinimidyl acetoacetate (265.4 mg, 1.33 mmol) was dissolved in CH2Cl2 (4 mL) at rt under
argon. 3-Mercaptopropionic acid (116 µL, 1.33 mmol) and N-methylmorpholine (292.8 µL, 2.66 mmol) were
added in one portion and the reaction medium was stirred overnight. Then, the solvent was evaporated and
the resulting yellow oil was purified by silica gel chromatography (ethyl acetate/cyclohexane : 4/6 v/v
containing 1 % acetic acid) to give 49.0 mg of AcA-MPA (19.4 %).
S32
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z
0
10
20
30
40
50
60
70
80
90
100 Relative Abundance 191.03650
85.02911
173.02618117.05522
m/z Intensity Relative Theo. Mass Delta (ppm) RDB equiv. Composition85,02911 52584900 41,66117,05519 6896370,5 5,46 117,05462 4,89 1,5 C5 H9 O3 173,02619 10185761 8,07 173,02669 -2,88 3,5 C7 H9 O3 S 191,03646 126233200 100 191,03726 -4,15 2,5 C7 H11 O4 S 192,03981 8157689,5 6,46
NO146_60_P1 #18 RT: 0.25 AV: 1 NL: 1.02E8T: FTMS + p ESI Full ms [50.00-400.00]
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
191.03650
85.02911
65.06052 173.02618117.05522
103.03958 208.06282
79.02195
235.05893125.06008 160.07954 359.31372279.03464 331.28244301.01614 376.33994
O
S
O
OH
O
65.06052- H2O
O
S
O
OH
O
H
OC
O
Figure S28. High resolution mass spectrometry of AcA-MPA (positive ion mode).
O
S
O
OH
O
Figure S29. 1H NMR spectrum (300.0 MHz, CDCl3) of AcA-MPA
S33
O
S
O
OH
O
Figure S30. 13C NMR spectrum (75 MHz, CDCl3) of AcA-MPA.
O
S
O
OH
O
Figure S31. 1H-1H COSY NMR spectrum of AcA-MPA.
S34
O
S
O
OH
O
Figure S32. 1H-13C HSQC NMR spectrum of AcA-MPA.
7.2.2 Synthesis of peptide 297.2.2.1 Synthesis of AcA-HGF 128-149-SEAoff
HGF 128-149: CIIGKGRSYK GTVSITKSGI K
0.1 M pH 7.2 sodium phosphate buffer was degassed during 30 min. Gdn.HCl (2.86 g, 0.03 mol) and
MPAA (168.3 mg, 1 mmol) were dissolved in this buffer (5 mL qsp). NaOH (6 M) was then added to adjust
the pH to 7-7.5.
Peptide CIIGKGRSYK GTVSITKSGI K-SEAoff (19.84 mg, 6.43 µmol) and AcA-MPA (1.47 mg, 7.72
µmol) were dissolved in the above solution (1.5 mL). The mixture was stirred at 37 °C under nitrogen
atmosphere overnight.
The reaction mixture was then acidified with glacial acetic acid (400 µL), diluted with water (6.1 mL) and
extracted with Et2O (3 x 2 mL) to remove MPAA. The crude peptide was purified by RP-HPLC (eluent A =
water containing 0.1 % TFA, eluent B = acetonitrile in water 4/1 containing 0.1 % TFA, 30 °C, detection at
215 nm, 6 mL/min, 0 to 20 % eluent B in 5 min, then 20 to 40 % eluent B in 60 min, C18 XBridge column) to
give 11.9 mg of peptide AcA-CIIGKGRSYK GTVSITKSGI K-SEAoff (62.4 %).
S35
Time (min)0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
0.000
100.000
200.000
300.000
400.000
500.000
600.000
700.000
800.000
900.000
1000.000
1100.000
1200.000
Intensity (light scattering, AU)
O OCIIGKGRSYKGTVSITKSGIK-SEAoff
m/z400 600 800 1000 1200 1400 1600 1800 2000
0
100
A: 2397.54±0.13
A3800.0
A4600.3
A21199.6
Intensity (AU)
Figure S33. LC-MS analysis of peptide AcA-HGF 128-149-SEAoff. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 x 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: [M+2H]2+ m/z calcd. 1199.98, obs. 1199.6, [M+3H]3+ m/z calcd. 800.32, obs. 800.0, [M+4H]4+ m/z calcd. 600.49, obs. 600.3.
