Supplementary Online Material
Sulfoxythiocarbamates Modify Cysteine Residues in HSP90 Causing
Degradation of Client Proteins and Inhibition of Cancer Cell Proliferation
Ying Zhang1,¶, Sharadha Dayalan Naidu1,¶, Kusal Samarasinghe2, Garrett C. VanHecke2,
Ashley Pheely1, Tatiana N. Boronina3, Robert N. Cole3, Ivor J. Benjamin4, Philip A. Cole5,
Young-Hoon Ahn2,*, and Albena T. Dinkova-Kostova1,5,*
1
Jacqui Wood Cancer Centre, Division of Cancer Research, Medical Research Institute,
University of Dundee, Dundee, Scotland, United Kingdom
2
Department of Chemistry, Wayne State University, Detroit, MI, USA
3
Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine,
Baltimore, MD 21205, USA
4
University of Utah School of Medicine Health Sciences Center, Salt Lake City, UT, USA
5
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of
Medicine, Baltimore, MD, USA
1
Supplementary Materials and Methods
Chemical Synthesis. The syntheses of STCA, K-1, K-3, K-5, K-7, S-1, S-2, S-3, and S-5
through S-10 have been reported previously (Ahn et al, 2010).
MsO
O
S
O
a, b
S
N
O
S
O
c
S
O
N
O
S
S-4
1
Reagents and conditions: (a) 4-Phenylbenzylamine, EtOH, 70oC. (b) S-phenylethyl
chlorothioformate, DIPEA, CH2Cl2, 0oC. (c) m-CPBA, CH2Cl2, -78oC.
Synthesis of 1. S-Phenethyl chlorothioformate was prepared in one step from 4phenylethanethiol: triphosgene (742 mg, 2.5 mmol) was dissolved in dichloromethane (10
mL) and cooled to 0oC. Phenylethanethiol (6.0 mmol) was added slowly, followed by a
solution of pyridine (0.5 mL, 6.2 mmol) in dichloromethane (4 mL). After stirring for 2 h at
0oC, the reaction was quenched by addition of water (5 mL). The reaction mixture was
diluted with dichloromethane (40 mL) and washed with ice-cold water (20 mL). The organic
layer was dried over sodium sulfate, and the filtrate was concentrated and used immediately
without further purification. 4-(Methylsulfinyl)-butyl methanesulfonate (Ahn et al., 2010)
(270 mg, 1.26 mmol) and 4-phenylbenzylamine (577 mg, 3.15 mmol) were dissolved in
ethanol (5 mL). The reaction mixture was heated at 75°C for 3 h and then concentrated and
dried under vacuum. This crude mixture was dissolved in dichloromethane (15 mL) and
cooled to 0°C. N,N-diisopropylethylamine (1.75 mL, 10.04 mmol) was added slowly,
followed by the addition of S-phenethyl chlorothioformate (1.24 g, 6.0 mmol). After stirring
for 1 h, the reaction mixture was diluted with dichloromethane, washed with 1N aq. HCl, sat.
aq. sodium bicarbonate, and brine. The organic layer was dried over sodium sulfate and
filtered. The filtrate was concentrated and purified by column chromatography, eluting with
methanol and dichloromethane (1:30, vol/vol) to give compound 1 (250 mg, 42.5%). 1HNMR (CDCl3) δ 7.55 (t, J = 5.6, 4H), 7.41 (t, J = 8.4, 2H), 7.33-7.17(m, br, 8H), 4.60 (d, br,
2H), 3.35 (d, br, 2H), 3.20 (t, J = 7.2, 2H), 2.94 (t, J = 8, 2H), 2.62 (s, br, 2H), 2.45 (s, 3H),
1.70 (s, br, 4H);
13
C-NMR (CDCl3) δ 168.59, 140.75, 140.49, 136.34, 135.47, 129.13,
128.98, 128.73, 128.56, 128.01, 127.62, 127.28, 126.70, 54.12, 51.30, 50.11, 47.06, 46.58,
38.78, 36.87, 32.20, 27.34, 26.85, 20.12, 14.52
2
Synthesis of S-4. Compound 1 (203 mg, 0.438 mmol) was dissolved in dichloromethane (5
ml) and cooled to -78°C. A solution of m-CPBA (95 mg, 0.42 mmol) in dichloromethane (5
mL) was then added slowly. After stirring for 15 min, the reaction was concentrated to
dryness. The reactions mixture was dissolved in ethyl acetate, and washed with ice-cold sat.
