Supplementary Material (ESI) for Chemical · PDF fileDaniel Moreno, José V. Cuevas,...

Supplementary Information

A Fluorescent Molecular Ruler as a Selective Probe for ω-Aminoacids

Daniel Moreno, José V. Cuevas, Gabriel García-Herbosa, and Tomás Torroba*

Department of Chemistry, Faculty of Science, University of Burgos, 09001 Burgos, Spain.

[email protected]

Experimental Part:

Materials and methods: The reactions performed with air sensitive reagents were conducted under dry

nitrogen. The solvents were previously distilled under nitrogen over calcium hydride or sodium filaments.

Melting points were determined in a Gallenkamp apparatus and are not corrected. Infrared spectra were

registered in a Nicolet Impact 410 spectrometer in potassium bromide tablets. NMR spectra were

recorded in Varian Mercury-300 and Varian Unity Inova-400 machines, in DMSO-d6, CDCl3, CD3CN,

CD3OD. Chemical shifts are reported in ppm with respect to residual solvent protons, coupling constants

(JX-X’) are reported in Hz. Elemental analyses of C, H and N were taken in a Leco CHNS-932. Mass

spectra were taken in a Micromass AutoSpec machine, by electronic impact at 70 eV. Quantitative UV-

visible measures were performed with a Varian, Cary 300 Bio UV spectrophotometer, in 1 cm UV cells at

25ºC. Fluorescence spectra were recorded in a Varian Cary Eclipse spectrofluorometer, in 1 cm quarz

cells at 25ºC.

Synthesis of 5-(4-aminophenyl)indan-1-one

Br

O

Pd(PPh3)4/CsCO3

O

H2N

3

+

B

NH2

O O

21

Pd(PPh3)4 (54 mg, 0.047 mmol) was added under nitrogen to a solution of 5-bromo-1-indanone 1 (100 mg

0.470 mmol) in toluene/butanol 4:1 mixture (15 mL) and the mixture was stirred for 30 minutes at room

temperature. Then solid 4-aminobenzeneboronic pinacol ester 2 (103 mg, 0.470 mmol) and cesium

carbonate (463 mg, 1.42 mmol) dissolved in H2O (2 mL) were added and the mixture was heated under

reflux for 16 h. Then the solvent was evaporated under reduced pressure and the residue was added to

water (20 mL) and extracted with CH2Cl2 (3 x 30 mL). The combined organic extracts were dried

Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2011

(Na2SO4) and evaporated and the residue was purified by flash chromatography (silica, 3 x 30 cm),

CH2Cl2, to get 5-(4-aminophenyl)indan-1-one 3 as a light yellow solid (95 mg, 91 %), mp 250-251 ºC.

IR (KBr, cm-1): 3429 and 3335 (NH2), 1678 and 1592 (C=O), 1297 and 1278, 811. 1H NMR (DMSO-d6, 400MHz) 7.71 (s, 1H, ArH), 7.60 (s, 2H, ArH), 7.48 (d, J = 6 Hz, 2H, ArH), 6.68 (d,

J = 6 Hz, 2H, ArH), 5.44 (s, br, exch, 2H, NH2), 3.10 (m, 2H, CH2), 2.61 (m, 2H, CH2). 13C NMR (DMSO-d6, 100MHz) : 205.44 (C=O), 156.14 (C-NH2), 149.53, 146.91, 134.05, 127.90 (CH),

125.84, 124.60 (CH), 123.25 (CH), 122.79 (CH), 114.13 (CH), 36.10 (CH2), 25.44 (CH2).

MS (EI) m/z (%): 224 (M++1, 17), 223 (M+, 100), 195 (18), 167 (8), 152 (9), 139 (4), 117 (7), 96 (3).

HRMS (EI): calcd. for C15H13NO: 223.0997 (M+); found: 223.0997.

Anal. Calcd for C15H13NO: C, 80.69; H, 5.87; N, 6.27. Found: C: 80.44; H: 5.95; N: 6.11.

1H-NMR spectrum (DMSO-d6, 400 MHz) of 5-(4-aminophenyl)indan-1-one


30405060708090100110120130140150160170180190200210f1 (ppm)

25

.44

36

.10

11

4.1

3

12

2.7

91

23

.25

12

4.6

01

25

.84

12

7.9

01

34

.05

14

6.9

11

49

.53

15

6.1

4

20

5.4

4

13C-NMR spectrum (DMSO-d6, 100 MHz) of 5-(4-aminophenyl)indan-1-one

DEPT-NMR spectrum (DMSO-d6, 100 MHz) of 5-(4-aminophenyl)indan-1-one


Synthesis of 1-dicyanomethylene-5-(4-aminophenyl)indane 4

Malononitrile (50 mg 0.76 mmol) and DABCO (30 mg, 0.23 mmol) were added under nitrogen to 5-(4-

aminophenyl)indan-1-one 3 (50 mg, 0.23 mmol) dissolved in toluene (10 mL). The mixture was heated

under reflux for 2 h and then added to water (30 mL), stirred for 5 minutes and extracted with

dichloromethane (5 x 20 mL). The combined organic extracts were dried (Na2SO4) and the solvent

evaporated under reduced pressure. The crude product was purified by flash chromatography (silica, 3 x

30 cm), CH2Cl2, to get 1-dicyanomethylene-5-(4-aminophenyl)indene 4 as an orange solid (59 mg, 95 %),

mp 320-322 ºC (decomp).