[M+H]+
2397.0
0
1000
2000
3000
500 1000 1500 2000 2500 3000m/z
Intensity (AU)
O OCIIGKGRSYKGTVSITKSGIK-SEAoff
2397.0
0
1000
2000
3000
2380 2385 2390 2395 2400 2405 2410 2415 2420m/z
Intensity (AU)
Figure S34. MALDI-TOF analysis of peptide AcA-HGF 128-149-SEAoff. Matrix, 2,5-dihydroxybenzoic acid (DHB) positive mode, [M+H]+ calcd. (monoisotopic) 2397.28, found 2397.0
S36
7.2.2.2 Exchange of the SEA group by MPA
A typical procedure for the exchange of the SEA group by MPA was described in detail elsewhere.4, 5 The
analytical HPLC and MS analyses of the purified thioester peptide 29 are shown below.
Yields for the HPLC purified MPA peptide thioester 29
Peptide 29: 6.41 mg (40% yield, 5.6 mol scale), MALDI-TOF calcd. for [M+H]+: 2368.3, observed mass: 2368.3 (monoisotopic).
Time (min)4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
0.000
200.000
400.000
600.000
800.000
1000.000
1200.000
1400.000
Intensity (light scattering, AU)
m/z400 600 800 1000 12000
100
A: 2368.39±0.03
A3790.393
A4593.105
A21185.156
Intensity (AU)
Figure S35. LC-MS analysis of peptide 29. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 x 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). MS trace: M calculated (mean) 2368.84, found 2368.39±0.03.
S37
Figure S36. MALDI-TOF analysis of thioester peptide 29. Matrix, 2,5-dihydroxybenzoic acid (DHB) positive mode. [M+H]+ calcd. (monoisotopic) 2368.27, found 2368.3.
7.2.3 Preparation of SeEAoff peptide 30 (one-pot process II)TCEP-HCl (2.89 mg, 0.1 mmol), selenide compound 7 (3.42 mg, 0.01 mmol, 5 eq) and metallic selenium
(0.237 mg, 1.5 equiv) were dissolved in 0.2 M pH 4.2 sodium acetate buffer (1 mL). NaOH (5 M) was then
added to adjust the pH to 5-5.5.
Peptide 29 (5.65 mg, 1.94 µmol) was dissolved in the above solution (972 µL, final peptide concentration 2
mM). The reaction mixture was shaken at 37°C under nitrogen atmosphere and monitored by LC-MS.
After 20 h of exchange, DMSO (105.8 µL) was added (10% of DMSO by vol) to inactivate the SeEA group
by formation of a diselenide bond. The reaction mixture was stirred 30 min at 37°C. Then, acetic acid
(275.7 µL) was added to have a final 20% acetic acid solution. 0.43 M hydroxylamine solution in water (45
µL) was then added to remove AcA protecting group. The reaction medium was heated at 37°C for 1 h 30
min and then diluted with water (11 mL final volume). The aqueous solution was extracted with diethyl ether
(3 x 3 mL) and then purified by RP-HPLC (eluent A = water containing 0.1% TFA, eluent B=acetonitrile in
water 4/1 containing 0.1% TFA, 30°C, detection at 215 nm, 6 mL/min, 0 to 20% eluent B in 5 min, then 20
to 40% eluent B in 60 min, C18XBridge column) to give 2.4 mg of SeEAoff peptide 30 (40%).
S38
A)
Time (min)0.00 5.00 10.00 15.00 20.00 25.00 30.00
0.0
200.0
400.0
600.0
800.0
1000.0
1200.0
Intensity (light scattering, AU)
m/z200 400 600 800 1000 1200
0
100 A: 2407.28±0.43A3
803.6
A4602.6 A2
1204.9
Intensity (AU)
B)
Figure S37. A) LC-MS analysis of peptide 30. LC trace, eluent A 0.10 % TFA in water, eluent B 0.10 % TFA in CH3CN/water: 4/1 by vol. C18 Xbridge BEH 300 Å 5 μm (4.6 x 250 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm).B) MALDI-TOF analysis of thioester peptide 30. Matrix, 2,5-dihydroxybenzoic acid (DHB) positive mode.
S39
7.3 One-pot assembly of peptide 31 (one-pot process III)
Step 1MPAA (3.34 mg, 0.2 mmol) and Se=TCEP (6.58 mg, 0.02 mmol) were dissolved in 6 M Gdn.HCl, 0.1 M pH
7.2 sodium phosphate buffer (100 µL). NaOH (5 M) was then added to adjust the pH to 7-7.5.
Peptide 26 (5.27 mg, 0.53 mol) and peptide 27 (2.51 mg, 0.53 mol) were dissolved in the above solution
(75 L, final peptide concentration 7 mM). The reaction mixture was shaken at 37°C under nitrogen
atmosphere and monitored by LC-MS.
Step 2
After 20 h, the reaction mixture was divided in two equal portions. To one of these, DTT (0.59 mg, 100 mM)
and SeEAoff peptide 30 (1.04 mg, 0.3 mol) were added in this order. The reaction mixture was shaken at
37°C under nitrogen atmosphere and monitored by LC-MS.
