Efficient reverse click labeling of azide oligonucleotides with multiple alkynyl Cy-Dyes applied to the synthesis of HyBeacon probes for genetic analysis

Article abstract of DOI:10.1016/j.tet.2011.11.041

A convenient method of oligonucleotide labeling using click chemistry has been developed. A 2′-mesyloxyethyl ribothymidine phosphoramidite monomer was incorporated into DNA at several loci during solid phase oligonucleotide synthesis and converted to 2′-a

Full text of DOI:10.1016/j.tet.2011.11.041

Efficient reverse click labeling of azide oligonucleotides with multiple alkynyl Cy-Dyes
applied to the synthesis of HyBeacon probes for genetic analysis
Marta Gerowska, Lucy Hall, James Richardson, Montserrat Shelbourne and Tom Brown
School of Chemistry, University of Southampton, Highfield, Southampton, SO17 1BJ, UK
Supporting information
Synthesis of Cyanine Dyes
All reagents were purchased from Aldrich, Fluka, Alfa Aesar or Apollo. The following solvents were purified by
distillation: DCM, Et3N, DIPEA, pyridine (over calcium hydride), and THF (over sodium wire/benzophenone). Thin
layer chromatography (TLC) was performed using Merck Kieselgel 60 F24 silica gel plates, 0.22 mm thickness,
aluminum backed. The compounds were visualized under UV lamp (254 and 365 nm) or by staining with ninhydrin.
Column chromatography was carried out under pressure (air/argon) using Fisher Scientific DAVISIL 60Å (35-70
micron) silica.
1H NMR spectra were measured at 300MHz on a Bruker AC300 spectrometer or at 400 MHz on a Bruker DPX400
spectrometer. 13C NMR spectra were measured at 75 MHz on a Bruker AC300 spectrometer or at 100 MHz on a Bruker
DPX400 spectrometer. Chemical shifts (in ppm) are given relative to tetramethylsilane. Spectra were internally
referenced to the appropriate residual undeuterated solvent signal (J. Org. Chem. 1997, 62, 21, 7512-7515). Coupling
1
1
constants are quoted in Hz. H and 13C assignments were supported by DEPT-135, H-1H COSY, HMQC, HMBC
experiments. Spectra were reprocessed using ACDlab program.
Low-resolution mass spectra were recorded using electrospray ionization on a Fisons VG platform instrument or a
Waters ZMD quadrupole mass spectrometer in HPLC grade acetonitrile or methanol. High-resolution mass spectra were
recorded using electrospray ionization on a Bruker APEX III FT-ICR mass spectrometer.
General synthetic procedures were taken from a combination of literature sources:
Indolium salts synthesis: Wang, J.; Cao, W. F.; Su, J. H.; Tian, H.; Huang, Y. H.; Sun, Z. R. Dyes Pigm. 2003, 57,
171-179
Cy5 synthesis: Kvach, M. V., Ustinov, A. V., Stepanova, I. A., Malakhov, A. D., Skorobogatyi, M., V., Shmanai, V.
V., Korshun, V. A., Eur. J. Org. Chem. 2008, 2107-2117
Cy3 hemicyanine: Mason, S. J.; Hake, J. L.; Nairne, J.; Cummin, W. J.; Balasubramanian, S. J. Org. Chem. 2005, 70,
2939-2949
Cy3 synthesis: Fegan, A.; Shirude, P. S.; Balasubramanian, S. Chem. Commun. 2008, 2004-2006
Intermediates are labeled with 1 – 10, the final compounds are labeled I – X
I. The synthesis of the indolenines and indolium salts used for the synthesis of Cy-dyes
1

1. Synthesis of 1,2,3,3-Tetramethyl-3H-indolinium iodide (1)
CH3I, CH3CN
N+
reflux, 7 h
N
I-
1
2,3,3-Trimethylindolenine (1.0 g, 1.01 mL, 6.28 mmol, 1 eq) was dissolved in acetonitrile (5 mL) and then methyl
iodide (0.43 mL, 6.91 mmol, 1.1 eq) was added. The reaction mixture was heated at reflux for 7 h, cooled to room
temperature and the precipitate was collected by filtration, washed with diethyl ether, and dried in vacuo over potassium
hydroxide pellets. Product 1 was obtained as pink crystals with 93 % yield (1.76 g, 5.84 mmol). Mw = 301.17 g/mol
(C12H16IN).
Rf:
0.55 [DCM/MeOH 95:5 v/v]
1H NMR
(300 MHz, DMSO) δ 7.96 - 7.89 (1 H, m, H-4/H-7), 7.87 - 7.79 (1 H, m, H-4/H-7), 7.68 - 7.57 (2 H,
m, H-5, H-6), 3.98 (3 H, s, N-CH3), 2.78 (3 H, s, 2-CH3), 1.53 (6 H, s, 3-CH3 ×2) ppm.
13C NMR
LRMS
(75 MHz, DMSO) δ 196.0 (C-2), 142.1, 141.6 (C-3a, C-7a), 129.3, 128.8 (C-5, C-6), 123.3 (C-4),
115.1 (C-7), 53.9 (C-3), 34.7 (N-CH3), 21.7 (2 C, 3-CH3 ×2), 14.2 (2-CH3) ppm.
[ESI+, MeOH] calculated mass: 174.13 for C12H16N+ [M-I]+; found m/z: 174.2 (100 %), 175.2
(13.4 %).
Data consistent with literature: Kvach, M. V., Ustinov, A. V., Stepanova, I. A., Malakhov, A. D., Skorobogatyi, M., V.,
Shmanai, V. V., Korshun, V. A., Eur. J. Org. Chem. 2008, 2107-2117
2. Synthesis of 5-iodo-2,3,3-trimethyl-3H-indolium iodide (2)
4-Iodophenylhydrazine (16.0 g, 68.36 mmol, 1 eq) was suspended in acetic acid (520 mL), then 3-methyl-2-butanone
(8.06 mL, 75.20 mmol, 1.1 eq) was added and the reaction was heated at reflux for 4 h, cooled to room temperature and
the solvent was removed under reduced pressure. The oily residue was dissolved in DCM and a large excess of hexane
was added. The purple oil with solid impurities precipitated below the yellow hexane layer. The hexane layer was
decanted off and the solvent was removed in vacuo to afford product 2 as a yellow oil in 96 % yield (18.65 g, 65.41
mmol). Mw = 285.12 g/mol (C11H12IN).
Rf:
0.71 [DCM/MeOH 95:5 v/v]
1H NMR
(300 MHz, CDCl3) δ 7.31 - 7.24 (2 H, m, H-4, H-6), 6.95 (1 H, d, J = 8.1 Hz, H-7), 1.92 (3 H, s, 2-
CH3), 0.95 (6 H, s, 3-CH3 ×2) ppm.
13C NMR
LRMS
(75 MHz, CDCl3) δ 188.3 (C-2), 153.4, 148.2 (C-7a, C-3a), 136.7, 130.7, 121.8 (C-4, C-6, C-7), 89.9
(C-5), 54.0 (C-3), 22.9 (2 C, 3-CH3 ×2), 15.4 (2-CH3) ppm.
[ESI+, MeOH] calculated mass: 285.0 for C11H12IN [M]+; found m/z: 286.0 (100 %), 287.1 (10.2 %)
[M+H]+.
Data consistent with literature: Tomasulo, M., Sortino, S., Raymo, F. M. J. Org. Chem. 2007, 73, 118-126
2

3. Synthesis of 5-Iodo-1,2,3,3-tetramethyl-3H-indolium iodide (3)
I
I
CH3I
N+
CH3CN, reflux,
N
I-
2 h,
3
2
5-Iodo-2,3,3-trimethylindolenine 2 (18.65 g, 65.41 mmol, 1 eq) was dissolved in acetonitrile (85 mL) then methyl
iodide (4.90 mL, 78.49 mmol, 1.2 eq) was added and the reaction mixture was heated at reflux for 2 h. After cooling to
room temperature the precipitate collected by filtration, washed with diethyl ether, and dried in vacuo. Product 3 was
obtained as an orange solid in 74 % yield (20.59 g, 48.20 mmol). Mw = 427.06 g/mol (C12H15I2N).
Rf:
0.58 [DCM/MeOH 95:5 v/v]
1H NMR
(300 MHz, DMSO) δ 8.29 (1 H, d, J = 1.1 Hz, H-4), 8.00 (1 H, dd, J = 8.4, 1.5 Hz, H-6), 7.72 (1 H, d,
J = 8.4 Hz, H-7), 3.94 (3 H, s, N-CH3), 2.74 (3 H, s, 2-CH3), 1.52 (6 H, s, 3-CH3 ×2) ppm.
13C NMR
LRMS
(75 MHz, DMSO) δ 196.1 (C-2), 143.7, 141.9 (C-3a, C-7a), 137.4, 132.2, 117.1 (C-4, C-6, C-7), 96.1
(C-5), 54.0 (C-3), 34.8 (N-CH3), 21.4 (2 C, 3-CH3 ×2), 14.2 (2-CH3) ppm.
[ESI+, MeOH] calculated mass: 300.02 for C12H15IN+ [M-I-]+; found m/z: 300.1 (100 %), 301.1
(14.0 %), 302.1 (1.7 %).
Data consistent with literature: Fegan, A., Shirude, P. S., Balasubramanian, S. Chem. Commun. 2008, 2004-2006
4. Synthesis of 1-Buthyl-5-iodo-2,3,3-trimethyl-3H-indolium iodide (4)
I
I
I
N+
CH3CN, reflux,
24 h
N
I-
2
4
5-Iodo-2,3,3-trimethylindolenine 2 (6.71 g, 23.54 mmol, 1 eq) was dissolved in acetonitrile (60 mL) then 1-iodobutane
(21.43 mL, 188.30 mmol, 8 eq) was added and the reaction mixture heated at reflux for 24 h. After the reaction was
cooled to room temperature, the precipitate was collected by filtration, washed with diethyl ether, and dried in vacuo.
Product 4 was obtained as pink crystals with 94 % yield (10.33 g, 22.03 mmol). Mw = 469.14 g/mol (C15H21I2N).
Rf:
0.71 [DCM/MeOH 95:5 v/v]
IR (neat):
1H NMR
3008 (w, C-H, aromatic), 2960, 2929 (s, C-H, alkyl), 1621 – 1539 (s, C-C, aromatic), 1463 (s, C-I).
(300 MHz, DMSO) δ 8.31 (1 H, d, J = 1.1 Hz, H-4), 7.99 (1 H, dd, J = 8.4, 1.1 Hz, H-6), 7.80 (1 H, d,
J = 8.4 Hz, H-7), 4.43 (2 H, t, J = 7.5 Hz, NCH2CH2CH2CH3), 2.83 (3 H, s, 2-CH3), 1.79 (2 H, quin, J
= 7.6 Hz, NCH2CH2CH2CH3), 1.53 (6 H, s, 3-CH3 ×2), 1.41 (2 H, dq, J = 15.2, 7.5 Hz,
NCH2CH2CH2CH3), 0.92 (3 H, t, J = 7.3 Hz, NCH2CH2CH2CH3) ppm.
3

13C NMR
(75 MHz, DMSO) δ 196.4 (C-2), 144.0, 140.9 (C-7a, C-3a), 137.5, 132.4, 117.5 (C-7, C-6, C-4), 96.3
(C-5), 54.2 (C-3), 47.6 (NCH2CH2CH2CH3), 29.2 (NCH2CH2CH2CH3), 21.8 (2 C, 3-CH3 ×2), 19.3
(NCH2CH2CH2CH3), 14.1, 13.6 (NCH2CH2CH2CH3, 2-CH3) ppm.
LRMS
HRMS
[ESI+, MeOH] calculated mass: 342.07 for C15H21IN+ [M-I-]+; found m/z: 342.1 (100 %), 343.2
(16.7 %), 344.1 (1.5 %).
[ESI+, MeOH] calculated mass: 342.0713 for C15H21IN+; found m/z: 342.0717
4

