Supplementary Material

A general modular approach for the solubility tagging of

BODIPY dyes

Alberto Blázquez-Moraleja, Delia Álvarez-Fernández, Ruth Prieto Montero,

Inmaculada García-Moreno, Virginia Martínez-Martínez,* Jorge Bañuelos, Inés Sáenz-

de-Santa-María, María D. Chiara, and Jose Luis Chiara*

Synthesis of the solubility tags

5,7-Dihydroxy-2,2-dimethyl-4H-benzo[d][1,3]dioxin-4-one (5). Trifluoroacetic

anhydride (28.7 mL, 206 mmol) and acetone (5 mL, 68.01 mmol) were added

sequentially to a stirred suspension of 2,4,6-trihidroxybenzoic acid monohydrate

(5.0 g, 29.57 mmol) in trifluoroacetic acid (38 mL, 496 mmol) maintained at 0 °C,

and the ensuing mixture was then left to warm to room temperature. After 20 h,

the brown solution obtained was diluted with toluene (50 mL) and concentrated

under reduced pressure. This step was repeated twice. The residue was diluted

with EtOAc (120 mL) and this solution was washed with NaHCO3 (2×30 mL). The

separate organic phase was dried (Na2SO4), filtered, and concentrated under

reduced pressure to afford crude 5 (4.22 g, 68%) as a light-yellow solid, which was

used in the next step without further purification. The combined aqueus phases

obtained were treated with an excess of concentrated HCl, and the precipitate so

formed was collected by filtration to recover unreacted 2,4,6-trihidroxybenzoic

acid (0.893 g).

1H NMR (DMSO, 300 MHz): = 10.88 (1H, s), 10.30 (1H, s), 6.02 (1H, dd, J = 2.1, 0.7

Hz), 5.94 (1H, dd, J = 2.1, 0.7 Hz), 1.66 (6H, s). 13C NMR (DMSO, 75 MHz): = 166.3,

163.5, 162.2, 156.9, 106.4, 97.3, 95.4, 91.8, 25.1. HRMS (API-ES+) calcd. for

C10H10NaO5 (M+Na)+ 233.0420, found 233.0428.

5,7-Bis((2,5,8,11-tetraoxatridecan-13-yl)oxy)-2,2-dimethyl-4H-benzo[d]

[1,3]dioxin-4-one (6a). Tetraethylene glycol monomethyl ether (700 µL, 3.60

mmol) and diisopropyl azodicarboxylate (DIAD) (844 µL, 4.29 mmol) were added

2

sequentially to a stirred suspension of 5 (300 mg, 1.43 mmol) and

triphenylphosphine (1.13 g, 4.27 mmol) in dry THF (5 mL) at 0 °C under an argon

atmosphere. After stirring for 2 h, the ice-water bath was removed and the ensuing

mixture was left to warm to room temperature for 18 h. The brown solution

obtained was concentrated under reduced pressure and the resulting residue was

purified by flash chromatography (EtOAc/MeOH 3:1) to afford 6a (622 mg, 74%)

as a light-yellow oil.

1H NMR (CDCl3, 400 MHz): = 6.18 (1H, d, J = 2.2 Hz, H6), 6.05 (1H, d, J = 2.2 Hz,

H8), 4.17 (2H, t, J = 5.0 Hz, H1´), 4.12 (2H, t, J = 4.8 Hz, H1´´), 3.93 (2H, t, J = 5.0 Hz,

H2´), 3.84 (2H, t, J = 4.8 Hz, H2´´), 3.80 (2H, t, J = 5.0 Hz, H3´), 3.70-3.61 (18H, m, H4

´-H7´ and H3´´-H7´´), 3.54-3.51 (4H, m, H8´ and H8´´), 3.36 (3H, s, H9´/H9´´), 3.33

(3H, s, H9´´/H9´), 1.67 (6H, s, 2 × CH32). 13C NMR (CDCl3, 100 MHz): = 165.6 (C7),

162.1 (C5), 159.3 (C3), 158.1 (C1), 105.1 (C2), 97.2 (C4), 95.4 (C6), 94.5 (C8), 72.0

(C8´/C8´´), 72.0 (C8´´/C8´), 71.1, 71.0, 70.7, 70.7, 70.7, 70.6, 70.6, 70.5, 70.5 (from

71.1 to 70.5: C3´-C7´ and C3´´-C7´´), 69.4 (C2´´), 69.2 (C2´), 69.1 (C1´), 68.0 (C1´´),

59.1 (C9´/C9´´), 59.1 (C9´´/C9´), 25.7 (2 × CH32). HRMS (API-ES+) calcd. for

C28H46NaO13 (M+Na)+ 613.2831; found 613.2839.

2,4-Bis((2,5,8,11-tetraoxatridecan-13-yl)oxy)-6-hydroxybenzoic acid (7a).

Compound 6a (135.9 mg, 0.23 mmol) was dissolved in 3.0 M aqueous KOH (4 mL).

After stirring the mixture for 1 h at room temperature, the solution was treated

with 3 M HCl until pH = 2. The aqueous phase was extracted with EtOAc (5 × 10

mL), and the combined organic phases were dried (Na2SO4), filtered, and

3

concentrated under reduced pressure to afford 7a (127.6 mg, 94%) as a light-

yellow oil.

1H NMR (CDCl3, 400 MHz): = 12.43 (1H, s, COOH1; OH2 not distinguishable), 6.14

(1H, s, H3), 6.03 (1H, s, H5), 4.24 (2H, br t, H1´), 4.10 (2H, br t, H1´´), 3.87 (2H, br t,

H2´), 3.82 (2H, br t, H2´´), 3.67-3.62 (20H, br m, H3´-H7´ and H3´´-H7´´), 3.51 (4H,

br m, H8´ and H8´´), 3.34 (6H, br s, H9´ and H9´´). 13C NMR (CDCl3, 100 MHz): =

171.0 (COOH1), 166.1 (C2), 164.7 (C4), 159.1 (C6), 95.9 (C1), 95.7 (C3), 93.1 (C5),

72.0 (C8´/C8´´), 72.0 (C8´´/C8´), 70.9, 70.8, 70.7, 70.7, 70.6, 70.6, 70.6, 70.5 (from

70.9 to 70.5: C3´-C7´ and C3´´-C7´´), 69.4 (C2´´), 69.1 (C1´), 68.4 (C2´), 67.9 (C1´´),

59.1 (C9´/C9´´), 59.0 (C9´´/C9´). HRMS (API-ES+) calcd. for C25H46NO13 (M+NH4)+

568.2964; found 568.2967.

