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 : oleg.melnyk@ibl.cnrs.frWeb 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 - jc.monbaliu@ulg.ac.be 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.