5. Synthesis of 1-(4-pentynyl)-2,3,3-trimethyl-3H-indolium iodide (5)
Cl
N+
I-
N
KI, CH3CN, reflux,
12 h,
5
Potassium iodide (11.50 g, 69.08 mmol, 2.2 eq) was suspended in acetonitrile (50 mL), then 5-chloro-1-pentyne (5.0
mL, 31.40 mmol, 1 eq) was added and the yellow suspension was stirred at 50 C. After 10 min 2,3,3-
trimethylindolenine (5.05 mL, 31.40 mmol, 1 eq) was added dropwise and the reaction mixture was heated at reflux
overnight then cooled to room temperature. The inorganic salt was filtered off, washed with DCM, and the filtrate was
evaporated to dryness. The residue was purified by column chromatography (0-10 % MeOH in DCM) and the pure
product 5 was obtained as a purple solid in 58 % yield (6.41 g, 18.15 mmol). Mw = 353.24 g/mol (C16H20IN).
Rf:
0.41 [DCM/MeOH 95:5 v/v]
1H NMR
(400 MHz, MeOD)  7.96 - 7.90, 7.82 - 7.77 (1 H, m, H-4; 1 H, m, H-7), 7.70 - 7.63 (2 H, m, H-5, H-
6), 4.66 (2 H, dd, J = 8.3, 7.0 Hz, NCH2CH2CH2C≡CH), 2.52 - 2.45 (3 H, m, NCH2CH2CH2C≡CH),
2.26 - 2.16 (2 H, m, NCH2CH2CH2C≡CH), 1.64 (6 H, s, 3-CH3 ×2) ppm. (*2-CH3 was not visible on
the spectrum)
13C NMR
(100 MHz, MeOD)  198.6 (C-2), 143.5, 142.7 (C-3a, C-7a), 131.4, 130.6 (C-5, C-6), 124.9 (C-4),
116.6 (C-7), 83.3 (C≡CH), 72.1 (C≡CH), 56.2 (C-3), 49.2 (NCH2CH2CH2C≡CH) 27.7
(NCH2CH2CH2C≡CH), 23.0 (2 C, 3-CH3 ×2), 16.7 (NCH2CH2CH2C≡CH) ppm. (*2-CH3 was not
visible on the spectrum)
LRMS
HRMS
[ESI+, MeOH] calculated mass: 226.16 for C16H20N+ [M-I]+; found m/z: 226.2 (100 %), 227.2
(18.2 %), 228.2 (1.8 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.8 (100 %).
[ESI+, MeOH] calculated mass: 226.1590 for C16H20N1 [M-I-]+; found m/z: 226.1590
6. Synthesis of 1,2,3,3-Tetramethylbenz[e]indolium iodide (6)
CH3I
N+
N
CH3CN, reflux,
15 min,
I-
6
2,3,3-Trimethylbenz[e]indole (10.0 g, 47.78 mmol, 1 eq) was dissolved in acetonitrile (50 mL) then methyl iodide (3.57
mL, 57.34 mmol, 1.2 eq) was added and the reaction mixture heated at reflux for 15 min. The reaction was cooled to
room temperature and the precipitate was collected by filtration, washed with diethyl ether, and dried in vacuo. Product
6 was obtained as a pale olive solid in 86 % yield (14.41 g, 41.01 mmol). Mw = 351.23 g/mol (C16H18IN).
Rf:
0.19 [DCM/MeOH 95:5 v/v]
1H NMR
(400 MHz, DMSO) δ 8.36 (1 H, d, J = 8.5 Hz, H-5), 8.29 (1 H, d, J = 9.0 Hz, H-10), 8.21 (1 H, d, J =
8.0 Hz, H-8), 8.11 (1 H, d, J = 9.0 Hz, H-11), 7.77 (1 H, dd, J = 6.8, 8.0 Hz, H-6), 7.73 (1 H, dd, J =
7.6, 6.8 Hz, H-7), 4.11 (3 H, s, N-CH3), 2.89 (3 H, s, 2-CH3), 1.76 (6 H, s, 3-CH3 ×2) ppm.
5

13C NMR
LRMS
(100 MHz, DMSO) δ 195.9 (C-2), 139.4 (C-11a), 136.5 (C-3a), 133.0 (2 C, C-4, C-9), 130.5 (C-10),
129.7 (C-8), 128.3 (C-6), 127.1 (C-7), 123.4 (C-5), 113.1 (C-11), 55.2 (C-3), 35.1 (N-CH3), 21.2 (2 C,
3-CH3 ×2), 14.1 (2-CH3) ppm.
[ESI+, MeOH] calculated mass: 224.14 for C16H18N+ [M-I]+; found m/z: 224.1 (5.3 %), 225.1
(53.4 %), 226.2 (22.3 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
HRMS
[ESI+, MeOH] calculated mass: 224.1434 for C16H18N+ [M-I]+; found m/z: 224.1436
Data consistent with literature: Wang, J.; Cao, W. F.; Su, J. H.; Tian, H.; Huang, Y. H.; Sun, Z. R. Dyes Pigm. 2003, 57,
171-179.
7. Synthesis of 1-(4-pentynyl)-2,3,3-trimethylbenz[e]indolium iodide (7)
Cl
N+
N
KI, CH3CN, reflux,
24 h,
I-
7
Potassium iodide (11.90 g, 71.67 mmol, 3 eq) was suspended in acetonitrile (20 mL) then 5-chloro-1-pentyne (3.80 mL,
35.83 mmol, 1.5 eq) was added and the reaction mixture was stirred at 50 C. After 10 min 2,3,3-trimethylbenz[e]indole
(5.0 g, 23.89 mmol, 1 eq) was added and reaction mixture heated at reflux for 24 h. The reaction was cooled to room
temperature, diluted with DCM and washed with water. The organic layer was separated, dried over sodium sulfate,
filtered and the solvent was removed in vacuo. The residue was dissolved in a small volume of DCM and precipitated
with diethyl ether. The solid was filtered off, washed with diethyl ether and dried in vacuo overnight. The compound 7
was obtained as a green solid with 69 % yield (6.62 g, 16.42 mmol). Mw = 403.30 g/mol (C20H22IN).
Rf:
0.23 [DCM/MeOH 95:5 v/v]
1H NMR
(400 MHz, DMSO) δ 8.42 (1 H, d, J = 8.5 Hz, H-5), 8.35 (1 H, d, J = 9.0 Hz, H-10), 8.27 (1 H, d, J =
8.0 Hz, H-8), 8.20 (1 H, d, J = 9.0 Hz, H-11), 7.87 - 7.74 (2 H, m, H-6, H-7), 4.68 (2 H, t, J = 7.5 Hz,
NCH2CH2CH2C≡CH), 3.02 - 3.00 (1 H, m, C≡CH), 3.01 (3 H, s, 2-CH3), 2.55 - 2.49 (2 H, m,
NCH2CH2CH2C≡CH), 2.20 – 2.16 (2 H, m, NCH2CH2CH2C≡CH), 1.82 (6 H, s, 3-CH3 ×2) ppm.
13C NMR
(100 MHz, DMSO) δ 196.8 (C-2), 138.5 (C-11a), 136.9 (C-3a), 133.0 (2 C, C-4, C-9), 130.7 (C-10),
129.7 (C-8), 128.4 (C-6), 127.2 (C-7), 123.4 (C-5), 113.1 (C-11), 83.1 (C≡CH), 72.4 (C≡CH), 55.6
(C-3), 47.1 (NCH2CH2CH2C≡CH), 26.2 (NCH2CH2CH2C≡CH), 21.6 (2 C, 3-CH3 ×2), 15.2
(NCH2CH2CH2C≡CH), 14.0 (2-CH3) ppm.
LRMS
HRMS
[ESI+, MeOH] calculated mass: 276.17 for C20H22N+ [M-I]+; found m/z: 276.3 (28.4 %), 277.3
(7.0 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ESI+, MeOH] calculated mass: 276.1747 for C20H22N+ [M-I-]+; found m/z: 276.1752
6

8. Synthesis of N-((1E)-3-(phenylimino)prop-1-enyl)benzenamine hydrochloride (malondialdehyde dianil
hydrochloride) (8)
An aqueous (171 mL) solution of concentrated hydrochloric acid (8.5 mL), and malondialdehyde bis(dimethyl acetal)
(10.50 mL, 0.06 mol, 1 eq) was heated to 50 °C, then a solution of water (210 mL), hydrochloric acid (15 mL), and
aniline (11.1 mL, 0.12 mol, 2 eq) was added dropwise. During the addition of the aniline solution the colorless reaction
mixture gradually changed to orange. The reaction had reached completion after 4 h. The precipitate was isolated by
filtration to give compound 8 as an orange solid with 72 % yield (11.22 g, 0.04 mol). Mw = 258.75 g/mol (C15H15ClN2).
Rf:
0.48 [DCM/MeOH 9:1 v/v]
1H NMR
(400 MHz, MeOD) δ 8.72 (2 H, d, J = 11.5 Hz, NCH=CH-CH=NH), 7.48 - 7.42 (4 H, m, H-2, H-2’,
H-6, H-6’), 7.41 - 7.37 (4 H, m, H-3, H-3’, H-5, H-5’), 7.30 - 7.24 (2 H, m, H-4, H-4’), 6.29 (1 H, t, J
= 11.5 Hz, NCH=CH-CH=NH) ppm.
13C NMR
LRMS
(100 MHz, MeOD) δ 159.7 (2 C, NCH=CH-CH=NH ), 139.8 (2 C, C-1, C-1’), 131.1 (4 C, C-2, C-2’,
C-6, C-6’), 127.6 (2 C, C-4, C-4’), 118.8 (4 C, C-3, C-3’, C-5, C-5’), 99.4 (1 C, s NCH=CH-CH=NH)
ppm.
[ESI+, MeOH] calculated mass: 223.12 for C15H15N2 [M-Cl-]+; found m/z : 223.2 (100 %), 224.2
+
(17.1 %), 225.2 (1.4 %).
Data consistent with literature: Jung, M. E., Kim, W. J., Bioorg. Med. Chem. 2006, 14, 92-97.
7

II. Cyanine Dyes synthesis
1. Synthesis of Ethynyl-Cy5 (I)
I
H
(i)
(ii)
N
Ph
N+
N
N+
I-
N+
I-
1
9
(hemicyanine
intermediate)
Si
(iii)
(iv)
N+
I-
N
N+
N
I-
10
I
Scheme S1. Synthesis of Ethynyl-Cy5 (I): (i) 8, Ac2O, reflux, 30 min; (ii) 4, Py, RT, 2 h, 75 %; (iii)
(trimethylsilyl)acetylene, CuI, Pd(PPh3)4, Et3N, DMF, 50 °C, 48 h, 58 %; (iv) TBAF, THF, RT, 2 h, 55 %.
1.1. Synthesis of 2-[5-(2,3-Dihydro-1,3,3-trimethyl-1H-indol-2-ylidene)-1,3-pentadienyl]-1-butyl-3,3-dimethyl-5-iodo-
3H-indolium Iodide (9)
4
7
4'
I
3a
3a'
5'
3
3'
5
6
2
2'
1'
6'
N+1
N
7a
7a'
7'
I-
9
1,2,3,3-Tetramethyl-3H-indolium iodide 1 (1.0 g, 3.32 mmol, 1 eq) and malondialdehyde bis(phenylimine)
monohydrochloride 8 (1.29 g, 4.98 mmol, 1.5 eq) in acetic anhydride (11 mL) were heated at reflux under argon. After
30 min of heating the reaction mixture was cooled to room temperature, then a solution of 1-butyl-5-iodo-2,3,3-
tetramethyl-3H-indolium iodide 4 (2.65 g, 5.64 mmol, 1.7 eq) in anhydrous pyridine (12 mL) was added and the
reaction mixture was stirred under argon at ambient temperature for 2 h. The solvent was removed in vacuo, the residue
was dissolved in chloroform and precipitated with hexane. The supernatant was decanted off and the residual oil was
dissolved in chloroform, washed with water and 0.1 M HCl. The organic layer was separated, dried over sodium sulfate
and the solvent was removed in vacuo. Column chromatography (0-5 % MeOH in DCM/EtOAc 1:1) afforded product 9
as a blue solid with 75 % yield (1.68 g, 2.48 mmol). Mw = 678.43 g/mol (C30H36I2N2).
Rf:
0.38 [DCM/MeOH 95:5 v/v]
1H NMR
(400 MHz, MeOD) δ 8.31 – 8.18 (2 H, m, CH=CH-CH=CH-CH), 7.79 (1 H, d, J = 1.6 Hz, H-4’),
7.71 (1 H, dd, J = 8.3, 1.6 Hz, H-6’), 7.52 (1 H, d, J = 7.5 Hz, H-4), 7.47 - 7.40 (1 H, m, H-6), 7.38 -
7.27 (2 H, m, H-5, H-7), 7.05 (1 H, d, J = 8.4 Hz, H-7’), 6.65 (1 H, t, J = 12.4 Hz, CH=CH-CH=CH-
CH), 6.36, 6.21 (1 H, d, J = 13.8 Hz; 1 H, d, J = 13.6 Hz, CH=CH-CH=CH-CH), 4.03 (2 H, t, J = 7.5
Hz, N’CH2CH2CH2CH3), 3.67 (3 H, s, N-CH3), 1.74, 1.71 (6 H, s, 3-CH3 ×2; 6 H, s, 3’-CH3 ×2), 1.79
- 1.75 (2 H, m, N’CH2CH2CH2CH3), 1.47 (2 H, dq, J = 15.2, 7.5 Hz, N’CH2CH2CH2CH3), 1.01 (3 H,
t, J = 7.4 Hz, N’CH2CH2CH2CH3) ppm.
13C NMR
(100 MHz, MeOD) δ 176.5 (C-2), 172.7 (C-2’), 156.2, 154.7 (CH=CH-CH=CH-CH), 144.7, 144.0,
143.7, 142.8 (C-3a, C-3a’, C-7a, C-7a’), 138.6 (C-6’), 132.5 (C-4’), 129.7 (C-6), 127.0 (CH=CH-
CH=CH-CH), 126.7 (C-5), 123.3 (C-4), 113.5 (C-7’), 112.2 (C-7), 105.5, 103.6 (CH=CH-CH=CH-
CH), 88.3 (C-5’), 50.8, 50.1 (C-3, C-3’), 44.6 (N’CH2CH2CH2CH3), 31.8 (N-CH3), 30.3
8