5,7-Bis((2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)oxy)-2,2-dimethyl-4H-

benzo[d][1,3]dioxin-4-one (6b). Octaethylene glycol monomethyl ether (463 µL,

1.26 mmol) and diisopropyl azodicarboxylate (DIAD) (310 µL, 1.5 mmol) were

added sequentially to a stirred suspension of 5 (105 mg, 0.49 mmol) and

triphenylphosphine (397 mg, 1.5 mmol) in dry THF (2 mL) at 0 °C under an argon

atmosphere. After stirring for 1 h, the ice-water bath was removed, and the

mixture was left to warm to room temperature for 23 h. The brown solution

obtained was concentrated under reduced pressure and the resulting residue was

purified by flash chromatography (EtOAc/MeOH 100:0 → 75:25) to afford 6b (395

mg, 83%) as a light-yellow oil.

4

1H NMR (CDCl3, 400 MHz): = 6.14 (1H, d, J = 1.9 Hz, H6), 6.02 (1H, d, J = 1.9 Hz,

H8), 4.12 (2H, t, J = 4.9 Hz, H1´), 4.08 (2H, t, J = 4.9 Hz, H1´´), 3.89 (2H, t, J = 4.9 Hz,

H2´), 3.80 (2H, t, J = 4.9 Hz, H2´´), 3.77 (2H, t, J = 4.9 Hz, H3´) 3.67-3.59 (50H, m, H4

´-H15´and H3´´-H15´´), 3.50-3.48 (4H, m, H16´and H16´´), 3.32 (6H, s, H17´ and H17

´´), 1.63 (6H, s, 2 × CH32). 13C NMR (CDCl3, 100 MHz): = 165.4 (C7), 162.0 (C5),

159.2 (C3), 157.7 (C1), 104.9 (C2), 97.0 (C4), 95.3 (C6), 94.3 (C8), 71.9 (C16´and

C16´´), 71.1 (C3´), 70.9 (C3´´), 70.7, 70.6, 70.6, 70.6, 70.5, 70.5, 70.5 (from 70.7 to

70.5: C4´-C15´ and C4´´-C15´´), 69.3 (C2´ and C2´´), 69.1 (C1´), 67.9 (C1´´), 59.0 (C17

´ and C17´´), 25.6 (2 × CH32). HRMS (API-ES+) calcd. for C44H82NO21 (M+NH4)+

960.5374; found 960.5365.

2,4-Bis((2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)oxy)-6-hydroxybenzoic

acid (7b). Compound 6b (59 mg, 0.06 mmol) was dissolved in 3.0 M aqueous KOH

(1.08 mL). After stirring the mixture for 1 h at room temperature, the solution was

treated with 3 M HCl until pH = 2. The aqueous phase was extracted with CH2Cl2 (5

× 5 mL), and the combined organic phases were dried (Na2SO4), filtered, and

concentrated under reduced pressure to afford 7b (119.6 mg, 100%) as a light-

yellow oil.

HO

HO

O

OO

OO

OO

OO

O

OO

OO

OO

OO

O2

1 6

5

43

1´

2´ 3´

4´ 5´

6´

1´´

2´´ 3´´

4´´ 5´´

6´´

7´

8´ 9´

10´ 11´

12´ 13´

14´ 15´

16´17´

7´´

8´´ 9´´

10´´ 11´´

12´´ 13´´

14´´ 15´´

16´´17´´

1H NMR (CDCl3, 400 MHz): = 12.42 (1H, s, COOH1), 11.24 (1H, br s, HO2), 6.14

(1H, d, J = 2.1 Hz, H3), 6.03 (1H, d, J = 2.1 Hz, H5), 4.24 (2H, t, J = 4.8 Hz, H1´), 4.10

5

(2H, t, J = 4.8 Hz, H1´´), 3.86 (2H, t, J = 4.8 Hz, H2´), 3.81 (2H, t, J = 4.8 Hz, H2´´) 3.67-

3.59 (52H, m, H3´-H15´ and H3´´-H15´´) 3.52-3.49 (4H, m, H16´ and H16´´), 3.33

(6H, s, H17´and H17´´). 13C NMR (CDCl3, 100 MHz): = 171.0 (COOH1), 166.1 (C2),

164.7 (C4), 159.1 (C6), 95.9 (C1), 95.7 (C3), 93.1 (C5), 72.0 (C16´ and C16´´), 70.9,

70.8, 70.7, 70.6, 70.6, 70.6, 70.6, 70.6, 70.5 (from 70.9 to 70.5: C3´-C15´ and C3´´-

C15´´), 69.3 (C2´´), 69.1 (C1´), 68.4 (C2´), 67.8 (C1´´), 59.0 (C17´ and C17´´). HRMS

(API-ES+) calcd. for C41H78NO21 (M+NH4)+ 920.5061; found 920.5074.

3-((2-(2-Hydroxyethoxy)ethyl)dimethylammonio)propane-1-sulfonate (8). 2-

[2-(dimethylamino)ethoxy]ethanol (275 µL, 1.86 mmol) was added to a stirred

solution of 1,3-propanesultone (208 mg, 1.69 mmol) in acetone (10 mL), under

argon atmosphere at room temperature for 3 d. The resultant white suspension

was cooled in an ice-water bath for 1 h, filtered and the precipitate was washed

with cold acetone (20 mL). The filtrate and washings were combined, concentrated

at reduced pressure, resuspended in acetone and kept at 0 °C overnight to recover

an additional amount of solid precipitate to afford 8 (430 mg, 99%) as a white

powder.

1H NMR (CD3OD, 400 MHz): = 3.94-3.93 (2H, br m, H1), 3.70 (2H, t, J = 4.8 Hz,

H2), 3.64-3.58 (6H, m, H4, H5 and H3), 3.18 (6H, s, (CH3)2N), 2.88 (2H, t, J = 6.9 Hz,

H7), 2.27-2.20 (2H, m, H6). 13C NMR (CD3OD, 100 MHz): = 73.8 (C2), 65.6 (C1),

65.2 (t, J =2.4 Hz , C4/C5), 64.6 (t, J = 2.8 Hz , C5/C4), 62.0 (C3), 52.4 (t, J = 3.8 Hz ,

(CH3)2N), 48.7 (C7), 20.0 (C6). HRMS (API-ES+) calcd. for C9H22NO5S (M+H)+

256.1213; found 256.1215.

