Synthesis and characterization of dimaleimide fluorogens designed for specific labeling of proteins

Article abstract of DOI:10.1021/ja045742x

A series of aromatic compounds were prepared bearing two maleimide groups attached directly to the fluorescent cores. The resulting derivatives do not fluoresce until the maleimide groups undergo their typical thiol addition reaction, thus removing their

Full text of DOI:10.1021/ja045742x

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Synthesis and Characterization
of Dimaleimide Fluorogens Designed for
Specific Labeling of Proteins
Stéphane Girouard‡, Marie-Hélène Houle‡, Alain Grandbois‡,
Jeffrey W. Keillor‡†* and Stephen W. Michnick†
‡Department of Chemistry and †Department of Biochemistry,
Université de Montréal, C.P. 6128, Succ. centre-ville,
Montréal, Québec H3C 3J7 Canada
Supporting Information

Supporting Information
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Experimental Section
Synthesis
Materials
The following material were obtained from Aldrich and were used without further purification:
2-hydroxy-5-nitrobenzaldehyde, N-acetylglycine, maleic anhydride, p-nitrophenylacetic acid, L-
aspartic acid dimethyl ester hydrochloride, citraconic anhydride, dimethyl L-tartrate, dimethyl
malonate. All reactions were carried out under an atmosphere of dry nitrogen employing
1
conventional bench-top techniques except for reactions with thiols. 13C-NMR and H-NMR
spectra were recorded on AMXR400 or AMX300 spectrometers and were referenced to the
13
residual proton or C signal of the solvent. Mass spectra were determined by FAB+ ionization
on an AutoSpecQ spectrometer at the Regional Mass Spectrometry Centre at the Université de
Montréal. Infrared spectra were recorded on a Perkin-Elmer (FTIR) spectrometer. Melting
points (uncorrected) were determined on a Unimelt Tomas-Hoover or on a Gallenkamp melting
point apparatus.
Methods
3-Acetamido-6-nitrochromen-2-one (1)
A dry 100-mL round bottomed flask was charged with 7.11 g (42.5 mmol) of 2-hydroxy-5-
nitrobenzaldehyde. To this flask were then added N-acetylglycine (4.98 g, 42.5 mmol) and
acetic anhydride (40.1 mL, 0.43 mol). In small aliquots, 1.71 g (42.5 mmol)of sodium hydride
(60% dispersion in mineral oil) was added to the flask. Reactants were observed to dissolve and
after 2-5 minutes, precipitation occurred. The reaction mixture was stirred for 20 h and 7.11 mL
of water were added. Following the addition of 43 mL acetic acid, the mixture was cooled to

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4°C for 4 h. The resulting precipitate was filtered and washed liberally with cold glacial acetic
acid. The acetic acid was then removed as an azeotrope upon addition of 250 mL toluene and
rotary evaporation to dryness, three times. The final residue was dried under vacuum to give
product 1 as a beige powder (6.40 g, 25.8 mmol) in a yield of 61%. mp 277-279 °C (lit1 278 °C)
-1
FTIR (KBr) (cm ) 3350, 3050, 1710, 1680, 1600, 1500, 1420, and 1335. 1H-NMR (DMSO-d6) :
d (ppm) 9.93 (s, 1H), 8.76 (s, 1H), 8.73 (d, J = 2.5 Hz, 1H), 8.28 (dd, J = 2.8 Hz, J = 9.1 Hz,
1H), 7.60 (d, J = 9.1 Hz, 1H), 2.19 (s, 3H). 13C-NMR (DMSO-d6 ): d (ppm) 171.47, 157.69,
145.07, 127.11, 124.96, 124.52, 122.79, 121.52, 118.26, 25.00. HRMS expected: 249.0524;
found: 249.0534. Elemental analysis for C H8N2O5, expected: C, 53.23; H, 3.25; :N, 11.27;
11
found: C, 53.19; H, 3.34; N, 11.06.
3-Acetamido-6-aminochromen-2-one (2)
Palladium 10 wt % on activated carbon (0.322 g, 1/10th the mass of 1) was placed in a 1-L round-
bottom flask and the flask was purged with nitrogen. A solution of sodium borohydride (1.21g,
32.9 mmol) in 22.6 mL water was added dropwise. A suspension of compound 1 (3.20 g, 12.9
mmol) in methanol (775 mL) was then added over a two hour period and allowed to stir at room
temperature an additional 30 min, when the reaction mixture was filtered through Celite and the
solvent was removed by rotary evaporation. Water (750 mL) was added to the residue and the
solution was cooled to 4°C for 20 h. The resulting precipitate was filtered and rinsed with cold
distilled water. The solid was recrystallized from ethanol to give 2 as a yellow solid (1.59 g, 7.3
-1
mmol) in 57 % yield. mp 250 – 252 °C (lit2 252 – 253 °C). FTIR (KBr) (cm ) : 3410, 3310,
1
1700, 1650, 1630. H-NMR (DMSO-d6) : d (ppm) 9.65 (s, 1H), 8.40 (s, 1H), 7.10 (dd, J = 9.1
Hz, J = 2.7 Hz, 1H), 6.73 (d, J = 9.1 Hz, 1H), 6.68 (d, J = 2.7 Hz, 1H), 5.19 (bs, 2H), 2.14 (s,