IR (KBr, cm-1): 3491 and 3382 (NH2), 2221 (CN), 1627, 1600 and 1561 (C=C). 1H NMR (DMSO-d6, 300MHz): 8.14 (d, J = 9 Hz, 1H, ArH), 7.75 (m, 2H, ArH), 7.54 (d, J = 9 Hz, 2H,

ArH), 6.66 (d, J = 9 Hz, 2H, ArH), 5.62 (s, br, exch, 2H, NH2), 3.23 (m, 2H, CH2), 3.23 (m, 2H, CH2). 13C NMR (DMSO-d6, 75 MHz): 179.83, 156.43, 150.35, 147.42, 132.53, 128.20 (CH), 125.34 (CH),

124.90 (CH), 124.72, 121.69 (CH), 114.36 (CN), 114.15 (CH), 113.95 (CN), 69.96 (C(CN)2), 34.66

(CH2), 29.41 (CH2).

MS (EI) m/z (%): 272 (M++1, 20), 271(M+, 100), 243 (3), 214 (2), 206 (1), 204 (2), 189 (2), 135 (2).

HRMS (EI): calcd. for C18H13N3: 271.1109 (M+); found: 271.1108

Anal. Calcd for C18H13N3: C, 79.68; H, 4.83; N, 15.49. Found: C: 79.77; H: 4.97; N: 15.35.


1H NMR spectrum (DMSO-d6, 300 MHz) of 1-dicyanomethylene-5-(4-aminophenyl)indane

13C NMR spectrum (DMSO-d6, 75 MHz) of 1-dicyanomethylene-5-(4-aminophenyl)indane


Expansion of the 13C NMR spectrum (DMSO-d6, 75 MHz) of 1-dicyanomethylene-5-(4-

aminophenyl)indane

DEPT-NMR spectrum (DMSO-d6, 75 MHz) of 1-dicyanomethylene-5-(4-aminophenyl)indane


Expansion of the DEPT-NMR spectrum (DMSO-d6, 75 MHz) of 1-dicyanomethylene-5-(4-

aminophenyl)indane


Synthesis of 1,3-bis[4-(1-dicyanomethyleneindan-5-yl)phenylureidomethyl)benzene 6

1-Dicyanomethylene-5-(4-aminophenyl)indane 4 (100 mg, 0.38 mmol) was added to m-xylylene

diisocyanate 5 (15 µL, 0.095 mmol) dissolved in CHCl3 (20 mL) and the mixture was stirred at room

temperature for 24 h. Then a second portion of m-xylylene diisocyanate (15 µL, 0.095 mmol) was added

and the mixture was stirred for additional 24 h. The solid formed after this time was filtered off, washed

with CHCl3 (2 portions of 2 mL) and dried under reduced pressure. Compound 6 was obtained as a

yellow solid (104 mg, 75%), mp 212-214ºC.

IR (KBr, cm-1): 3328 (br, NH), 2221(C≡N), 1658, 1604 and 1557 (C=O), 1320, 1219, 1184. 1H NMR (DMSO-d6 , 400MHz) 8.82 (s, 2H, NH) 8.17 (d, J = 6 Hz, 2H, ArH), 7.82 (s, 2H, ArH), 7.79 (d,

J = 6 Hz, 2H, ArH), 7.65 (d, J = 6 Hz, 4H, ArH), 7.52 (d, J = 6 Hz, 4H, ArH), 7.29 (m, 2H, ArH), 7.20

(m, 2H, ArH), 6.71 (t, J = 6 Hz, 2H, NH), 4.32 (d, J = 6 Hz, 4H, CH2) , 3.28 (m, 4H, CH2), 3.15 (m, 4H,

CH2). 13C NMR (DMSO-d6 , 100MHz) 179.60, 155.78, 154.63, 146.04, 141.21, 140.00, 133.07, 130.10, 128.22

(CH), 127.58 (CH), 125.72, 125.67 (CH), 125.55 (CH), 125.22 (CH), 122.60 (CH), 117.54 (CH), 113.66

(CN), 113.25 (CN), 70.61, 42.32 (CH2), 34.24 (CH2), 29.04 (CH2).

MS (FAB) m/z (+): 731.40 (M++1). ESI MS m/z (%):753.28 (M+Na+), 731.29 (M++1)

Anal. Calcd for C46H34N8O2: C, 75.60; H, 4.69; N, 15.33. Found: C, 75.42; H, 4.84; N, 15.09.


1H NMR spectrum (DMSO-d6, 400 MHz) of 1,3-bis[4-(1-dicyanomethyleneindan-5-yl)phenylureidomethyl)benzene

13C NMR spectrum (DMSO-d6, 100 MHz) of 1,3-bis[4-(1-dicyanomethyleneindan-5-yl)phenylureidomethyl)benzene


DEPT-NMR spectrum (DMSO-d6, 100 MHz) of 1,3-bis[4-(1-dicyanomethyleneindan-5-yl)phenylureidomethyl)benzene


Synthesis of 1-benzyl-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea 8:

1-Dicyanomethylene-5-(4-aminophenyl)indane 4 (50 mg, 0.18 mmol) was added to benzyl isocyanate 7

(25 mg, 0.18 mmol) dissolved in CHCl3 (10mL) and the mixture was stirred at room temperature for 48 h.

Hexane (2 mL) was then added and the solid formed was filtered off, washed with CHCl3 (2 portions of 2

mL) and dried. Compound 8 was obtained as a yellow solid (56 mg, 75%), mp 189-190 ºC.

IR (KBr, cm-1) 3336 (br, NH), 2221 (C≡N), 1639 and 1565 (C=O), 1328, 1227. 1H NMR (DMSO-d6 , 400MHz): 8.74 (s, 1H, NH) 8.25 (d, J = 8 Hz, 1H, ArH), 7.87 (s, 1H, ArH), 7.83 (d,

J = 8 Hz, 1H, ArH), 7.71 (d, J = 8 Hz, 2H, ArH), 7.56 (d, J = 8 Hz, 2H, ArH), 7.34-7.32 (m, 4H, ArH),

7.25 (m, 1H, ArH), 6.66 (t, J = 6 Hz, 1H, NH), 4.33 (d, J = 6 Hz, 2H, CH2), 3.30 (m, 2H, CH2), 3.19 (m,

2H, CH2). 13C NMR (DMSO-d6, 100MHz) 179.68, 155.96, 154.82, 146.36, 141.44, 139.96, 133.40, 130.55, 128.08,

127.44, 126.96, 126.57, 125.63, 125.17, 122.87 (CHAr), 117.85, 113.81 (CN), 113.40 (CN), 71.00, 42.69

(CH2), 34.50 (CH2), 29.28 (CH2).