aq. sodium bicarbonate and ice-cold brine. The organic layer was dried over sodium sulfate,
and the filtrate was concentrated and purified by column chromatography, eluting with ethyl
acetate and hexane (2:1, vol/vol) to yield S-4 (40 mg, 19%). 1H-NMR (CDCl3) δ 7.58 (t, J =
8.4, 4H), 7.45 (q, J = 6.6, 2H), 7.39-7.16(m, 8H), 4.92-4.56 (m, 2H), 3.76-3.02 (m, 6H), 2.702.63 (m, br, 2H), 2.55 (d, J = 1.6, 3H), 1.85-1.74 (br, 4H); 13C-NMR (CDCl3) δ 169.30,
168.86, 141.71, 141.50, 140.54, 140.38, 138.56, 138.49, 134.59, 134.09, 129.15, 129.12,
129.10, 129.09, 129.05, 128.97, 128.92, 128.07, 128.03, 128.01, 127.93, 127.84, 127.30,
127.19, 127.1553.89, 53.79, 53.31, 53.28, 53.14, 51.32, 51.30, 49.53, 49.50, 48.07, 48.03,
45.19, 45.13, 38.88, 38.84, 28.92, 28.23, 28.13, 26.27, 26.21, 20.11, 20.07, 19.98, 19.87. ESIHRMS (M+Na) calculated for C27H31NO3NaS2 504.1643, found m/z 504.1650.
O
S
O
O
O
N
K-1
a
O
O
N
S
S
O
O
b
N
2
O
O
N
S
O
S
O
O
N
K-2
Reagents and conditions: (a) 1,3-propanedithiol, DIPEA, acetone/H2O, RT. (b) m-CPBA,
CH2Cl2, -78oC.
Synthesis of 2. To a solution of K-1 (97 mg, 0.33 mmol) in acetone (3 mL) and H2O (0.3
mL) were added 1,3-propanedithiol (16.6 μL, 0.17 mmol) and N,N-diisopropylethylamine
(64.4 μL, 0.36 mmol). The mixture was stirred for 5 h at RT. The reaction mixture was then
diluted with dichloromethane, and washed with 1N HCl, sat. aq. sodium bicarbonate, and
brine. The organic layer was concentrated and purified by column chromatography eluting
with ethyl acetate and hexane (1:2, vol/vol) giving compound 2 as clear oil (53 mg, 28%).
1
H-NMR (CDCl3) δ 7.32-7.21 (m, 10H), 4.57 (d, br, 4H), 3.27 (d, br, 4H), 3.03 (t, J = 6.8,
4H), 2.41(t, J = 6.4, 4H), 2.10 (s, 6H), 2.05-1.97 (m, 2H), 1.52 (s, br, 8H); 13C-NMR (CDCl3)
δ 208.55, 168.43, 168.32, 137.30, 136.45, 128.92, 128.08, 127.91, 127.73, 127.45, 51.43,
50.21, 47.15, 46.84, 43.22, 37.65, 37.09, 30.88, 30.19, 29.81, 29.68, 29.34, 28.38, 27.51,
27.01, 21.02
3
Synthesis of K-2. Compound K-2 was prepared from compound 2 in a similar procedure to
that of compound S-4. Yield (30%). Column chromatography eluting with ethyl acetate and
hexane (1:6, vol/vol); 1H-NMR (CDCl3) δ 7.41-7.22 (m, br, 10H), 4.81-4.54 (m, br, 4H),
3.46-2.98 (m, br, 8H), 2.43 (t, J = 6.8, 4H), 2.12 (s, 6H), 1.62-1.52(m, br, 10H). ESI-HRMS
(M+Na) calculated for C31H42N2O6NaS2 625.2382, found m/z 625.2376.
O
O
O
O
a, b
S
O
N
O
c
S
Cl
3
O
N
4
O
d
S
O
O
N
K-4
Reagents and conditions: (a) 4-phenylbenzylamine, K2CO3, NaI, DMF. (b) S-phenethyl
chlorothioformate, DIPEA, CH2Cl2, 0oC. (c) 3 N HCl, THF. (d) m-CPBA, CH2Cl2, -78oC.