(N’CH2CH2CH2CH3) 27.9, 27.6 (2 C, 3-CH3 ×2; 2 C, 3’-CH3 ×2), 21.1 (N’CH2CH2CH2CH3), 14.1
(N’CH2CH2CH2CH3) ppm.
+
LRMS
HRMS
[ESI+, MeOH] calculated mass: 551.19 for C30H36IN2 [M-I-]+; found m/z: 551.2 (100 %), 552.3
(30.1 %), 553.3 (4.3 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ESI+, MeOH] calculated mass: 551.1918 for C30H36IN2+ [M-I-]+; found m/z: 551.1921
1.2. Synthesis of 2-[5-(2,3-Dihydro-1,3,3-trimethyl-1H-indol-2-ylidene)-1,3-pentadienyl]-1-butyl-3,3-dimethyl-5-
(trimethylsilyl)ethynyl-3H-indolium Iodide (10)
Si
4
4'
3a'
7a'
3a
7a
5'
3
3'
5
6
2
2'
6'
N+1
1' N
I-
7'
7
10
Iodo-Cy5 9 (6.02 g, 8.87 mmol, 1 eq) was dissolved in anhydrous DMF (30 mL), then anhydrous triethylamine (30
mL), copper (I) iodide (0.34 g, 1.77 mmol, 0.2 eq), and (trimethylsilyl)acetylene (6.27 mL, 44.37 mmol, 5 eq) were
added successively under argon. After ten min of stirring the tetrakis(triphenylphosphine)palladium (0) (1.03 g, 0.89
mmol, 0.1 eq) was added to the reaction mixture and it was stirred under argon at room temperature overnight. After 24
h another aliquot of CuI (0.34 g, 1.77 mmol, 0.2 eq) and (trimethylsilyl)acetylene (6.27 mL, 44.37 mmol, 5 eq) were
added and the reaction mixture was heated at 50 °C overnight. After a total of 48 h the reaction mixture was cooled to
room temperature and solvents were removed under reduced pressure. The residue was purified by column
chromatography (0-4 % MeOH/DCM) to give the product 10 with 58 % yield (3.32 g, 5.12 mmol). Mw = 648.74 g/mol
(C35H45IN2Si).
Rf:
0.67 [DCM/MeOH 9:1 v/v]
1H NMR
(400 MHz, CDCl3) δ 8.18 - 8.05 (2 H, m, CH=CH-CH=CH-CH), 7.72 - 7.63 (1 H, m, H-4’), 7.51 -
7.44 (1 H, m, H-6’), 7.44 - 7.36 (2 H, m, H-4, H-6), 7.31 - 7.28 (1 H, m, H-5), 7.18 (1 H, d, J = 7.8
Hz, H-7), 6.97 (1 H, d, J = 8.2 Hz, H-7’), 6.88 (1 H, t, J = 12.6 Hz, CH=CH-CH=CH-CH), 6.47, 6.25
(1 H, d, J = 13.8 Hz; 1 H, d, J = 13.4 Hz, CH=CH-CH=CH-CH), 4.01 (2 H, t, J = 7.5 Hz,
N’CH2CH2CH2CH3), 3.77 (3 H, s, N-CH3), 1.81 - 1.77 (2 H, m, N’CH2CH2CH2CH3), 1.76, 1.75 (6 H,
s, 3-CH3 ×2; 6 H, s, 3’-CH3 ×2), 1.48 (2 H, dq, J = 15.2, 7.4 Hz, N’CH2CH2CH2CH3), 1.00 (3 H, t, J =
7.3 Hz, N’CH2CH2CH2CH3), 0.27 (9 H, s, Si(CH3)3) ppm.
13C NMR
(100 MHz, CDCl3) δ 174.4, 171.5 (C-2, C-2’), 154.1, 152.8 (CH=CH-CH=CH-CH), 142.5, 142.2,
141.1, 141.0 (C-3a, C-3a’, C-7a, C-7a’), 132.6 (C-6’), 128.7 (C-6), 126.7 (CH=CH-CH=CH-CH),
125.7 (C-5), 122.2 (C-4), 119.8 (C-4’), 119.1 (C-5’), 110.9 (C-7), 109.9 (C-7’), 105.1, 103.4
(CH=CH-CH=CH-CH), 104.5 (C≡C-Si(CH3)3), 95.1 (C≡C-Si(CH3)3), 49.6, 48.8 (C-3, C-3’), 44.1
(N’CH2CH2CH2CH3), 32.8 (N-CH3), 29.3 (N’CH2CH2CH2CH3), 28.1, 27.9 (2 C, 3-CH3 ×2; 2 C, 3’-
CH3 ×2), 20.2 (N’CH2CH2CH2CH3), 13.8 (N’CH2CH2CH2CH3), -0.1 (3 C, Si(CH3)3) ppm.
LRMS
[ESI+, MeOH] calculated mass: 521.33 for C35H45N2Si+ [M-I-]+; found m/z: 521.4 (100 %), 525.5
(41.6 %), 523.4 (12.3 %), 524.4 (1.8 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.8 (100 %).
9

1.3. Synthesis of 2-[5-(2,3-Dihydro-1,3,3-trimethyl-1H-indol-2-ylidene)-1,3-pentadienyl]-1-butyl-3,3-dimethyl-5-
ethynyl-3H-indolium Iodide (I)
Silyl-ethynyl-Cy5 10 (3.18 g, 4.90 mmol, 1 eq) was dissolved in freshly distilled THF (135 mL), then
tetrabutylammonium fluoride (1 M solution in THF) (7.35 mL, 7.35 mmol, 1.5 eq) was added and the reaction was
carried out under argon at room temperature. After 2 h the reaction mixture was diluted with DCM and washed with
saturated aqueous sodium bicarbonate. The DCM layer was dried over anhydrous sodium sulfate, filtered, and the
solvent was removed in vacuo. The green residue was purified by column chromatography (0-10 % MeOH/DCM) to
afford the product I as a red-gold foam in 55 % yield (1.54 g, 2.67 mmol). Mw = 576.55 g/mol (C32H37IN2).
Rf:
IR
0.38 [DCM/MeOH 9:1 v/v]
(neat): 3235 (br, ≡CH, alkyne), 3050 (w, C-H, aromatic), 2958 (m, =CH, alkene), 2929 (m, C-H,
alkyl), 2130 (w, C≡C, alkyne), 1672, 1599 (m, C=C, alkene), 1537-1449 (s, C-C, aromatic) cm-1.
1H NMR
(400 MHz, CDCl3) δ 8.16 - 7.97 (2 H, m, CH-CH=CH-CH=CH), 7.70 - 7.58 (2 H, m, H-4’, H-5),
7.49 - 7.34 (3 H, m, H-4, H-6, H-6’), 7.16 (1 H, d, J = 7.5 Hz, H-7’), 6.99 - 6.93 (1 H, m, H-7), 6.83 (1
H, t, J = 12.3 Hz, CH-CH=CH-CH=CH), 6.42 (1 H, d, J = 14.1 Hz, CH-CH=CH-CH=CH), 6.20 (1 H,
d, J = 13.6 Hz, CH-CH=CH-CH=CH), 3.98 (2 H, t, J = 7.3 Hz, N’CH2CH2CH2CH3), 3.74 (3 H, s, N-
CH3), 3.13 (1 H, s, C≡CH), 1.73 (12 H, s, 3-CH3 ×2, 3’-CH3 ×2), 1.70 - 1.62 (2 H, m,
N’CH2CH2CH2CH3), 1.51 - 1.36 (2 H, m, N’CH2CH2CH2CH3), 1.03 - 0.92 (3 H, m,
N’CH2CH2CH2CH3) ppm.
13C NMR
(100 MHz, CDCl3) δ 174.8, 171.6 (C-2, C-2’), 154.4, 153.0 (CH-CH=CH-CH=CH), 142.6, 142.6,
141.2, 138.5 (C-7a, C-7a’, C-3a, C-3a’), 133.0 (C-4’), 132.2, 128.6 (C-6, C-6’) 126.9 (CH-CH=CH-
CH=CH), 125.9, 122.4, (C-4, C-5), 118.2 (C-5’), 111.1, 110.1 (C-7, C-7’), 105.3 (CH-CH=CH-
CH=CH), 103.5 (CH-CH=CH-CH=CH), 83.3 (C≡CH), 78.0 (C≡CH), 49.8, 49.0 (C-3, C-3’), 44.3
(N’CH2CH2CH2CH3), 33.0 (N-CH3), 29.5 (N’CH2CH2CH2CH3), 28.3, 28.0 (2 C, 3-CH3 ×2; 2 C, 3’-
CH3 ×2), 24.4 (N’CH2CH2CH2CH3), 13.9 (N’CH2CH2CH2CH3) ppm.
+
LRMS
HRMS
[ESI+, MeOH] calculated mass: 449.30 for C32H37N2 [M-I-]+; found m/z: 449.3 (34.5 %), 450.4
(12.0 %), 451.5 (2.0 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.8 (100 %).
[ESI+, MeOH] calculated mass: 449.2951 for C32H37N2+ [M-I-]+; found m/z: 449.2949
10

2. Synthesis of N-pentynyl-Cy5 (II)
(ii)
(i)
NHPh
N+
N+
N+
I-
N
I-
hemicyanine intermediate
5
II
Scheme S2. Synthesis of N-pentynyl-Cy5 (II): (i) 8, Ac2O, reflux, 30 min; (ii) 1, Py, RT, 15 h, 83 %.
2.1. Synthesis of 2-[5-(2,3-Dihydro-1-(4-pentynyl)-3,3-dimethyl-1H-indol-2-ylidene)-1,3-pentadienyl]-1,3,3-trimethyl-
3H-indolium Iodide (II)
1-(4-Pentynyl)-2,3,3-trimethyl-3H-indolium iodide 5 (1.76 g, 5.0 mmol, 1 eq) and malondialdehyde bis(phenylimine)
monohydrochloride 8 (1.68 g, 6.50 mmol, 1.3 eq) were suspended in acetic anhydride (17 mL) and heated at reflux
under argon. After 30 min the reaction mixture was cooled to room temperature and the solution of 1,2,3,3-tetramethyl-
3H-indolium iodide 1 (3.01 g, 10.0 mmol, 2 eq) in anhydrous pyridine (21 mL) was added and the reaction mixture
stirred under argon at ambient temperature for 15 h. After precipitation with cold diethyl ether, the green solution was
decanted off and the precipitate was purified by column chromatography (0-5 % MeOH in EtOAc) to give the product
II as a green-gold foam with 83 % yield (2.34 g, 4.16 mmol). Mw = 562.53 g/mol (C31H35IN2).
Rf:
0.36 [EtOAc/MeOH 95:5 v/v]
1H NMR
(400 MHz, CD3CN) δ 8.12, 8.11 (1 H, dd, J = 13.2, 12.8; 1 H, dd, J = 13.2, 12.8, Hz, CH=CH-
CH=CH-CH), 7.49, 7.47 (1 H, d, J = 7.2 Hz; 1 H, d, J = 6.8 Hz; H-4, H-4’), 7.43 - 7.34 (2 H, m, H-6,
H-6’), 7.30 - 7.19 (4 H, m, H-5, H-5’, H-7, H-7’), 6.58 (1 H, t, J = 12.5 Hz, CH=CH-CH=CH-CH),
6.29, 6.26 (1 H, d, J = 13.6 Hz; 1 H, d, J = 14.0 Hz, CH=CH-CH=CH-CH) 4.11 (2 H, t, J = 8.0 Hz,
NCH2CH2CH2C≡CH), 3.57 (3 H, s, N’-CH3), 2.41 - 2.32 (3 H, m, NCH2CH2CH2C≡CH), 1.99 - 1.94
(2 H, m, NCH2CH2CH2C≡CH), 1.68 (12 H, s, 3-CH3 ×2, 3’-CH3 ×2) ppm.
13C NMR
(100 MHz, CD3CN) δ 175.5, 174.0 (C-2, C-2’), 155.2, 154.7 (CH=CH-CH=CH-CH), 144.0, 143.4,
142.5, 142.3 (C-3a, C-3a’, C-7a, C-7a’), 129.5, 129.6 (C-6, C-6’), 126.2 (2 C, C-5, C-5’), 125.8
(CH=CH-CH=CH-CH), 123.3, 123.2 (C-4, C-4’), 112.0, 111.7 (C-7, C-7’), 104.7, 103.8 (CH=CH-
CH=CH-CH), 84.1 (C≡CH), 71.3 (C≡CH), 50.3, 50.1 (C-3, C-3’), 43.8 (NCH2CH2CH2C≡CH), 32.4
(N’-CH3), 27.9, 27.6 (2 C, 3-CH3 ×2; 2 C, 3’-CH3 ×2), 26.9 (NCH2CH2CH2C≡CH), 16.5
(NCH2CH2CH2C≡CH) ppm.
+
LRMS
HRMS
[ESI+, MeOH] calculated mass: 435.28 for C31H35N2 [M-I-]+; found m/z: 435.2 (100 %), 436.3
(32.3 %), 437.3 (5.0 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.8 (100 %).
[ES] calculated mass: 435.2795 for C31H35N2+ [M-I-]+; found m/z: 435.2789
11