MS (FAB) m/z (+): 405.06 (M++1). MS (EI) m/z (%): 404 (M+, 10), 297 (95), 271 (100).

HRMS (EI): calcd. for C26H20N4O: 404.1637 (M+); found: 404.1633.

Anal. Calcd for C26H20N4O: C, 77.21; H, 4.98; N, 13.85. Found: C, 77.06; H, 5.12; N, 13.67.


1H NMR spectrum (DMSO-d6, 400 MHz) of 1-benzyl-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea

13C NMR spectrum (DMSO-d6, 100 MHz) of 1-benzyl-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea


112113114115116117118119121123125127129131133135f1 (ppm)

An expansion of the 13C NMR spectrum (DMSO-d6, 100 MHz) of 1-benzyl-3-[4-(1-(dicyanomethyleneindan-

5-yl)phenyl]urea


Solvatochromism of 1-dicyanomethylene-5-(4-aminophenyl)indane 4:

(1) (2) (3) (4) (5) (6) (7) 10-4 M solutions of 4 in (1) Hexane, (2) Et2O, (3) THF, (4) CH2Cl2, (5) MeCN, (6) MeOH, (7) DMSO

0

0.2

0.4

0.6

0.8

1

350 400 450 500 550 600wavelength (nm)

no

rmal

ized

ab

sorb

ance

HEXANE

ETHER

THF

DICHLOROMETANE

ACETONITRILE

METHANOL

DMSO

Normalized absorbance spectra of 10-4 M solutions of 4 in Hexane, Et2O, THF, CH2Cl2, MeCN, MeOH, DMSO

H2N

NC

CN

4 (1) (2) (3) (4) (5) (6) (7) λexc = 366 nm 10-4 M solutions of 4 in (1) Hexane, (2) Et2O, (3) THF, (4) CH2Cl2, (5) MeCN, (6) MeOH, (7) DMSO

0

0.2

0.4

0.6

0.8

1

375 425 475 525 575 625 675Wavelength (nm)

I/Io

HEXANE

ETHER

THF

DICHLOROMETHANE

ACETONITRILE

METHANOL

DMSO

Normalized fluorescente spectra of 10-4 M solutions of 4 in Hexane, Et2O, THF, CH2Cl2, MeCN,

MeOH, DMSO; λexc = 366 nm.


Behavior of 1,3-bis[4-(1-dicyanomethyleneindan-5-yl)phenylureidomethyl)benzene 6 in MeCN and

DMSO in the presence of common anions:

Ref. F- Cl- Br- I- BzO- NO3- H2PO4

- HSO4- AcO- CN- SCN-

10-4 M Solutions of 6 in MeCN +2 equivalents of anion: F-, Cl-, Br-, I-, BzO-, NO3-, H2PO4

-, HSO4-, AcO-,

CN-, SCN-



10-4 M Solutions of 6 in MeCN +2 equivalents of anion: F-, Cl-, Br-, I-, BzO-, NO3-, H2PO4

-, HSO4-, AcO-,

CN-, SCN-; λexc = 366nm



10-4 M Solutions of 6 in DMSO +2 equivalents of anion: F-, Cl-, Br-, I-, BzO-, NO3-, H2PO4

-, HSO4-, AcO-,

CN-, SCN-



10-4 M Solutions of 6 in DMSO +2 equivalents of anion: F-, Cl-, Br-, I-, BzO-, NO3-, H2PO4

-, HSO4-, AcO-,

CN-, SCN-; λexc = 366 nm


Fluorimetric titrations of 1,3-bis[4-(1-dicyanomethyleneindan-5-yl)phenylureidomethyl)benzene 6

in DMSO with common anions:

F-DMSO

0

10

20

30

40

50

60

70

80

400 450 500 550 600 650Wavelength (nm)

Inte

nsi

ty (

a.u

.)

0.00000 0.00005 0.00010 0.00015 0.00020 0.0002510

20

30

40

50

60

70

80

complex 1:1 Chi^2 = 3.81884R^2 = 0.99156 Ao 78.32304 ±1.15304Af 11.15866 ±2.668Co 0.00009 ±5.5948E-6K 146856.21076 ±64388.08958

Inte

nsi

ty

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO) with F- (excitation = 290 nm).

Right: Titration profile fitted to a 1:1 model (λmax = 515 nm) for the titration of 6 with F-

BzO- DMSO

0

20

40

60

80

100

120

140

450 500 550 600 650 700Wavelength (nm)

Inte

nsi

ty (

a.u

.)

0.0000 0.0001 0.0002 0.0003 0.00040

20

40

60

80

100

complejo 1:1 Quitando los 2 primeros puntos Chi^2 = 0.0917R^2 = 0.99977 Ao 158.11626 ±3.15139Af 7.99681 ±0.16131Co 0.00001 ±8.0069E-7K 131190.53066 ±1787.80021

Inte

nsity

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO) with BzO- (excitation = 396

nm). Right: Titration profile fitted to a 1:1 model (λmax = 555 nm) for the titration of 6 with BzO-

H2PO4- DMSO

0

10

20

30

40

50

60

70

80

450 500 550 600Wavelength (nm)

Inte

ns

ity

(a

.u.)