Synthesis of 4. To a solution of 4-phenylbenzylamine (487 mg, 2.66 mmol) and 2-(4chlorobutyl)-2-methyl-1,3-dioxolane (Ahn et al., 2010) (260 mg, 1.45 mmol) in DMF (10
mL) were added potassium carbonate (367 mg, 2.61 mmol) and sodium iodide (272 mg, 1.81
mmol). The mixture was heated at 90°C for 5 h. After cooling to RT, the reaction mixture
was diluted with ethyl acetate, washed with H2O (40 mL x 3) and brine (50 mL x 2). The
organic layer was dried over sodium sulfate and filtered. The filtrate was concentrated and
dried under vacuum. The crude material was dissolved in dichloromethane (10 mL) and
cooled to 0°C. N,N-diisopropylethylamine (1.39 mL, 8 mmol) was added slowly, followed
by the addition of S-phenethyl chlorothioformate (1.20 g, 6 mmol). After stirring for 1 h, the
mixture was diluted with dichloromethane, and washed with 1N HCl, sat. aq. sodium
bicarbonate and brine. The organic layer was dried over sodium sulfate and filtered. The
filtrate was concentrated and purified using column chromatography, eluting with ethyl
acetate and hexane (1:10, vol/vol), yielding compound 3 (345 mg, 48%). To compound 3
(345 mg, 0.70 mmol) in THF (20 mL) was added 3 N HCl (aq., 2 mL). After stirring for 3 h
at RT, the mixture was diluted with ethyl acetate, and washed with sat. aq. sodium
bicarbonate and brine. The organic layer was dried over sodium sulfate and the filtrate was
concentrated and purified by column chromatography, eluting with ethyl acetate and hexane
(1:10, vol/vol) to give compound 4 (300 mg, 92%); 1H-NMR (CDCl3) δ 7.60 (t, J =7.2, 4H),
7.46 (t, J = 7.2, 2H), 7.39-7.23 (m, 8H), 4.69-4.61 (d, br, 2H), 3.41-3.29 (d, br, 2H), 3.24 (t, J
= 7.6, 2H), 2.99 (t, J = 7.6, 2H), 2.45 (m, br, 2H), 2.13 (s, 3H), 1.58 (m, br, 4H); 13C-NMR
(CDCl3) δ 208.91, 208.58, 168.57, 140.89, 140.59, 136.42, 135.59, 129.09, 129.01, 128.55,
4
127.94, 127.68, 127.32, 126.66, 51.18, 49.95, 47.28, 46.92, 43.26, 36.94, 32.19, 30.24, 27.60,
27.12, 21.06
Synthesis of K-4. Compound K-4 was prepared from compound 4 in a similar procedure to
that of S-4. Yield (19%), eluting with acetone and ethyl acetate (1:1, vol/vol); 1H-NMR
(CDCl3) δ 7.58 (t, J = 8.4, 4H), 7.45 (q, J = 6.6, 2H), 7.39-7.16 (m, 8H), 4.92-4.56 (m, 2H),
3.76-3.02 (m, 6H), 2.70-2.63 (m, br, 2H), 2.55 (d, J = 1.6, 3H), 1.85-1.74 (m, br, 4H); 13CNMR (CDCl3) δ 169.30, 168.86, 141.71, 141.50, 140.54, 140.38, 138.56, 138.49, 134.59,
134.09, 129.15, 129.12, 129.10, 129.09, 129.05, 128.97, 128.92, 128.07, 128.03, 128.00,
127.94, 127.84, 127.31, 127.19, 127.15, 53.89, 53.79, 53.75, 53.31, 53.28, 53.14, 51.32,
51.30, 49.54, 49.50, 48.07, 48.03, 45.19, 45.13, 38.88, 38.84, 28.92, 28.87, 28.23, 28.13,
26.27, 26.21, 20.11, 20.07, 19.98, 19.87. ESI-HRMS (M+Na) calculated for C28H31NO3NaS
484.1922, found m/z 484.1933.
O
HS
NH2
O
O
a
c
b
S
NH
5
S
N
O S
6
N
K-6
Reagents and conditions: (a) triphosgene, DIPEA, CH2Cl2, 0oC. (b) NaH, benzyl bromide,
THF, 0oC. (c) m-CPBA, CH2Cl2, -78oC.