6

Compound 6c. A solution of 8 (362 mg, 1.42 mmol), 5 (99.82 mg, 0.47 mmol)

and triphenylphosphine (503.30 mg, 1.9 mmol) in dry DMSO was stirred at room

temperature under argon atmosphere for 30 minutes. Diisopropyl

azodicarboxylate (DIAD) (393 µL, 1.9 mmol) was added dropwise to the stirred

solution; a color change from light-yellow to purple was immediately observed.

The resulting reaction mixture was stirred at room temperature for 2 d. The purple

solution was diluted with acetone (15 mL) and centrifuged at 8000 rpm/4 °C for

30 minutes. After removing the supernatant, this treatment was repeated twice.

The resulting grey precipitate was purified by reversed-phase column

chromatography (H2O/MeCN 100:0 → 0:100). The fractions containing product

were combined and lyophilized to afford 6c (213.2 mg, 65%) as a yellow oil.

1H NMR (CD3OD, 400 MHz): = 6.39 (1H, d, J =2.0 Hz , H6), 6.23 (1H, d, J = 2.0 Hz,

H8), 4.27 (4H, br t, H1´ and H1´´), 4.12 (2H, br m, H3´/H3´´), 4.01 (2H, br m,

H3´´/H3´), 3.94 (2H, t, J =4.1 Hz, H2´/H2´´), 3.89 (2H, t, J = 4.1 Hz, H2´´/H2´), 3.64-

3.57 (8H, m, H4´, H5´, H4´´ and H5´´), 3.18 (6H, s, (CH3)2N), 3.17 (6H, s, (CH3)2N)),

2.84 (2H, t, J = 6.8 Hz, H7´/H7´´), 2.76 (2H, t, J = 6.8 Hz, H7´´/H7´), 2.26-2.17 (4H, m,

H6´ and H6´´), 1.68 (6H, s, 2 × CH32). 13C NMR (CD3OD, 100 MHz): = 167.5 (C7),

163.4 (C5), 160.8 (C3), 160.1 (C1), 106.6 (C2), 97.5 (C4), 96.1 (C6), 95.9 (C8), 70.3

(C2´ and C2´´), 70.2 (C1´/C1´´), 69.1 (C1´´/C1´), 66.1 (C3´/C3´´), 65.8 (C3´´/C3´´),

65.1 (br s, C4´/C4´´/C5´/C5´´), 64.9 (br s, C4´´/C5´/C5´´/C4´), 64.7 (br s,

C5´/C5´´/C4´/C4´´), 64.5 (br s, C5´´/C4´/C4´´/C5´), 52.6 (br m, 2 × (CH3)2N), 48.7

7

(C7´ and C7´´), 25.7 (2 × CH3C2), 20.1 (C6´ and C6´´). HRMS (API-ES+) calcd. for

C28H49N2O13S2 (M+H)+ 685.2671; found 685.2662.

Compound 7c. Compound 6c (228 mg, 0.33 mmol) was dissolved in 3.0 M

aqueous KOH (5.77 mL). After stirring the mixture for 2 h at room temperature,

the solution was treated at 0 °C with 3 M HCl until pH = 2. The solution was

lyophilized and the resulting crude solid was purified by reversed-phase C18

column chromatography (H2O/MeCN 100:0 → 0:100). The fractions containing

product were combined and lyophilized to afford 7c (195 mg, 92%) as a white

solid.

1H NMR (CD3OD, 400 MHz): = 6.25 (1H, br d, H5), 6.19 (1H, d, J = 1.7 Hz, H3) 4.37

(2H, t, J = 4.2 Hz, H1´), 4.23 (2H, t, J = 4.2 Hz, H1´´), 4.04 (2H, br m, H3´/H3´´), 4.00

(2H, br m, H3´´/H3´), 3.94 (2H, t, J = 4.2 Hz, H2´), 3.88 (2H, t, J = 4.2 Hz, H2´´), 3.63-

3.58 (8H, m, H4´, H5´, H4´´and H5´´), 3.17 (6H, s, (CH3)2N)), 3.17 (6H, s, (CH3)2N)),

2.84 (4H, q, J = 6.7 Hz, H7´ and H7´´), 2.26-2.18 (4H, m, H6´ and H6´´). 13C NMR

(CD3OD, 100 MHz): = 172.9 (COOH1), 166.9 (C2), 166.1 (C4), 161.8 (C6), 97.4

(C1), 96.3 (C3), 93.9 (C5), 70.4 (C2´´), 70.3 (C1´), 70.1 (C2´), 68.7 (C1´´), 65.8

(C3´/C3´´), 65.7 (C3´´/C3´), 65.0 (br s, C4´/C4´´/C5´/C5´´), 64.9 (br s, C5´´/C4´/C4

´´/C5´), 64.7 (br s, C5´/C5´´/C4´/C4´´), 64.5 (br s, C4´´/C5´/C5´´/C4´), 52.6, 52.6,

52.6, 52.5 (from 52.6 to 52.5: 2 × (CH3)2N), 48.7 (C7´ and C7´´), 20.1 (C6´/C6´´), 20.0

(C6´´/C6´). HRMS (API-ES+) calcd. for C25H43N2O13S2 (M–H)– 643.2212; found

643.2201.

8

Figure S1. Height normalized absorption (left) and emission (right) spectra for compound 2a in water at different concentration; a: 1.910–5 M (1mm), b: 1.210–

4 M (0.1 mm), c: 3.110–4 M (0.1 mm), and d: 7.110–4 M (0.1 mm) recorded in different optical path cuvettes.

Figure S2. Height normalized absorption (left) and emission (right) spectra for compound 2b in water at different concentrations: a: 5.910–6 M (1 mm), b: 1.010-4 M (1 mm), c: 1.510–4 M (0.1 mm), d: 510–4 M (0.1 mm), and e: 7.210–4

M (0.1 mm).

9

Figure S3. Height normalized absorption (left) and emission (right) spectra for compound 3a in water at different concentration; a: 9.710–6 M, b: 1.510–5 M, c: 2.710–5 M, and d: 4.610–5 M, recorded in 1-mm optical path cuvette.

Figure S4. Height normalized absorption (left) and emission (eight) spectra for compound 3b in water at different concentration; a: 9.410–6 M, b: 5.010–5 M, c: 1.010–4 M, recorded in 1-mm optical path cuvette, and d: 3.610–4 M and e: 5.410–4 M, recorded in 0.1 mm optical path cuvette.

10

Figure S5. Time-dependent absorption spectra of 3b (7 10–5 M) in cell culture media with 10% fetal bovine serum (pH 7.4) at room temperature in 0.1 cm cuvette. Time zero (black), after 1 day (red) and 7 days (blue).

