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Li, 2020 - Metodologia Cloração 2,3-Dihidroxiquinoxalina - SupInf
Li, 2020 - Metodologia Cloração 2,3-Dihidroxiquinoxalina - SupInf
Li, 2020 - Metodologia Cloração 2,3-Dihidroxiquinoxalina - SupInf
anie_201913620_sm_miscellaneous_information.pdf
Table of Contents
1. General methods S2
2. Synthetic procedures and characterization S3
2.1. List of abbreviations used in the synthetic procedures S3
2.2. Synthesis and characterizations S3
3. Optimization of conditions S62
4. Investigations on side reactions S69
5. Competitive effect of glutathione on conjugations S72
6. Investigations on stability of reagents and conjugates S73
6.1. CQ-DT reactions in cell growth medium S73
6.2. Investigations on stability of o-dithiophenols S74
6.3. Investigations on stability of conjugates S75
7. Kinetic measurements S75
8. Fluorescence investigations of the conjugates S77
9. Bioorthogonal ligations and cleavages with model peptide and protein S77
9.1. Bioorthogonal ligations with model peptide S77
9.2. Bioorthogonal ligations with model protein S93
9.3. Bioorthogonal ligations and cleavages with model protein S103
9. References S114
10. 1H, 13C NMR spectra of the synthesized small molecules S115
-S1 -
1. General methods
Unless otherwise stated, all reagents were obtained from commercial sources and used
directly without further purification. Solvents were freshly distilled over sodium (THF),
calcium hydride (CH2Cl2 and DMF) or phosphorus pentoxide (MeCN). Deionized
water was used for chemical reactions and Milli-Q purified water for protein
manipulations. All reactions were carried out in oven-dried glassware (110 °C) and
monitored by thin layer chromatography (TLC). The products were obtained by flash
column chromatography on silica gel or recrystallization from a suitable solvent. Proton
(1H NMR) and carbon (13C NMR) nuclear magnetic resonance spectra were recorded
on a JEOL ECS-400 or JNM-ECA 600 spectrometers. Chemical shifts were reported
in parts per million (ppm), 1H NMR chemical shifts were internally referenced to
tetramethylsilane (TMS) (1H NMR: TMS references at 0.00 ppm) and residual proton
signals of solvents (1H NMR: CDCl3 at 7.26 ppm, DMSO-d6 at 2.50 ppm, and CD3OD
at 3.31 ppm), and 13C NMR chemical shifts were internally referenced to carbon signals
of solvents (13C NMR: CDCl3 at 77.16 ppm, DMSO-d6 at 39.52 ppm, and CD3OD at
49.00 ppm). Coupling constants (J) were reported in Hz with the following splitting
abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd =
doublet of doublets, ddd = doublet of doublet of doublets, td = triplet of doublets, and
br = broad. High-resolution mass spectra (HRMS) were recorded on an Agilent Q-
TOF 6540 and low-resolution mass spectra (LRMS) were recorded on a ThermoFisher
Scientific MSQ using electrospray ionization (ESI). High-performance liquid
chromotography (HPLC) was performed with an Agilent Eclipse XDB-C8 column (5
m, 4.6 mm ×250 mm) and a Shiseido Proteonavi column (5 m, 4.6 mm ×250 mm).
All the yields determined by HPLC were calculated via external standard method. UV-
Vis spectra were obtained using a TU-1901 spectrometer. Fluorescence spectra were
measured using an Edinburgh FLSP920 spectrometer. Fluorescence gel images were
taken using ProteinSimple FCM.
-S2 -
2. Synthetic procedures and characterization
-S3 -
(1) Synthesis of 2,3-dichloroquinoxaline (1a)
1a
S2
Prepared according to the procedures reported.[2] To 80 mL of vigorously boiling water
was added 2,3-dichloroquinoxaline (1a) (2.0 g, 10 mmol), and then KMnO4 (10.2 g, 64
mmol) was added slowly in small portions over 30 min, keeping the mixture 95-98 °C
for 2 h. The solid MnO2 was removed by filtration from the hot suspension and rinsed
with boiling water for three times. The combined aqueous filtrate was acidified with 3
mL of concentrated aqueous HCl and concentrated under reduced pressure, and the
solid residue was washed with two portions of acetone. The filtrate was concentrated to
give the crude product that was recrystallized from acetone to afford S2 as an off white
solid (1.33 g, 56% yield). 13C NMR (100 MHz, DMSO-d6) δ 142.73, 147.19, 164.12.
1d
Prepared according to the procedures reported.[2] A mixture of 5,6-dichloropyrazine-
2,3-dicarboxylic acid (S2) (474 mg, 2 mmol) and SOCl2 (4 mL) was refluxed for 30
-S4 -
min. The volatiles were evaporated, and the crystalline residue was dried in vacuo.
Mixture of the crystalline residue and CH3NH2HC1 (270 mg, 4.0 mmol) in Ac2O (1.0
mL) was heated in a sealing tube at 120 °C for 1 h. The volatiles were evaporated in
vacuo and the residue was purified by column chromatography on silica gel (PE/EtOAc
= 10:1, v/v) to afford 1d as an orange solid (246 mg, 47% total yield of two steps). 1H
NMR (400 MHz, CDCl3) δ 4.03 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 53.90, 141.97,
148.96, 162.99.
1b
S4
In a 250 mL round bottom flask equipped with a reflux condenser, 3,4-diaminobenzoic
acid (S3) (4.0 g, 26 mmol) in diethyl oxalate (120 mL) was heated at 160 C for 12 h.
The reaction mixture was cooled to RT, and filtered. The flask and filter cake were
washed with Et2O for three times, and the combined solution was concentrated to give
2,3-dihydroxyquinoxaline-6-carboxylic acid (S4) as a brown solid that was directly
used in the next step without further purification.
-S5 -
(6) Synthesis of 2,3-dichloroquinoxaline-6-carbonyl chloride (S5)
S5
A suspension of S4 (4.95 g, 24 mmol) in trichloroethylene (50 mL) was treated with a
catalytic amount of DMF, and the solution was heated to reflux. A solution of SOCl2
(15 ml, 206 mmol) in trichloroethylene (30 mL) was added dropwise over a period of
2 h, and the reaction mixture was stirred under reflux for an additional 4 h. After being
cooled to RT, the clear yellow solution was concentrated, and the crude product was
purified by recrystallization from n-hexane to give 2,3-dichloro-6-quinoxalinecarbonyl
chloride (S5) as a tan solid (4.96 g, 79% yield). 1H NMR (400 MHz, CDCl3) 8.12 (d,
J = 8.8 Hz, 1H), 8.37 (dd, J = 8.8, 1.6 Hz, 1H), 8.86 (d, J = 1.6 Hz, 1H). 13C NMR (100
MHz, CDCl3) δ 129.28, 130.79, 133.63, 135.19, 139.77, 143.41, 147.63, 149.05, 167.36.