Synthesis of 5. To a solution of triphosgene (133 mg, 0.45 mmol) in dichloromethane was
added cysteamine (100 mg, 1.23 mmol) at 0°C. After stirring for 5 min, N,Ndiisopropylethylamine (0.85 mL, 2.58 mmol) was added dropwise to the mixture. The
reaction mixture was then stirred for 2 h at 0°C and at RT overnight. The reaction mixture
was diluted with ethyl acetate, and washed with 1 N HCl, sat. sodium bicarbonate and brine.
The organic layer was concentrated and purified by column chromatography, eluting with
ethyl acetate and hexane (1:2, vol/vol) to yield compound 5 (20 mg, 20%). 1H-NMR (CDCl3)
δ 6.49 (s, br 1H), 3.63 (m, 2H), 3.41 (t, J = 7.2, 2H);
13
C-NMR (CDCl3) δ 176.84, 45.03,
43.63, 43.57, 42.14, 30.31, 30.24, 28.89, 28.82
Synthesis of 6. To a solution of compound 5 (95 mg, 0.92 mmol) in THF (5 mL) was added
sodium hydride (53 mg, 2.21 mmol) at 0oC, followed by benzyl bromide (0.263 mL, 2.21
mmol). After stirring at RT overnight, the reaction was quenched by adding water. The
reaction mixture was diluted with ethyl acetate, and washed with 1 N HCl, sat. aq. sodium
5
bicarbonate and brine. The organic layer was dried over sodium sulfate, and the filtrate was
concentrated and purified by column chromatography, eluting with ethyl acetate and hexane
(1:10, vol/vol) to yield compound 6 (81 mg, 46%). 1H-NMR (CDCl3) δ 7.37-7.25 (m, 5H),
4.48 (s, 2H), 3.50 (t, J = 7.2, 2H), 3.22 (t, J = 8, 2H)
Synthesis of K-6. Compound K-6 was prepared from compound 6 in a similar procedure to
that of S-4. Yield (49%), column chromatography eluting with ethyl acetate and hexane (2:1,
vol/vol), 1H-NMR (CDCl3) δ 7.35-7.24 (m, 5H), 4.54 (q, J = 14.5, 2H), 3.71 (m, 1H), 3.51 (m,
1H), 3.45 (m, 1H), 3.18 (m, 1H); 13C-NMR (CDCl3) δ 168.65, 134.02, 129.18, 128.62, 128.41,
49.35, 46.82, 42.09. ESI-MS (M+H) calculated for C10H12NO2S 210.05, found m/z 210.06
O
Cl
O
O
O
O
a, b
S
O
N
S
O
O
O
N
O
O
7
O
d
S
N
O
c
8
O
O
K-8
O
Reagents and conditions: (a) 4-(aminomethyl)-benzoate, K2CO3, DMF. (b) S-ethyl
chlorothioformate, DIPEA, CH2Cl2, 0oC. (c) 3 N HCl, THF. (d) m-CPBA, CH2Cl2, -78oC.
Synthesis of 8. To a solution of methyl 4-(aminomethyl)-benzoate (332 mg, 2.01 mmol) and
2-(4-chlorobutyl)-2-methyl-1,3-dioxolane (Ahn et al., 2010) (0.3 g, 1.68 mmol) in DMF (6
mL) was added potassium carbonate (298 mg, 2.01 mmol). The mixture was heated at 80°C
for 6 h. After cooling to RT, the reaction mixture was diluted with ethyl acetate, and washed
with H2O (50 mL x 2) and brine (50 mL x 3). The organic layer was dried over sodium
sulfate, and the filtrate was concentrated and dried under vacuum. The crude product was
dissolved in dichloromethane (10 mL) and cooled to 0°C. N,N-Diisopropylethylamine (1.05
mL, 6.04 mmol) was added, followed by addition of S-ethyl chlorothioformate (0.42 mL,
4.03 mmol). After stirring for 1 h, the mixture was diluted with dichloromethane and washed
with aq. 1 N HCl, sat. aq. sodium bicarbonate, and brine sequentially. The organic layer was
dried over sodium sulfate, and the filtrate was concentrated and purified by column
chromatography, eluting with ethyl acetate and hexane (1:7, vol/vol) to yield compound 7
(220 mg, 30%). To compound 7 (200 mg, 0.51mmol) in THF (20 mL) was added 3 N aq.