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3H). 13C-NMR (DMSO-d6 ) : d (ppm) 171.12, 158.78, 146.92, 142.52, 125.26, 124.98, 120.82,
117.54, 117.15, 110.66, 24.93. HRMS expected, 219.0777; found, 219.0770. Elemental
analysis for C11H10N2O3 : expected : C, 60.55; H, 4.62; N, 12.84; found : C, 60.15; H, 4.50; N,
12.62.
3,6-Diaminochromen-2-one (3)
To a solution composed of 12.63 mL of 12 M hydrochloric acid in 6.32 mL ethanol was added
0.919 g (4.2 mmol) of compound 2. The solution was heated to reflux for 30 min and then
cooled to 4 °C overnight. The resulting precipitate was removed by filtration and dissolved in a
minimal volume of distilled water. The solution was neutralized by addition of 25% ammonium
hydroxide and cooled at 4 °C for three hours. The resulting precipitate was removed by filtration
and recrystallized from water to give a yellow compound (3) (0.484 g, 2.7 mmol) in 65% yield.
-1
mp 183–186 °C (lit1 183 - 184°C). FTIR (KBr) (cm ) : 3420, 3340, 1690, 1635. 1H-NMR
(DMSO-d6) : d (ppm) 6.95 (d, J = 8.5 Hz, 1H), 6.56 (s, 1H), 6.49 (d, J = 2.4 Hz, 1H), 6.46 (dd, J
= 10.0 Hz, J = 2.6 Hz, 1H), 5.48 (bs, 2H), 5.00 (bs, 2H). 13C-NMR (DMSO-d6) : d (ppm)
159.95, 146.50, 141.19, 134.13, 123.04, 116.75, 113.70, 109.30, 108.42. HRMS expected :
177.0592; found : 177.0598.
3,6-Dimaleimidylchromen-2-one (4)
Diamine 3 (200 mg, 1.14 mmol) and maleic anhydride (335 mg, 3.42 mmol) were placed in a dry
50-mL round bottom flask. Chloroform (11.5 mL) was added and the solution was heated to
reflux for 20 h. The mixture was then filtered and the recovered solid was rinsed liberally with
chloroform and then dried under vacuum. To this solid was added acetic anhydride (9 mL, 96

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mmol) and sodium acetate (35 mg, 0.427 mmol) and the reaction was stirred vigorously for
another 30 min. The mixture was then cooled to 4 °C for 4 h and filtered. The beige solid thus
obtained was dried over vacuum to give compound 4 in 20% yield (76 mg, 0.226 mmol). mp :
-1
1
>296 °C (dec). FTIR (KBr) (cm ) : 1700, 1620. H-NMR (CDCl ) : d (ppm) 7.78 (s,1H), 7.66
3
(dd, J = 9.8 Hz, J = 2.5 Hz, 1H), 7.63 (d, J = 2.5 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 6.93 (s, 2H),
6.91 (s, 2H). 13C-NMR (DMSO-d6) : d (ppm) 1699.99, 169.06, 156.80, 151.50, 143.62, 135.82,
134.89, 131.61, 128.63, 127.11, 118.60, 118.13, 117.58. HRMS : expected, 337.0463; found,
337.0461.
3,6-Di-(3'-methoxycarbonylmaleimidyl)chromen-2-one (5)
To a suspension of diamine 3 (268 mg, 1.52 mmol) in dry chloroform (15.4 mL) was added á-
carbomethoxymaleic anhydride (712 mg, 4.56 mmol). The solution was heated to reflux for 20
h, then cooled to room temperature and filtered. The solid thus obtained was rinsed liberally
with chloroform and dried under reduced pressure. It was then added to 12 mL acetic anhydride
and 47 mg of sodium acetate (0.56 mmol). The mixture was then heated to 100 °C for 90 min
and then allowed to cool to room temperature for 24 h. The resulting precipitate was recovered
by filtration and rinsed with water, giving the desired product 5 in 22 % yield (151 mg, 0.33
-1
1
mmol). mp >230 °C (dec). FTIR (KBr) (cm ) : 1720, 1670. H-NMR (DMSO-d6) : d (ppm)
8.24 (s, 1H, ), 8.04 (s, 2H), 7.66 (dd, J = 8.97 Hz, J = 2.43 Hz, 1H), 7.42 (d, J = 8.96 Hz, 1H),
7.40 (d, J = 2.42 Hz, 1H), 4.04 (s, 3H), 4.00 (s, 3H). 13C-NMR (DMSO-d6) : d (ppm) 171.02,
163.38, 132.09, 159.92, 145.83, 142.80, 142.31, 135.32, 128.06, 124.83, 120.92, 119.92, 119.27,
117.16, 48.83. HRMS expected for C21H12N2O10 : 452.0492; found: 452.0491.