After 42 h, the reaction mixture was diluted with 5 % aqueous acetic acid (4 mL), extracted with Et2O (5 x 2
mL) to remove MPAA and purified by RP-HPLC (eluent A = water containing 0.1 % TFA, eluent
B=acetonitrile in water 4/1 containing 0.1 % TFA, 50 °C, detection at 215 nm, 6 mL/min, 0 to 20 % eluent B
in 5 min, then 20 to 50 % eluent B in 90 min, C18 XBridge column) to give 0.94 mg of SeEAoff peptide 31
(21 % overall starting from peptide 26).
A)
Time(min)0.00 5.00 10.00 15.00 20.00 25.00 30.00
0.0
40.0
80.0
120.0
160.0
200.0
240.0
280.0
320.0
360.0
Intensity (light scattering, AU)
S40
B)
m/z600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
0
100 A: 13722.96±0.44A18
763.3
A19723.2
A24571.0
A17808.2
A16858.7
A15915.9
A14981.2
A131056.6
A121144.5 A11
1248.5 A101373.3
A91525.8
Intensity (AU)
C)
mass13700 13702 13704 13706 13708 13710 13712 13714 13716 13718 13720 13722 13724 13726 13728 13730 13732 13734 13736 13738 13740 13742 137440
100mass
13700 13702 13704 13706 13708 13710 13712 13714 13716 13718 13720 13722 13724 13726 13728 13730 13732 13734 13736 13738 13740 13742 137440
100
13722.09
M 13722.00
Calculated
Observed after deconvolution
Figure S38. LC-MS analysis of thioester peptide 31. A) LC trace, eluent C 0.10 % FA in water, eluent D 0.10 % FA in CH3CN/water: 4/1 by vol. C3 Zorbax 300SB 3.5 µm (4.6 x 150 mm) column, gradient 0-100 % D in 30 min (1 mL/min, detection 215 nm). B) ESI MS trace: M calculated (mean) 13722.89, found 13722.96. C) High resolution ESI MS with experimental and calculated profile after deconvolution.
S41
7.4 One-pot synthesis of NK1-B (one-pot process IV)TCEP.HCl (2.87 mg, 0.01 mmol), MPAA (1.29 mg, 0.01 mmol) and Se=TCEP (2.63 mg, 0.008 mmol) were
dissolved in 6 M guanidine-HCl, 0.1 M pH 7.2 sodium phosphate buffer (100 µL). NaOH (5 M) was then
added to adjust the pH to 7-7.5.
Peptide 31 (0.82 mg, 0.047 mol) and peptide 18 (0.23 mg, 0.057 mol, 1.2 equiv) were dissolved in the
above solution (12 L, final peptide concentration 4 mM). The reaction mixture was shaken at 37°C under
nitrogen atmosphere and monitored by LC-MS.
After 18 h, TCEP.HCl (2.87 mg, 0.01 mmol) and MPAA (1.29 mg, 0.01 mmol) were dissolved in 6 M
Gdn.HCl, 0.1 M pH 7.2 sodium phosphate buffer (100 µL). NaOH (5 M) was then added to adjust the pH to
5.
Peptide 33 (0.43 mg, 0.095 µmol, 2 equiv) was dissolved in the above solution (12 µL) and then added to
the reaction mixture which was shaken at 37°C under nitrogen atmosphere.
After 72 h, the reaction mixture was diluted with 5 % aqueous acetic acid (4 mL), extracted with Et2O (3 x 2
mL) to remove MPAA and purified by RP-HPLC (eluent C = water containing 0.1 % formic acid (FA), eluent
D=acetonitrile in water 4/1 containing 0.1 % FA, 50 °C, detection at 215 nm, 6 mL/min, 0 to 20 % eluent B
in 5 min, then 20 to 50 % eluent B in 90 min, C3 Zorbax column) to give 335 µg of NK1-B (32% overall
starting from peptide 31).