HCl (2 mL). After stirring for 3 h at RT, the mixture was diluted with ethyl acetate, and
washed with sat. aq. sodium bicarbonate and brine. The organic layer was dried over sodium
sulfate, and the filtrate was concentrated and purified by column chromatography, eluting
with ethyl acetate and hexane (1:5, vol/vol) to give compound 8 (164 mg, 98%). 1H-NMR
6
(CDCl3) δ 7.92 (d, J = 8, 2H), 7.22 (d, J = 8, 2H), 4.56 (s, br, 2H), 3.82 (s, 3H), 3.23 (d, br,
2H), 2.86 (q, J = 7.2, 2H), 2.36 (t, J = 6.8, 2H), 2.04 (s, 3H), 1.46 (br, 4H), 1.22(t, J = 7.6,
3H); 13C-NMR (CDCl3) δ 208.39, 168.93, 166.89, 142.77, 141.99, 130.14, 129.53, 127.73,
127.24, 52.29, 51.11, 49.96, 47.51, 47.02, 43.07, 30.12,27.50, 27.07, 20.88, 15.50
Synthesis of K-8. Compound K-8 was prepared from compound 8 in a similar procedure to
that of S-4. Yield (68%). Column chromatography eluting with ethyl acetate and hexane (2:1,
vol/vol). 1H-NMR (CDCl3) δ 8.02, 7.99 (two d, J = 8.0, 2H), 7.32 (d, J = 8.0, 2H), 5.08-4.60
(m, 2H), 3.90, 3.89 (two s, 3H), 3.62-3.27 (m, 2H), 3.11-2.92 (m, 2H), 3.11-2.92 (m, 2H),
2.42 (m, 2H), 2.10, 2.09 (two s, 3H), 1.62-1.50 (m, 4H), 1.35 (t, J = 7.6, 3H); 13C-NMR
(CDCl3) δ 208.40, 208.21, 168.87, 168.39, 166.54, 166.46, 140.64, 140.38, 130.90, 130.25,
130.14, 129.80, 128.12, 127.27, 52.19, 50.65, 48.67, 47.87, 45.52, 45.46, 42.59, 42.45, 29.97,
28.06, 26.07, 20.39, 20.20, 6.91, 6.90. ESI-MS (M+H) calculated for C18H26NO5S 368.15,
found m/z 368.15
O
O
NH2
OH
b
a
c, d
O
OH
9
O
O
11
10
Reagents and conditions: (a) 3-chloromethylbut-1-yne, K2CO3, KI, DMF, 80oC. (b) NaBH4,
EtOH, 0oC. (c) MsCl, DIPEA, CH2Cl2. (d) NH3, MeOH.
Synthesis of 10. To a solution of 4-hydroxybenzaldahyde (2.0 g, 16.37 mmol) in DMF (20
mL) were added potassium carbonate (4.52 g, 32.7 mmol), potassium iodide (1.35 g, 8.2
mmol), and 3-chloro-methylbut-1-yne (2.76 mL, 24.6 mmol). The mixture was heated at
80°C for 2 days. Additional potassium carbonate (2.26 g, 16.37 mmol), potassium iodide
(0.68 g, 4.09 mmol), and 3-chloro-methylbut-1-yne (1.38 mL, 12.27 mmol) were added to the
mixture, and the reaction mixture was heated at 80°C overnight. After cooling to RT, the
mixture was diluted with ethyl acetate, and washed with H2O and brine. The organic layer
was dried over sodium sulfate and filtered. The filtrate was concentrated and purified by
column chromatography, eluting with ethyl acetate and hexane (1:50, vol/vol) to yield
compound 9 (630 mg, 20%). Compound 9 (750 mg, 4.0 mmol) was dissolved in ethanol (25
mL). Sodium borohydride (128 mg, 3.25 mmol) was then added at 0oC. After stirring for 30
min, the mixture was diluted with ethyl acetate, and washed with H2O and brine. The organic
layer was dried over sodium sulfate and filtered. The filtrate was concentrated and dried
7
under vacuum, yielding compound 10 (710 mg, 94%); 1H-NMR (CDCl3) δ 7.26 (d, J = 8.8,
2H), 7.19 (d, J = 8.4, 2H), 4.62 (s, br, 2H), 2.56 (s, 1H), 1.64 (s, 6H); 13C-NMR (CDCl3) δ
155.24, 135.54, 128.11, 121.77, 86.19, 74.15, 72.67, 65.23, 29.75
Synthesis of 11. Compound 10 was dissolved in dichloromethane (10 mL) and cooled to
0°C. To the mixture were added N,N-diisopropylethylamine (0.792 mL, 4.5 mmol) and
methanesulfonyl chloride (0.33 mL, 4.3 mmol). After stirring for 1 h, the mixture was
diluted with dichloromethane, and washed with cold 1 N HCl, cold sat. aq. sodium carbonate
and brine. The organic layer was dried over sodium sulfate, and filtered. The filtrate was
concentrated to approximately 20 mL at which point the solution was quickly cooled to 0°C,
and 7 N ammonia in methanol (25 mL) was added. After stirring overnight, the mixture was
concentrated and purified by column chromatography, eluting with methanol and
dichloromethane (1:10, vol/vol) to yield compound 11 (240 mg, 34%). 1H-NMR (CDCl3) δ
7.24 (d, J = 8.8, 2H), 7.17 (d, J = 8.4, 2H), 3.82 (s, br, 2H), 2.56(s, 1H), 1.62 (s, 6H).