Figure S6. Time-dependent absorption spectra of 3b (8 10–5 M) in pH 6.0 phosphate buffered saline at room temperature in 0.1 cm cuvette. Time zero (black), after 1 day (red) and 7 days (blue).

11

Figure S7. Time-dependent absorption spectra of 3b (1.6 10–5 M) in pH 8.0 phosphate buffered saline at room temperature in 0.1 cm cuvette. Time zero (black), after 1 day (red) and 7 days (blue).

0.000 1.000 2.000 3.000 4.000 5.000 6.000

-1.000

-0.500

0.000

0.500

1.000

1.500

f(x) = 0.420427712444 x − 1.175045572976R² = 0.956419839883554

Log P

Figure S8. Calibration curve of log k (retention factor, k = (tR – t0)/t0)) vs. log P for the reference substances indicated in Table 3, using the RP-HPLC method.

12

Table S1. Photophysical properties of newly synthesized dyes derived from BODIPY skeleton 1 in different solventsa.

Dye Solvent λabb

(nm)εmax

c

(104 M–1 cm–1)λfl

d

(nm)Фfl

e τ(ns)f

1a CHCl3 507.0 4.8 520.0 0.17 0.45

EtOH 504.0 4.4 518.0 0.03 <0.15

H2O 503.0 3.2 515.0 <0.02 <0.15

1b CHCl3 507.0 5.8 521.0 0.06 0.49

EtOH 505.0 5.1 514.5 0.02 0.11

H2O 503.0 4.0 514.0 <0.01 <0.15aDye concentration: 210–6 M; bAbsorption peak wavelength; cMolar extinction coefficient at peak; dFluorescence peak wavelength, upon excitation at 490 nm; eUsing quantum yield PM546 (Фfl= 0.91) in cyclohexane as reference; fFluorescence lifetime. The fluorescence data are recorded under right angle configuration and using 1-cm optical path cuvette.

Table S2. Photophysical properties of newly synthesized dyes derived from BODIPY skeleton 2 in different solvents.a

Dye Solvent λab

(nm)εmax

(104 M–1 cm–1)λfl

b

(nm)Фflu

b τb

(ns)

2a CHCl3 507.0 7.6 516.0 0.88 6.04

EtOH 504.0 7.9 512.5 0.85 6.58

H2O 501.0 4.0 512.5 0.66 6.86

2b CHCl3 507.0 7.8 515.5 0.86 5.82

EtOH 504.0 6.9 514.5 0.79 6.60

H2O 502.0 4.2 511.5 0.78 6.40

2c CHCl3 507.0 8.1 517.0 0.71 5.72

EtOH 504.0 6.5 514.5 0.84 5.99

H2O 501.0 6.4 510.5 0.85 6.31aDye concentration: 210–6 M and using quantum yield of PM546 (Фfl = 0.91) in cyclohexane as reference. The fluorescence data are recorded under right angle configuration and using a 1-cm optical pathway cuvette.

13

Table S3. Photophysical properties of newly synthesized dyes derived from BODIPY skeleton 3 in different solvents.a

Dye Solvent λab

(nm)εmax

(104 M–1 cm–1)λfl

b

(nm)Фflu

b τb

(ns)

3a CHCl3 527.0 6.3 548.5 0.72 6.99

EtOH 523.0 6.2 546.0 0.75 7.42

H2O 519.0 4.1 540.5 0.71 7.55

3b CHCl3 527.0 6.2 547.0 0.73 6.85

EtOH 523.0 4.5 545.5 0.72 7.28

H2O 520.0 4.9 540.0 0.71 7.77

3c CHCl3 528.0 4.6 546.0 0.72 6.87

EtOH 523.0 5.5 543.0 0.76 7.39

H2O 519.0 4.5 538.0 0.78 7.50aDye concentration: 210–6 M and using PM567 ( = 0.84) in ethanol as reference; bThe fluorescence data are recorded under right angle configuration and using a 1-cm optical pathway cuvette.

Table S4. Dependence of the fluorescence quantum yield and lifetimea of new synthesized dyes derived from BODIPY skeleton 2 on the dye concentration in water.

2a 2b 2c[c]104

(M) b c (ns)b b c (ns)b b c (ns)b

0.02 - - - 0.78 - 6.30 0.85 - 5.890.05 - - - 0.78 - 6.40 0.85 - 6.090.06 0.66 - 6.06 0.78 - 6.40 0.85 - 6.200.1 - - - 0.78 - 6.59 0.86 - 6.200.2 0.66 - 6.14 - - - 0.85 - 6.360.3 0.64 - 6.14 - - - 0.83 - -0.7 0.63 - 6.17 0.76 - 7.35 0.87 - -0.95 0.60 - 6.22 0.75 - - 0.85 - -1.2 0.57 0.58 6.29 0.42 0.55 7.79 0.45 0.58 6.813.0 0.38 0.45 6.41 - - - 0.19 - -5.0 0.19 0.20 6.41 0.14 0.20 - - - -7.0 <0.1 <0.1 6.45 0.05 - - - - -aUpon excitation at 490 nm using PM546 (Фfl = 0.91) in cyclohexane as reference; b,cEvaluated under front-face configuration and using 1 and 0.1-mm optical pathway cuvette, respectively.

14

Table S5. Dependence of the fluorescence quantum yield and lifetimea of new synthesized dyes derived from BODIPY skeleton 3 on the dye concentration in water.

3a 3b 3c[c]104

(M)

b (ns)b b c (ns)b (ns)c b c (ns)b (ns)c

0.02 - - 0.72 - 7.52 - 0.78

- 7.25 -

0.05 0.71 7.30 - - - - 0.78

- 7.27 -

0.06 0.71 7.33 - - - - - - - -0.1 0.71 7.42 0.72 - 7.58 - 0.7

8- 7.36 -

0.2 0.73 7.44 0.73 - 7.78 - 0.76

- 7.44 -

0.3 0.72 7.47 0.75 - 7.81 - 0.78

- - -

0.5 0.33 - 0.74 - 8.03 - 0.68

0.77 7.88 -

0.9d - - - - - - 0.63

0.75 - -

1.3 - - 0.65 0.75

8.22 7.67 0.35

0.48 8.22 7.48

2.5 - - 0.31 0.57

7.85 7.42 0.22

0.34 8.24 7.47

3.6 - - 0.17 0.55

7.52 7.39 0.06

0.24 7.83 7.21

5.5 - - 0.08 0.33

7.53 7.42 0.01

0.09 - -

aUpon excitation at 490 nm and using PM567 (Фfl = 0.84) in ethanol as reference; b,cEvaluated under front-face configuration and using a 1 and 0.1-mm optical pathway cuvette, respectively. dDye 3c not soluble beyond this concentration.