0.0000 0.0001 0.0002 0.0003 0.0004 0.000510

20

30

40

50

60

70

80complejo 1:1 DEJANDO c=CTE

Chi^2 = 1.99719R^2 = 0.99453 Ao 78.39944 ±0.56336Af 17.3972 ±0.68597Co 0.0001 ±0K 92906.65808 ±10696.21957

Inte

nsity

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO) with H2PO4- (exc = 290 nm).

Right: Titration profile fitted to a 1:1 model (λmax = 515 nm) for the titration of 6 with H2PO4-


AcO- DMSO

0

20

40

60

80

100

120

140

400 450 500 550 600 650Wavelength (nm)

Inte

nsi

ty (

a.u

.)

0.0000 0.0001 0.0002 0.0003 0.0004 0.0005

40

60

80

100

120

140

160

complex 1:1 Chi^2 = 14.31229R^2 = 0.98801 Ao 160.10738 ±1.47945Af 40 ±0Co 0.0001 ±0K 31868.16814 ±1766.98407

Inte

nsity

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO) with AcO- (exc = 290 nm).

Right: Titration profile fitted to a 1:1 model (λmax = 515 nm) for the titration of 6 with AcO-

CN- DMSO

0

10

20

30

40

50

60

70

80

420 470 520 570 620Wavelength (nm)

Inte

nsi

ty (

a.u

.)

0.0000 0.0001 0.0002 0.0003 0.0004 0.0005

10

20

30

40

50

60

70

80

90

complex 1:1

Chi^2 = 4.68425R^2 = 0.99192 Ao 89.28482 ±1.48214Af 14.8314 ±0.98686Co 0.00008 ±2.6546E-6K 1051857.84153±535334.04094

Inte

nsity

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO) with CN- (exc = 290 nm).

Right: Titration profile fitted to a 1:1 model (λmax = 515 nm) for the titration of 6 with CN-

BzO- DMSO:H2O(9:1)

0

10

20

30

40

50

60

450 500 550 600 650Wavelength (nm)

Inte

ns

ity

0.0000 0.0001 0.0002 0.0003 0.0004 0.000510

20

30

40

50

60

complex 1:1 Chi^2 = 3.83587R^2 = 0.97474 Ao 54.88309 ±0.77198Af 17.22144 ±0.91885Co 0.0001 ±0K 167026.89433 ±54717.90414

Inte

nsity

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO:H2O (9:1)) with BzO- (exc =

396 nm). Right: Titration profile fitted to a 1:1 model (λmax = 555 nm) for the titration of 6 with BzO-


AcO- DMSO:H2O(9:1)

0

10

20

30

40

50

60

450 500 550 600 650 700Wavelength (nm)

Inte

nsi

ty (

a.u

.)

0.0000 0.0001 0.0002 0.0003 0.0004 0.00050

10

20

30

40

50

60complex 1:1 Chi^2 = 0.59147R^2 = 0.99799 Ao 61.56569 ±0.41839Af 5.96347 ±0.57055Co 0.0001 ±4.2261E-6K 57097.03899 ±6778.11165

Inte

nsity

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO:H2O (9:1)) with AcO- (exc =

396 nm). Right: Titration profile fitted to a 1:1 model (λmax = 555 nm) for the titration of 6 with AcO-

CN- DMSO:H2O(9:1)

0

50

100

150

200

250

450 500 550 600 650 700Wavelength (nm)

Inte

nsi

ty (

a.u

.)

0.0000 0.0001 0.0002 0.0003 0.0004 0.00050

50

100

150

200

250complex 1:1 Chi^2 = 26.64141R^2 = 0.99379 Ao 231.84437 ±2.09644Af 32.00637 ±1.74664Co 0.00009 ±0K 480213.59697 ±105297.44565

Inte

nsity

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO:H2O (9:1)) with CN- (exc =

396 nm). Right: Titration profile fitted to a 1:1 model (λmax = 555 nm) for the titration of 6 with CN-

F- DMSO:H2O (9:1)

0

10

20

30

40

50

450 500 550 600 650Wavelength (nm)

Inte

nsi

ty (

a.u

.)

0.0000 0.0001 0.0002 0.0003 0.0004 0.0005

15

20

25

30

35

40

45

50

55complex 1:1 Chi^2 = 0.66835R^2 = 0.99426 Ao 50.77066 ±0.32986Af 16.90096 ±0.44674Co 0.0001 ±0K 73740.60155 ±8926.16566

Inte

nsi

ty

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO:H2O (9:1)) with F- (exc = 396

nm). Right: Titration profile fitted to a 1:1 model (λmax= 555 nm) for the titration of 6 with F-


Job's plot analysis of fluorescence titrations of 6 with F-

Job's plot analysis of fluorescence titrations of 6 with F- revealed a maximum at a 50% mole fraction, in

accord with the proposed 1:1 binding stoichiometry

-5

0

5

10

15

20

0 0.2 0.4 0.6 0.8 1

XF-

XF-x

Inte

ns

ity

Job's plot analysis of fluorescence titrations of 6 with F- in MeCN (exc = 396 nm) (em = 557 nm)

Complexation constants for compound 6 and common anions

Anions Complex logK1 R2

BzO- 1:1 5.118 ± 0.006 0.999 AcO- 1:1 4.503 ± 0.023 0.988 CN- 1:1 6.022 ± 0.179 0.992 F- 1:1 5.169 ± 0.158 0.991

H2PO4- 1:1 4.968 ± 0.047 0.994

Complexation constants for compound 6 in DMSO and common anions

Anions Complex logK1 R2

BzO- 1:1 5.118 ± 0.006 0.999 AcO- 1:1 4.503 ± 0.023 0.988 CN- 1:1 6.022 ± 0.179 0.992 F- 1:1 5.169 ± 0.158 0.991

H2PO4- 1:1 4.968 ± 0.047 0.994

Complexation constants for compound 6 in DMSO:H2O (9:1) and common anions



in DMSO with α-aminoacids:

0

10

20

30

40

50

60

420 470 520 570 620

Wavelength (nm)

Inte

nsi

ty (

a.u

.)