O
O
O
O
a, b
Cl
S
O
O
O
c
S
N
O
N
O
O
O
12
O
d
S
O
N
13
K-9
Reagents and conditions: (a) 11, K2CO3, NaI, DMF. (b) S-ethyl chlorothioformate, DIPEA,
CH2Cl2, 0oC. (c) 3 N HCl, THF, (d) m-CPBA, CH2Cl2, -78oC.
Synthesis of 12. Compound 12 was prepared from 2-(4-chlorobutyl)-2-methyl-1,3-dioxolane
and amine compound 11 in a similar procedure to that of compound 7. Yield (26%), column
chromatography eluting with ethyl acetate and hexane (1:10, vol/vol); 1H-NMR (CDCl3) δ
7.13 (s, 4H), 4.49 (br, 2H), 3.89 (m 2H), 3.87 (m, 2H), 3.23 (br, 2H), 2.92 (q, J = 7.2, 2H),
2.55 (s, 1H), 1.61 (s, 6H), 1.55 (m, 4H), 1.32 (m, 2H), 1.28 (t, J = 7.6, 3H), 1.26 (s, 3H); 13CNMR (CDCl3) δ 168.74, 168.60, 155.10, 131.79, 128.76, 128.22, 121.64, 110.07, 86.22,
74.21, 72.56, 64.86, 50.97, 49.50, 47.19, 46.87, 38.95, 29.78, 28.11, 27.72, 25.05, 23.99,
21.55, 15.62
Synthesis of 13. Compound 13 was prepared from compound 12 in a similar procedure to
that of compound 8. Yield (88%), column chromatography eluting with ethyl acetate and
hexane (1:8, vol/vol); 1H-NMR (CDCl3) δ 7.14 (s, br, 4H), 4.49 (br, 2H), 3.24 (br, 2H), 2.92
8
(q, J = 7.2, 2H), 2.55 (s, 1H), 2.39 (t, J = 6.8, 2H), 2.09 (s, 3H), 1.61 (s, 6H), 1.49 (s, br, 4H),
1.28 (t, J = 7.6, 3H);
13
C-NMR (CDCl3) δ 208.53, 168.59, 155.19, 131.66, 130.74, 128.28,
121.65, 86.21, 74.21, 72.5850.92, 49.52, 46.95, 46.38, 43.24, 30.18, 29.77, 27.49, 27.02,
25.13, 21.01, 15.59
Synthesis of K-9. Compound K-9 was prepared from compound 13 in a similar procedure to
that of S-4. Yield (62%), column chromatography eluting with ethyl acetate and hexane (1:1,
vol/vol); 1H-NMR (CDCl3) δ 7-24-7.13 (m, 4H), 4.82-4.87 (m, 2H), 3.57-3.32 (m, 2H), 3.092.86 (m, 2H), 2.56 (s, 1H), 2.44-2.41 (m, 2H), 2.11, 2.10 (two s, 3H), 1.62 (s, 6H), 1.60-1.52
(m, 4H), 1.35, 1.34 (two t, J = 7.6, 3H); 13C-NMR (CDCl3) δ 208.58, 208.40, 168.74, 168.66,
156.25, 156.11, 129.69, 129.19, 128.55, 128.22, 121.59, 121.45, 74.20, 74.12, 50.31, 48.72,
47.44, 45.50, 45.41, 44.86, 42.73, 42.61, 30.01, 29.54, 28.06, 26.15, 20.50, 20.35, 7.02, 6.97.