1e
Hydrolysis of S5 in water provided 1e in almost quantitative yield. 1H NMR (400 MHz,
DMSO-d6) δ 8.14 (d, J = 8.8 Hz, 1H), 8.34 (dd, J = 8.8, 1.6 Hz, 1H), 8.50 (d, J = 1.6
Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 128.40, 129.36, 130.96, 133.85, 139.49,
141.84, 145.89, 146.67, 166.20.
(8) General procedure for the synthesis of chloroquinoxaline derivatives (1f, S6,
1h, 1j, S8, 1o, 1p and 1r-1x)
To a solution of 2,3-dichloroquinoxaline (1a) (398 mg, 2.0 mol, 1 equiv) in anhydrous
DMF (10 mL) were added RH (2.0 mmol, 1 equiv) and K2CO3 (552 g, 4.0 mmol, 2
equiv). The reaction mixture was stirred at RT for 2 h or 12 h (for 1f and S6, 2 h; for
1h, 1j, S8, 1o, 1p and 1r-1x, 12 h) under argon atmosphere and then concentrated under
reduced pressure. The residue was partitioned with EtOAc/H2O solution. The organic
layer was collected, and the aqueous layer was extracted with two additional portions
of EtOAc. The combined organic phase was washed with H2O and brine, dried over
anhydrous MgSO4, and filtered. The filtrate was concentrated under reduced pressure,
and the crude was purified by flash chromatography on silica gel to afford the
-S6 -
corresponding product (1f, S6, 1h, 1j, S8, 1o, 1p and 1r-1x).
2-Chloro-3-(1H-imidazol-1-yl)quinoxaline (1f)
1f
Eluent: PE/EA (1:1, v/v). Yield 249 mg (54%). Yellow solid. 1H NMR (400 MHz,
CDCl3) δ 7.29 (s, 1H), 7.81 (s, 1H), 7.84-7.90 (m, 2H), 8.10 (m, 2H), 8.45 (s, 1H). 13C
NMR (100 MHz, CDCl3) δ 119.39, 128.21, 128.62, 130.17, 131.44, 131.70, 137.57,
139.59, 139.64, 140.89, 141.21.
S6
Eluent: DCM/MeOH (96:4, v/v). Yield 462 mg (48%). White solid. 1H NMR (400 MHz,
CDCl3) δ 1.43 (s, 9H), 3.17 (m, 2H), 3.74 (s, 3H), 4.64 (m, 1H), 5.82 (d, J = 8.0 Hz,
1H), 7.58 (s, 1H), 7.80-7.87 (m, 2H), 8.00-8.08 (m, 2H), 8.33 (s, 1H). 13C NMR (100
MHz, CDCl3) δ 28.46, 30.45, 52.45, 53.41, 79.86, 116.91, 128.28, 128.63, 131.46,
131.79, 137.44, 138.67, 139.44, 139.63, 140.91, 141.00, 155.64, 172.50. HRMS (ESI+):
m/z calcd for C20H23ClN5O4 ([M+H]+) 432.1439; found 432.1434.
To a cooled (0 ºC) solution of S6 (241 mg, 0.5 mmol) in anhydrous DCM (2 ml) was
added TFA (1 ml). The reaction mixture was allowed to warm to RT and stirred for
additional 3 h. The volatiles were removed by rotary evaporation to dryness. The
residue was partitioned with EtOAc and saturated aqueous Na2CO3. The organic layer
was collected, and the aqueous layer was extracted twice with EtOAc. The combined
organic phase was washed with saturated aqueous NaCl, dried over MgSO4, filtered,
and the filtrate was concentrated under reduced pressure. The crude product was
-S7 -
purified by flash chromatography on silica gel (DCM/MeOH = 95:5, v/v) to afford 1g
as a waxy solid (151 mg, 91% yield). 1H NMR (400 MHz, CDCl3) δ 1.91 (s, 2H), 2.94
(m, 1H), 3.12 (m, 1H), 3.72 (s, 3H), 3.89 (t, J = 1.6 Hz, 1H), 7.60 (s, 1H), 7.77-7.82 (m,
2H), 8.02 (m, 2H), 8.32 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 33.36, 52.20, 54.30,
116.92, 128.23, 128.58, 131.36, 131.72, 137.34, 139.41, 139.45, 139.60, 140.82, 141.02,
175.53. HRMS (ESI+): m/z calcd for C15H15ClN5O2 ([M+H]+) 332.0914; found
332.0913.
1h
Eluent: DCM/MeOH (99:1, v/v). Yield 659 mg (97%). White solid. 1H NMR (400 MHz,
CDCl3) δ 1.93 (s, 3H), 3.74 (m, 4H), 3.95 (m, 1H), 5.03 (m, 1H), 6.57 (d, J = 7.2 Hz,
1H), 7.67 (m, 1H), 7.73 (m, 1H), 7.96 (m, 2H). 13C NMR (100 MHz, CDCl3 + CD3OD)
δ 22.49, 32.32, 52.36, 52.95, 127.98, 128.42, 129.93, 131.20, 139.79, 141.56, 145.35,
-S8 -
154.83, 171.74, 172.28. HRMS (ESI+): m/z calcd for C14H15ClN3O3S ([M+H]+)
340.0523; found 340.0522.
1i
To a solution of 2,3-dichloroquinoxaline (1a) (398 mg, 2.0 mol) in anhydrous DMF (10
mL) were added L-cysteine methyl ester hydrochloride (344 mg, 2.0 mmol) and DIPEA
(340 L, 4.0 mmol). The reaction mixture was stirred at RT for 2 h under argon
atmosphere and then concentrated under reduced pressure. The residue was partitioned
with EtOAc/H2O solution. The organic layer was collected, and the aqueous layer was
extracted with two additional portions of EtOAc. The combined organic phase was
washed with H2O and brine, dried over anhydrous MgSO4, and filtered. The filtrate was
concentrated under reduced pressure, and the crude was purified by flash
chromatography on silica gel (DCM/MeOH = 97:3, v/v) to afford 1i as a faint yellow
solid (456 mg, 77% yield). 1H NMR (400 MHz, CDCl3) δ 1.81 (s, 2H), 3.49 (m, 1H),
3.76 (s, 3H), 3.81 (m, 1H), 3.94 (m, 1H), 7.63 (td, J = 8.0, 1.6 Hz, 1H), 7.69 (td, J =
13
8.0, 1.6 Hz, 1H), 7.92 (m, 2H). C NMR (100 MHz, CDCl3) δ 35.40, 52.54, 53.78,
127.55, 128.32, 129.19, 130.47, 139.37, 141.05, 145.17, 154.62, 174.26. HRMS (ESI+):
m/z calcd for C12H13ClN3O2S ([M+H]+) 298.0417; found 298.0416.