0.0000 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007

30

35

40

45

50

55

60

65

complejo1:1 Chi 2 = 0.52045R 2 = 0.99418 Ao 63.33815 ±0.68132Af 24.58799 ±1.12648Co 0.0001 ±0K 9478.00549 ±1199.41294

Inte

nsity

(a.

u.)

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO) with L-arginine in water (exc

= 306 nm). Right: Titration profile fitted to a 1:1 model (λmax = 511 nm) for the titration of 6 with L-

arginine

0

20

40

60

80

100

120

140

160

180

420 470 520 570 620Wavelength (nm)

Inte

ns

ity

(a

.u.)

0.0000 0.0002 0.0004 0.0006 0.0008

80

100

120

140

160

180complejo1:1 Chi 2 = 5.24383R^2 = 0.9946 Ao 175.46372 ±1.5268Af 62.09588 ±2.63464Co 0.0001 ±0K 8535.51692 ±844.18975

Inte

nsity

(a.

u.)

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO) with L-asparagine in water

(exc = 306 nm). Right: Titration profile fitted to a 1:1 model (λmax = 511 nm) for the titration of 6 with L-

asparagine.

0 equiv

6 equiv

0 equiv

8 equiv


0

10

20

30

40

50

60

70

80

90

400 450 500 550 600 650 700Wavelength (nm)

Inte

ns

ity

(a

.u.)

0.0000 0.0001 0.0002 0.0003 0.0004 0.0005

30

40

50

60

70

80

90

complejo1:1 Chi 2 = 0.96289R 2 = 0.99598 Ao 80.40854 ±0.68865Af 25.94513 ±1.21473Co 0.0001 ±0K 14641.88662 ±1544.09989

Inte

nsi

ty (

a.u

.)

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO) with L-glutamine in water

(exc = 306 nm). Right: Titration profile fitted to a 1:1 model (λmax = 511 nm) for the titration of 6 with L-

glutamine.

0

10

20

30

40

50

60

70

425 475 525 575 625 675Wavelength (nm)

Inte

ns

ity

(a

.u.)

0.0000 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007

35

40

45

50

55

60

65

70 complejo1:1 Chi 2 = 0.37202R 2 = 0.99663 Ao 69.06404 ±0.40442Af 23.51426 ±1.66134Co 0.0001 ±0K 4602.27513 ±477.19618

Inte

nsity

(a.

u.)

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO) with L-lysine in water (exc =

306nm). Right: Titration profile fitted to a 1:1 model (λmax = 511 nm) for the titration of 6 with L-lysine

0 equiv

5 equiv

0 equiv

6 equiv


Job's plot analysis of fluorescence titrations of 6 with L-lysine and L-asparagine

Job's plot analysis of fluorescence titrations of 6 with L-lysine and L-asparagine revealed maximum peaks

at 50% mole fraction, in accord with the proposed 1:1 binding stoichiometry

0

10

20

30

40

50

0 0.2 0.4 0.6 0.8 1Xlysine

Xly

sin

exIn

ten

sity

0

5

10

15

20

25

0 0.2 0.4 0.6 0.8 1Xasparagine

Xa

sp

ara

gin

exIn

ten

sity

Left: Job's plot analysis of fluorescence titrations of 6 (10-4 M in DMSO) with L-lysine in water (exc =

350 nm) (em = 515 nm). Right: Job's plot analysis of fluorescence titrations of 6 (10-4 M in DMSO) with

L-asparagine in water (exc = 396 nm) (em = 557 nm)



in DMSO with ω-aminoacids:

0

10

20

30

40

50

60

70

80

450 500 550 600 650 700Wavelength (nm)

Inte

nsit

y (a

.u.)

0.0000 0.0002 0.0004 0.0006 0.00080.0

0.2

0.4

0.6

0.8

1.0 Model: complejo 2:1 Chi 2 = 0.00006R^2 = 0.99888 Co 0.0001 ±0K1 30661.1342 ±1331.09082K2 8433.74738 ±1872.32341Ao 1.01019 ±0.00672Af 0.16621 ±0.00992b -160.76936 ±166.02104

I/Io

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4M in DMSO) with β-alanine in water (exc=

396 nm). Right: Titration profile fitted to a 2:1 model (λmax = 562 nm) for the titration of 6 with β-alanine.

0

50

100

150

200

250

450 500 550 600 650 700

Wavelength (nm)

Inte

nsi

ty (

a.u

.)

0.0000 0.0002 0.0004 0.0006 0.00080.0

0.2

0.4

0.6

0.8

1.0

Model: complex 2:1 Chi^2 = 0.00004R^2 = 0.99933 Co 0.0001 ±0K1 14046.16698 ±644.88932K2 12683.49195 ±2084.75033Ao 1.00998 ±0.00556Af 0.13514 ±0.00854b -180.80111 ±436.82904I/

Io

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4M in DMSO) with γ-aminobutiric acid in

water (exc = 396 nm). Right: Titration profile fitted to a 2:1 model (λmax = 562 nm) for the titration of 6

with γ-aminobutiric acid.

0 equiv

8 equiv

0 equiv

8 equiv


0

20

40

60

80

100

120

140

450 500 550 600 650 700

Wavelength (nm)

Inte

nsi

ty (

a.u

.)

0.0000 0.0002 0.0004 0.0006 0.00080.0

0.2

0.4

0.6

0.8

1.0

Model: complejo 2:1 Chi^2 = 0.00002R^2 = 0.99973 Co 0.0001 ±0K1 9596.28128 ±815.54057K2 147933.57307 ±16545.99678Ao 1.00253 ±0.00381Af 0.09748 ±0.00202b -429.44422 ±714.22269I/

Io

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4M in DMSO) with 5-aminovaleric acid in


with 5-aminovaleric acid.