ESI-MS (M+H) calculated for C21H30NO4S 392.18, found m/z 392.19
Liquid chromatography tandem mass spectrometry (LC-MS/MS). Eluted peptides were
identified by liquid chromatography tandem mass spectrometry (LCMS/MS) using an LTQ
Orbitrap Velos MS (ThermoFisher Scientific, www.thermofisher.com) interfaced with a 2D
nanoLC system (Eksigent, www.eksigent.com) and an Agilent 1100 autosampler
(www.agilent.com). Peptides were fractionated by reverse-phase HPLC on a 75 μm x 100
mm C18 column (YMC ODS-AQ 3μm, 120) with a 10 μm emitter using 7-40-90%
acetonitrile/0.1% formic acid gradient over 30 min at 300 nl/min. Peptide sequences were
identified using Mascot (www.matrixscience.com) or Bioworks (v3.3,ThermoFisher) to
search raw MS data against the IPI Mouse v3.26 fasta database with using the following
criteria: variable modifications of oxidation on Met, carboamidomethylation on Cys and
STCA (+771.41 Da) on Cys; peptide and fragment tolerances 1.5 Da and 0.8 Da,
respectively; and trypsin protease with up to 2 missed cleavages.
Reference
Ahn YH, Hwang Y, Liu H, Wang XJ, Zhang Y, Stephenson KK, Boronina TN, Cole RN,
Dinkova-Kostova AT, Talalay P, Cole PA (2010) Electrophilic tuning of the chemoprotective
natural product sulforaphane. Proc Natl Acad Sci U S A 107(21): 9590-5
9
Supplementary Figure 1. Purified recombinant HSP90β was incubated with vehicle for 2 h,
dialyzed, subject to click reaction with biotin azide, and digested by trypsin. The tryptic
digest was incubated with avidin-coated beads. The captured peptides were eluted by 0.5 M
acetic acid and analyzed by MALDI mass spectroscopy.
10
A
B
C
11
Supplementary Figure 2. MS/MS fragmentation spectra of STCA-modified peptides from
HSP90β. (A) Cys412 modification of Peptide C412LELFSELAEDKENYK (MH+ 2703 Da),
which shows b-ion m/z 559.27 with STCA modification. (B) Cys589 or Cys590 modification
of Peptide LVSSPC589C590IVTSTYGWTANME (MH+ 3146 Da). (C) Cys564 modification
of FENLC564K (MH+ 1524). The identified b and y ion are indicated.
Supplementary Table 1. Modified peptides identified by liquid chromatography tandem
mass spectrometry after incubation of recombinant HSP90β with STCA followed by click
reaction with biotin azide, trypsin digestion, and capture with avidin-coated beads.
Sequence
Protein
#
PSMs
Group
Value
Accessions
AKFENLcK
LVSSPccIVTSTYGWTANmER
12
4
HSP90
HSP90
LVSSPccIVTSTYGWTANMER
4
HSP90
FENLcK
q-
Modifications
PEP
XCorr
Charge
MH+
ΔM
RT
[Da]
[ppm]
[min]
C7(STCA)
C6(Carbamidomethyl);
C7(STCA);
M19(Oxidation)
C6(Carbamidomethyl);
C7(STCA)
0
0
0.009327
0.096694
1.59
2.72
3
3
1723.893
3162.465
1.64
-3.47
30.09
33.50
0
0.009275
3.04
3
3146.480
-0.48
33.16
15
HSP90
C5(STCA)
0
0.050164
1.21
2
1524.759
0.58
32.17
KcLELFSELAEDK
1
HSP90
C2(STCA)
0
0.059827
1.80
3
2296.160
-0.04
32.99
cLELFSELAEDKENYK
4
HSP90
C1(STCA)
0
9.04E-06
4.50
3
2702.311
0.70
33.11
12
Western Blot Densitometry
Figure 1B
13
Figure 1C
14
Figure 1D
15
Figure 1E
16
Figure 1F
17
Figure 1G
18
Figure 5B
19
Figure 7B
20
Figure 7C
21
Figure 7D
22
Figure 7E
23
Figure 7F
24
25