15

Table S6. Atom coordinates and total energy (in hartrees) in the ground state (B3LYP/6-311+G* in water) for the representative compounds 1a and its methylated analog 2a.

1a C 6.55625600 -0.64613100 0.92760700 N 5.28685800 -0.22563900 0.51896600 B 4.91806900 0.28313800 -0.91659400 N 6.25154900 0.31452600 -1.70425600 C 7.49556500 -0.11156600 -1.23516100 C 7.65790900 -0.59453700 0.06735600 C 6.40954200 0.72483900 -2.97443700 C 7.75078200 0.58232100 -3.36920300 C 8.43523800 0.05705200 -2.28080400 C 6.46434600 -1.06822000 2.27643100 C 5.14472000 -0.89922300 2.67155300 C 4.45362200 -0.37864200 1.56343800 C 10.64102600 -2.79369700 0.82460700 C 9.38137300 -2.39359400 0.37295300 C 9.00249700 -1.04747700 0.53896300 C 9.87523400 -0.12547900 1.14812600 C 11.12439700 -0.57477600 1.58291700 C 11.52867000 -1.90201400 1.42942600 C 8.45885300 -3.40038700 -0.27416600 C 9.48785700 1.32250700 1.33975600 C 12.89606500 -2.35462900 1.88006800 C 0.72897400 0.91463900 -1.09008200 C -0.08908500 -0.12793500 -1.51879600 C 0.48083700 -1.31869900 -1.98267800 C 1.86668800 -1.48152000 -1.97510200 C 2.68148800 -0.44110200 -1.54601700 C 2.13997600 0.80205500 -1.12292200 C 3.05781900 1.94098700 -0.92872900 O 0.06726900 2.03193700 -0.67502100 O 4.01180700 -0.64112100 -1.58589700 O -0.22567200 -2.38649000 -2.43565100 O 2.72068000 3.10828200 -0.87235700 O 4.36811500 1.63678800 -0.85556000 C -1.65414400 -2.33255500 -2.53404500 C -2.31568000 -2.72862700 -1.21965900 O -3.71227900 -2.73738000 -1.45429500 C -4.47015900 -3.11267500 -0.31474900 C -5.93537200 -3.08896300 -0.71108200 O -6.69245700 -3.45935100 0.42837900 C 0.33057100 2.57186200 0.63418900 C -1.00929400 2.74873200 1.32994400 O -0.74116400 3.40694500 2.56212800 C -1.81873900 3.43692800 3.48967800 C -2.84360100 4.52576600 3.22851100 O -3.66267300 4.17142000 2.12219300 C -4.63766500 5.15416700 1.81731600 C -8.09077900 -3.48629400 0.18890200 C -8.76635000 -3.88055200 1.49887100 O -10.18268200 -3.89322100 1.38841600 C -5.45787200 4.64572500 0.64542600 O -6.45084500 5.61745100 0.36431600 C -7.28808000 5.26033700 -0.72260900 C -8.30150400 6.37463100 -0.91106500 O -9.13542800 6.01758800 -1.99796900 C -10.12997500 6.99049300 -2.26639500 C -10.72718300 -5.09936600 0.86908600 C -12.23904700 -4.97071700 0.91592800 O -12.78758500 -6.16709900 0.39424300 C -14.20445000 -6.16100100 0.38597800 H 5.57189500 1.09893600 -3.54460000 H 8.15242100 0.83937400 -4.33770300 H 9.48598100 -0.18306600 -2.21752900 H 7.28587700 -1.44404700 2.86765500 H 4.71295300 -1.11497700 3.63719100 H 3.40803200 -0.11695500 1.48755400 H 10.93396700 -3.83261400 0.70044800 H 11.79790900 0.13450800 2.05607000 H 8.92539000 -4.38563600 -0.31318700 H 8.20104900 -3.11343400 -1.29760300

2a C 7.06026100 -1.18810700 -0.91940600 N 5.68883800 -1.06429700 -1.20017600 B 4.66565600 -0.25773100 -0.34580700 N 5.41275800 0.16622300 0.95328500 C 6.79224300 0.01635400 1.17922200 C 7.60609100 -0.64857600 0.25254400 C 4.87923700 0.80869100 2.01945400 C 5.89641400 1.08633600 2.94612000 C 7.09954400 0.60551100 2.44598200 C 7.66465700 -1.92836100 -1.98406500 C 6.63904700 -2.22927500 -2.87072700 C 5.44117300 -1.69017000 -2.37505800 C 11.32881400 0.03348900 0.34154800 C 9.96677200 0.19117600 0.07537900 C 9.07100200 -0.79697100 0.52387600 C 9.54114400 -1.92063900 1.22805600 C 10.91184000 -2.03546700 1.47131300 C 11.82387800 -1.07251000 1.03535200 C 9.48409500 1.40843200 -0.67869200 C 8.59818100 -2.99219300 1.72388700 C 13.30380500 -1.23493400 1.28326800 C 8.41328200 0.71928900 3.15756400 C 3.43662000 1.15352400 2.17413600 C 9.09291200 -2.33839700 -2.17672400 C 4.10326500 -1.78099400 -3.02841400 C 0.56937700 0.63741500 -1.32088800 C -0.41031100 -0.29388100 -0.99194100 C -0.05293600 -1.47942000 -0.33853300 C 1.27850900 -1.72195600 -0.00502900 C 2.25838400 -0.79430400 -0.34511700 C 1.93527700 0.40923700 -1.02403900 C 3.02559200 1.30126800 -1.46607500 O 0.13474500 1.74613200 -1.99554000 O 3.52337900 -1.10189400 -0.01346200 O -0.93804000 -2.44037700 0.02265600 O 2.87825100 2.30529700 -2.13831100 O 4.26725200 0.94575900 -1.09437500 C -2.32295900 -2.27140200 -0.29394200 C -3.03631000 -3.51029800 0.21601900 O -4.40791900 -3.35190400 -0.09618200 C -5.20694900 -4.44671200 0.32280300 C -6.64033400 -4.14535600 -0.07573200 O -7.43959200 -5.23801600 0.34339000 C 0.20907900 3.02005600 -1.33115200 C -1.18031100 3.41357300 -0.85019000 O -1.06151400 4.70971100 -0.27694000 C -2.19861800 5.15296600 0.44964500 C -3.29841600 5.69746800 -0.45698500 O -4.33235400 6.17993600 0.38622000 C -5.43230800 6.72015000 -0.32634800 C -8.81119200 -5.08051400 0.02162500 C -9.54555100 -6.30998300 0.52514300 O -10.91619300 -6.15250200 0.2011500 C -6.45254900 7.19232300 0.69385600 O -7.55367100 7.73084400 -0.01757800 C -8.58383900 8.21082700 0.82994900 C -9.69216700 8.75557300 -0.05269000 O -10.71991200 9.23481500 0.7948230 C -11.81731100 9.76901000 0.07507900 C -11.71764600 -7.24399500 0.6209620 C -13.15022100 -6.94755800 0.2159240 O -13.94980600 -8.03772000 0.6357980 C -15.31753600 -7.87182200 0.3048800 H 5.75107000 1.59096900 3.89107000 H 6.73159500 -2.78547400 -3.79310500 H 12.02061300 0.79788200 -0.0018990 H 11.27501200 -2.90138100 2.0182130 H 10.31865200 2.05970600 -0.9423090 H 8.77526000 1.99665800 -0.08853800