0

50

100

150

200

450 500 550 600 650 700Wavelength (nm)

Inte

ns

ity

(a.u

.)

0.0000 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.00070.0

0.2

0.4

0.6

0.8

1.0

Model: complejo 2:1 Chi^2 = 0.00001R^2 = 0.9998 Co 0.0001 ±0K1 4963.08488 ±792.53822K2 378157.69636 ±67242.39871Ao 1.00021 ±0.00338Af 0.09165 ±0.00192b -2665.67851 ±1499.6116I/

Io

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4M in DMSO) with 6-aminohexanoic acid in

water (exc = 396 nm). Right: Titration profile fitted to a 2:1 model (λmax= 562 nm) for the titration of 6

with 6-aminohexanoic acid.

0 equiv

8 equiv

0 equiv

6 equiv


0

10

20

30

40

50

60

70

80

90

100

450 500 550 600 650 700Wavelength (nm)

Inte

nsi

ty (

a.u

.)

0.0000 0.0002 0.0004 0.0006 0.00080.0

0.2

0.4

0.6

0.8

1.0 Model: complejo 2:1 Chi^2 = 0.00002R^2 = 0.99955 Co 0.0001 ±0K1 15193.02869 ±1825.44451K2 321035.15614 ±49096.59229Ao 0.99983 ±0.00442Af 0.08496 ±0.00168b 986.08083 ±949.92876

I/Io

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4M in DMSO) with 7-aminoheptanoic acid in


with 7-aminoheptanoic acid.

Job's plot analysis of fluorescence titrations of 6 with 6-aminohexanoic acid

Job's plot analysis of fluorescence titrations of 6 with 6-aminohexanoic acid revealed a maximum at 66%

mole fraction, in accord with the proposed 2:1 binding stoichiometry

0

10

20

30

40

50

60

70

0 0.2 0.4 0.6 0.8 1X5-aminohexanoic

X5-

amin

oh

exan

oicxI

nte

nsi

ty

Job's plot analysis of fluorescence titrations of 6 (10-4 M in DMSO) with 6-aminohexanoic acid in water

(exc = 350 nm) (em = 515 nm).

0 equiv

8 equiv



in DMSO with gabapentin and pregabalin in water:

0

10

20

30

40

50

60

70

80

450 500 550 600 650 700

Wavelength (nm)

Inte

ns

ity

(a

.u.)

0.0000 0.0005 0.0010 0.0015 0.0020 0.002530

40

50

60

70

80 complejo 2:1 Chi^2 = 1.03872R^2 = 0.99369 Co 0.0001 ±0K1 8048.57001 ±7489.43154K2 1028.00328 ±830.97575Ao 79.23813 ±0Af 26.20715 ±3.83709b 593356.59337 ±152143.42604

Inte

nsity

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4M in DMSO) with gabapentin in water (exc

= 428 nm). Right: Titration profile fitted to a 2:1 model (λmax = 555 nm) for the titration of 6 with

gabapentin.

0

10

20

30

40

50

60

70

80

450 500 550 600 650 700Wavelength (nm)

Inte

ns

ity

(a

.u.)

0.0000 0.0003 0.0006 0.0009 0.0012 0.0015 0.0018

10

20

30

40

50

60

70

80complex 2:1 Chi^2 = 0.66205R^2 = 0.99817 Co 0.0001 ±0K1 799.52569 ±49.47501K2 1139.54936 ±246.55148Ao 76.1475 ±0Af 11.3929 ±0b -323927.50203 ±28491.10108

Inte

nsity

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4M in DMSO) with pregabalin in water (exc =

426 nm). Right: Titration profile fitted to a 2:1 model (λmax= 554 nm) for the titration of 6 with

pregabalin.

0 equiv

20 equiv

0 equiv

18 equiv


Job's plot analysis of fluorescence titrations of 6 with gabapentin

Job's plot analysis of fluorescence titrations of 6 with gabapentin acid revealed a maximum at 66% mole

fraction, in accord with the proposed 2:1 binding stoichiometry

0

10

20

30

40

50

0 0.2 0.4 0.6 0.8 1Xgabapentin

Xg

ab

ap

en

tinx

Inte

nsi

ty

Job's plot analysis of fluorescence titrations of 6 (10-4 M in DMSO) with gabapentin in water (exc = 350

nm) (em = 515 nm).


in DMSO with creatine and L-carnitine:

0

10

20

30

40

50

60

70

80

90

450 500 550 600 650 700

Wavelength (nm)

Inte

nsi

ty (

a.u

.)

0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030

0

10

20

30

40

50

60

70

80

90Data: Data1_BModel: complejo 1:1 Chi^2 = 1.70395R^2 = 0.99641 Ao 78.93344 ±0.38829Af 5.03749 ±1.11732Co 0.0001 ±0K 2130.14674 ±90.24296

Inte

nsity

(a.

u.)

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4M in DMSO) with creatine in water (exc =

428 nm). Right: Titration profile fitted to a 1:1 model (λmax= 557 nm) for the titration of 6 with creatine.

0 equiv

30 equiv


0

10

20

30

40

50

60

70

80

450 500 550 600 650 700

Wavelength (nm)

Inte

nsi

ty (

a.u

.)

0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030

0

10

20

30

40

50

60

70

80

complex 2:1 Chi^2 = 0.87142R^2 = 0.99845 Co 0.0001 ±0K1 12584.13228 ±4321.36026K2 6244.93566 ±881.17155Ao 77.78475 ±0.70153Af 2.05354 ±0.65023b 573609.90847 ±117217.05574

Inte

nsity

c(M)

Left: Emission spectra for the fluorimetric titration of 6 (10-4 M in DMSO) with L-carnitine in water

(exc= 426 nm). Right: Titration profile fitted to a 2:1 model (λmax = 555 nm) for the titration of 6 with L-

carnitine.