3. Synthesis of Ethynyl-Cy5.25 (III)
Scheme S3. Synthesis of ethynyl-Cy5.25 (III): (i) 8, Ac2O, reflux, 30 min; (ii) 3, Py, RT, 24 h, 81 %; (iii)
(trimethylsilyl)acetylene, Et3N, CuI, Pd(PPh3)4, DMF, RT, 48 h, 69 %; (iv) TBAF, THF, RT, 15 min, 82 %.
3.1. Synthesis of 2-[5-(2,3-Dihydro-1,3,3-trimethyl-1H-benz[e]indol-2-ylidene)-1,3-pentadienyl]-5-iodo-1,3,3-
trimethyl-3H-indolium Iodide (11)
1,2,3,3-Tetramethylbenz[e]indolium iodide 6 (5.0 g, 14.24 mmol, 1 eq) and malondialdehyde bis(phenylimine)
monohydrochloride 8 (4.42 g, 17.08 mmol, 1.2 eq) in acetic anhydride (42 mL) were heated at reflux under argon. After
30 min of heating the reaction mixture was cooled to room temperature and a solution of 5-iodo-1,2,3,3-tetramethyl-3H-
indolium iodide 3 (7.30 g, 17.08 mmol, 1.2 eq) in anhydrous pyridine (36 mL) was added. The reaction mixture was
stirred under argon at ambient temperature for 24 h then precipitated with diethyl ether. The solid was filtered off,
washed with diethyl ether and dried in vacuo. The crude product was purified by column chromatography (0-2 %
MeOH/DCM) to give compound 11 in 81 % yield (7.90 g, 11.51 mmol). Mw = 686.41 g/mol (C31H32I2N2).
Rf:
0.57 [DCM/MeOH 9:1 v/v]
1H NMR
(400 MHz, DMSO) δ 8.43 (2 H, dd, J = 13.8, 12.3 Hz, CH-CH=CH-CH=CH), 8.23 (1 H, d, J = 8.0
Hz, H-5), 8.09 (1 H, d, J = 9.0 Hz, H-10), 8.05 (1 H, d, J = 8.0 Hz, H-8), 7.97 (1 H, d, J = 2.0 Hz, H-
4’), 7.78 (1 H, d, J = 9.0 Hz, H-11), 7.71 - 7.63 (2 H, m, H-6, H-6’), 7.51 (1 H, t, J = 7.8 Hz, H-7),
7.14 (1 H, d, J = 8.5 Hz, H-7’), 6.55 (1 H, t, J = 12.3 Hz, CH-CH=CH-CH=CH), 6.40, 6.15 (1 H, d, J
= 14.1 Hz, CH-CH=CH-CH=CH; 1 H, d, J = 13.6 Hz, CH-CH=CH-CH=CH), 3.79, 3.52 (3 H, s, N-
CH3; 3 H, s, N’-CH3), 1.91, 1.65 (6 H, s, 3-CH3 ×2; 6 H, s, 3’-CH3 ×2) ppm.
13C NMR
(100 MHz, DMSO) δ 175.6, 170.8 (C-2, C-2’), 153.9, 152.7 (CH-CH=CH-CH=CH), 143.2, 142.8 (C-
3a’, C-7a’), 140.2 (C-11a), 136.7 (C-6’), 133.6 (C-3a), 131.5 (C-9), 130.9, 130.2, 129.9 (C-4’, C-10,
C-8), 127.8 (CH-CH=CH-CH=CH), 127.3 (C-4), 125.4, 125.0 (C-7, C-6), 122.2 (C-5), 112.6, 111.8
(C-11, C-7’), 104.3, 102.3 (CH-CH=CH-CH=CH), 87.9 (C-5’), 51.0, 48.4 (C-3, C-3’), 31.9, 30.9 (N-
CH3, N’-CH3), 26.9, 26.4 (2 C, 3-CH3 ×2; 2 C, 3’-CH3 ×2) ppm.
[ESI+, MeOH] calculated mass: 559.16 for C31H32IN2 [M-I-]+; found m/z: 559.2 (100 %), 560.3
+
LRMS
(29.6 %), 561.3 (4.6 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
12

HRMS
[ESI+, MeOH] calculated mass: 559.1605 for C31H32IN2+ [M-I-]+; found m/z: 559.1599
3.2.
Synthesis
of
2-[5-(2,3-Dihydro-1,3,3-trimethyl-1H-benz[e]indol-2-ylidene)-1,3-pentadienyl]-5-
(trimethylsilyl)ethynyl-1,3,3-trimethyl-3H-indolium Iodide (12)
Iodo-Cy5.25 11 (7.80 g, 11.36 mmol, 1 eq) was dissolved in anhydrous DMF (57 mL) and triethylamine (40 mL), then
copper (I) iodide (0.43 g, 2.27 mmol, 0.2 eq) and (trimethylsilyl)acetylene (8.03 mL, 56.80 mmol, 5 eq) were added.
The reaction mixture was stirred for 10 min, before tetrakis(triphenylphosphine)palladium (0) (1.31 g, 1.14 mmol, 0.1
eq) was added and the reaction mixture was stirred at room temperature, under argon for 14 h. The reaction was not yet
completed and additional amount of (trimethylsilyl)acetylene (8.03 mL, 56.80 mmol, 5 eq), and copper (I) iodide (0.43
g, 2.27 mmol, 0.2 eq) were added and the reaction continued for 48 h in total. The mixture was filtered through Celite,
then diluted with DCM and washed with sodium bicarbonate and brine. The organic layer was dried over sodium
sulfate, filtered, and the solvent was removed in vacuo. The crude mixture was purified by column chromatography (0-
5 % MeOH/DCM) to afford the product 12 with 69 % yield (5.13 g, 7.81 mmol). Mw = 656.71 g/mol (C36H41IN2Si).
Rf:
0.54 [DCM/MeOH 9:1 v/v]
1H NMR
(400 MHz, DMSO) δ 8.46 (2 H, t, J = 12.8 Hz, CH-CH=CH-CH=CH), 8.24 (1 H, d, J = 8.5 Hz, H-5),
8.10 (1 H, d, J = 9.0 Hz, H-10), 8.05 (1 H, d, J = 7.5 Hz, H-8), 7.81 (1 H, dd, J = 8.8, 1.8 Hz, H-11),
7.70 (1 H, s, H-4’), 7.67 (1 H, d, J = 8.5 Hz, H-6), 7.52 (1 H, t, J = 6.8 Hz, H-7), 7.44 (1 H, d, J = 8.0
Hz, H-6’), 7.28 (1 H, d, J = 8.0 Hz, H-7’), 6.57 (1 H, t, J = 11.8 Hz, CH-CH=CH-CH=CH), 6.46 (1
H, d, J = 13.6 Hz, CH-CH=CH-CH=CH), 6.16 (1 H, d, J = 13.1 Hz, CH-CH=CH-CH=CH), 3.8 (3 H,
s, N-CH3), 3.54 (3 H, s, N’-CH3), 1.91 (6 H, s, 3-CH3 ×2), 1.66 (6 H, s, 3’-CH3 ×2), 0.21 (9 H, s,
Si(CH3)3) ppm.
13C NMR
(100 MHz, DMSO) δ 176.0, 171.0 (C-2, C-2’), 154.1, 152.6 (CH-CH=CH-CH=CH), 143.4 (C-7a’),
141.0 (C-3a’), 140.1 (C-11a), 133.8 (C-3a), 132.1 (C-6’), 131.6 (C-9), 130.2, 129.9 (C-10, C-8),
127.8 (C-6), 127.3 (C-4), 125.7, 125.6 (C-7, C-4’), 125.1 (CH-CH=CH-CH=CH), 122.2 (C-5), 117.0
(C-5’), 111.9 (C-11), 110.4 (C-7’), 105.5 (C≡C-Si(CH3)3), 104.8, 102.5 (CH-CH=CH-CH=CH), 93.7
(C≡C-Si(CH3)3), 51.1, 48.1 (C-3, C-3’), 32.0 (N-CH3), 30.8 (N’-CH3), 27.0 (2 C, 3-CH3 ×2), 26.3 (2
C, 3’-CH3 ×2), -0.1 (3 C, C≡C-Si(CH3)3) ppm.
LRMS
HRMS
[ESI+, MeOH] calculated mass: 529.30 for C36H41N2Si+ [M-I-]+; found m/z: 529.4 (100 %), 530.5
(42.3 %), 531.5 (8.4 %), 531.5 (4.7 %) 532.5 (1.4 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ESI+, MeOH] calculated mass: 529.3034 for C36H41N2Si+ [M-I-]+; found m/z: 529.3031
13

3.3. Synthesis of 2-[5-(2,3-Dihydro-1,3,3-trimethyl-1H-benz[e]indol-2-ylidene)-1,3-pentadienyl]-5-ethynyl-1,3,3-
trimethyl-3H-indolium Iodide (III)
Silyl-ethynyl-Cy5.25 12 (4.89 g, 7.44 mmol, 1 eq) was dissolved in anhydrous THF (70 mL), then 1 M solution of
tetrabutylammonium fluoride in THF (11.16 mL, 11.16 mmol, 1.5 eq) was added and the reaction stirred under argon,
at room temperature for 15 min. The mixture was diluted with DCM, washed with saturated sodium hydrogen carbonate
and potassium iodide aqueous solutions, dried over sodium sulfate, filtered, and the solvent was removed in vacuo. The
crude product was purified by column chromatography (0-3 % MeOH/DCM) to give compound III as a red-gold foam
with 82 % yield (3.58 g, 6.12 mmol). Mw = 584.53 g/mol (C33H33IN2).
Rf:
0.46 [DCM/MeOH 9:1 v/v]
1H NMR
(400 MHz, DMSO) δ 8.47 (2 H, t, J = 13.0 Hz, CH-CH=CH-CH=CH), 8.26 (1 H, d, J = 7.0 Hz, H-5),
8.11 (1 H, dd, J = 8.8, 2.3 Hz, H-10), 8.07 (1 H, d, J = 8.5 Hz, H-8), 7.80 (1 H, d, J = 8.8, Hz, H-11),
7.73 (1 H, s, H-4’), 7.69 (1 H, t, J = 8.0 Hz, H-6), 7.57 - 7.45 (2 H, m, H-7, H-6’), 7.29 (1 H, dd, J =
8.0, 2.5 Hz, H-7’), 6.59 (1 H, t, J = 12.3 Hz, CH-CH=CH-CH=CH), 6.46 (1 H, d, J = 14.6 Hz, CH-
CH=CH-CH=CH), 6.17 (1 H, d, J = 13.6 Hz, CH-CH=CH-CH=CH), 4.18 (1 H, d, J = 2.5 Hz,
C≡CH), 3.80 (3 H, s, N-CH3), 3.54 (3 H, s, N’-CH3), 1.94 (6 H, s, 3-CH3 ×2), 1.68 (6 H, s, 3’-CH3 ×2)
ppm.
13C NMR
(100 MHz, DMSO) δ 176.0, 171.0 (C-2, C-2’), 154.0, 152.5 (CH-CH=CH-CH=CH), 143.4 (C-7a’),
141.0 (C-3a’), 140.1 (C-11a), 133.8 (C-3a), 132.2 (C-6’), 131.6 (C-9), 130.2, 129.9 (C-10, C-8),
127.8 (C-6), 127.3 (C-4), 125.6, 125.5 (C-7, C-4’), 125.1 (CH-CH=CH-CH=CH), 122.2 (C-5), 116.6
(C-5’), 111.8 (C-11), 110.4 (C-7’), 104.7, 102.4 (CH-CH=CH-CH=CH), 83.7 (C≡CH), 80.5 (C≡CH),
51.1, 48.1 (C-3, C-3’), 32.0, 30.8 (N-CH3, N’-CH3), 27.0, 26.4 (2 C, 3-CH3 ×2; 2 C, 3’-CH3 ×2) ppm.
+
LRMS
HRMS
[ESI+, MeOH] calculated mass: 457.26 for C33H33N2 [M-I-]+; found m/z: 457.3 (100 %), 458.3
(33.7 %), 459.3 (6.0 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ESI+, MeOH] calculated mass: 457.2638 for C33H33N2+ [M-I-]+; found m/z: 457.2628
4. Synthesis of N-pentynyl-Cy5.5 (IV)
Scheme S4. Synthesis of N-pentynyl-Cy5.5 (IV): (i) 8, Ac2O, reflux, 30 min; (ii) 7, Py, RT, 12 h, 53 %.
14

