A versatile Near Infrared Asymmetric Tricarbocyanine for Zinc
Ion Sensing in Water
Guillermo O. Menéndez, Cecilia Samaniego López, Elizabeth A. Jares-Erijman and
Carla C. Spagnuolo*
CIHIDECAR-CONICET, Dpto. de Química Orgánica, Fac. de Ciencias Exactas y
Naturales, Universidad de Buenos Aires. 3er piso, Pabellón II, Ciudad Universitaria (1428)
Ciudad Autónoma de Buenos Aires, Argentina
carlacs@qo.fcen.uba.ar
S1
Supplementary Materials
Contents
Synthesis of compound 1…………………………………………S3
Synthesis of DPEN…………………………………………….… S4
NMR spectra of compounds 1, 2, 3, 4, 5…………………………S6
Calculation of Kd…………………………………….……………S9
Spectra of compound 5 in different solvents……………………S10
S2
Synthesis of compound 1
chloro-1-formyl-3-(hydroxymethylen)-1-cyclohexen (S1)
A solution of POCl3 (0.198 mmol) in DCM was added dropwise to a solution of DMF (20
ml) in DCM (20 ml) in an ice bath. Then a solution of cycloheaxone (0.050 mmol) was
added dropwise and the ice bath was removed and a the mixture was refluxed for 3 hours.
A red solution was obtained and cold to 0ºC. Cold water was added dropwise to this
solution and it was stirred for 15 min. The biphasic system so obtained was kept at 4ºC
overnight. Yellow needles were obtained through filtration, washing with wáter and ether
and dried in vacuo. Yield 48%.
NMR‐1H (200 MHz, DMSO‐d6): δ 2.36 (t, J = 6.1 Hz, 4H), 1.58 (quin, J = 6.0 Hz, 2H).
NMR‐13C (50 MHz, DMSO‐d6) δ 191.4, 136.4, 119.9, 26.2, 22.1.
HRMS (EI+): (M+H)+ calc. C8H9ClO2 172.0291; found 172.0299.
N-[5-anilin-3-chloro-2,4-(propan-1,3-diyl)-2,4-pentadien-1-yliden]aniline chlorhydrate
(S2)
A solution of POCl3 (0.198 mmol) in DCM was added dropwise to a solution of DMF (20
ml) in DCM (20 ml) in an ice bath. Then a solution of cyclohexanone (0.050 mmol) was
added dropwise and the ice bath was removed and a the mixture was refluxed for 3 hours.
The reaction was heat to 20ºC and a mix of etanol/aniline (1:1) was added dropwise and
S3
stand for 1 h at RT. Then the reaction was placed in an ice bath and 20 ml of HCl 10% was
added. The reaction was kept at 4ºC overnight. A purple solid was obtained after work-up
(30%).
NMR‐1H (300 MHz, CD3OD): δ 8.63 (s, 2H), 7.75 – 7.33 (m, 8H), 7.37 – 7.09 (m, 2H),
2.71 (t, J = 6.1 Hz, 4H), 1.98 (quin, J = 5.7 Hz, 2H).
NMR‐13C (75 MHz, CD3OD) δ 150.4, 140.7, 131.1, 128.0, 120.0, 116.4, 25.6, 21.0.
HRMS (ESI+): (M+H)+ calc. C20H19N2Cl 323.1310; found 323.1308.
N-(3-sulfopropyl)-1,1,2-trimethylbenzo[e]indolenine (S3)
A solution of 1,1,2-trimethylbenzo[e]indol (9.7 mmol) and 1,3-propanesultone (14.5
mmol) in anhydrous toluene (10 ml) was refluxed for 18 hs under Ar. The reaction was
cold to 4ºC and a purple solid was obtained through filtration and washing with ether.
Yield: 87%.
NMR‐1H (300 MHz, CD3OD): δ 8.30 (d, J = 8.4 Hz, 1H), 8.19 (d, J = 9.0 Hz, 1H), 8.11 (d,
J= 8.9 Hz, 2H), 7.78 (t, J = 7.1 Hz, 1H), 7.68 (t, J = 7.5 Hz, 1H), 4.75 (t, 2H, J = 7.2Hz),
3.30(dd, J = 3.2, 1.6 Hz, 2H), 2.43 (dt, J = 14.5, 7.7 Hz, 2H), 1.82 (d, J = 3.6 Hz, 6H).
NMR‐13C (75 MHz, CD3OD): δ 164.2, 143.7, 142.8, 131.4, 130.8, 124.8, 116.9, 116.6,
98.2, 70.8, 56.3, 48.2, 26.5, 25.4, 24.9, 23.0.
HRMS (ESI‐): (M‐H)‐ calc. C18H21NO3S 330.1169; found 330.1171.
Compound 1
Method A: S1 (0.63 mmol) and S3 (1.26 mmol) were mixed in 28 ml of benzene/n-butane
(3:7). The mixture was refluxed for 1h. The reaction was partially evaporated in vacuo until
a precipitate was obtained. A green solid was obtained (62%).
Method B: S2 (0.62 mmol) and S3 (0.31 mmol) and anhydrous sodium acetate (0.72 mmol)
were refluxed in absolute etanol (15 ml) for 3.5 h. The solvent was evaporated in vacuo and
a green solid was obtained and recristalized from ether/metanol.