2-Chloro-3-(p-tolyloxy)quinoxaline (1j)
1j
Eluent: PE/EtOAc (50:1, v/v). Yield 524 mg (97%). White solid. 1H NMR (400 MHz,
CDCl3) δ 2.42 (s, 3H), 7.17 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 8.4 Hz, 1H), 7.59-7.66 (m,
2H), 7.74 (m, 1H), 7.98 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 21.31, 121.62, 127.61,
128.23, 128.43, 130.46, 130.64, 135.73, 139.43, 139.53, 139.72, 150.57, 153.28.
Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-((3-chloroquinoxalin-2-
yl)oxy)phenyl)propanoate (S8)
-S9 -
S8
Eluent: PE/EtOAc (3:1, v/v). Yield 669 mg (73%). White solid. 1H NMR (400 MHz,
CDCl3) δ 1.44 (s, 9H), 3.14 (m, 2H), 3.74 (s, 3H), 4.63 (q, J = 6.8 Hz, 1H), 5.06 (d, J =
8.0 Hz, 1H), 7.23 (s, 4H), 7.59-7.66 (m, 2H), 7.72 (dd, J = 7.6, 2.0 Hz, 1H), 7.97 (dd, J
= 7.6, 2.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 28.44, 37.99, 52.42, 54.55, 80.16,
121.73, 127.36, 128.06, 128.39, 130.52, 130.61, 133.74, 139.22, 139.30, 139.44, 151.67,
152.79, 155.20, 172.36. HRMS (ESI+): m/z calcd for C23H25ClN3O5 ([M+H]+)
458.1483; found 458.1486.
To a cooled (0 ºC) solution of S8 (229 mg, 0.5 mmol) in anhydrous DCM (2 ml) was
added TFA (1 ml). The reaction mixture was allowed to warm to RT and stirred for
additional 3 h. The volatiles were removed by rotary evaporation to dryness. The
residue was partitioned with EtOAc and saturated aqueous Na2CO3. The organic layer
was collected, and the aqueous layer was extracted twice with EtOAc. The combined
organic phase was washed with saturated aqueous NaCl, dried over MgSO4, filtered,
and the filtrate was concentrated under reduced pressure. The crude product was
purified by flash chromatography on silica gel (DCM/MeOH = 95:5, v/v) to afford 1k
as a white solid (160 mg, 90% yield). 1H NMR (400 MHz, CDCl3) δ 2.93 (m, 1H), 3.14
(m, 1H), 3.73 (s, 3H), 3.78 (m, 1H), 7.24 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H),
7.60 (td, J = 7.6, 2.0 Hz, 1H), 7.64 (td, J = 7.6, 2.0 Hz, 1H),7.71 (dd, J = 7.6, 2.0 Hz,
1H), 7.96 (dd, J = 7.6, 2.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 40.58, 52.07, 55.86,
121.65, 127.24, 127.94, 128.26, 130.42, 130.48, 134.86, 139.10, 139.16, 139.32, 151.39,
152.71, 175.38. HRMS (ESI+): m/z calcd for C18H17ClN3O3 ([M+H]+) 358.0958; found
358.0958.
-S10 -
(13) Synthesis of 2-chloro-3-(1H-indol-1-yl)quinoxaline (1l)
1l
To a solution of 2,3-dichloroquinoxaline (1a) (199 mg, 1.0 mmol) in anhydrous DMF
(10 mL) were added indole (117 mg, 1.0 mmol) and K2CO3 (276 mg, 2.0 mmol). The
reaction mixture was stirred at 80 C for 4 h under argon atmosphere and then
concentrated under reduced pressure. The residue was partitioned with EtOAc/H2O
solution. The organic layer was collected, and the aqueous layer was extracted with two
additional portions of EtOAc. The combined organic phase was washed with H2O and
brine, dried over anhydrous MgSO4, and filtered. The filtrate was removed under
reduced pressure, and the crude product was purified by column chromatography on
silica gel (PE/EtOAc = 20:1, v/v) to afford 1l as a yellow solid (154 mg, 55% yield). 1H
NMR (400 MHz, CDCl3) δ 6.80 (d, J = 3.6 Hz, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.32 (t, J
= 7.2 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.75 (m, 2H), 7.81-7.86 (m, 2H), 8.01-8.12 (m,
2H). 13C NMR (100 MHz, CDCl3) δ 106.07, 112.71, 121.25, 122.04, 123.41, 127.63,
128.15, 128.41, 129.67, 130.67, 131.19, 136.44, 139.89, 140.47, 142.12, 143.94.
HRMS (ESI+): m/z calcd for C16H11ClN3 ([M+H]+) 280.0642; found 280.0639.
1m
Prepared according to the procedure reported.[5] A mixture of sodium (46 mg, 2.0
mmol) and alcohol (6 mL) was stirred for 15 min at RT. Then 2,3-dichloroquinoxaline
(1a) (398 mg, 2.0 mmol) was added to the mixture, and the reaction was performed
until the complete consumption of the starting materials monitored by TLC. After
evaporation of the solvent, the resulting precipitate was partitioned with EtOAc/H2O
solution. The organic layer was collected, and the aqueous layer was extracted with two
additional portions of EtOAc. The combined organic phase was washed with H2O and
saturated aqueous NaCl, dried over anhydrous MgSO4, and filtered. The filtrate was
removed under reduced pressure, and the crude product was purified by column
chromatography on silica gel (PE/EtOAc = 20:1, v/v) to afford 1m as a white solid (404
-S11 -
mg, 97% yield). 1H NMR (400 MHz, CDCl3) δ 1.51 (t, J = 7.2 Hz, 3H), 4.58 (q, J = 7.2
Hz, 2H), 7.54 (t, J = 8.4 Hz, 1H), 7.63 (t, J = 8.4 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.89
13
(d, J = 8.4 Hz, 1H). C NMR (100 MHz, CDCl3) δ 14.36, 63.95, 126.83, 127.31,
128.03, 130.19, 138.27, 139.62, 139.73, 153.15.