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3,6-Di-(3'-methylmaleimidyl)chromen-2-one (6)
To a suspension of diamine 3 (268 mg, 1.52 mmol) in 15.4 mL of dry chloroform was added
citraconic anhydride (410 mL, 4.56 mmol). The mixture was heated to reflux for 20 h. The
mixture was cooled to room temperature and filtered, and the recovered solid was rinsed well
with chloroform. After drying, it was added to a solution of sodium acetate (47 mg, 0.56 mmol)
in acetic anhydride (12 mL). The mixture was heated to 100 °C for 90 min and then cooled to
room temperature and allowed to stand for 24 h. The resulting precipitate was then filtered and
rinsed with water to give the desired product 6 in 20% yield (147 mg, 0.3 mmol). mp >230 °C
-1
1
(dec). FTIR (KBr) (cm ) : 1720, 1654. H-NMR (CDCl ) : d (ppm) 8.52 (s, 1H), 7.88 (d, J =
3
2.29 Hz, 1H), 7.63 (s, 1H), 7.52 (dd, J = 9.26 Hz, J = 2.29 Hz, 1H), 7.45 (s, 1H), 7.34 (d, J =
8.83 Hz, 1H), 2.07 (d, J = 6.92 Hz, 3H), 1.91 (d, J = 7.02 Hz, 3H). 13C-NMR (DMSO-d6) : d
(ppm) 170.00, 169.02, 158.90, 146.22, 137.32, 126.04, 122.06, 120.43, 118.24, 117.16, 24.80.
HRMS expected for C19H12N2O6 : 364.0695; found: 364.0693.
3,6-Di(3'-ethylthiosuccinimidyl)chromen-2-one (7)
To a suspension of 4 (40 mg, 0.12 mmol ) in 3 mL dry chloroform was added 27 mL (0.36 mmol)
of ethanethiol. The solution was stirred at room temperature for 12 h, then concentrated under
reduced pressure and purified by flash chromatography (30:70 EtOAc:hexane). Compound 7
was thus obtained as a yellow solid in 85% yield ( 47 mg, 0.10 mmol). mp 128-131 °C. FTIR (
-1
1
KBr ) ( cm ): 3000, 1700, 1620, 600. H-NMR (CDCl ) : d ( ppm ) 7.46 (m, 2H), 7.42 (d, J =
3
11.0 Hz, 1H) 7.39 (s, 1H), 3.90 (d, J = 5.6 Hz, 1H), 3.88 (d, J = 5.6 Hz, 1H), 3.35 (q, J = 9.7 Hz,
2H), 2.94 (m, 2H), 2.80 (m, 2H), 2.69 (d, J = 5.6 Hz, 1H), 2.64 (d, J = 5.6 Hz, 1H), 1.29 (t, J =
8.0 Hz, 6H). 13C-NMR (CDCl ) : d ( ppm ) 175.20, 173.42, 167.90, 155.98, 151.07, 136.55,
3

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130.14, 128.85, 128.25, 125.39, 118.72, 117.35, 38.72, 35.96, 26.04, 20.40, 14.03. HRMS
calculated: 461.0851; found: 461.0849. Elemental analysis for C21H20N2O6S2 expected: C,
54.77; H, 4.38; N, 6.08; S, 13.92; found: C, 54.49; H, 4.35; N, 5.99; S, 13.50.
3,6-Di-(3'-ethylthio-4'-methoxycarbonylsuccinimidyl)chromen-2-one (8)
The reaction of 54 mg (0.12 mmol) of compound 5 in 3 mL dry DMSO with 27 mL ethanethiol
(0.36 mmol) was carried out according to the same protocol used to obtain compound 7, giving
-1
1
compound 8 in 83 % yield (58 mg, 0.1 mmol). FTIR (KBr) (cm ) : 3000, 1727, 1622, 600. H-
NMR (DMSO-d6) : d (ppm) 8.24 (s, 1H), 7.53 (d, J = 2.63, 1H), 7.36 (dd, J = 9.24 Hz, J = 2.67
Hz, 1H), 7.24 (d, J = 9.24 Hz, 1H), 4.38 (d, 1H), 4.32 (s, 1H), 4.00 (s, 3H), 3.98 (d, J = 5.6 Hz,
3H), 3.92 (d, J = 5.4 Hz, 1H), 3.87 (d, J = 5.6 Hz, 1H), 2.80 (m, 4H), 1.30 (t, J = 7.24 Hz, 6H).
13C-NMR (DMSO-d6) : d (ppm) 175.06, 172.67, 168.06, 158.44, 150.63, 137.28, 128.83, 128.32,
124.22, 119.69, 118.47, 117.21, 50.01, 46.23, 42.16, 39.12, 14.24, 12.02. HRMS expected for
C25H24N2O10S2 : 576.0872; found: 576.0874.
3,6-Di-(3'-ethylthio-4'-methylsuccinimidyl)chromen-2-one (9)
The reaction of 44 mg (0.12 mmol) of compound 6 in 3 mL dry DMSO with 27mL ethanethiol
(0.36 mmol) was carried out according to the same protocol used to obtain compound 7, giving
-1
1
compound 9 in 84% yield (49mg, 0.1 mmol). FTIR (KBr) (cm ) : , 1720, 1620, 600. H-NMR
(CDCl ) : d (ppm) 8.21 (s, 1H), 7.57 (d, J = 2.53 Hz, 1H), 7.39 (dd, J = 8.47 Hz, J = 2.67 Hz,
3
1H), 7.26 (d, J = 8.35 Hz, 1H), 3.84 (s, 1H), 3.12 (s, 1H), 3.07 (s, 1H, 2.83 (m, 4H), 1.93 (d, J =
7.08 Hz, 3H), 1.90 (d, J = 7.25 Hz, 3H), 1.30 (t, J = 7.28 Hz, 6H). 13C-NMR (DMSO-d6) : d
(ppm) 175.04, 172.32, 167.92, 156.34, 150.73, 137.21, 129.87, 128.65, 124.34, 118.03, 117.04,