Job's plot analysis of fluorescence titrations of 6 with L-carnitine

Job's plot analysis of fluorescence titrations of 6 with L-carnitine revealed a maximum at a 66% mole

fraction, in accord with the proposed 2:1 binding stoichiometry

0

10

20

30

40

50

60

70

0 0.2 0.4 0.6 0.8 1Xcarnitine

Xca

rnit

inex

Inte

nsi

ty

Job's plot analysis of fluorescence titrations of 6 (10-4 M in DMSO) with L-carnitine in water (exc = 350

nm) (em = 515 nm).

0 equiv

28 equiv


1H NMR titration experiments of 1,3-bis[4-(1-dicyanomethyleneindan-5-yl)phenylureidomethyl)-

benzene 6 (10-1 M solution in DMSO) with β-alanine in DMSO.

By addition of increasing amounts of β-alanine, the two urea NH signals at δ 8.8 and 6.7 move to lower

field and progressively disappear as the H-bonds are formed. The aromatic signals become more

complicate but their chemical shifts remain unchanged.

8 6 4 2 0

1H NMR titration of 6 (10-1 M solution in DMSO-d6) with β-alanine in DMSO-d6.

2.0 equiv 1.5 equiv 1.0 equiv 0.5 equiv 0.3 equiv 0.1 equiv 0.0 equiv

δ (ppm)


The blue emission: deconvolution of the emission bands after titration:

Deconvolution of emission bands

0

10

20

30

40

50

60

70

80

90

100

400 450 500 550 600 650 700

Wavelength (nm)

Inte

nsi

ty (

a.u

.)

mixture 366nm (22,3eq)

mixture 390nm (22,3eq)

BLUE Emission

YELLOW emission

0eq 390nm

0 eq. 390 nm: the original fluorescence spectrum at λexc = 390 nm of 6 (10-4 M in DMSO).

Mixture 390 nm (22.3 equiv): Fluorescence spectrum at λexc = 390 nm after addition of 22.3 equivalents

of L-carnitine to the previous solution.

Mixture 366 nm (22.3 equiv): Fluorescence spectrum at λexc = 366 nm of the same mixture used in the

acquisition of the previous spectrum.

BLUE Emission: Fluorescence spectrum of the blue emission obtained by substraction of the yelow

emission spectrum at λexc = 390 nm from the bluish emission spectrum obtained at λexc = 366 nm from the

mixture of 6 and 22.3 equivalents of the analite (λmax = 475 nm).

YELLOW Emission: Fluorescence spectrum of the yellow emission obtained by substraction of the blue

emission spectrum at λexc = 366 nm from the emission spectrum obtained at λexc = 366 nm from the

mixture of 6 and 22.3 equivalents of the analite.


Calculations:

All calculations were performed with the Gaussian 03 set of programs using the ONIOM method.1

Geometry optimizations were conducted using a two-level ONIOM procedure2, 3 in which the hybrid

functional B3LYP4, 5 along with the basis set 6-31G+(d,p) was used as the higher level of theory and the

semiempirical AM1 model6 was used as the lower level. After the optimization, analytical computations

of the Hessian matrix were performed to compute the nature of the located minima on the potential

energy surface. The real and model systems used in the two-layer ONIOM(MO:MO) calculations are

shown in the figure.

NH HN

NH HN

O O

N

N N

N

Computational ModelB3LYP/AM1

O O

R

References: 1 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. J. A.

Montgomery, T. Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B.

Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota,

R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E.

Knox, H. P. Hratchian, J. B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A.

J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J.

J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick, A.

D. Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski,

B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A.

Al-Laham, C. Y. Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W.

Wong, C. Gonzalez, J. A. Pople, Gaussian 03, Revision C.02, Gaussian, Inc., Wallingford CT, 2004.

2 S. Humbel, S. Sieber, K. Morokuma, J. Chem. Phys. 1996, 105, 1959.

3 F. Maseras, K. Morokuma, J. Comput. Chem. 1995, 16, 1170.

4 A. D. Becke, J. Chem. Phys. 1993, 98, 5648.

5 C. T. Lee, W. T. Yang, R. G. Parr, Phys. Rev. B 1988, 37, 785.

6 M. J. S. Dewar, E. G. Zoebisch, E. F. Healy, J. J. P. Stewart, J. Am. Chem. Soc. 1985, 107, 3902.


Optimized geometry of the 6:GABA·3DMSO complex at the ONIOM (B3LYP/6-31G*:AM1) in

GAUSSIAN 03.

Optimized geometry of the 6:PREGABALIN·3DMSO complex at the ONIOM (B3LYP/6-31G*:AM1) in

GAUSSIAN 03.

NH HNO OHN

CN

CN

NH

CN

NC

O O

NH3

3·[DMSO]

NH HNO OHN

CN

CN

NH

CN

NC

O O

NH3

3·[DMSO]


INTERFERENCE EXPERIMENTS:

We realized some interference experiments by adding 5 equivalents of interference compounds and then

succesive equivalents of the measured analyte (0.5 equiv, 1 equiv, 2 equiv, 3 equiv, 5 equiv, 7 equiv) and

represented in the plots the variation of the fluorescence intensity in each case.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

6

crea

tine(

1 eq

uiv)

L-ca

rnitin

e(1

equiv

)

sarc

osin

e+cr

eatin

ine(5

equ

iv ea

ch)

(sar

cosin

e+cr

eatini

ne)+

crea

tine(

1 eq

uiv)

(S+C

t)+cr

eatin

e(1.5

equ

iv)

(S+C

t)+cr

eatin

e(2.0

equ

iv)

(S+C

t)+cr

eatin

e(3.0

equ

iv)

(S+C

t)+cr

eatin

e(5.0

equ

iv)

(sar

cosin

e+cr

eatini

ne)+

L-ca

rnitin

e(1 e

quiv)