1n
Prepared according to the procedure reported.[6] To a solution of 2,3-
dichloroquinoxaline (1a) (199 mg, 1.0 mmol) in anhydrous EtOH was added n-
butylamine (198 L, 2.0 mmol). The reaction mixture was heated to reflux for 4 h and
then concentrated under reduced pressure. The resulting precipitate was dissolved in
EtOAc, and the solution was washed with H2O and saturated aqueous NaCl, dried over
anhydrous MgSO4, and filtered. The filtrate was removed under reduced pressure, and
the crude product was purified by column chromatography on silica gel (PE/EtOAc =
10:1, v/v) to afford 1n as a white solid (198 mg, 84% yield). 1H NMR (400 MHz,
CDCl3) δ 1.09 (t, J =7.2 Hz, 3H), 1.56 (m, 2H), 1.79 (m, J = 7.2 Hz, 2H), 3.68 (q, J =
6.8 Hz, 2H), 5.62 (s, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.66 (t, J = 8.0 Hz, 1H), 7.80 (d, J =
13
8.4 Hz, 1H), 7.87 (d, J = 8.4 Hz, 1H). C NMR (100 MHz, CDCl3) δ 13.97, 20.32,
31.35, 41.36, 124.88, 126.04, 127.96, 130.14, 136.39, 137.99, 141.57, 148.21.
2-Chloro-3-(p-tolylthio)quinoxaline (1o)
1o
Eluent: PE/EtOAc (30:1, v/v). Yield 545 mg (95%). 1H NMR (400 MHz, CDCl3) δ 2.45
(s, 3H), 7.29 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.58-763 (m, 2H), 7.71 (m,
13
1H), 7.92 (m, 1H). C NMR (100 MHz, CDCl3) δ 21.60, 124.64, 128.13, 129.17,
130.16, 130.25, 135.59, 139.78, 140.00, 141.41, 144.51, 155.76.
2-Chloro-3-(pyridin-2-ylthio)quinoxaline (1p)
1p
-S12 -
Eluent: PE/EtOAc (5:1, v/v). Yield 318 mg (58%). White solid. 1H NMR (400 MHz,
CDCl3) 7.33 (ddd, J = 6.8, 4.8, 2.0 Hz, 1H), 7.59-7.67 (m, 2H), 7.7.68-7.81 (m, 3H),
13
7.90-7.94 (m, 1H), 8.65 (d, J = 4.8 Hz, 1H). C NMR (100 MHz, CDCl3) δ 123.57,
128.17, 128.19, 129.80, 130.11, 130.36, 137.31, 140.01, 141.17, 145.12, 150.72,
152.64, 154.14. HRMS (ESI+): m/z calcd for C13H9ClN3S ([M+H]+) 274.0206; found
274.0202.
1q
To a solution of 2,3-dichloroquinoxaline (1a) (398 mg, 2.0 mol) in anhydrous DMF (10
mL) were added 2-mercaptoethanol (140 L, 2.0 mmol) and K3PO4 (425 mg, 2.0
mmol). The reaction mixture was stirred at RT for overnight under argon atmosphere
and then concentrated under reduced pressure. The residue was partitioned with
EtOAc/H2O solution. The organic layer was collected, and the aqueous layer was
extracted with two additional portions of EtOAc. The combined organic phase was
washed with H2O and brine, dried over anhydrous MgSO4, and filtered. The filtrate was
concentrated under reduced pressure, and the crude was purified by flash
chromatography on silica gel (PE/EtOAc (2:1, v/v) to afford 1q as an off white solid
(340 mg, 71% yield). 1H NMR (400 MHz, CDCl3) δ 2.84 (t, J = 5.6 Hz, 1H), 3.54 (t, J
= 5.6 Hz, 2H), 4.03 (q, J = 5.6 Hz, 2H), 7.65 (td, J = 8.0, 1.6 Hz, 1H), 7.71 (td, J = 8.0,
1.6 Hz, 1H), 7.93 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 33.66, 61.51, 127.12, 128.09,
129.07, 130.42, 139.07, 140.70, 145.07, 155.11.
2-Chloro-3-(pyridin-2-yloxy)quinoxaline (1r)
1r
Eluent: PE/EtOAc (5:1, v/v). Yield 320 mg (62%). White solid. 1H NMR (400 MHz,
CDCl3) 7.16-7.25 (m, 2H), 7.65-7.72 (m, 2H), 7.81-7.87 (m, 2H), 7.98-8.04 (m, 1H),
8.29 (dd, J = 4.8, 1.6 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 114.33, 121.14, 127.88,
128.13, 129.31, 130.60, 139.52, 140.06, 140.12, 140.77, 148.24, 151.96, 160.90.
-S13 -
HRMS (ESI+): m/z calcd for C13H9ClN3O ([M+H]+) 258.0434; found 258.0434.
2-(4-Azidophenoxy)-3-chloroquinoxaline (1s)
1s
Eluent: PE/EtOAc (10:1, v/v). Yield 405 mg (68%). White solid. 1H NMR (400 MHz,
CDCl3) δ 7.11-7.15 (m, 2H), 7.28-7.32 (m, 2H), 7.63 (td, J = 7.2, 1.6 Hz, 1H), 7.66 (td,
J = 7.2, 1.6 Hz, 1H), 7.72 (m, 1H), 7.99 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 120.19,
123.24, 127.33, 128.09, 128.51, 130.64, 137.61, 139.13, 139.23, 139.35, 149.44, 152.82.
HRMS (ESI+): m/z calcd for C14H9ClN5O ([M+H]+) 298.0496; found 298.0495.
(4-((3-Chloroquinoxalin-2-yl)oxy)phenyl)methanol (1t)
1t
Eluent: PE/EtOAc (1:1, v/v). Yield 436 mg (76%). White solid. 1H NMR (400 MHz,
CDCl3) δ 1.76 (t, J = 5.2 Hz, 1H), 4.77 (d, J = 5.2 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H),
7.48 (d, J = 8.4 Hz, 2H), 7.60-7.67 (m, 2H), 7.70-7.76 (m, 1H), 7.95-8.01 (m, 1H). 13C
-S14 -
NMR (100 MHz, DMSO-d6) δ 62.45, 121.31, 126.84, 127.65, 127.89, 128.52, 130.92,
138.54, 138.65, 138.90, 140.20, 151.06, 152.84. HRMS (ESI+): m/z calcd for
C15H12ClN2O2 ([M+H]+) 287.0587; found 287.0587.