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38.53, 34.29, 26.02, 20.68, 14.27, 11.67. HRMS expected for C23H24N2O6S2 : 488.1076; found:
488.1072.
3,6-(4,4'-bis(methylmercapto-3-succinimidyl)benzophenone)chromen-2-one (10)
Compound 4 (42 mg, 0.13 mmol) and BMMB (34 mg, 0.13 mmol) were placed in a 10-mL
round bottom flask. Chloroform (2.5 mL) was then added with stirring. To this suspension
triethylamine ( 52 mL, 0.39 mmol) was then added and the reaction mixture was heated to reflux
for 12 h. The mixture was cooled to room temperature. A 50-mL aliquot of water was then
added and the solution was extracted 4 times with 20 mL dichloromethane. The organic layers
were combined, dried over MgSO4 and removed under reduced pressure. The resulting product
was then purified by flash chromatography (20:80 EtOAc:hexane). Compound 10 was thus
obtained as a beige solid in 78% yield (61 mg, 0.10 mmol). mp: >220 °C (dec). FTIR (KBr)
-1
1
(cm ) : 1720, 1654, 1605, 602. H-NMR (CDCl ) : d (ppm) 7.81 (d, J = 8.0 Hz, 4H), 7.73 (d, J
3
= 7.9 Hz, 4H), 7.60 (d, J = 9.4 Hz, 1H), 7.52 (s, 1H), 7.45 (d, J = 9.6 Hz, 1H), 7.39 (s, 1H), 4.40
(d, J = 5.6 Hz, 1H), 3.96 (d, J = 5.5 Hz, 1H), 3.68 (s, 4H), 3.26 (q, J = 8.8 Hz, 2H), 2.65 (d, J =
5.6 Hz, 2H). 13C-NMR (CDCl ) : d (ppm) 195.92, 176.77, 175.24, 159.32, 148.90, 147.76,
3
143.07, 136.90, 136.51, 130.96, 130.25, 129.88, 129.38, 127.62, 127.08, 121.77, 117.69, 105.43,
36.70, 35.34, 28.47. HRMS expected for C32H22N2O7S2 : 611.0925; found : 611.0927.
6-Nitro-3-p-nitrophenylchromen-2-one (11)
2-Hydroxy-5-nitrobenzaldehyde (1.00 g, 6.00 mmol) and p-nitrophenylacetic acid (1.08 g, 6.00
mmol) were placed in a dry 25-mL round bottom flask. Acetic anhydride (11.2 mL, 120 mmol)
was then added with stirring. To this suspension sodium hydride (240 mg, 6.00 mmol) was then