4.1. Synthesis of 2-[5-(2,3-Dihydro-1,3,3-trimethyl-1H-benz[e]indol-2-ylidene)-1,3-pentadienyl]-3,3-dimethyl-1-(4-
pentynyl)-3H-benz[e]indolium Iodide (IV)
1,2,3,3-Tetramethylbenz[e]indolium iodide 6 (5.0 g, 14.24 mmol, 1 eq) and malondialdehyde bis(phenylimine)
monohydrochloride 8 (4.42 g, 17.08 mmol, 1.2 eq) in acetic anhydride (42 mL) was heated at reflux under argon. After
30 min the reaction mixture was cooled to room temperature and
a solution of 1-pentynyl-2,3,3-
trimethylbenz[e]indolium iodide 7 (6.0 g, 14.88 mmol, 1.05 eq) in anhydrous pyridine (36 mL) was added and the
reaction mixture stirred under argon at ambient temperature overnight. The reaction mixture was precipitated with
diethyl ether and the solid was filtered off, washed with diethyl ether and dried in vacuo. Column chromatography (0-
2 % MeOH/DCM) provided product IV as a red-gold foam in 53 % yield (5.02 g, 7.57 mmol). Mw = 662.64 g/mol
(C39H39IN2).
Rf:
0.53 [DCM/MeOH 9:1 v/v]
1H NMR
(400 MHz, DMSO) δ 8.46 (2 H, td, J = 13.1, 7.0 Hz, CH-CH=CH-CH=CH), 8.26, 8.24 (1 H, d, J =
7.2 Hz; 1 H, d, J = 8.4 Hz, H-5, H-5’), 8.12 - 8.02 (4 H, m, H-8, H-8’, H-10, H-10’), 7.76, 7.73 (1 H,
d, J = 8.8 Hz; 1 H, d, J = 9.0 Hz, H-11, H-11’), 7.71 - 7.64 (2 H, m, H-6, H-6’), 7.54 - 7.46 (2 H, m,
H-7, H-7’), 6.62 (1 H, t, J = 12.3 Hz, CH-CH=CH-CH=CH), 6.39 (1 H, d, J = 5.0 Hz, CH-CH=CH-
CH=CH), 6.36 (1 H, d, J = 5.5 Hz, CH-CH=CH-CH=CH), 4.27 (2 H, t, J = 7.0 Hz,
NCH2CH2CH2C≡CH), 3.76 (3 H, s, N’-CH3), 2.98 (1 H, t, J = 2.8 Hz, C≡CH), 2.39 (2 H, td, J = 7.0,
2.5 Hz, NCH2CH2CH2C≡CH), 1.97, 1.96 (6 H, s, 3-CH3 ×2; 6 H, s, 3’-CH3 ×2), 1.93 - 1.90 (2 H, m,
NCH2CH2CH2C≡CH) ppm.
13C NMR
(100 MHz, DMSO) δ 174.7, 173.2 (C-2, C-2’), 153.1, 152.6 (CH-CH=CH-CH=CH), 140.3, 139.7 (C-
11a, C-11a’), 133.2, 132.8 (C-3a, C-3a’), 131.4, 131.3 (C-9, C-9’), 130.2 (2 C, C-10, C-10’), 129.9 (2
C, C-8, C-8’), 128.6 (2 C, C-6, C-6’), 127.7 (2 C, C-4, C-4’), 125.5 (CH-CH=CH-CH=CH), 124.8,
124.6 (C-7, C-7’), 122.2, 122.0 (C-5, C-5’), 111.6, 111.2 (C-11, C-11’), 103.5, 102.3 (CH-CH=CH-
CH=CH), 83.5 (C≡CH), 72.2 (C≡CH), 50.7, 50.5 (C-3, C-3’), 42.5 (NCH2CH2CH2C≡CH), 31.7 (N’-
CH3), 26.8, 26.6 (2 C, 3-CH3 ×2; 2 C, 3’-CH3 ×2), 26.1 (NCH2CH2CH2C≡CH), 15.2
(NCH2CH2CH2C≡CH) ppm.
+
LRMS
HRMS
[ESI+, MeOH] calculated mass: 535.31 for C39H39N2 [M-I-]+; found m/z: 535.4 (100 %), 536.4
(38.9 %), 537.5 (7.8 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ESI+, MeOH] calculated mass: 535.3108 for C39H39N2+ [M-I-]+; found m/z: 535.3100
15

5. Synthesis of Phenyl-ethynyl-Cy5.25 (V)
(i)
Si
N+
N
I-
III
(ii)
N+
N
I-
13
N+
N
I-
V
Scheme S5. Synthesis of phenyl-ethynyl-Cy5.25 (V): (i) (4-iodophenyl)-trimethylsilane, CuI, Pd(PPh3)4, Et3N, DMF,
RT, 24 h, 60 %; (ii) TBAF, THF, RT, 10 min, 54 %
5.1.
Synthesis
of
2-[5-(2,3-Dihydro-1,3,3-trimethyl-1H-benz[e]indol-2-ylidene)-1,3-pentadienyl]-5-(4-
trimethylsilylethynylphenyl)ethynyl-1,3,3-trimethyl-3H-indolium Iodide (13)
Ethynyl-Cy5.25 III (1.0 g, 1.71 mmol, 1 eq) was dissolved in anhydrous DMF (9 mL) and triethylamine (6 mL), then
copper (I) iodide (0.07 g, 0.34 mmol, 0.2 eq) and (4-iodophenyl)-trimethylsilane (2.05 g, 6.84 mmol, 4 eq) were added.
The reaction mixture was stirred for 10 min before tetrakis(triphenylphosphine)palladium (0) (0.20 g, 0.17 mmol, 0.1
eq) was added and the reaction was stirred at room temperature under argon for 24 h. The mixture was filtered through
Celite to remove palladium, diluted with DCM and washed with water and brine. The organic layer was separated, dried
over sodium sulfate, filtered, and the solvent removed in vacuo. Column chromatography (0-3 % MeOH/DCM)
afforded the product 13 in 60 % yield (0.78 g, 1.03 mmol). Mw = 756.83 g/mol (C44H45IN2Si).
Rf:
0.36 [DCM/MeOH 95:5 v/v]
1H NMR
(400 MHz, DMSO) δ 8.34 - 8.28 (2 H, m, CH-CH=CH-CH=CH), 8.25 (1 H, d, J = 8.6 Hz, H-5), 8.11
(1 H, d, J = 8.6 Hz, H-10), 8.06 (1 H, d, J = 7.1 Hz, H-8), 7.84 - 7.78 (2 H, m, H-11, H-4’), 7.72 -
7.65 (1 H, m, H-6), 7.58 - 7.53 (1 H, m, H-6’), 7.53 - 7.46 (5 H, m, H-7, H-13 ×2, H-14 ×2), 7.35 (1
H, d, J = 8.1 Hz, H-7’), 6.58 (1 H, t, J = 12.4 Hz, CH-CH=CH-CH=CH), 6.46, 6.18 (1 H, d, J = 14.2
Hz; 1 H, d, J = 13.1 Hz, CH-CH=CH-CH=CH), 3.82, 3.56 (3 H, s, N-CH3; 3 H, s, N’-CH3), 1.90,
1.69 (6 H, s, 3-CH3 ×2; 6 H, s, 3’-CH3 ×2), 0.24 (9 H, s, Si(CH3)3) ppm.
13C NMR
(100 MHz, DMSO) δ 176.1, 170.8 (C-2, C-2’), 154.1, 152.4 (CH-CH=CH-CH=CH), 143.6 (C-7a’),
141.2 (C-3a’), 140.1 (C-11a), 133.9 (C-3a), 132.0 (C-6’), 131.9, 131.4 (2 C, C-13 ×2; 2 C, C-14 ×2),
131.6 (C-9), 130.1, 129.9 (C-8, C-10), 127.8 (C-6), 127.3 (C-4), 125.8, 125.4, 125.0 (CH-CH=CH-
16

CH=CH, C-7, C-4’), 122.9 (C-5), 122.3, 122.0 (C-12, C-15), 116.7 (C-5’), 111.9, 110.6 (C-11, C-7’),
104.9 (C≡C-Si(CH3)3), 104.6, 102.6 (CH-CH=CH-CH=CH), 96.5 (C≡C-Si(CH3)3), 92.2 (C5’-C≡C-
C12), 88.8 (C5’-C≡C-C12), 51.2, 48.1 (C-3, C-3’), 32.0, 30.7 (N-CH3, N’-CH3), 27.1, 26.4 (2 C, 3-CH3
×2; 2 C, 3’-CH3 ×2), -0.2 (3 C, Si(CH3)3) ppm.
LRMS
HRMS
5.2.
[ESI+, MeOH] calculated mass: 629.33 for C44H45N2Si+ [M-I-]+; found m/z: 629.5 (100 %), 630.6
(54.1 %), 631.6 (15.5 %), 632.6 (3.7 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ESI+, MeOH] calculated mass: 629.3347 for C44H45N2Si+ [M-I-]+; found m/z: 629.3349
Synthesis
of
2-[5-(2,3-Dihydro-1,3,3-trimethyl-1H-benz[e]indol-2-ylidene)-1,3-pentadienyl]-5-(4-
ethynylphenyl)ethynyl-1,3,3-trimethyl-3H-indolium Iodide (V)
Silyl-ethynyl-Cy5.25 13 (0.82 g, 1.09 mmol, 1 eq) was dissolved in anhydrous THF (13 mL), then a 1 M solution of
tetrabutylammonium fluoride in THF (1.63 mL, 1.63 mmol, 1.5 eq) was added and the reaction was stirred under argon,
at room temperature for 10 min. The mixture was diluted with DCM, washed with sodium hydrogen carbonate and
aqueous potassium iodide, dried over sodium sulfate, filtered, and the solvent was removed in vacuo. The crude product
was purified by column chromatography (0-2 % MeOH/DCM) to give the compound V as a red-gold foam in 54 %
yield (0.40 g, 0.58 mmol). Mw = 684.65 g/mol (C41H37IN2).
Rf:
0.16 [DCM/MeOH 95:5 v/v]
1H NMR
(400 MHz, DMSO) δ 8.51 - 8.38 (2 H, m, CH-CH=CH-CH=CH), 8.25 (1 H, d, J = 8.4 Hz, H-5), 8.11
(1 H, d, J = 9.1 Hz, H-10), 8.09 - 8.04 (1 H, m, H-8), 7.84 - 7.78 (2 H, m, H-11, H-4’), 7.65 - 7.59 (1
H, m, H-6), 7.59 – 7.54 (1 H, m, H-6’), 7.53 – 7.48 (5 H, m, H-7, H-13 ×2, H-14 ×2), 7.35 (1 H, d, J =
8.6 Hz, H-7’), 6.58 (1 H, t, J = 12.1 Hz, CH-CH=CH-CH=CH), 6.45, 6.18 (1 H, d, J = 14.2 Hz; 1 H,
d, J = 13.1 Hz, CH-CH=CH-CH=CH), 4.37 (1 H, s, C≡CH), 3.81, 3.56 (3 H, s, N-CH3; 3 H, s, N’-
CH3), 1.93, 1.70 (6 H, s, 3-CH3 ×2; 6 H, s, 3’-CH3 ×2) ppm.
13C NMR
(100 MHz, DMSO) δ 176.1, 170.8 (C-2, C-2’), 154.1, 152.4 (CH-CH=CH-CH=CH), 143.5 (C-7a’),
141.1 (C-3a’), 140.1 (C-11a), 133.9 (C-3a), 132.0, 131.3 (2 C, C-13 ×2; 2 C, C-14 ×2), 131.6, 131.4
(C-9, C-6’), 130.3, 129.9 (C-8, C-10), 127.8 (C-4), 127.3 (C-6), 125.4, 125.1 (C-7, C-4’), 125.8 (CH-
CH=CH-CH=CH), 122.9, 121.6 (C-12, C-15), 122.3 (C-5), 116.7 (C-5’), 111.9 (C-11), 110.5 (C-7’),
104.8, 102.5 (CH-CH=CH-CH=CH), 91.9 (C5’-C≡C-C12), 88.7 (C5’-C≡C-C12), 83.0, 82.9 (C≡CH),
51.2, 48.1 (C-3, C-3’), 32.0, 30.8 (N-CH3, N’-CH3), 27.1, 26.4 (2 C, 3-CH3 ×2; 2 C, 3’-CH3 ×2) ppm.
+
LRMS
HRMS
[ESI+, MeOH] calculated mass: 557.30 for C41H37N2 [M-I-]+; found m/z: 557.4 (100 %), 558.5
(43.7 %), 559.5 (8.1 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ESI+, MeOH] calculated mass: 557.2951 for C41H37N2+ [M-I-]+; found m/z: 557.2948
17

6. Synthesis of Ethynyl-Cy3 (VI)
I
I
I
(i)
(ii)
NHPh
N+
N+
N+
N
I-
I-
I-
4
15
14
Si
(iii)
(iv)
N+
N+
N
N
I-
I-
VI
16
Scheme S6. Synthesis of ethynyl-Cy3 (VI) (i) N,N’-diphenylformamidine, triethylorthoformate, ethanol, reflux, 6 h,
88 %; (ii) 1, Py, acetic anhydride, 50 °C, 18 h, 95 %; (iii) (trimethylsilyl)acetylene, Et3N, CuI, Pd(PPh3)4, DMF, RT, 20
h, 53 %; (iv) TBAF, THF, RT, 30 min, 63 %.
6.1. Synthesis of 5-Iodo-1,3,3-trimethyl-2-[(E)-2-(phenylamino)ethenyl]-3H-indolium Iodide (14)
1-Butyl-5-iodo-2,3,3-trimethyl-3H-indolium iodide 4 (2.74 g, 5.84 mmol, 1 eq) and N,N’-diphenylformamidine (1.26 g,
6.42 mmol, 1.1 eq) were suspended in ethanol (18 mL), then triethylorthoformate (1.10 mL, 6.42 mmol, 1.1 eq) was
added and the suspension heated to reflux. After 6 h, the reaction mixture was cooled to room temperature then kept on
ice for 5 min to complete precipitation. The precipitate was filtered off, washed with diethyl ether, and dried in vacuo to
give the product 14 as a red solid with 88 % yield (2.93 g, 5.13 mmol). Mw = 572.26 g/mol (C22H26I2N2).
Rf:
0.58 [DCM/MeOH 9:1 v/v]
1H NMR
(400 MHz, DMSO) δ 11.97 (1 H, br. s., N-H), 8.68 (1 H, dd, J = 12.3, 1.76 Hz, CH=CH-NHPh),
8.09 (1 H, s, H-4), 7.82 - 7.75 (1 H, m, H-6), 7.58 - 7.42 (4 H, m, H-9 ×2, H-10 ×2), 7.38 (1 H, dd, J =
8.5, 2.0 Hz, H-7), 7.33 - 7.26 (1 H, m, H-11), 6.19 (1 H, dd, J = 12.1, 1.5 Hz, CH=CH-NHPh), 4.06 (2
H, t, J = 6.8 Hz, NCH2CH2CH2CH3), 1.70 (3 H, s, 3-CH3), 1.69 (3 H, s, 3-CH3), 1.75 - 1.63 (2 H, m,
NCH2CH2CH2CH3), 1.46 - 1.35 (2 H, m, NCH2CH2CH2CH3), 0.94 (3 H, td, J = 7.3, 2.0 Hz,
NCH2CH2CH2CH3) ppm.
13C NMR
LRMS
(100 MHz, DMSO) δ 176.9 (C-2), 152.1 (CH=CH-NHPh), 143.4, 141.4 (C-3a, C-7a), 138.3 (C-8),
137.0 (C-6), 131.2 (C-4), 129.8 (2 C, C-10 ×2), 126.3 (C-11), 118.4 (2 C, C-9 ×2), 113.9 (C-7), 91.2
(CH=CH-NHPh), 90.2 (C-5), 49.5 (C-3), 44.0 (NCH2CH2CH2CH3), 28.6 (NCH2CH2CH2CH3), 27.5
(2 C, 3-CH3 ×2), 19.5 (NCH2CH2CH2CH3), 13.7 (NCH2CH2CH2CH3) ppm.
[ESI+, MeOH] calculated mass: 445.11 for C22H26IN2 [M-I-]+; found m/z: 445.0 (100 %), 446.1
+
(22.5 %), 447.1 (2.5 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
18