NMR‐1H (500 MHz, DMSO‐d6): δ 8.32 (d, J = 14.1 Hz, 2H), 8.25 (d, J = 8.5 Hz, 2H), 8.05
(d, J = 8.9 Hz, 2H), 8.01 (d, J = 8.3 Hz, 2H), 7.82 (d, J = 9.0 Hz, 2H), 7.60 (td, J = 6.8, 1.2
Hz, 2H), 7.47 (dt, J = 6.8, 1.2 Hz, 2H), 6.52 (d, J = 14.3 Hz, 2H), 4.47 (t, 4H), 2.74 (t, J =
5.6 Hz, 4H), 2.60 (t, J = 6.7 Hz, 4H), 2.05 (quin, J = 7.2 Hz, 4H), 1.90 (s, 12H), 1.82 (quin,
J = 5.8 Hz, 3H).
NMR‐13C (125 MHz, DMSO‐d6) δ 173.8, 147.8, 142.6, 140.2, 134.1, 132.0, 130.9, 130.4,
128.2, 128.0, 127.0, 125.4, 122.7, 112.3, 102.0, 51.1, 49.1, 48.2, 43.5, 27.5, 26.5, 24.2,
21.09.
HRMS (ESI‐): (M‐H)‐ calc. C44H47N2O6S2Cl 797.2491; found 797.2493.
Synthesis of N,N-di(2-picolyl)ethylendiamine (DPEN)
S4
6 ml of ethylendiamine and 0.6 ml of triethylamine in ethanol was held in an ice bath and
BOC2O (46 mmol) in ethanol (2 ml) was added dropwise. The reaction was stirred for 2.5 h
and the solvent evaporated in vacuo. The oil residue was dissolved in 30 ml of DCM and
extracted with HAcO 1N (3x15 ml). The acqueous phase was washed with DCM and
neutralized with NaOH 2N and extracted with DCM. After drying with Na2SO4 the solvent
was removed and S4 was obtained as a pale yellow oil whch was further used without
purification.
4 mmol of S4, 9.14 mmol of 2-chloromethylpyridin chlorihydrate and 1.7 g of Na2CO3 in
25 ml of ethanol were refluxed for 7.5 h, the solvent removed and the residue redisolved in
NaOH 2N. After extraction with DCM and drying wth Na2SO4 the solvent was evaporated
yielding a brown oil that was purified through column chromatography (AcOEt/cHex, 1:9).
A yellow oil was obtained (66%). This oil was dissolved in DCM and placed in an ice bath.
TFA (10 ml) was added dropwise and the reaction was left up to RT for 1.5h. The residue
obtained after evaporation was redissolved in NaOH 2N, extracted with DCM, dried with
Na2SO4 and evaporated to yield quantitevely S5 as a yellow oil.
NMR‐1H (200 MHz, CDCl3): δ 8.54 (d, J = 4.3 Hz, 2H), 7.63 (dt, J = 7.6, 1.5 Hz, 2H), 7.41
(d, J = 7.7 Hz, 2H), 7.15 (dd, J = 6.7, 5.4 Hz, 2H), 3.86 (s, 4H), 3.21 (t, J = 5.5 Hz, 2H),
2.70 (t, J = 5.7 Hz, 2H), 2.00 (s, 2H).
NMR‐13C (50 MHz, CDCl3): δ 157.8, 144.1, 137.4, 121.4, 120.9, 64.5, 51.8, 38.1.
S5
NMR spectra of compounds 1, 2, 3, 4, 5: Chemical shifts () are indicated in
ppm and referenced to TMS or to the corresponding deuterated solvent signal:
MeOD for compounds 2, 3, 4 and 5 and DMSO-d6 for compound 1.
Compound 1
a
Compound 2
b
b
d
c
d
c
a
S6
5
6
4
7
Compound 3
2’
1
1’
11
1’’’
9
12
9
8
6’ 2’
4
13
6,
4’’
8’’
10
8
4’’ 5’’
6’’
6’
7’’
7’ 1’’
3’’’
9’’ 10’’
2’’’
11’’ 12’’
13’’
10
8’’
7’’
7
6’’
5’’
1’
7’
5
11
Compound 4
S7
9’’
13
1’’’
10’’
3’’’ 13’’ 2’’’ 12’’
12
11’’
Compound 5
Compound 3
S8
Calculation of Kd
Fractional Change Abs650nm
Fractional Change Fl805nm
Determination of Kd for compound 2 with the fractional change method (1) from emission
(A) and absorbance (B) changes and for conjugate 5/SAv (C).
A
0.74
B
0.74
Kd = 330 ± 30 nM
0.37
0.37
R2 = 0.998
0.00
0.00
0.0
3.4
2+
[Zn ] (M)
Kd = 300 ± 40 nM
6.8
R2 = 0.989
0
2
2+
4
6
8
[Zn ] (M)
Fractional Change Fl770nm
1.0
C
0.8
0.6
0.4
Kd = 470 ± 40 nM
0.2
R2 = 0.999
0.0
0
2
4
2+
6
[Zn ] (M)
8
10
(1) Atar, D.; Backx,P.; Appel, M.M., et al. J. Biol. Chem. 1995, 270, 2473-7
Spectra of compound 5 in different solvents
UV-Vis spectra of compound 5 were measured in PBS showing a band at 780 nm and at
660 nm corresponding to aggregates and the monomer, respectively. In order to minimize
the formation of dye aggregrates, different solvents were tested: in all cases the absorption
band of the aggregates disappeared along with a bathochromic shift of the monomer
absorption.
S9
Normalized Absorbance
1.0
DMSO
PBS
PBS:AcCN
PBS-SAv
0.8
0.6
0.4
0.2
0.0
500
600
700
800
Wavelength / nm
S10
900