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added in small aliquots (60% oil suspension) and the reaction mixture was stirred for 3 h at room
temperature. The resulting beige powder was collected by filtration and washed with diethyl
ether, then dried under vacuum to give compound 11 in 95 % yield (1.77 g, 5.67 mmol). mp :
-1
250 °C (dec). FTIR (KBr) (cm ) : 1620, 1480, 1750, 1540, 1350. 1H-NMR (DMSO-d6) : d
(ppm) 8.80 (d, J = 2.6 Hz, 1H), 8.62 (s, 1H), 8.47 (dd, J = 9.1 Hz, J = 2.7 Hz, 1H), 8.37 (dd, J =
8.9 Hz, J = 2.0 Hz, 1H), 8.02 (dd, J = 8.9 Hz, J = 2.0 Hz, 2H), 7.71 (d, J = 9.1 Hz, 1H). 13C-
NMR (DMSO-d6 ) : d (ppm) 159.43, 157.87, 148.46, 144.68, 142.30, 141.50, 130.85, 127.92,
125.77, 124.51, 120.53, 118.64. HRMS : expected, 312.0382; found, 312.0391. Elemental
analysis for C H8N2O6 : expected : C, 57.70; H, 2.58; N, 8.97; found : C, 57.37; H, 2.62; N,
15
8.78.
6-Amino-3-p-aminophenylchromen-2-one (12)
Following the same protocol used to obtain diamine 2, palladium on carbon (10% water) (0.322
g, 1/10th of the mass of 11) and a solution of sodium borohydride (1.21 g, 32.9 mmol) in water
(22.6 mL) were used to reduce a suspension of compound 11 (4.03 g, 12.9 mmol) in methanol
(775 mL). In this way, compound 12 (2.04 g, 8.1 mmol) was obtained as a yellow solid in 63 %
-1
1
yield. FTIR (KBr) (cm ) : 3180, 3120, 1700, 1610, 850. H-NMR (DMSO-d6) : d (ppm) 7.84
(s, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.08 (d, J = 8.6 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.77 (s, 1H),
6.60 (d, J = 7.8 Hz, 2H), 5.32 (ds, 3H), 5.15 (ds, 3H). 13C-NMR (DMSO-d6) : d (ppm) 163.34,
150.20, 146.43, 145.43, 137.99, 130.38, 127.88, 123.18, 11.33, 118.85, 116.94, 114.39, 111.04.
HRMS : expected , 252.0899; found, 252.0898. Elemental analysis for C15H12N2O2 : expected :
C, 71.42; H, 4.79; N, 11.10; found : C, 71.30; H, 4.58; N, 10.92.

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6-Maleimidyl-3-p-maleimidylphenylchromen-2-one (13)
The protocol used to obtain compound 4 was followed, using diamine 12 (400 mg, 1.59 mmol)
and maleic anhydride (777 mg, 4.77 mmol) in 15 mL chloroform and then 15 mL acetic
anhydride (0.16 mol) and 130 mg sodium acetate (1.59 mmol). In this way, 13 was obtained as a
beige solid and dried over vacuum to give 13 in 20 % yield (131 mg, 0.32 mmol). mp : >250 °C
-1
1
(dec). FTIR (KBr) (cm ) : 2923, 1721, 1615, 1520, 1347, 853. H-NMR (DMSO-d6) : d (ppm)
8.36 (s, 1H), 7.87 (dd, J = 8.9 Hz, J = 2.0 Hz, 2H), 7.85 (d, J = 8.6 Hz, 1H), 7.60 (dd, J = 8.6 Hz,
J = 2.6 Hz, 1H), 7.59 (d, J = 2.6 Hz, 1H), 7.45 (dd, J = 8.9 Hz, J = 2.0 Hz, 2H), 7.24 (d, 4H).
13C-NMR (DMSO-d6) : d (ppm) 169.94, 159.60, 152.04, 141.00, 134.90, 133.82, 131.96, 130.50,
130.11, 129.25, 127.88, 126.95, 126.50, 119.77, 117.40, 104.30. HRMS : expected, 412.0695;
found, 412.0695. Elemental analysis for C H12N2O6 : expected: C, 66.99; H, 2.93; N, 6.79;
23
found: C, 66.13; H, 2.84; N, 6.64.
6-(3'-Ethylthiosuccinimidyl)-3-p-(3'-ethylthiosuccinimidyl)phenylchromen-2-one (14)
To a solution of compound 13 (20 mg, 0.049 mmol) in 2.5 mL of dry DMSO was added 110 mL
of ethanethiol (0.147 mmol ) and the solution was allowed to stir overnight at room temperature.
A 100-mL aliquot of water was then added and the solution was extracted 4 times with 20 mL
dichloromethane. The organic layers were combined, dried over MgSO4 and removed under
reduced pressure, apart from traces of residual DMSO, giving compound 14 in 89 % yield (22
-1
1
mg, 0.044 mmol). FTIR (KBr) ( cm ) : 2950, 1713, 1602, 835, 756. H-NMR (DMSO–d6) : d
(ppm 8.39 (s, 1H), 7.86 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 2.5 Hz, 1H), 7.60 (d, J = 9.1 Hz, 1H),
7.55 (dd, J = 8.9 Hz, J = 2.5 Hz, 1H), 7.38 (d, J = 8.6 Hz, 2H), 4.17 (m, 2H), 3.42 (dd, J = 8.2
Hz, J = 2.0 Hz, 1H), 3.38 (dd, J = 8.2 Hz, J = 2.0 Hz, 1H), 2.84 (m, 4H), 2.76 (d, J = 5.6 Hz,