(4-((3-Chloroquinoxalin-2-yl)oxy)-3-nitrophenyl)methanol (1u)
1u
Eluent: PE/EtOAc (1:1, v/v). Yield 524 mg (79%). White solid. 1H NMR (400 MHz,
DMSO-d6) δ 4.68 (d, J = 5.6 Hz, 2H), 5.63 (t, J = 5.6 Hz, 1H), 7.67-7.72 (m, 2H), 7.75-
7.80 (m, 2H), 7.86 (dd, J = 8.4, 2.0 Hz, 1H), 8.02-8.08 (m, 1H), 8.19 (d, J = 2.0 Hz,
1H). 13C NMR (100 MHz, DMSO-d6) δ 61.47, 123.29, 125.32, 126.79, 127.76, 129.10,
131.32, 133.44, 138.04, 138.13, 138.81, 141.15, 142.60, 143.26, 151.87. HRMS (ESI+):
m/z calcd for C15H11ClN3O4 ([M+H]+) 332.0438; found 332.0436.
7-((3-Chloroquinoxalin-2-yl)oxy)-2H-chromen-2-one (1v)
1v
Eluent: PE/DCM (1:1, v/v). Yield 428 mg (66%). White solid. 1H NMR (400 MHz,
CDCl3) δ 6.88 (s, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.64 (t, J = 8.0
Hz, 1H), 7.80 (m, 2H), 7.96 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 8.07 (d, J =
13
8.0 Hz, 1H). C NMR (101 MHz, CDCl3) δ 110.40, 116.12, 116.74, 118.34, 127.38,
128.18, 128.94, 129.02, 130.93, 138.88, 139.06, 139.64, 143.07, 152.05, 155.02, 155.15,
160.53. HRMS (ESI+): m/z calcd for C17H10ClN2O3 ([M+H]+) 325.0380; found
325.0381.
4-((3-Chloroquinoxalin-2-yl)oxy)-2H-chromen-2-one (1w)
1w
Eluent: PE/EtOAc (5:1, v/v). Yield 396 mg (61%). White solid. 1H NMR (400 MHz,
CDCl3) δ 6.88 (s, 1H), 7.40 (m, 2H), 7.64 (t, J = 8.0 Hz, 1H), 7.80 (m, 2H), 7.96 (d, J
= 8.0 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H). 13C NMR (100 MHz,
-S15 -
CDCl3) δ 101.16, 115.34, 117.08, 123.26, 124.59, 127.79, 128.24, 130.28, 131.46,
133.03, 138.43, 139.05, 140.12, 149.68, 153.64, 160.37, 161.88. HRMS (ESI+): m/z
calcd for C17H10ClN2O3 ([M+H]+) 325.0380; found 325.0383.
1-((3-Chloroquinoxalin-2-yl)oxy)pyrrolidine-2,5-dione (1x)
1x
Eluent: PE/EtOAc (1:1, v/v). Yield 380 mg (68%). White solid. 1H NMR (400 MHz,
CDCl3) δ 2.99 (s, 4H), 7.67-7.74 (m, 2H), 7.77-7.81 (m, 1H), 7.99-8.03 (m, 1H). 13C
NMR (100 MHz, CDCl3) δ 25.80, 127.46, 128.32, 129.64, 131.18, 135.83, 138.26,
140.68, 149.71, 169.12. HRMS (ESI+): m/z calcd for C12H9ClN3O3 ([M+H]+) 278.0332;
found 278.0336.
S12
S16
S17
Methyl 3,4-bis((dimethylcarbamothioyl)oxy)benzoate was synthesized and purified
according to the procedures described.[8] To an ice-cold mixture of methyl-3,4-
dihydroxybenzoate (S15) (10.08 g, 60 mmol) and DABCO (26.94 g, 240 mmol) in
anhydrous DMF (60 mL) was slowly added a solution of S12 (29.58 g, 240 mmol) in
DMF (20 mL), and then the reaction mixture was stirred for additional 30 min at RT.
The resulting white solid was dissolved in 400 mL of water. The product was extracted
with EtOAc (3240 mL) and the combined organic phase was dried over anhydrous
MgSO4, and the solvent was removed under reduced pressure. The crude product was
purified by column chromatography on silica gel (PE/EtOAc = 3:1, v/v) to give S17 as
a white solid (18.0 g, 88% yield). 1H NMR (400 MHz, CDCl3) δ 3.26 (s, 3H), 3.27 (s,
3H), 3.39 (s, 3H), 3.40 (s, 3H), 3.90 (s, 3H) 7.25 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 2.0
Hz, 1H), 7.99 (dd, J = 8.4, 2.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 38.60, 38.67,
-S17 -
43.07, 43.13, 52.14, 124.17, 125.59, 127.77, 128.33, 145.34, 149.15, 165.31, 185.61,
186.02.
S18
Prepared according to the method for synthesis of S17, with S16 (10.2 g, 56 mmol),
DABCO (25.2 g, 224 mmol), and S12 (27.6 g, 224 mmol). Purification by column
chromatography on silica gel (PE/EtOAc = 3:1, v/v) afforded S18 as a white solid (16.5
g, 83% yield). 1H NMR (400 MHz, CDCl3) δ 3.23 (s, 6H), 3.37 (s, 6H), 3.61 (s, 2H),
3.66 (s, 3H) 7.09 (m, 2H), 7.19 (dd, J = 8.0, 2.4 Hz, 1H). 13C NMR (100 MHz, CDCl3)
δ 38.71, 40.43, 43.22, 52.07, 124.03, 125.06, 127.33, 132.40, 144.53, 145.25, 171.11,
186.55. HRMS (ESI+): m/z calcd for C15H21N2O4S2 ([M+H]+) 357.0943; found
357.0941.
S19
Methyl 3,4-bis((dimethylcarbamothioyl)oxy)benzoate (S17) (6.0 g, 17.5 mmol) was
heated at 240 C in diphenyl ether (60 mL) for 30 min. The reaction mixture was cooled
to RT and purified directly by column chromatography on silica gel using a gradient
(PE/EtOAc = 98:2 to 60:40, v/v) to afford methyl 2-oxobenzo[d][1,3]dithiole-5-
carboxylate (S19) as a white solid (1.86 g, 47% yield). 1H NMR (400 MHz, CDCl3) δ
3.95 (s, 3H), 7.56 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 84 Hz, 1H), 8.16 (s, 1H). 13C NMR
(100 MHz, CDCl3) δ 52.64, 122.98, 124.16, 127.85, 129.14, 133.01, 137.99, 165.73,
189.00.
-S18 -
S20
Prepared according to the method for synthesis of S19, with S18 (3.6 g, 10 mmol).