(sar

c+cr

eatn

)+L-

carn

t(1.5

equ

iv)

(sar

c+cr

eatn

)+Lc

arnt

(2.0

equ

iv)

(sar

c+cr

eatn

)+Lc

arnt

(3.0

equ

iv)

(sar

c+cr

eatn

)+Lc

arnt

(5.0

equ

iv)

(sar

c+cr

eatn

)+Lc

arnt

(7.0

equ

iv)

1-I/I

o

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

6

Taurin

e+Glyc

ine+

Met

hionin

e(5

equiv

eac

h)

(T+G

+M)+

crea

tine(

1 eq

uiv)

(T+G

+M)+

crea

tine(

1.5

equiv

)

(T+G

+M)+

crea

tine(

2.0

equiv

)

(T+G

+M)+

crea

tine(

3.0

equiv

)

(T+G

+M)+

crea

tine(

5.0

equiv

)

(T+G

+M)+

Lcar

nitine

(1 e

quiv)

(T+G

+M)+

Lcar

nitine

(1.5

equ

iv)

(T+G

+M)+

Lcar

nitine

(2.0

equ

iv)

(T+G

+M)+

Lcar

nitine

(3.0

equ

iv)

(T+G

+M)+

Lcar

nitine

(5.0

equ

iv)

1-I

/Io


Ultraviolet-visible titration of 1,3-bis[4-(1-dicyanomethyleneindan-5-yl)phenylureidomethyl)-

benzene 6 in DMSO with L-lysine in water:

lysine

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

280 330 380 430 480

Wavelength (nm)

Ab

sorb

ance

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.2

1.4

1.6

2

2.5

3

3.5

4

5

400 nm

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0 1 2 3 4 5 6

equivalents of lysine

Ab

sorb

ance

Up: UV-visible absorption spectra for the colorimetric titration of 6 (10-5 M in DMSO) with L-lysine in

water. Down: Titration profile of the λmax = 400 nm for the UV-visible titration of 6 with L-lysine

0 equiv

5 equiv


Titration Materials and Methods.

5×10-2 M, 5×10-3 M, 5×10-4 M solutions of every salt or aminoacid were prepared, then a 10-4 M solution of the compound

under study was prepared. For qualitative experiments, 2 mL solution of the compound under study were measured and the

corresponding amount of salt or aminoacid was added by micropipette.

Eppendorf Research micropipette characteristics:

MODEL Ep T.I.P.S. Volume Systematic error of measurement

Random error of measurement (CV)

2 - 20μL 2 - 200 2 μL ± 5.0 % ≤ 1.5 %

10 μL ± 1.2 % ≤ 0.6 %

20 μL ± 1.0 % ≤ 0.3 %

10 - 100μL 2 - 200 10 μL ± 3.0 % ≤ 1.0 %

50 μL ± 1.0 % ≤ 0.3 %

100 L ± 0.8 % ≤ 0.2 %

100 - 1000μL 50 - 1000 100 μL ± 3.0 % ≤ 0.6 %

5000 μL ± 1.0 % ≤ 0.2 %

1000 μL ± 0.6 % ≤ 0.2 %

500 - 5000μL 100 - 5000 500 μL ± 2.4 % ≤ 0.6 %

2500 μL ± 1.2 % ≤ 0.25 %

5000 μL ± 0.6 % ≤ 0.15 %

Quantitative studies:

As a general procedure, to a 5000 μl solution of complex, the corresponding amount (μL) of solution of the corresponding salt

(5×10-2 M, 5×10-3 M, 5×10-4 M solutions) was added, using the minor amount of solvent.

A table, corresponding to the number of equivalents and the corresponding added volumes is included:

Vtotal (µL) equiv C

5000 0 0

5005 0.05 4.995E-06

5010 0.1 9.98E-06

5015 0.15 1.4955E-05

5020 0.2 1.992E-05

5025 0.25 2.4876E-05

5030 0.3 2.9821E-05

5035 0.35 3.4757E-05

5040 0.4 3.9683E-05

5045 0.45 4.4599E-05

5050 0.5 4.9505E-05



5055 0.55 5.4402E-05

5060 0.6 5.9289E-05

5065 0.65 6.4166E-05

5070 0.7 6.9034E-05

5075 0.75 7.3892E-05

5080 0.8 7.874E-05

5085 0.85 8.3579E-05

5090 0.9 8.8409E-05

5095 0.95 9.3229E-05

5100 1 9.8039E-05

5105 1.05 0.00010284

5110 1.1 0.00010763

5115 1.15 0.00011241

5120 1.2 0.00011719

5125 1.25 0.00012195

5130 1.3 0.00012671

5135 1.35 0.00013145

5140 1.4 0.00013619

5145 1.45 0.00014091

5150 1.5 0.00014563

5155 1.55 0.00015034

5160 1.6 0.00015504

5165 1.65 0.00015973

5170 1.7 0.00016441

5180 1.8 0.00017375

5190 1.9 0.00018304

5200 2 0.00019231

5210 2.1 0.00020154

5220 2.2 0.00021073

5230 2.3 0.00021989

5240 2.4 0.00022901

5250 2.5 0.0002381

5260 2.6 0.00024715

5270 2.7 0.00025617

5280 2.8 0.00026515

5300 3 0.00028302



5320 3.2 0.00030075

5340 3.4 0.00031835

5360 3.6 0.00033582

5380 3.8 0.00035316

5400 4 0.00037037

5405 4.5 0.00041628

5410 5 0.00046211

5415 5.5 0.00050785

5420 6 0.00055351

5430 7 0.00064457

5440 8 0.00073529

5450 9 0.00082569

5460 10 0.00091575


Supplementary Material (ESI) for Chemical · PDF fileDaniel Moreno, José V. Cuevas,...

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Transcript of Supplementary Material (ESI) for Chemical · PDF fileDaniel Moreno, José V. Cuevas,...