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1H), 2.71 (d, J = 5.5 Hz, 1H), 1.25 (t, J = 7.3 Hz, 6H). 13C-NMR (DMSO–d6) : d ( ppm )
176.96, 175.33, 160.45, 153.44, 141.38, 135.52, 133.56, 131.38, 130.26, 129.42, 127.89, 127.79,
120.76, 117.79, 37.26, 25.82, 20.49, 19.31, 15.31. HRMS expected for C27H24O6S2 : 537.1168;
found: 537.1164.
1,5-Dimaleimidylnaphthalene (15)
The protocol used to obtain compound 4 was followed, using a suspension of 300 mg of 1,5-
diaminonaphtalene (1.90 mmol) in 20 mL of dry chloroform, 560 mg (5.70 mmol) maleic
anhydride, and then 15 mL acetic anhydride and 59 mg (0.70 mmol) of sodium acetate.
Compound 15 was thus obtained as a yellow solid in 71% yield (428 mg, 1.35 mmol). mp >230
-1
1
°C (dec). FTIR (KBr) (cm ) : 3070, 1705. H-NMR (DMSO-d6) : d (ppm) 7.87 (d, J = 7.80 Hz,
1H), 7.70 (t, J = 8.49 Hz, 1H), 7.62 (d, J = 7.38 Hz, 1H), 7.32 (s, 4H). 13C-NMR (DMSO-d6) : d
(ppm) 170.03, 134.61, 130.70, 128.24, 127.62, 126.31, 123.83. HRMS expected: 318.0641;
found : 318.0641. Elemental analysis for C H10N2O4 expected: C, 67.93; H, 3.17; N, 8.80;
18
found: C, 67.05; H, 3.13; N, 8.62.
1,5-Di(3'-ethylthiosuccinimidyl)naphthalene (16)
The same protocol used to obtain 14 was followed, using 38 mg of compound 15 (0.12 mmol) in
3 mL anhydrous DMSO (3 mL) with 27 mL of ethanethiol (0.36 mmol) to give compound 16 in
-1
1
87 % yield (46 mg, 0.10 mmol). FTIR (KBr) (cm ) : 3000, 1620, 600. H-NMR (DMSO-d6) : d
(ppm) 8.00 (d, J = 8.16 Hz, 1H), 7.78 (t, J = 7.82 Hz, 1H), 7.75 (m, 2H), 7.57 (d, J = 8.04 Hz,
1H), 7.51 (d, J = 8.02 Hz, 2H), 4.42 (d, J = 5.20 Hz, 1H), 4.22 (d, J = 5.00 Hz, 1H), 3.64 (m,
1H), 3.42 (m, 1H), 3.34 (m, 6H), 1.89 (t, 6H). 13C-NMR (DMSO-d6) : d (ppm) 175.95, 174.40,

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162.92, 129.81, 129.56, 128.99, 128.84, 128.35, 127.06, 126.63, 126.31, 125.82, 124.01, 122.48,
36.43, 35.91, 24.70, 24.40, 13.86. HRMS expected for C22H22N2O4S2 : 442.1021; found:
442.1023.
3,6-Dinitro-1,8-naphthalic anhydride (17)3
Sulfuric acid (40.0 mL, 0.75 mol) was placed in a 100-mL flask to which was added 1,8-
naphtalic anhydride (10.0g, 50.5 mmol). The solution was cooled to 5 °C in an ice bath. Nitric
acid (10mL, 0.22 mol) was added drop by drop, taking care to not allow the temperature to
exceed 20 °C. The solution was then heated to 60 °C for 90 min and then cooled again to 4 °C
for 24 h to induce precipitation. The resulting precipitate was removed by filtration and washed
with cold glacial acetic acid. The solid was then washed with toluene (3 × 50 mL) that was
subsequently removed by rotary evaporation, repeating this process two more times to remove
trace acetic acid, giving 17 as a beige powder (10.31g, 37.8 mmol) in 72 % yield. mp 208-210
-1
1
°C (lit3 208 °C. FTIR (KBr) (cm ) : 3098, 2897, 1707, 1617, 1552, 1336, 1260. H-NMR
(acetone-d6) : d (ppm) 9.80 (d, J = 2.23 Hz, 2H), 9.05 (d, J = 2.13 Hz, 2H). 13C-NMR (DMSO-
d6) : d (ppm) 159.75, 148.14, 134.62, 133.28, 131.62, 128.06, 123.07. HRMS expected :
288.0019; found : 288.0014.
N-(3,6-Dinitro-1,8-naphthalyl)-L-aspartic acid dimethyl ester (18)
Compound 17 (300 mg, 1.06 mmol) and L-aspartic acid dimethyl ester hydrochloride (217 mg,
1.06 mmol) were added to a 25-mL flask containing 10 mL of dry acetonitrile. Triethylamine
(180 mL, 1.27 mmol) was added drop by drop to give a violet solution that was heated to 90 °C
for 60 h. After cooling the solution to room temperature, the solvent was removed through