Purification by column chromatography on silica gel (PE/EtOAc = 98:2 to 60:40, v/v)
afforded S20 as a white solid (577 mg, 24% yield). 1H NMR (400 MHz, CDCl3) δ 3.65
(s, 2H), 3.72 (s, 3H), 7.24 (dd, J = 8.4, 1.6 Hz, 1H), 7.44 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 40.69, 52.38, 123.17, 123.80, 128.27, 131.45, 133.04, 133.31, 171.29, 189.99.
HRMS (ESI+): m/z calcd for C10H9O3S2 ([M+H]+) 240.9993; found 240.9995.
2c
An aqueous solution of NaOH (50 mL, 1 M) was added to methyl 2-
oxobenzo[d][1,3]dithiole-5-carboxylate (S19) (1.2 g, 5.3 mmol). The resulting mixture
was heated at 70 C under argon atmosphere for 6 h. The reaction mixture was cooled
to RT and adjusted pH to 3-4 with HCl (1N). The white precipitate was collected by
filtration, washed three times with H2O, and dried to afford 3,4-dimercaptobenzoic acid
(2c) as an off white solid (0.9 g, 91% yield). 1H NMR (400 MHz, CD3OD) δ 7.44 (d, J
= 8.0 Hz, 1H), 7.65 (dd, J = 8.0, 1.6 Hz, 1H), 7.99 (d, J = 1.6 Hz, 1H). 13C NMR (100
MHz, CD3OD) δ 128.36, 129.46, 130.44, 131.45, 132.84, 140.42, 168.91.
2d
Prepared according to the method for synthesis of 2c, with S20 (481 mg, 2.0 mmol) and
NaOH (20 mL, 1 M) to afford 2d as an off white solid (344 mg, 86% yield). 1H NMR
(400 MHz, CDCl3) δ 3.56 (s, 2H), 3.69 (s, 1H), 3.74 (s, 1H), 6.99 (dd, J = 8.0, 1.6 Hz,
1H), 7.30 (d, J = 1.6 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H). 13C NMR (150 MHz, CD3OD)
δ 41.02, 128.47, 130.52, 131.75, 132.32, 132.68, 134.46, 175.13. HRMS (ESI+): m/z
calcd for C8H9O2S2 ([M+H]+) 201.0044; found 201.0041.
-S19 -
Scheme S3. Reactions of chloroquinoxalines and ortho-dithiophenols
3a
Prepared according to the method for synthesis of 3a, with 2,3-dichloroquinoxaline (1a)
(99.5 mg, 0.5 mmol), toluene-3,4-dithiol (2b) (78 mg, 0.5 mmol) and K2HPO4 (174 mg,
1.0 mmol). Purification by column chromatography on silica gel (PE/EtOAc = 10:1,
v/v) afforded 3b as a yellow solid (134 mg, 95% yield). 1H NMR (400 MHz, CDCl3) δ
2.35 (s, 3H), 7.10 (dd, J = 8.0, 1.2 Hz, 1H), 7.31 (d, J = 1.2 Hz, 1H), 7.31 (d, J = 8.0
-S20 -
Hz, 1H), 7.68 (m, 2H), 7.95 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 21.06, 127.95,
128.44, 128.68, 129.39, 130.13, 130.15, 131.40, 138.81, 140.72, 152.71, 152.83.
3c
A mixture of 2,3-dichloroquinoxaline (1a) (99.5 mg, 0.5 mmol), 2c (93 mg, 0.5 mmol)
and K2HPO4 (261 mg, 1.5 mmol) in DMF/H2O (10 mL, 4/1, v/v) was stirred at RT for
10 min. The reaction mixture was poured to dilute hydrochloric acid solution (25 mL,
0.1 M), and the solution was extracted with EtOAc (3 20 mL) and dried over
anhydrous MgSO4. After filtration, the filtrate was evaporated in vacuo. The crude
product was purified by column chromatography on silica gel (DCM/MeOH = 9:1, v/v)
to afford 3c as a yellow solid (151 mg, 97% yield). 1H NMR (400 MHz, DMSO-d6) δ
7.69 (d, J = 8.0 Hz, 1H), 7.79-7.83 (m, 2H), 7.89 (d, J = 8.0 Hz, 1H), 7.92-8.00 (m, 2H),
8.04 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 127.75, 128.34, 128.44, 128.61, 128.66,
128.89, 130.39, 130.43, 131.17, 135.30, 139.91, 139.97, 150.21, 150.41, 165.62.
HRMS (ESI+): m/z calcd for C15H9N2O2S2 ([M+H]+) 313.0105; found 313.0105.
3d
Prepared according to the method for synthesis of 3c, with 2,3-dichloroquinoxaline (1a)
(99.5 mg, 0.5 mmol), 2d (100 mg, 0.5 mmol) and K2HPO4 (261 mg, 1.5 mmol).
Purification by column chromatography on silica gel (DCM/MeOH = 9:1, v/v) afforded
3d as a yellow solid (158 mg, 95% yield). 1H NMR (400 MHz, DMSO-d6) δ 3.51 (s,
-S21 -
2H), 7.26 (d, J = 7.2 Hz, 1H), 7.48 (m, 2H), 7.78 (m, 2H), 7.94 (m, 2H). 13C NMR (100
MHz, DMSO-d6) δ 41.57, 127.37, 127.79, 128.08, 129.29, 129.66, 129.79, 130.31,
137.85, 139.83, 151.28, 151.38, 173.20. HRMS (ESI+): m/z calcd for C16H11N2O2S2
([M+H]+) 327.0262; found 327.0259.
3k
Prepared according to the method for synthesis of 3a, a mixture of 1c (74.5 mg, 0.5
mmol), benzene-1,2-dithiol (2b) (71 mg, 0.5 mmol) and K2HPO4 (174 mg, 1.0 mmol)
in DMF/H2O (5.0 mL, 4/1, v/v) (10 mL) was stirred at RT for 30 min. The reaction
mixture was poured to water (25 mL), and the solution was extracted with EtOAc (3
10 mL) and dried over anhydrous MgSO4. After filtration, the filtrate was evaporated
in vacuo. The crude product was purified by column chromatography on silica gel
(PE/EtOAc, 10:1) to afford 3k as a white solid (91 mg, 78% yield). 1H NMR (400 MHz,
CDCl3) δ 2.33 (s, 3H), 7.09 (d, J = 8.0 Hz, 1H), 7.28 (s, 1H), 7.33 (d, J = 8.0 Hz, 1H),
13
8.27 (s, 3H). C NMR (100 MHz, CDCl3) δ 20.98, 128.64, 128.67, 129.39, 129.46,
132.12, 138.84, 141.65, 153.63, 153.80.