16

H 7.51787900 -3.49894600 0.27450100 H 10.28800000 1.87647000 1.83247200 H 8.58710200 1.42360300 1.95172600 H 9.28048500 1.81534800 0.38563800 H 12.89262000 -3.40509400 2.17941600 H 13.62911400 -2.25073700 1.07268300 H 13.25786400 -1.76174600 2.72300600 H -1.15739300 0.01852700 -1.45022100 H 2.30906100 -2.41104700 -2.31091900 H -1.99032700 -1.34768400 -2.86516200 H -1.91407400 -3.05653600 -3.30604800 H -2.06280500 -2.02230600 -0.41800300 H -1.96293600 -3.72333100 -0.91555300 H -4.18874300 -4.11911500 0.02306900 H -4.29426200 -2.41480100 0.51490200 H -6.11064100 -3.78910300 -1.53935900 H -6.21483300 -2.08325500 -1.05371500 H 0.97323200 1.89687600 1.20393300 H 0.84189100 3.52684000 0.52737400 H -1.47905200 1.77255600 1.51086300 H -1.68470000 3.33777400 0.70254000 H -2.31908500 2.46013400 3.52622900 H -1.36296500 3.62172500 4.46561100 H -2.33056300 5.47796100 3.03368000 H -3.46442500 4.65489700 4.12792200 H -5.29366500 5.32982000 2.68132800 H -4.16102000 6.10909500 1.55677200 H -8.44954400 -2.49815700 -0.13055700 H -8.32511100 -4.20403300 -0.60897900 H -8.51994800 -3.14310200 2.26617200 H -8.39213400 -4.85633800 1.83333100 H -4.81104400 4.48872000 -0.22868700 H -5.91883300 3.68205500 0.90230700 H -6.69856000 5.13122300 -1.64072900 H -7.80654800 4.31341100 -0.51834700 H -7.78181300 7.32248900 -1.11189300 H -8.89093000 6.50321400 0.00809800 H -10.71577300 6.62885900 -3.11142300 H -10.79398600 7.13288900 -1.40372400 H -9.68293100 7.95869300 -2.52741000 H -10.40750300 -5.27706100 -0.16494700 H -10.40590900 -5.95684900 1.47541100 H -12.56849600 -4.80805300 1.95209100 H -12.55888000 -4.10265200 0.32174000 H -14.52907900 -7.11504600 -0.02927400 H -14.61041600 -6.05403900 1.40042700 H -14.59763900 -5.34613000 -0.23595300

H 8.96978700 1.13409400 -1.60442600 H 8.04434100 -3.45689100 0.90288800 H 7.85294200 -2.58899500 2.41581900 H 9.14379800 -3.78019000 2.24489000 H 13.78587000 -1.75973700 0.4512540 H 13.80207600 -0.26833300 1.3864880 H 13.49798700 -1.81602900 2.1874780 H 8.27557400 1.25167400 4.10040800 H 8.84713300 -0.25722400 3.38451300 H 9.15615500 1.26283000 2.56964500 H 3.07116600 1.77088100 1.35035200 H 2.81075400 0.25887900 2.21131100 H 3.28852200 1.70912600 3.10000700 H 9.44605800 -2.99467300 -1.37791700 H 9.19702500 -2.87528600 -3.12130900 H 9.76973000 -1.48156600 -2.20007300 H 4.19032500 -2.32152300 -3.97085900 H 3.38080800 -2.30703200 -2.40064500 H 3.68770500 -0.79405300 -3.24686500 H -1.43408100 -0.06275800 -1.24955500 H 1.55612500 -2.63521600 0.50640500 H -2.72149900 -1.37737700 0.19482300 H -2.45980000 -2.17453900 -1.37502500 H -2.89016700 -3.61023000 1.29986000 H -2.62559800 -4.40689300 -0.26711300 H -4.87179700 -5.37676200 -0.15575400 H -5.13988100 -4.58167400 1.41073400 H -6.70597200 -4.01025200 -1.16401100 H -6.97399100 -3.21368000 0.40138200 H 0.90502400 2.98560900 -0.49263500 H 0.58360500 3.74206300 -2.05582000 H -1.54374800 2.69728400 -0.10117200 H -1.87923900 3.40596500 -1.69530200 H -2.59819000 4.34509800 1.07605900 H -1.85085100 5.95181700 1.10831700 H -3.68754100 4.91617800 -1.12390600 H -2.89686500 6.50651900 -1.08281400 H -5.11241900 7.56273700 -0.95457900 H -5.88149800 5.96138700 -0.98176400 H -9.22065100 -4.17856100 0.49678700 H -8.94659300 -4.98166200 -1.06414200 H -9.41132900 -6.40825300 1.61118200 H -9.13491300 -7.21205800 0.05105500 H -6.00366900 7.95178600 1.34874000 H -6.77071600 6.34950600 1.32283000 H -8.97326200 7.40230100 1.46384100 H -8.20589300 9.00442400 1.48912900 H -10.06859200 7.96114300 -0.7129980 H -9.30111000 9.56304900 -0.68822000 H -12.54924200 10.10899200 0.8076820 H -11.51147200 10.61937600 -0.5483940 H -12.28032100 9.01081500 -0.5700310 H -11.38060400 -8.17723900 0.1491280 H -11.65575900 -7.37474000 1.7100900 H -13.21063100 -6.81525800 -0.8738740 H -13.48577100 -6.01230700 0.6866400 H -15.84641200 -8.75386000 0.6658560 H -15.73989300 -6.97869400 0.7837480 H -15.45940400 -7.78705200 -0.7805130

E -2785.86793 E -2943.173931

17

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)

6.05

1.01

1.01

1.04

1.00

1.66

2.50

dm

so

5.93

5.93

5.94

5.94

6.01

6.01

6.02

6.02

10.3

0

10.8

8

1H NMR (CDCl3, 400 MHz) spectrum of compound 518

0102030405060708090100110120130140150160170180190200f1 (ppm)

25.1

38.7

dm

so39

.0 d

mso

39.2

dm

so39

.5 d

mso

39.8

dm

so40

.1 d

mso

40.4

dm

so

91.8

95.4

97.3

106.