HRMS
[ESI+, MeOH] calculated mass: 445.1135 for C22H26IN2+ [M-I-]+; found m/z: 445.1140
6.2. Synthesis of 2-[3-(1-Butyl-2,3-dihydro-3,3-dimethyl-5-iodo-1H-indol-2-ylidene)-1-propenyl]-1,3,3-trimethyl-3H-
indolium Iodide (15)
Hemicyanine 14 (2.84 g, 4.97 mmol, 1 eq) and 1,2,3,3-tetramethyl-3H-indolium iodide 1 (2.25 g, 7.46 mmol, 1.5 eq)
were suspended in anhydrous pyridine (25 mL) and then acetic anhydride (4.7 mL) was added. The reaction mixture
was stirred at 50 °C for 18 h, then cooled to room temperature and precipitated with cold diethyl ether. The precipitate
was filtered off and washed with diethyl ether. Column chromatography (5 % MeOH/EtOAC) afforded the product 15
in 95 % yield (3.10 g, 4.74 mmol). Mw = 652.39 g/mol (C28H34I2N2).
Rf:
0.52 [DCM/MeOH 9:1 v/v]
1H NMR
(400 MHz, DMSO) δ 8.32 (1 H, t, J = 13.3 Hz, CH=CH-CH), 8.05 - 7.01 (1 H, m, H-4), 7.76 (1 H,
dd, J = 8.3, 1.3 Hz, H-6), 7.65 (1 H, d, J = 7.0 Hz, H-4’), 7.53 - 7.42 (2 H, m, H-6’, H-7’), 7.32 (1 H,
t, J = 7.0 Hz, H-5’), 7.27 (1 H, d, J = 8.5 Hz, H-7), 6.59 (1 H, d, J = 13.6 Hz, CH=CH-CH), 6.51 (1
H, d, J = 13.1 Hz, CH=CH-CH), 4.07 (2 H, t, J = 7.5 Hz, NCH2CH2CH2CH3), 3.68 (3 H, s, N’-CH3)
1.71 – 1.64 (14 H, m, 3-CH3 ×2, 3’-CH3 ×2, NCH2CH2CH2CH3), 1.47 - 1.34 (2 H, m,
NCH2CH2CH2CH3), 0.93 (3 H, t, J = 7.5 Hz, NCH2CH2CH2CH3) ppm.
13C NMR
(100 MHz, DMSO) δ 175.0 (C-2’), 172.4 (C-2), 149.6 (CH=CH-CH), 143.4, 143.02, 142.4, 141.2 (C-
3a, C-3a’, C-7a, C-7a’), 137.1 (C-6), 131.1 (C-4), 128.6 (C-6’), 125.5 (C-5’), 122.4 (C-4’), 113.5 (C-
7), 111.8 (C-7’), 103.8 (CH=CH-CH), 102.1 (CH=CH-CH), 89.2 (C-5), 49.0, 48.7 (C-3, C-3’), 43.6
(NCH2CH2CH2CH3), 31.7 (N’-CH3), 29.0 (NCH2CH2CH2CH3), 27.3, 27.1 (2 C, 3-CH3 ×2; 2 C, 3’-
CH3 ×2), 19.5 (NCH2CH2CH2CH3), 13.8 (NCH2CH2CH2CH3) ppm.
+
LRMS
HRMS
[ESI+, MeOH] calculated mass: 525.18 for C28H34IN2 [M-I-]+; found m/z: 525.1 (100 %), 526.2
(26.0 %), 527.3 (2.9 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ESI+, MeOH] calculated mass: 525.1761 for C28H34IN2+ [M-I-]+; found m/z: 525.1754
19

6.3. Synthesis of 2-[3-(1-Butyl-2,3-dihydro-3,3-dimethyl-5-(trimethylsilyl)ethynyl-1H-indol-2-ylidene)-1-propenyl]-
1,3,3-trimethyl-3H-indolium Iodide (16)
Iodo-Cy3 15 (3.0 g, 4.60 mmol, 1 eq) was dissolved in anhydrous DMF (27 mL) and triethylamine (16 mL), then the
copper (I) iodide (0.18 g, 0.92 mmol, 0.2 eq) and (trimethylsilyl)acetylene (3 mL, 21.16 mmol, 4.6 eq) were added. The
reaction mixture was stirred for 10 min, before tetrakis(triphenylphosphine)palladium (0) (0.53 g, 0.46 mmol, 0.1 eq)
was added. The reaction was stirred at room temperature for a further 20 h, filtered through Celite to remove palladium
then diluted in DCM, and washed with aqueous sodium bicarbonate and brine. The organic layer was separated, dried
over sodium sulfate, filtered, and the solvent was removed in vacuo. The residual oil was purified by column
chromatography (0-5 % MeOH in DCM/EtOAc 1:1) to give the product 16 in 53 % yield (1.51 g, 2.43 mmol). Mw =
622.70 g/mol (C33H43IN2Si).
Rf:
0.51 [10 % MeOH in DCM/EtOAc 1:1 v/v]
1H NMR
(400 MHz, DMSO) δ 8.33 (1 H, t, J = 13.5 Hz, CH=CH-CH), 7.77 (1 H, d, J = 1.3 Hz, H-4), 7.68 -
7.58 (1 H, m, H-4’), 7.58 - 7.50 (2 H, m, H-6, H-7’), 7.50 - 7.45 (1 H, m, H-6’), 7.42 (1 H, d, J = 8.3
Hz, H-7), 7.34 (1 H, t, J = 7.3 Hz, H-5’), 6.61 (1 H, d, J = 13.7 Hz, CH=CH-CH), 6.52 (1 H, d, J =
13.3 Hz, CH=CH-CH), 4.08 (2 H, t, J = 7.3 Hz, NCH2CH2CH2CH3), 3.70 (3 H, s, N’-CH3), 1.69 –
1.65 (2 H, m, NCH2CH2CH2CH3), 1.69 (6 H, s, 3-CH3 ×2), 1.68 (6 H, s, 3’-CH3 ×2), 1.48 - 1.36 (2 H,
m, NCH2CH2CH2CH3), 0.94 (3 H, t, J = 7.3 Hz, NCH2CH2CH2CH3), 0.25 (9 H, s, Si(CH3)3) ppm.
13C NMR
(100 MHz, DMSO) δ 175.3, 172.9 (C-2, C-2’), 149.7 (CH=CH-CH), 142.5, 142.4 (C-7a, C-7a’),
140.8 (2 C, C-3a, C-3a’), 132.5 (C-6), 128.8 (C-6’), 125.7 , 125.7 (C-4, C-5’), 122.4 (C-4’), 118.1 (C-
5), 111.9 (C-7’), 111.4 (C-7), 105.1 (C≡C-Si(CH3)3), 104.2 (CH=CH-CH), 102.4 (CH=CH-CH), 94.4
(C≡C-Si(CH3)3), 49.1, 48.5 (C-3, C-3’), 43.6 (NCH2CH2CH2CH3), 31.7 (N’-CH3), 29.0
(NCH2CH2CH2CH3), 27.4, 27.1 (2 C, 3-CH3 ×2; 2 C, 3’-CH3 ×2), 19.5 (NCH2CH2CH2CH3), 13.8
(NCH2CH2CH2CH3), -0.1 (3 C, Si(CH3)3) ppm.
LRMS
HRMS
[ESI+, MeOH] calculated mass: 495.32 for C33H43N2Si+ [M-I-]+; found m/z: 495.3 (100 %), 496.4
(39.3 %), 497.4 (10.3 %), 498.4 (1.8 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ESI+, MeOH] calculated mass: 495.3190 for C33H43N2Si+ [M-I-]+; found m/z: 495.3180
6.4. Synthesis of 2-[3-(1-Butyl-2,3-dihydro-3,3-dimethyl-5-ethynyl-1H-indol-2-ylidene)-1-propenyl]-1,3,3-trimethyl-
3H-indolium Iodide (VI)
Silyl-ethynyl-Cy3 16 (1.45 g, 2.33 mmol, 1 eq) was dissolved in anhydrous THF (60 mL), then a 1 M solution of
tetrabutylammonium fluoride in THF (7.0 mL, 6.98 mmol, 3 eq) was added and the reaction was stirred under argon at
room temperature for 30 min. The mixture was diluted with DCM and washed with aqueous sodium bicarbonate and
20

brine. The organic layer was separated, dried over sodium sulfate, filtered, and the solvent was removed in vacuo.
Column chromatography (0-1 % MeOH in DCM/EtOAc 1:1) afforded the product VI as a green-gold solid in 63 %
yield (0.81 g, 1.47 mmol). Mw = 550.52 g/mol (C30H35IN2).
Rf:
0.41 [10 % MeOH in DCM/EtOAc 1:1 v/v]
1H NMR
(400 MHz, DMSO) δ 8.33 (1 H, t, J = 13.5 Hz, CH=CH-CH), 7.78 (1 H, d, J = 1.4 Hz, H-4), 7.69 -
7.58 (1 H, m, H-4’), 7.58 - 7.51 (1 H, m, H-6), 7.51 - 7.46 (2 H, m, H-6’, H-7’), 7.46 - 7.40 (1 H, m,
H-7), 7.34 (1 H, t, J = 7.3, Hz, H-5’), 6.60 (1 H, d, J = 13.7 Hz, CH=CH-CH), 6.51 (1 H, d, J = 13.2
Hz, CH=CH-CH), 4.25 (1 H, s, C≡CH), 4.09 (2 H, t, J = 7.3 Hz, NCH2CH2CH2CH3), 3.70 (3 H, s,
N’-CH3), 1.69 (12 H, s, 3-CH3 ×2, 3’-CH3 ×2), 1.73 - 1.63 (2 H, br. s., NCH2CH2CH2CH3) 1.47 -
1.36 (2 H, m, NCH2CH2CH2CH3), 0.94 (3 H, t, J = 7.3 Hz, NCH2CH2CH2CH3) ppm.
13C NMR
(100 MHz, DMSO) δ 175.2, 173.0 (C-2, C-2’), 149.7 (CH=CH-CH), 142.5, 142.4 (C-7a, C-7a’),
140.8 (2 C, C-3a, C-3a’), 132.2 (C-6), 128.8 (C-6’), 125.8, 125.6 (C-4, C-5’), 122.4 (C-4’), 117.7 (C-
5), 111.9, 111.4 (C-7, C-7’), 104.1 (CH=CH-CH), 102.3 (CH=CH-CH), 83.4 (C≡CH), 81.0 (C≡CH),
49.1, 48.5 (C-3, C-3’), 43.6 (NCH2CH2CH2CH3), 31.7 (N’-CH3), 29.0 (NCH2CH2CH2CH3), 27.4,
27.1 (2 C, 3-CH3 ×2; 2 C, 3’-CH3 ×2), 19.5 (NCH2CH2CH2CH3), 13.8 (NCH2CH2CH2CH3) ppm.
+
LRMS
HRMS
[ESI+, MeOH] calculated mass: 423.28 for C30H35N2 [M-I-]+; found m/z: 423.4 (100 %), 424.4
(33.2 %), 425.4 (5.1 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ESI+, MeOH] calculated mass: 423.2795 for C30H35N2+ [M-I-]+; found m/z: 423.2793
21