3l
Prepared according to the method for synthesis of 3a, with 1d (131 mg, 0.5 mmol),
toluene-3,4-dithiol (2b) (78 mg, 0.5 mmol) and K2HPO4 (174 mg, 1.0 mmol).
Purification by column chromatography on silica gel (PE/EtOAc = 10:1, v/v) afforded
3l as a yellow solid (166 mg, 96% yield). 1H NMR (400 MHz, CDCl3) δ 2.33 (s, 3H),
3.99 (s, 6H), 7.11 (d, J = 8.0, 2.0 Hz, 1H), 7.22 (d, J = 2.0 Hz, 1H), 7.28 (d, J = 8.0 Hz,
13
1H). C NMR (100 MHz, CDCl3) δ 20.95, 53.54, 126.91, 128.66, 129.41, 129.92,
130.39, 139.51, 141.46, 141.50, 155.47, 155.66, 164.28.
-S22 -
(32) Synthesis of 3m and 3′m
Prepared according to the method for synthesis of 3c, with 1e (121.5 mg, 0.5 mmol),
toluene-3,4-dithiol 2b (78 mg, 0.5 mmol) and K2HPO4 (261 mg, 1.5 mmol). Purification
by column chromatography on silica gel (DCM/MeOH = 9:1, v/v) afforded a mixture
of region-isomers 3m and 3′m as a yellow solid (160 mg, 98% yield). 1H NMR (400
MHz, DMSO-d6) δ 2.25 (s, 3H), 7.14 (d, J = 8.0 Hz, 1H), 7.35 (s, 1H), 7.39 (dd, J =
8.0, 2.8 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.36 (s, 1H). 13C
NMR (100 MHz, DMSO-d6, 1/1 = regioisomeric equivalents) δ 20.32, 125.92/126.01,
128.18, 128.22/128.26, 128.74/128.77, 129.33/129.41, 129.46/129.50, 129.56, 129.63,
132.08/132.10, 138.73/138.77, 139.22, 141.72, 152.54/152.67, 153.75/153.88, 166.28.
HRMS (ESI+): m/z calcd for C16H11N2O2S2 ([M+H]+) 327.0262; found 327.0265.
Prepared according to the method for synthesis of 3c, with 1e (121.5 mg, 0.5 mmol),
toluene-3,4-dithiol (2a) (71 mg, 0.5 mmol) and K2HPO4 (261 mg, 1.5 mmol).
Purification by column chromatography on silica gel (DCM/MeOH = 9:1, v/v) afforded
3n as a yellow solid (148 mg, 95% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.39-7.44
(m, 2H), 7.59-7.64 (m, 2H), 8.01 (d, J = 8.8 Hz, 1H), 8.21 (dd, J = 8.8, 1.6 Hz, 1H),
13
8.41 (d, J = 1.6 Hz, 1H). C NMR (100 MHz, DMSO-d6) δ 128.26, 128.65, 128.67,
128.90, 128.93, 129.60, 129.65, 129.73, 129.86, 132.51, 139.36, 141.80, 152.41, 153.58,
166.38. HRMS (ESI+): m/z calcd for C15H9N2O2S2 ([M+H]+) 313.0105; found 313.0109.
-S23 -
Scheme S4. Reactions of 1a, 1f and 1j with 4a
S21
To a solution of 1a (100 mg, 0.5 mol) in anhydrous DMF (10 mL) was added N-Ac-
Cys-OMe (4a) (178 mg, 1.0 mmol) and K2CO3 (512 g, 2.0 mmol). The reaction mixture
was stirred at RT for overnight under argon atmosphere and then concentrated under
reduced pressure. H2O (20 mL) and EtOAc (20 mL) were added, and the organic layer
was separated. The aqueous layer was extracted with EtOAc (220 mL), and the
combined organic phase was washed with saturated aqueous NaCl, dried over
anhydrous MgSO4 and concentrated under reduced pressure. The residue was purified
by column chromatography on silica gel (DCM/MeOH = 96:4, v/v) to afford S21 as a
white solid (221 mg, 92% yield). 1H NMR (400 MHz, DMSO-d6) δ 1.85 (s, 6H), 3.56
(s, 6H), 3.66 (s, 6H), 3.90 (m, 2H), 4.73 (m, 2H), 7.68-7.72 (m, 2H), 7.87-7.92 (m, 2H),
8.34 (d, J = 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 22.31, 31.06, 51.12, 52.28,
127.27, 129.03, 139.00, 152.94, 169.57, 170.94. HRMS (ESI+): m/z calcd for
C20H25N4O6S2 ([M+H]+) 481.1216; found 481.1219.
-S24 -
(35) Synthesis of methyl N-acetyl-S-(3-(p-tolyloxy)quinoxalin-2-yl)-L-cysteinate
(S22)
S22
Prepared according to the method for synthesis of S21, with 1j (135 mg, 0.5 mol), 4a
(89 mg, 0.5 mmol) and K2CO3 (256 g, 1.0 mmol). Purification by column
chromatography on silica gel (DCM/MeOH = 98:2, v/v) afforded S22 as a white solid
(177 mg, 86% yield). 1H NMR (400 MHz, CDCl3) δ 1.94 (s, 3H), 2.39 (s, 3H), 3.75 (s,
3H), 3.81 (m, 1H), 3.91 (m, 1H), 5.01 (m, 1H), 7.02 (d, J = 6.8 Hz, 1H), 7.15 (d, J =
8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.53 (m, 2H), 7.68 (d, J = 8.0 Hz, 1H), 7.90 (d, J
= 8.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 21.06, 23.18, 30.61, 52.84, 53.06, 121.44,
126.76, 127.53, 127.77, 128.68, 130.15, 135.37, 138.01, 139.38, 148.73, 150.17, 153.61,
170.14, 171.06. HRMS (ESI+): m/z calcd for C21H22N3O4S ([M+H]+) 412.1331; found
412.1332.
S23
Prepared according to the method for synthesis of S21, with 1f (115 mg, 0.5 mol), 4a
(89 mg, 0.5 mmol) and K2CO3 (256 g, 1.0 mmol). Purification by column
chromatography on silica gel (DCM/MeOH = 95:5, v/v) afforded S23 as a white solid
(150 mg, 81% yield). 1H NMR (400 MHz, CDCl3) δ 1.95 (s, 3H), 3.73 (s, 3H), 3.3.74-
3.80 (m, 1H), 4.04 (m, 1H), 5.06 (m, 1H), 6.61 (d, J = 7.2 Hz, 1H), 7.27 (s, 1H), 7.64
(s, 1H), 7.73 (ddd, J = 8.4, 7.2, 1.6 Hz, 1H), 7.79 (ddd, J = 8.4, 7.2, 1.6 Hz, 1H), 8.01
(dd, J = 8.4, 1.6 Hz, 1H), 8.05 (dd, J = 8.4, 1.6 Hz, 1H), 8.23 (s, 1H). 13C NMR (100
MHz, CDCl3) δ 23.03, 32.23, 51.91, 52.85, 119.11, 127.41, 128.78, 129.87, 129.96,
131.04, 137.14, 138.24, 141.28, 141.74, 149.47, 170.29, 170.98. HRMS (ESI+): m/z
calcd for C17H18N5O3S ([M+H]+) 372.1130; found 372.1134.