4

156.

9

162.

216

3.5

166.

3

13C NMR (75 MHz, DMSO) spectrum of compound 519

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)

6.03

2.92

3.01

4.00

18.5

44.

001.

932.

021.

95

0.98

0.96

1.67

3.33

3.36

3.51

3.52

3.53

3.53

3.53

3.54

3.61

3.62

3.62

3.63

3.65

3.66

3.66

3.67

3.68

3.69

3.70

3.70

3.79

3.80

3.81

3.83

3.84

3.85

3.91

3.93

3.94

4.10

4.12

4.13

4.16

4.17

4.18

5.29

6.05

6.05

6.18

6.18

7.26

CD

Cl3

20

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)

6.03

2.92

3.01

4.00

18.5

44.

001.

932.

021.

95

0.98

0.96

1.67

3.33

3.36

3.51

3.52

3.53

3.53

3.53

3.54

3.61

3.62

3.62

3.63

3.65

3.66

3.66

3.67

3.68

3.69

3.70

3.70

3.79

3.80

3.81

3.83

3.84

3.85

3.91

3.93

3.94

4.10

4.12

4.13

4.16

4.17

4.18

5.29

6.05

6.05

6.18

6.18

7.26

CD

Cl3

21

0102030405060708090100110120130140150160170180190200f1 (ppm)

25

.7

59

.15

9.1

68

.06

9.1

69

.26

9.4

70

.57

0.5

70

.67

0.6

70

.77

0.7

70

.77

1.0

71

.17

2.0

72

.07

6.8

CD

Cl3

77

.2 C

DC

l37

7.5

CD

Cl3

94

.59

5.4

97

.2

10

5.1

15

8.1

15

9.3

16

2.1

16

5.6

13C NMR (100 MHz, CDCl3) spectrum of compound 6a22

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.012.513.0f1 (ppm)

5.95

4.21

20.6

12.

292.

031.

992.

09

1.02

1.02

1.02

3.34

3.51

3.62

3.65

3.67

3.82

3.87

4.10

4.24

6.03

6.14

7.26

CD

Cl3

12.4

3

1H NMR (CDCl3, 400 MHz) spectrum of compound 7a23

13C NMR (100 MHz, CDCl3) spectrum of compound 7a24

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)

6.00

3.00

5.60

4.01

18.5

64.

031.

931.

952.

00

0.98

0.99

1.94

1.88

1.95

1.90

2.11

2.14

2.36

3.36

3.36

3.52

3.54

3.54

3.55

3.62

3.63

3.63

3.63

3.63

3.64

3.64

3.64

3.64

3.65

3.65

3.65

3.65

3.65

3.66

3.66

3.66

3.67

3.67

3.67

3.67

3.68

3.68

3.68

3.68

3.68

3.69

3.69

3.70

3.70

3.70

3.71

3.71

3.72

3.72

3.72

3.83

3.84

3.85

3.86

3.87

3.89

3.98

3.99

4.00

4.10

4.11

4.12

4.20

4.22

4.23

6.08

6.09

6.16

6.17

6.39

6.39

6.40

6.40

6.67

6.68

6.68

6.69

6.95

7.26

cdc

l37.

69

1H NMR (CDCl3, 400 MHz) spectrum of compound 1a25

0102030405060708090100110120130140150160170180190200f1 (ppm)

20.2

20.3

21.3

59.2

59.2

67.7

69.2

69.4

69.6

70.6

70.6

70.7

70.7

70.8

70.8

70.8

70.8

71.0

71.2

72.1

76.8

cdc

l377

.2 c

dcl3

77.5

cdc

l394

.395

.699

.2

118.

8

128.

212

8.4

129.

713

0.6

135.

313

6.3

136.

713

9.0

144.

7

147.

9

160.

816

2.2

162.

316

4.6

13C NMR (100 MHz, CDCl3) spectrum of compound 1a26

1H NMR (CDCl3, 400 MHz) spectrum of compound 2a27

13C NMR (100 MHz, CDCl3) spectrum of compound 2a28

1H NMR (CDCl3, 400 MHz) spectrum of compound 3a29

0102030405060708090100110120130140150160170180190200f1 (ppm)

13.0

14.7

15.1

17.3

17.4

59.2

59.2

67.6

69.1

69.4

69.6

70.7

70.7

70.8

70.8

70.9

71.0

71.2

72.1

76.8

CD

Cl3

77.2

CD

Cl3

77.5

CD

Cl3

93.6

94.7

100.

0

132.

413

3.0

137.

014

0.0

152.

5

161.

116

2.3

163.

516

4.6

13C NMR (100 MHz, CDCl3) spectrum of compound 3a30

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.012.5f1 (ppm)

5.99

5.70

3.90

50.2

34.

332.

064.

22

0.97

1.00

1.63

3.32

3.48

3.49

3.50

3.50

3.59

3.60

3.62

3.63

3.65

3.66

3.67

3.76

3.77

3.78

3.79

3.80

3.81

3.88

3.89

3.91

4.07

4.08

4.09

4.11

4.12

4.13

5.26

6.01

6.02

6.14

6.14

7.26

CD

Cl3

1H NMR (CDCl3, 400 MHz) spectrum of compound 6b

CH

2Cl 2

31

0102030405060708090100110120130140150160170180190200f1 (ppm)

25.6

53.5

59.0

67.9

69.1

69.3

70.5

70.5

70.5

70.6

70.6

70.6

70.7

70.9

71.1

71.9

77.2

CD

Cl3

94.3

95.3

97.0

104.

9

157.

715

9.2

162.

016

5.4

13C NMR (100 MHz, CDCl3) spectrum of compound 6bC

H2C

l 232

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.012.513.013.514.014.5f1 (ppm)

6.14

4.26

53.2

42.

092.

012.

002.