7. Synthesis of N-pentynyl-Cy3 (VII)
Scheme S7. Synthesis of N-pentynyl-Cy3 (VII): (i) N,N’-diphenylformamidine, triethylorthoformate, ethanol, 97 °C, 15
h, 84 %; (ii) 1, Py, acetic anhydride, 50 °C, 24 h, 61 %.
7.1 Synthesis of 3,3-Dimethyl-1-(4-pentynyl)-2-[(E)-2-(phenylamino)ethenyl]-3H-indolium Iodide (17)
1-(4-Pentynyl)-2,3,3-trimethyl-3H-indolium iodide 5 (1.27 g, 3.60 mmol, 1 eq) and N,N’-diphenylformamidine (0.78 g,
3.96 mmol, 1.1 eq) were suspended in ethanol (11 mL), then triethylorthoformate (0.66 mL, 3.96 mmol, 1.1 eq) was
added and the suspension was heated at reflux for 15 h. The reaction mixture was cooled to room temperature, and kept
for 15 min on ice to improve precipitation. The precipitate was filtered off, washed with diethyl ether, and dried in
vacuo to give the product 17 as a red solid in 84 % yield (1.38 g, 3.02 mmol). Mw = 456.36 g/mol (C23H25IN2).
Rf:
0.43 [DCM/MeOH 9:1 v/v]
1H NMR
(400 MHz, CD3CN) δ 8.61 (1 H, d, J = 12.6 Hz, CH=CH-NHPh), 7.56 - 7.42 (6 H, m, H-4, H-6, H-9
×2, H-10 ×2), 7.40 - 7.26 (3 H, m, H-5, H-7, H-11), 6.98 (1 H, d, J = 12.6 Hz, CH=CH-NHPh), 4.16
(2 H, t, J = 8.0 Hz, NCH2CH2CH2C≡CH), 2.46 (2 H, td, J = 7.0, 2.5 Hz, NCH2CH2CH2C≡CH), 2.33
(1 H, t, J = 2.5 Hz, NCH2CH2CH2C≡CH), 2.04 (2 H, quin, J = 7.4 Hz, NCH2CH2CH2C≡CH), 1.70 (6
H, s, 3-CH3 ×2), ppm.
13C NMR
(100 MHz, CD3CN) δ 179.4 (C-2), 152.8 (CH=CH-NHPh), 142.9, 142.2 (C-3a, C-7a), 139.6 (C-8),
130.8 (2 C, C-10 ×2), 129.7, 127.4, 127.0, 123.4 (C-4, C-5, C-6, C-11), 119.1 (2 C, C-9 ×2), 112.5
(C-7), 92.2 (CH=CH-NHPh), 83.9 (NCH2CH2CH2C≡CH), 71.3 (NCH2CH2CH2C≡CH), 50.8 (C-3),
44.8 (NCH2CH2CH2C≡CH), 28.6 (2 C, 3-CH3 ×2), 26.8 (NCH2CH2CH2C≡CH), 16.8
(NCH2CH2CH2C≡CH) ppm.
[ESI+, MeOH] calculated mass: 329.20 for C23H25N2 [M-I-]+; found m/z: 329.3 (100 %), 330.3
+
LRMS
(29.7 %), 331.3 (7.5 %).
22

7.2. Synthesis of 2-[3-(2,3-dihydro-3,3-dimethyl-1-(4-pentynyl)-1H-indol-2-ylidene)-1-propenyl]-1,3,3-trimethyl-3H-
indolium Iodide (VII)
Hemicyanine 17 (1.34 g, 2.93 mmol, 1 eq) and 1,2,3,3-tetramethyl-3H-indolium iodide 1 (1.32 g, 4.39 mmol, 1.5 eq)
were suspended in anhydrous pyridine (15 mL) and the acetic anhydride (2.76 mL, 29.25 mmol, 10 eq) was added. The
reaction mixture was stirred at 50 °C for 24 h, then cooled to room temperature and precipitated with cold diethyl ether.
The yellow solution was decanted off and the precipitate was purified by column chromatography (DCM, DCM/EtOAc
1:1, DCM/EtOAc 1:2, and 1-5 % MeOH in DCM respectively). The pure compound VII was obtained as a green-gold
foam in 61 % yield (0.96 g, 1.79 mmol). Mw = 536.49 (C29H33IN2).
Rf:
0.34 [DCM/MeOH 9:1 v/v]
1H NMR
(400 MHz, CDCl3)  8.44 (1 H, t, J = 13.3 Hz, CH=CH-CH), 7.47, 7.46 (1 H, d, J = 13.6 Hz; 1 H, d,
J = 13.6 Hz CH=CH-CH), 7.42 - 7.34 (4 H, m, H-4, H-4’, H-6, H-6’), 7.28 - 7.21 (3 H, m, H-5, H-5’,
H-7/H-7’), 7.13 (1 H, d, J = 8.0 Hz, H-7/H-7’), 4.40 (2 H, t, J = 8.0 Hz, NCH2CH2CH2CCH), 3.81
(3 H, s, N’-CH3), 2.62 (2 H, td, J = 6.5, 2.5 Hz, NCH2CH2CH2CCH), 2.16 - 2.10 (1 H, m, CCH),
2.16 - 2.09 (2 H, m, NCH2CH2CH2CCH), 1.71, 1.67 (6 H, s, 3-CH3 ×2; 6 H, s, 3’-CH3 ×2) ppm.
13C NMR
(100 MHz, CDCl3)  174.4, 173.8 (C-2, C-2’), 151.0 (CH=CH-CH), 142.8, 142.3 (C-7a, C-7a’),
140.6 (2 C, C-3a, C-3a’), 129.0, 129.0 (C-6, C-6’), 125.5 (2 C, C-5, C-5’), 122.2, 122.1 (C-4, C-4’),
111.0, 110.9 (C-7, C-7’), 105.4, 104.9 (CH=CH-CH), 83.6 (CCH), 69.8 (CCH), 48.9 (2 C, C-3, C-
3’), 43.7 (NCH2CH2CH2CCH), 32.9 (N’-CH3), 28.3, 28.2 (2 C, 3-CH3 ×2; 2 C, 3’-CH3 ×2), 26.2
(NCH2CH2CH2CCH), 16.5 (NCH2CH2CH2CCH) ppm.
+
LRMS
HRMS
[ESI+, MeOH] calculated mass: 409.26 for C29H33N2 [M-I-]+; found m/z: 409.2 (100 %), 410.2
(31.6 %), 411.2 (4.7 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ES+] calculated mass: 409.2638 for C29H33N2+ [M-I-]+; found m/z: 409.2637
23

8. Synthesis of Ethynyl-Cy3.25 (VIII)
I
(i)
(ii)
NHPh
N+
I-
N+
N+
N
I-
I-
6
18
19
Si
(iii)
(iv)
N+
N+
N
N
I-
I-
VIII
20
Scheme S8. Synthesis of ethynyl-Cy3.25 (VIII): (i) N,N’-diphenylformamidine, CH(OEt)3, EtOH, reflux, 24 h, 99 %;
(ii) 3, Ac2O, Py, 50 °C, 48 h, 50 %; (iii) (trimethylsilyl)acetylene, Et3N, CuI, Pd(PPh3)4, DMF, RT, 48 h, 80 %; (iv)
TBAF, THF, RT, 15 min, 71 %.
8.1. Synthesis of 1,3,3-Trimethyl-2-[(E)-2-(phenylamino)ethenyl]-3H-indolium Iodide (18)
1,2,3,3-Tetramethylbenz[e]indolium iodide 6 (10.0 g, 28.47 mmol, 1 eq) and N,N’-diphenylformamidine (6.71 g, 34.17
mmol, 1.2 eq) were suspended in ethanol (85 mL), then triethylorthoformate (5.68 mL, 34.17 mmol, 1.2 eq) was added
and the reaction mixture was heated at reflux overnight. The reaction was cooled to room temperature, the solvent was
removed under reduced pressure, and the oily residue was dissolved in DCM and precipitated with diethyl ether. The
resulting green solid was collected by filtration, washed with ether and dried in vacuo to give the product 18 in 99 %
yield (12.82 g, 28.22 mmol). Mw = 454.35 g/mol (C23H23IN2).
Rf:
0.28 [DCM/MeOH 95:5 v/v]
1H NMR
(400 MHz, MeOD) δ 8.78 (1H, d, J = 12.6 Hz, CH=CH-NHPh), 8.29 (1 H, d, J = 8.0 Hz, H-5), 8.08,
8.04 (1 H, d, J = 9.2 Hz; 1 H, d, J = 8.4 Hz, H-8, H-10), 7.75 - 7.63 (2 H, m, H-6, H-11), 7.57 - 7.42
(5 H, m, H-7, H-13, H-14, H-16, H-17), 7.31 (1 H, t, J = 7.0 Hz, H-15), 6.25 (1 H, d, J = 12.6 Hz,
CH=CH-NHPh), 3.86 (3 H, s, N-CH3), 2.01 (6 H, s, 3-CH3 ×2) ppm.
13C NMR
LRMS
(100 MHz, MeOD) δ 182.1 (C-2), 152.5 (CH=CH-NHPh), 141.2 (C-11a), 140.2 (C-12), 135.3 (C-3a),
134.0 (2 C, C-4, C-9), 132.0, 131.3 (C-10, C-8), 131.3 (2 C, C-14, C-16), 129.1 (C-6), 127.6 (C-15),
126.8 (C-7), 123.5 (C-5), 119.1 (2 C, C-13, C-17), 112.4 (C-11), 91.6 (CH=CH-NHPh), 53.3 (C-3),
33.1 (N-CH3), 28.4 (2 C, 3-CH3 ×2) ppm.
[ESI+, MeOH] calculated mass: 327.19 for C23H23N2 [M-I-]+; found m/z: 327.3 (100 %), 328.3
+
(24.6 %), 329.3 (3.1 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
24

8.2. Synthesis of 2-[3-(2,3-Dihydro-1,3,3-trimethyl-1H-benz[e]indol-2-ylidene)-1-propenyl]-5-iodo-1,3,3-trimethyl-
3H-indolium Iodide (19)
Hemicyanine 18 (2.60 g, 5.72 mmol, 1 eq) and 5-iodo-1,2,3,3-tetramethyl-3H-indolium iodide 3 (3.70 g, 8.58 mmol,
1.5 eq) were suspended in anhydrous pyridine (26 mL), then acetic anhydride (5.40 mL) was added. The reaction
mixture was stirred at 50 °C for 48 h, then cooled to room temperature and precipitated with cold diethyl ether. The
yellow solution was decanted off and the precipitate was dissolved in DCM and evaporated to dryness. Column
chromatography (0-5 % MeOH/EtOAc) afforded product 19 in 50 % yield (1.90 g, 2.88 mmol). Mw = 660.37 g/mol
(C29H30I2N2).
Rf:
0.65 [DCM/MeOH 9:1 v/v]
1H NMR
(400 MHz, DMSO) δ 8.43 (1 H, t, J = 13.6 Hz, CH=CH-CH), 8.31 (1 H, d, J = 8.5 Hz, H-5), 8.09 (1
H, d, J = 8.8 Hz, H-10), 8.05 (1 H, d, J = 8.0 Hz, H-8), 8.04 (1 H, s, H-4’), 7.82 (1 H, d, J = 8.8 Hz,
H-11), 7.76 (1 H, dd, J = 8.0, 1.6 Hz, H-6’), 7.70 - 7.63 (1 H, m, H-6), 7.54 (1 H, t, J = 7.3 Hz, H-7),
7.25 (1 H, d, J = 8.4 Hz, H-7’), 6.59 (1 H, d, J = 13.6 Hz, CH=CH-CH), 6.45 (1 H, d, J = 13.1 Hz,
CH=CH-CH), 3.81 (3 H, s, N’-CH3), 3.60 (3 H, s, N-CH3), 1.95 (6 H, s, 3-CH3 ×2), 1.71 (6 H, s, 3’-
CH3 ×2) ppm.
13C NMR
(100 MHz, DMSO) δ 176.5 (C-2), 172.7 (C-2’), 148.8 (CH=CH-CH), 142.7, 140.1, 133.5, 131.6 (C-
11a, C-3a, C-7a’, C-3a’), 137.0 (C-6’), 131.0, 130.3, 129.87, 127.8, 122.3 (C-5, C-6, C-7, C-8, C-10),
127.2 (2 C, C-4, C-9), 125.2 (C-4’), 113.3, 111.9 (C-11, C-7’), 103.2, 102.2 (CH=CH-CH), 89.0 (C-
5’), 50.7, 48.5 (C-3, C-3’), 32.1, 31.3 (N-CH3, N’-CH3), 27.2, 26.7 (2 C, 3-CH3 ×2; 2 C, 3’-CH3 ×2)
ppm.
+
LRMS
HRMS
[ESI+, MeOH] calculated mass: 533.14 for C29H30IN2 [M-I-]+; found m/z: 533.2 (100 %), 534.3
(30.3 %), 535.3 (4.3 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ES+] calculated mass: 533.1448 for C29H30IN2+ [M-I-]+; found m/z: 533.1452
8.3. Synthesis of 2-[3-(2,3-Dihydro-1,3,3-trimethyl-1H-benz[e]indol-2-ylidene)-1-propenyl]-5-(trimethylsilyl)ethynyl-
1,3,3-trimethyl-3H-indolium Iodide (20)
6
5
7
8
Si
4'
7'
4
3a
5'
3
3a'
7a'
3'
9
2
2'
6'
10
N+1
11a
1'N
I-
11
20
Iodo-Cy3.25 19 (3.15 g, 4.77 mmol, 1 eq) was dissolved in anhydrous DMF (25 mL) and triethylamine (17 mL).
Copper (I) iodide (0.18 g, 0.95 mmol, 0.2 eq) and (trimethylsilyl)acetylene (3.37 mL, 23.85 mmol, 5 eq) were then
added. The reaction mixture was stirred for 10 min before tetrakis(triphenylphosphine)palladium (0) (0.55 g, 0.48
mmol, 0.1 eq) was added and then the reaction was stirred at room temperature for 14 h. The reaction was not complete
and a further amount of (trimethylsilyl)acetylene (3.37 mL, 23.85 mmol, 5 eq), and copper (I) iodide (0.18 g, 0.95
mmol, 0.2 eq) were added and the reaction was left for a further 24 h. The mixture was filtered through Celite to remove
palladium and the residue was dissolved in DCM and washed with aqueous sodium bicarbonate and brine. The organic
25