A)
Time (min)0.00 5.00 10.00 15.00 20.00 25.00 30.00
0.0
2.0e-1
4.0e-1
6.0e-1
8.0e-1
Abs (UV, 215 nm)
NK1-B
S42
B)
m/z600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650
%
0
100XE000077SJ 1357 (11.653) Cm (1348:1363) TOF MS ES+
3.06e6A: 20924.24±0.87
A28A29
A30
A31
A32
A27
A26
A25
A24
A23
A22
1000 12001100900800700600 1300 1400 1500 1600
32+
31+
30+
29+ 27+28+
24+
25+
26+
23+
22+21+
20+ 19+ 18+17+
16+15+
m/z
Intensity (AU)
NK1-B
C)
mass20911 20913 20915 20917 20919 20921 20923 20925 20927 20929 20931 20933 20935 20937 209390
10020911 20913 20915 20917 20919 20921 20923 20925 20927 20929 20931 20933 20935 20937 209390
10020924.5059
Experimental profileFound 20924.24±0.87
Intensity (AU)
Theoretical profile
Figure S39. A) LC analysis of NK1-B. A) LC trace, eluent A 0.10 % formic acid (FA) in water, eluent B 0.10 % FA in CH3CN/water: 4/1 by vol. C3 Zorbax 300SB 3.5 µm (4.6 x 150 mm) column, gradient 0-100 % B in 30 min (1 mL/min, detection 215 nm). B) ESI MS high resolution spectrum. C) Deconvoluted spectrum and comparison with the theoretical profile.
S43
8. Computational analysis
Quantum chemical calculations were performed using the Gaussian 09 package of programs.10 DFT
computations were carried out using the B3LYP hybrid functional employing the 6-31+G* basis set with 5
pure d functions. Gradient techniques using internal coordinates with very tight optimization convergence
criteria (each component of the first energy derivative below 2.0 10-6Hartree/Bohr or radian) were used for
both geometry optimization and computation of vibrational properties. The transitions states were localized
using the Newton-Raphson algorithm, and the nature of the stationary points was determined by analysis of
the Hessian. The activation and reaction energies were calculated from the thermochemical output
(298.150 Kelvin, 1 atm) computed for the reagents, transition states and products, using standard
thermochemistry as implemented in Gaussian 09. Intrinsic reaction coordinate (IRC) calculations were
performed in the gas phase to localize the nearest local minima on the reactant and product sides of the
reaction coordinate.11 Solvent effects (water) were taken into account using Tomasi’s polarizable continuum
model (PCM).12
TS SEA TS SEA_2 TS SEA_3
H = -1330.731085 HartreeG = -1330.789404 Hartree
H = -1330.703070 HartreeG = -1330.763103 Hartree
H = -1331.221528 HartreeG = -1331.281239 Hartree
Figure S40 Structures and absolute energies for anionic (TS SEA, TS SEA_2) and neutral (TS SEA_3) transitions states (gas phase). TS SEA is discussed in the main manuscript (one neutral 2-mercaptoethyl limb protonates the amide nitrogen, while the other is anionic and attacks the amide carbonyl). In the other anionic transition state TS SEA_2, one neutral 2-mercaptoethyl limb protonates the amide nitrogen, while the other is anionic and spectator. The third transition state TS SEA_3 is neutral (one neutral 2-mercaptoethyl limb protonates the amide nitrogen, while the other is neutral too and spectator). The activation barrier for TS SEA is about 20 kcal mol-1 less than for TS SEA_2 and TS SEA_3.
References
1. N. Ollivier, J. Dheur, R. Mhidia, A. Blanpain and O. Melnyk, Org. Lett., 2010, 12, 5238-5241.2. W. Amelung and S. Brodowski, Anal. Chem., 2002, 74, 3239-3246.
S44
3. N. Ollivier, J. Vicogne, A. Vallin, H. Drobecq, R. Desmet, O. El-Mahdi, B. Leclercq, G. Goormachtigh, V. Fafeur and O. Melnyk, Angew. Chem., Int. Ed., 2012, 51, 209-213.
4. N. Ollivier, L. Raibaut, A. Blanpain, R. Desmet, J. Dheur, R. Mhidia, E. Boll, H. Drobecq, S. L. Pira and O. Melnyk, J. Pept. Sci., 2014, 20, 92–97.
5. J. Dheur, N. Ollivier, A. Vallin and O. Melnyk, J. Org. Chem., 2011, 76, 3194-3202.6. L. Raibaut, H. Drobecq and O. Melnyk, Org. Lett., 2015, 17, 3636-3639.7. L. Henriksen, in The Chemistry of Organic Selenium and Tellurium Compounds, ed. S. Patai, John
Wiley & Sons, Chichester, 1987, vol. 2, pp. 393-420.8. N. Ollivier, A. Blanpain, L. Raibaut, H. Drobecq and O. Melnyk, Org. Lett., 2014, 16, 3032-4035.9. L. Raibaut, J. Vicogne, B. Leclercq, H. Drobecq, R. Desmet and O. Melnyk, Bioorg. Med. Chem.,
2013, 21, 3486-3494.10. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani,
V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, Izmaylov A. F., J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, J. Peralta, J. E., F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox, Gaussian 09. Revision A.02, Gaussian, Inc., Wallingford (CT, USA), 2009.
11. K. Fukui, Acc. Chem. Res., 1981, 14, 363-368.12. J. Tomasi and M. Persico, Chem. Rev., 1994, 94, 2027-2094.