04

1.00

1.00

0.89

0.98

3.33

3.49

3.50

3.51

3.52

3.59

3.60

3.61

3.63

3.64

3.64

3.65

3.66

3.67

3.67

3.80

3.81

3.82

3.85

3.86

3.87

4.08

4.10

4.11

4.23

4.24

4.25

5.27

6.02

6.03

6.13

6.14

7.26

CD

Cl3

11.2

4

12.4

2

1H NMR (CDCl3, 400 MHz) spectrum of compound 7b

CH

2Cl 2

33

0102030405060708090100110120130140150160170180190200f1 (ppm)

53.5

59.0

67.8

68.4

69.1

69.3

70.5

70.6

70.6

70.6

70.6

70.6

70.7

70.8

70.9

72.0

76.8

CD

Cl3

77.2

CD

Cl3

77.5

CD

Cl3

93.1

95.7

95.9

159.

1

164.

716

6.1

171.

0

13C NMR (100 MHz, CDCl3) spectrum of compound 7b

CH

2Cl 2

34

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)

6.02

3.00

5.59

4.71

50.3

94.

382.

182.

042.

00

0.93

0.92

1.95

1.91

1.98

1.90

2.11

2.14

2.36

3.37

3.53

3.54

3.55

3.56

3.57

3.58

3.62

3.64

3.65

3.66

3.68

3.68

3.70

3.71

3.73

3.82

3.84

3.86

3.87

3.88

3.88

3.90

3.98

3.99

4.01

4.09

4.11

4.12

4.19

4.21

4.23

6.08

6.09

6.16

6.16

6.39

6.39

6.40

6.41

6.67

6.68

6.69

6.69

6.96

7.26

7.69

1H NMR (CDCl3, 400 MHz) spectrum of compound 1b

CD

Cl 3

35

0102030405060708090100110120130140150160170180190200f1 (ppm)

20.2

20.3

21.3

59.2

67.7

69.2

69.4

69.6

70.6

70.7

70.7

70.8

70.8

70.9

71.0

71.3

72.1

76.8

CD

Cl3

77.2

CD

Cl3

77.5

CD

Cl3

94.3

95.6

99.2

118.

8

128.

312

8.4

129.

713

0.6

135.

313

6.3

136.

713

9.0

144.

7

147.

9

160.

716

2.2

162.

316

4.6

13C NMR (100 MHz, CDCl3) spectrum of compound 1b36

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.012.5f1 (ppm)

6.05

3.05

3.03

6.01

3.02

6.00

4.15

48.6

52.

394.

172.

062.

092.

10

1.99

1.00

1.05

2.00

1.37

2.10

2.14

2.16

2.19

2.33

3.36

3.52

3.53

3.54

3.55

3.62

3.63

3.64

3.67

3.67

3.68

3.70

3.71

3.71

3.72

3.82

3.83

3.85

3.85

3.86

3.87

3.97

3.98

3.99

4.08

4.09

4.10

4.17

4.18

4.19

5.29

5.92

6.04

6.04

6.09

6.10

6.95

7.26

CD

Cl3

1H NMR (CDCl3, 400 MHz) spectrum of compound 2b

CH

2Cl 2

C3H

6O

37

0102030405060708090100110120130140150160170180190200f1 (ppm)

13.6

15.5

19.7

19.8

21.3

31.0

59.2

67.6

69.2

69.3

69.6

70.6

70.6

70.7

70.7

70.8

70.8

71.0

71.2

72.1

76.8

CD

Cl3

77.5

CD

Cl3

93.7

94.5

99.7

121.

8

129.

112

9.2

131.

213

1.2

134.

913

5.3

138.

814

2.0

142.

8

155.

8

161.

216

2.2

163.

516

4.7

38

0102030405060708090100110120130140150160170180190200f1 (ppm)

13.6

15.5

19.7

19.8

21.3

31.0

59.2

67.6

69.2

69.3

69.6

70.6

70.6

70.7

70.7

70.8

70.8

71.0

71.2

72.1

76.8

CD

Cl3

77.5

CD

Cl3

93.7

94.5

99.7

121.

8

129.

112

9.2

131.

213

1.2

134.

913

5.3

138.

814

2.0

142.

8

155.

8

161.

216

2.2

163.

516

4.7

39

1H NMR (CDCl3, 500 MHz) spectrum of compound 3b40

41

13C NMR (100 MHz, CDCl3) spectrum of compound 3b42

43

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.012.5f1 (ppm)

2.00

2.02

6.11

6.11

2.06

2.03

1.01

0.02

2.20

2.21

2.23

2.24

2.26

2.27

2.86

2.88

2.89

3.18

3.31

MeO

D3.

583.

603.

613.

623.

643.

693.

703.

713.

933.

933.

944.

83 M

eOD

1H NMR (CD3OD, 500 MHz) spectrum of sulfobetine 844

0102030405060708090100110120130140150160170180190200210f1 (ppm)

20.0

48.4

MeO

D48

.6 M

eOD

48.7

48.8

MeO

D49

.0 M

eOD

49.2

MeO

D49

.4 M

eOD

49.6

MeO

D52

.452

.452

.562

.064

.664

.664

.665

.165

.265

.265

.673

.8

64.364.564.764.965.165.365.5f1 (ppm)

64.6

64.6

64.6

65.1

65.2

65.2

52.052.152.252.352.452.552.652.752.8f1 (ppm)

52.4

52.4

52.5

13C NMR (CD3OD, 100 MHz) spectrum of sulfobetine 845

1H NMR (CD3OD, 400 MHz) spectrum of compound 6c46

47

13C NMR (CD3OD, 100 MHz) spectrum of compound 6c48

49

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.012.5f1 (ppm)

4.08

4.07

12.0

0

8.21

4.19

4.10

1.93

2.02

0.81

0.81

2.18

2.20

2.22

2.23

2.26

2.81

2.82

2.84

2.85

3.17

3.17

3.31

MeO

D3.

583.

593.

603.

623.

633.

873.

883.

893.

933.

943.

954.

004.

044.

214.

234.

244.

354.

374.

374.

83 M

eOD

6.18

6.19

6.25

1H NMR (CD3OD, 400 MHz) spectrum of compound 7c50

0102030405060708090100110120130140150160170180190200f1 (ppm)

20.0

20.1

48.4

MeO

D48

.6 M

eOD

48.7

48.8

MeO

D49

.0 M

eOD

49.2

MeO

D49

.4 M

eOD

49.6

MeO

D52

.552

.652

.664

.564

.764

.965

.065

.765

.868

.770

.170

.370

.4

93.9

96.3

97.4

161.

816

6.1

166.

9

172.

9

13C NMR (CD3OD, 100 MHz) spectrum of compound 7c51

52