Supporting Information
S13
rotary evaporation and the solid obtained was purified by flash chromatography (70:30
hexane :EtOAc). The solvent was evaporated to give compound 18 as a beige powder (229 mg,
-1
0.53 mmol) in 50 % yield. mp : 128-129 °C. FTIR (KBr) (cm ) : 3083, 2954, 1740, 1678, 1536,
1
1319, 748. H-NMR (CDCl ) : d (ppm) 9.44 (d, J = 2.08 Hz, 2H), 9.39 (d, J = 2.07 Hz, 2H),
3
6.17 (dd, J = 7.88 Hz, J = 6.29 Hz, 1H), 3.72 (s, 3H), 3.63 (s, 3H), 3.44 (dd, J = 16.84 Hz, J =
6.26 Hz, 1H), 3.01 (dd, J = 16.84, J = 7.94 Hz, 1H). 13C-NMR (CDCl ) : d (ppm) 170.53,
3
168.37, 160.99, 147.46, 131.97, 130.90, 130.80, 127.58, 124.54, 52.98, 52.00, 50.12, 33.35.
HRMS expected for C18H14N3O10: 432.0679; found : 432.0688.. a D= -33° (c = 1, CHCl ).
3
N-(3,6-Diamino-1,8-naphthalyl)-L-aspartic acid dimethyl ester (19)
Compound 18 (220 mg, 0.51 mmol) was placed in a 50-mL flask and 20 mL of THF and
palladium on carbon (10% wet) (22 mg, 1/10th the mass of 18) were added. The reaction mixture
was then placed in a hydrogenation reactor and left under 150 psi pressure for 24 h. The mixture
was then filtered through Celite and the filtrate was evaporated under reduced pressure. The
residue was purified by flash chromatography (70:30 EtOAc:CHCl ). The solvent was then
3
removed by rotary evaporation to give compound 19 (92.5 mg, 0.25 mmol) as an orange powder
-1
1
in 49 % yield. mp : 175-177 °C. FTIR (KBr) (cm ) : 3380, 1748, 1625, 1316, 797. H-NMR
(DMSO-d6) : d (ppm) 7.59 (d, J = 1.90 Hz, 2H), 6.97 (d, J = 1.90 Hz, 2H), 6.02 (dd, J = 8.00 Hz,
J = 5.43 Hz, 1H), 5.74 (s, 4H), 3.61 (s, 6H), 3.28 (dd, J = 16.49 Hz, J = 8.59 Hz, 1H), 2.85 (dd, J
= 16.53 Hz, J = 5.00 Hz, 1H). 13C-NMR (DMSO-d6) : d (ppm) 171.81, 170.66 , 164.56, 148.76,
136.60, 122.74, 118.43, 115.51, 111.18, 111.11, 53.50, 50.73, 49.52, 34.76. HRMS expected for
C18H17N3O6 : 371.1117; found : 371.1135. a D= -150° (c = 0.1, DMSO).

Supporting Information
S14
N-(3,6-Dimaleimidyl-1,8-naphthalyl)-L-aspartic acid dimethyl ester (20)
Compound 19 (200 mg, 0.54 mmol) and maleic anyhdride (159 mg, 1.62 mmol) were placed in a
dry 50-mL round-bottom flask. Chloroform (15 mL) was added and the solution was heated to
reflux for 24 h. The solution was then cooled to room temperature and filtered. The brown
precipitate thus recovered was dried under reduced pressure. The solid was then placed in a 25-
mL round-bottom flask to which sodium acetate (21 mg, 0.37 eq) and acetic anhydride (10 mL)
were added. The solution was heated to 100 °C for 90 min. The solvent was then removed
through evaporation and the resulting solid was dissolved in ethyl acetate. The solution was
cooled to 4°C for 48 h and the recovered precipitate was washed with 25 mL of 80:20 water-
DMSO and then 25 mL of water. The solid was then dissolved in a minimal volume of hot ethyl
acetate and hexane was added to induce precipitation of the expected product (20) (53 mg, 0.10
-1
mmol) as an orange powder in 19 % yield. mp >230 °C (dec). FTIR (KBr) (cm ) : 3102, 1717,
1
1669, 1413. H-NMR (DMSO-d6) : d (ppm) 8.60 (d, J = 1.83 Hz, 2H), 8.58 (d, J = 1.86 Hz, 2H),
7.32 (s, 4H), 6.12 (dd, J = 7.96 Hz, J = 5.38 Hz, 1H), 3.64 (s, 3H), 3.63 (s, 3H), 3.32 (dd, J =
16.52 Hz, J = 8.60 Hz, 1H), 2.97 (dd, J = 16.53 Hz, J = 5.03 Hz, 1H). 13C-NMR (DMSO-d6) : d
(ppm) 171.77, 170.63, 170.33, 163.45, 136.08, 132.70, 132.35, 132.16, 130.80, 125.85, 123.47,
53.72, 52.83, 50.16, 34.46. HRMS expected: 532.0992; found: 532.0980. a D = -85° (c = 0.1,
DMSO).
N-(3,6-Di(3'-ethylthiosuccinimidyl)-1,8-naphthalyl)-L-aspartic acid dimethyl ester (21)
The reaction of 26 mg (0.49 mmol) of compound 20 in 2.5 mL dry DMSO with 110 mL
ethanethiol (0.147 mmol) was carried out according to the same protocol used to obtain
-1
compound 7, giving compound 21 in 89% yield (29 mg, 0.044 mmol). FTIR ( KBr ) ( cm ) :