layer was dried over sodium sulfate, filtered, and the solvent was removed in vacuo. Column chromatography (0-5 %
MeOH/DCM) afforded product 20 in 80 % yield (2.42 g, 3.83 mmol). Mw = 630.68 g/mol (C34H39IN2Si).
Rf:
0.55 [DCM/MeOH 9:1 v/v]
1H NMR
(400 MHz, DMSO) δ 8.45 (1 H, t, J = 13.6 Hz, CH=CH-CH), 8.32 (1 H, d, J = 8.5 Hz, H-5), 8.14 (1
H, d, J = 9.0 Hz, H-10), 8.09 (1 H, d, J = 8.0 Hz, H-8), 7.85 (1 H, d, J = 9.0 Hz, H-11), 7.81 - 7.77 (1
H, m, H-4’), 7.70 - 7.64 (1 H, m, H-6), 7.58 - 7.50 (2 H, m, H-7, H-6’), 7.41 (1 H, d, J = 8.0 Hz, H-
7’), 6.63 (1 H, d, J = 14.1 Hz, CH=CH-CH), 6.47 (1 H, d, J = 13.1 Hz, CH=CH-CH), 3.83, 3.62 (3 H,
s, N-CH3; 3 H, s, N’-CH3), 1.96, 1.72 (6 H, s, 3-CH3 ×2; 6 H, s, 3’-CH3 ×2), 0.25 (9 H, s, Si(CH3)3)
ppm.
13C NMR
(100 MHz, DMSO) δ 176.8, 173.2 (C-2, C-2’), 148.9 (CH=CH-CH), 143.2 (C-7a’), 140.7 (C-3a’),
140.1 (C-9), 133.7 (C-11a), 132.4 (C-6’), 131.7 (C-3a), 130.4 (C-10), 129.9 (C-8), 127.8 (C-6), 127.2
(C-4), 125.7 (C-4’), 125.2 (C-7), 122.3 (C-5), 117.9 (C-5’), 111.9 (C-11), 111.2 (C-7’), 105.3 (C≡C-
Si(CH3)3), 103.7, 102.5 (CH=CH-CH), 94.2 (C≡C-Si(CH3)3), 50.8, 48.4 (C-3, C-3’), 32.1, 31.2 (N-
CH3, N’-CH3), 27.2, 26.7 (2 C, 3-CH3 ×2; 2 C, 3’-CH3 ×2), -0.1 (3 C, Si(CH3)3) ppm.
LRMS
HRMS
[ESI+, MeOH] calculated mass: 503.29 for C34H39N2Si+ [M-I-]+; found m/z: 503.4 (100 %), 504.4
(41.2 %), 505.4 (11.3 %), 506.5 (2.3 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ESI+, MeOH] calculated mass: 503.2877 for C34H39N2Si+ [M-I-]+; found m/z: 503.2872
8.4. Synthesis of 2-[3-(2,3-Dihydro-1,3,3-trimethyl-1H-benz[e]indol-2-ylidene)-1-propenyl]-5-ethynyl-1,3,3-trimethyl-
3H-indolium Iodide (VIII)
Silyl-ethynyl-Cy3.25 20 (2.17 g, 3.45 mmol, 1 eq) was dissolved in distilled THF (40 mL), then 1 M solution of
tetrabutylammonium fluoride in THF (5.17 mL, 5.17 mmol, 1.5 eq) was added and the reaction was stirred under argon
at room temperature for 15 min. The mixture was diluted with DCM, washed with saturated sodium hydrogen carbonate
and aqueous potassium iodide solutions. The organic layer was separated, dried over sodium sulfate, filtered, and the
solvent was removed in vacuo. The crude product was purified by column chromatography (0-3 % MeOH/DCM) to
give compound VIII as a dark gold foam in 71 % yield (1.37 g, 2.45 mmol). Mw = 558.50 g/mol (C31H31IN2).
Rf:
0.51 [DCM/MeOH 9:1 v/v]
1H NMR
(400 MHz, DMSO) δ 8.45 (1 H, dd, J = 14.0, 12.0 Hz, CH=CH-CH), 8.32 (1 H, d, J = 9.0 Hz, H-5),
8.14 (1 H, dd, J = 8.8, 2.3 Hz, H-10), 8.09 (1 H, d, J = 9.0 Hz, H-8), 7.83 (1 H, dd, J = 8.5, 2.5 Hz, H-
11), 7.80 (1 H, s, H-4’), 7.68 (1 H, t, J = 7.0 Hz, H-6), 7.58 - 7.51 (2 H, m, H-6’, H-7), 7.41 (1 H, dd,
J = 8.5, 2.5 Hz, H-7’), 6.61 (1 H, dd, J = 13.8, 2.3 Hz, CH=CH-CH), 6.46 (1 H, dd, J = 13.3, 2.3 Hz,
CH=CH-CH), 4.25 (1 H, d, J = 2.5 Hz, C≡CH), 3.82, 3.62 (3 H, s, N-CH3; 3 H, s, N’-CH3), 1.96, 1.72
(6 H, s, 3-CH3 ×2; 6 H s, 3’-CH3 ×2) ppm.
13C NMR
(100 MHz, DMSO) δ 176.7, 173.2 (C-2, C-2’), 148.9 (CH=CH-CH), 143.1, 140.6, 140.1, 133.6 (C-
11a, C-3a, C-3a’, C-7a’), 132.4 (C-7), 131.7, 127.2 (C-9, C-4), 130.3, 129.9 (C-8, C-10), 127.8 (C-6),
125.7 (C-4’), 125.2 (C-6’), 122.3 (C-5), 117.5 (C-5’), 111.9 (C-7’), 111.2 (C-11), 103.5, 102.4
(CH=CH-CH), 83.5 (C≡CH), 80.9 (C≡CH), 50.8, 48.4 (C-3, C-3’), 32.1, 31.3 (N-CH3, N’-CH3),
27.3, 26.7 (2 C, 3-CH3 ×2; 2 C, 3’-CH3 ×2) ppm.
26

+
LRMS
HRMS
[ESI+, MeOH] calculated mass: 431.25 for C31H31N2 [M-I-]+; found m/z: 431.4 (100 %), 432.4
(32.5 %), 433.4 (5.4 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ESI+, MeOH] calculated mass: 431.2482 for C31H31N2+ [M-I-]+; found m/z: 431.2495
9. Synthesis of N-pentynyl-Cy3.5 (IX)
Scheme S9. Synthesis of N-pentynyl-Cy3.5 (IX): (i) N,N’-diphenylformamidine, CH(OEt)3, EtOH, reflux, 24 h, 99 %;
(ii) 7, Ac2O, Py, 50 °C, 48 h, 20 %.
9.1. Synthesis of 2-[3-(2,3-Dihydro-1,3,3-trimethyl-1H-benz[e]indol-2-ylidene)-1-propenyl]-3,3-dimethyl-1-(4-
pentynyl)-3H-benz[e]indolium Iodide (IX)
Hemicyanine 18 (5.0 g, 11.01 mmol, 1 eq) and 1-pentynyl-2,3,3-trimethylbenz[e]indolium iodide 12 (5.33 g, 13.21
mmol, 1.2 eq) were suspended in anhydrous pyridine (50 mL), then acetic anhydride (10.40 mL, 110.05 mmol, 10 eq)
was added. The reaction mixture was stirred at 50 °C for 48 h, then cooled to room temperature and precipitated with
cold diethyl ether. The yellow solution was decanted off and the precipitate was dissolved in DCM and evaporated to
dryness to give the crude product as a violet foam. Column chromatography (0-5 % MeOH/EtOAc) afforded compound
IX as a brown-gold foam in 20 % yield (1.37 g, 2.15 mmol). Mw = 636.61 g/mol (C37H37IN2).
Rf:
0.38 [EtOAc/MeOH 9:1 v/v]
1H NMR
(400 MHz, DMSO) δ 8.59 (1 H, t, J = 13.6 Hz, CH=CH-CH), 8.30, 8.29 (1 H, d, J = 8.4 Hz; 1 H, d, J
= 8.8 Hz H-5, H-5’), 8.17 - 8.05 (4 H, m, H-8, H-8’, H-10, H-10’), 7.82, 7.79 (1 H, d, J = 9.2 Hz; 1 H,
d, J = 8.4 Hz, H-11, H-11’), 7.73 - 7.64 (2 H, m, H-6, H-6’), 7.59 - 7.48 (2 H, m, H-7, H-7’), 6.64,
6.56 (1 H, d, J = 13.6 Hz; 1 H, d, J = 13.6 Hz, CH=CH-CH), 4.32 (2 H, t, J = 7.0 Hz,
NCH2CH2CH2C≡CH), 3.82 (3 H, s, N-CH3), 2.99 - 2.93 (1 H, m, C≡CH), 2.52 - 2.48 (2 H, m,
NCH2CH2CH2C≡CH), 2.43 (2 H, td, J = 7.0, 2.0 Hz, NCH2CH2CH2C≡CH), 2.02, 2.0 (6 H, s, 3-CH3
×2; 6 H, s, 3’-CH3 ×2) ppm.
13C NMR
(100 MHz, DMSO) δ 175.8, 174.9 (C-2, C-2’), 148.3 (CH=CH-CH), 140.2, 139.6 (C-11a, C-11a’),
133.1, 132.9 (C-3a, C-3a’), 131.5, 131.4 (C-9, C-9’), 130.4, 130.3 (C-10, C-10’), 129.9, 129.8 (C-8,
C-8’), 127.9 (2 C, C-6, C-6’), 127.4, 127.3 (C-4, C-4’), 125.0, 124.9 (C-7, C-7’), 122.1 (2 C, C-5, C-
5’), 111.8, 111.4 (C-11, C-11’), 102.6, 101.8 (CH=CH-CH), 83.4 (C≡CH), 72.2 (C≡CH), 50.6, 50.5
27

(C-3, C-3’), 43.0 (NCH2CH2CH2C≡CH), 31.9 (N-CH3), 27.1, 26.9 (2 C, 3-CH3 ×2; 2 C, 3’-CH3 ×2),
26.7 (NCH2CH2CH2C≡CH), 15.4 (NCH2CH2CH2C≡CH) ppm.
+
LRMS
HRMS
[ESI+, MeOH] calculated mass: 509.30 for C37H37N2 [M-I-]+; found m/z: 509.3 (100 %), 510.4
(37.3 %), 511.4 (7.0 %).
[ESI-, MeOH] calculated mass: 126.91 for [I]-; found m/z: 126.9 (100 %).
[ESI+, MeOH] calculated mass: 509.2951 for C37H37N2+ [M-I-]+; found m/z: 509.2958
10. Synthesis of Ethynyl-Cy3B (X) is described in a separate manuscript (in preparation).
Oligonucleotides - Supplementary Data
Figure S1. Reversed –phase C8 HPLC (left) and capillary electrophoresis (right) of oligonucleotides after mesyl to
azide conversion.
Figure S2. Example of the capillary electrophoresis of labeled oligonucleotides.
28

Table S1. Electrospray ionization Mass Spectrometry MS [ES-] of the labeled oligonucleotides.
Dye
Oligonucleotide
Name
Calculated mass
Found mass
m/z
Ethynyl-Cy5 (1)
HyB-3-EtCy5
HyB-5-EtCy5
HyB-7-EtCy5
7832
7832
7832
7804
7804
7804
7848
7848
7848
8004
8004
8004
8049
8049
8049
7781
7781
7781
7753
7753
7753
7796
7796
7796
7952
7952
7952
7829
7829
7829
7831.38
7831.37
7830.38
7806.30
7802.33
7803.36
7847.30
7846.28
7846.26
8003.35
8003.37
8002.37
8046.48
8046.46
8047.44
7781.50
7782.70
7782.10
7754.80
7755.30
7754.20
7795.16
7795.17
7794.20
7950.27
7951.34
7950.30
7828.10
7828.10
7829.60
N-pentynyl-Cy5 (2)
Ethynyl-Cy5.25 (3)
N-pentynyl-Cy5.5 (4)
Phenyl-ethynyl-Cy5 (5)
Ethynyl-Cy3 (6)
HyB-3-NpCy5
HyB-5-NpCy5
HyB-7-NpCy5
HyB-3-EtCy5.25
HyB-5-EtCy5.25
HyB-7-EtCy5.25
HyB-3-NpCy5.5
HyB-5-NpCy5.5
HyB-7-NpCy5.5
HyB-3-PhCy5.25
HyB-5-PhCy5.25
HyB-7-PhCy5.25
HyB-3-EtCy3
HyB-5-EtCy3
HyB-7-EtCy3
N-pentynyl-Cy3 (7)
Ethynyl-Cy3.25 (8)
N-pentynyl-Cy3.5 (9)
Ethynyl-Cy3B (10)
HyB-3-NpCy3
HyB-5-NpCy3
HyB-7-NpCy3
HyB-3-EtCy3.25
HyB-5-EtCy3.25
HyB-7-EtCy3.25
HyB-3-NpCy3.5
HyB-5-NpCy3.5
HyB-7-NpCy3.5
HyB-3-EtCy3B
HyB-5-EtCy3B
HyB-7-EtCy3B
29

Fluorescence melting curves and derivatives of all HyBeacons made in this study
Figure S3. Melting curves and melting derivatives of oligonucleotides labeled with ethynyl-Cy5 (1). WT – wild type
(thick line), MT – mutant type (thin line), 3 base pair separation (black), 5 base pair separation (red), 7 base pair
separation (blue).
Figure S4. Melting curves and melting derivatives of oligonucleotides labeled with N-pentynyl-Cy5 (2). WT – wild
type (thick line), MT – mutant type (thin line), 3 base pair separation (black), 5 base pair separation (red), 7 base pair
separation (blue).
Figure S5. Melting curves and melting derivatives of oligonucleotides labeled with ethynyl-Cy5.25 (3). WT – wild type
(thick line), MT – mutant type (thin line), 3 base pair separation (black), 5 base pair separation (red), 7 base pair
separation (blue).
30