Supporting Information
S15
1
3104, 1715, 1671, 1528, 756. H-NMR (DMSO–d6) : d ( ppm ) 8.59 (d, J = 1.82 Hz, 2H), 8.52
(d, J = 1.78 Hz, 2H), 6.08 (dd, J = 7.86, J = 5.29 Hz, 1H), 4.20 (q, J = 8.66 Hz, 2H), 3.61 (m, 10
H), 3.41 (dd, J = 16.46, J = 8.56 Hz, 1H), 2.88 (m, 5H), 1.25 (t, J = 8.27 Hz, 6H). 13C-NMR
(DMSO–d6) : d (ppm) 175.86, 173.73, 170.26, 169.15, 168.80, 161.87, 134.58, 131.96, 131.20,
130.68, 129.30, 127.72, 127.34, 126.98, 124.74, 124.46, 121.98, 121.73, 52.18, 51.32, 48.64,
32.93, 24.44, 18.98, 17.78, 13.84. HRMS expected for C30H29O10S2 : 655.1294; found:
655.1290.

Supporting Information
S16
Scheme 1
O
O2N
NHAc
O
NHAc
O
H2N
b
O2N
H
OH
a
O
O
1
2
c
R
R
O
O
O
NH2
O
H2N
d
N
O
N
O
O
O
3
(R = H)
(R = CO2Me)
(R = Me)
4
5
6
R
R
O
O
O
e
f
SEt
SEt
N
N
O
O
O
O
(R = H)
(R = CO2Me)
7
8
9 (R = Me)
S
S
O
O
N
O
N
O
O
O
10
(a) N-acetylglycine, NaH, Ac2O, rt, 61%; (b) Pd/C (10% H O), NaBH4, MeOH, 57%; (c) EtOH,
2
HCl, reflux, 65%; (d) (i) maleic anhydride, CHCl , reflux, (ii) Ac2O, AcONa, 100 °C, 20% (2
3
steps); (e) EtSH, CHCl or DMSO, rt, 83-85%; (f) BMMB, NEt3, CHCl , reflux, 78%.
3
3

Supporting Information
S17
Scheme 2
NO2
O
O2N
O2N
H
a
OH
O
O
11
b
NH2
H2N
c
O
O
12
O
O
N
O
N
O
O
O
13
SEt
O
O
EtS
O
d
O
N
N
O
O
14
(a) 4-nitrophenylacetic acid, Ac2O, NaH, rt, 95%; (b) Pd/C (10% H O), NaBH4, MeOH, 63%; (c)
2
(i) maleic anhydride, CHCl , reflux, (ii) Ac2O, AcONa, 100°C, 20% (2 steps) (d) EtSH, DMSO,
3
rt, 89%.

Supporting Information
S18
Scheme 3
SEt
O
N
O
O
N
O
N
O
NH2
N
O
a
b
NH2
O
O
15
16
EtS
(a) (i) maleic anhydride, CHCl , reflux, (ii) Ac2O, AcONa, 100 °C, 75% (2 steps) (b) EtSH,
3
DMSO, rt, 87%.

Supporting Information
S19
Scheme 4
H3C
O
N
O
O
O
CH3
O
O
O
O
O
O
O
O
a
b
O2N
NO2
17
O2N
NO2
18
c
H3C
O
O
O
H3C
O
O
O
O
CH3
O
CH3
O
O
O
O
N
N
d
O
O
H2N
NH2
N
O
N
19
O
20
H3C
O
N
O
O
CH3
O
O
e
O
O
O
N
O
N
SEt
EtS
O
21
(a) H2SO4, HNO3, 10-20 °C, 72% (b) CH3CN, TEA, L-aspartic acid dimethyl ester•HCl, 90 °C,
45% (c) H /Pd/C, 150 psi, THF, 49% (d) (i) maleic anhydride,CHCl , reflux, (ii) Ac2O, AcONa,
2
3
100 °C, 19% (2 steps). (e) EtSH, DMSO, rt, 89%.

Supporting Information
S20
References
1
Trivedi, K.; Sethna, S. J. Org. Chem. 1960, 25, 1817.
2
Kokotos, G.; Tzougrakic, C. J. Heterocyclic Chem. 1986, 23, 87.
3
Braña, M. F.; Castellano, J. M.; Morán, M.; Pérez de Vega, M. J.; Romerdahl, C. R.; Qian,
X.-D.; Bousquet, P.; Emiling, F.; Schlick, E.; Keilhauer, G. Anti-cancer Drug Design 1993,
8, 257.