-S25 -
Scheme S5. Synthesis of compounds 5 and S32
S24
Prepared according to the similar procedures reported by Yang et al.[9] To a stirred
solution of TCCA (328 mg, 1.4 mmol) in anhydrous ACN (80 mL) at -20 C in a
tetrachloromethane/liquid nitrogen bath was added 2c powder (373 mg, 2.0 mmol).
After the mixture turned deep yellow (approximately 30 min), a solution of 2-methyl-
2-propanethiol (450 uL, 4.0 mmol) in anhydrous ACN (20 mL) was added quickly to
the mixture. The reaction mixture was kept stirring for 5 min and then evaporated under
reduced pressure. The residue was partitioned with EtOAc/H2O solution and the
combined organic layer was washed with brine, dried over anhydrous MgSO4, and
filtered. The filtrate was concentrated under reduced pressure, and the crude was
purified by flash chromatography on silica gel (DCM/MeOH = 98:2, v/v) to afford S24
as a white solid (580 mg, 80% yield). 1H NMR (400 MHz, DMSO-d6) δ 1.27 (s, 18H),
7.82 (dd, J = 8.0, 1.6 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 8.29 (d, J = 1.6 Hz, 1H). 13C
NMR (100 MHz, DMSO-d6) δ 29.38, 29.48, 49.89, 50.14, 127.39, 128.28, 129.50,
129.72, 136.21, 142.62, 166.44. HRMS (ESI+): m/z calcd for C15H23O2S4 ([M+H]+)
-S26 -
363.0581; found 363.0582.
S26
Prepared according to the procedures described.[10] Powdered potassium hydroxide
(11.2 g, 200 mmol) was added portionwise to a cool (0 C) solution of tetraethylene
glycol (S25) (4.32 mL, 25 mmol) and p-toluenesulfonyl chloride (TsCl, 9.5 g, 50 mmol)
in DCM (30 mL). The reaction mixture was stirred for 3 h at 0 C. Water (30 mL) was
then added, and the solution was extracted with DCM (2×15 mL). The combine organic
phase was dried over anhydrous MgSO4 and concentrated under reduced pressure. The
residue was purified by column chromatography on silica (DCM/MeOH = 98:2, v/v) to
afford S26 as colorless oil (12.1 g, 96% yield). 1H NMR (400 MHz, CDCl3) δ 2.44 (s,
6H), 3.56 (m, 8H), 3.68 (m, 4H), 4.15 (m, 4H), 7.33 (d, J = 8.0 Hz, 4H), 7.79 (d, J =
8.0 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ 21.68, 68.69, 69.35, 70.56, 70.74, 127.98,
129.89, 132.96, 144.90.
S27
Prepared according to the procedures described.[10] To a solution of S26 (12.1 g, 24
mmol) in DMF (35 mL) were added TBAI (448 mg, 1.2 mmol) and NaN3 (6.24 g, 96
mmol). The reaction mixture was stirred overnight at 80 C. The solvent was then
evaporated under reduced pressure, and the residue was suspended in Et2O generating
a white precipitate. The precipitate was then removed by filtration, and the filtrate was
concentrated under reduced pressure. The procedure was repeated twice, and the
residue was purified by column chromatography on silica gel (DCM/MeOH = 98:2, v/v)
to give S27 as a colorless liquid (5.4 g, 92% yield). 1H NMR (400 MHz, CDCl3) δ 3.39
(t, J = 5.2 Hz, 4H), 3.68 (m, 12H). 13C NMR (100 MHz, CDCl3) δ 50.70, 70.05, 70.73.
S28
-S27 -
Prepared according to the procedures described.[10] A solution of PPh3 (5.2 g, 20 mmol)
in Et2O (30 mL) was added dropwise to a solution of S27 (4.88 g, 20 mmol) in a mixture
of Et2O/THF/1 M HCl (5:1:4, 60 mL). The reaction was vigorously stirred overnight at
RT. A 4 M HCl aqueous solution (20 mL) was then added to the reaction mixture, and
the aqueous layer was extracted with Et2O (3×10 mL), and adjusted to pH 14 by
addition of sodium hydroxide pellets. The aqueous layer was then extracted with DCM
(3×20 mL). The combined organic layer was dried over MgSO4, concentrated under
reduced pressure. The residue was purified by column chromatography on silica gel
(DCM/MeOH = 9:1, v/v) to give S28 as a colorless liquid (2.7 g, 68% yield). 1H NMR
(400 MHz, CDCl3) δ 1.60 (s, 2H), 2.87 (t, J = 5.2 Hz, 2H), 3.52 (t, J = 5.2 Hz, 2H),
3.62-3.70 (m, 10H). 13C NMR (100 MHz, CDCl3) δ 41.70, 50.61, 69.98, 70.21, 70.58,
70.59, 70.64, 73.28.
S29
Prepared according to the procedures described.[10] To an ice-cold solution of S28 (218
mg, 1.0 mmol) and TEA (280 L, 2.0 mmol) in anhydrous DCM (4 mL) was added
(Boc)2O (241 mg, 1.1 mmol). The reaction mixture was warmed to RT and stirred for
15 h. The solvent was removed under reduced pressure. The crude product was purified
by silica gel chromatography (EtOAc: cyclohexane 1:1) to yield S29 as a colorless oil
(235 mg, 74% yield). 1H NMR (400 MHz, CDCl3) δ 1.45 (s, 9H), 3.31 (q, J = 5.2 Hz,
2H), 3.40 (t, J = 5.2 Hz, 2H), 3.54 (t, J = 5.2 Hz, 2H), 3.61-3.70 (m, 10H), 5.06 (br s,
1H). 13C NMR (100 MHz, CDCl3) δ 28.49, 40.43, 50.74, 70.13, 70.13, 70.29, 70.32,
70.68, 70.70, 70.77, 79.21, 156.06.
S30
Prepared according to the procedures described.[10] S29 (637 mg, 2.0 mmol) was
dissolved in MeOH (20 mL), and the flask was charged with 10% Pd/C (150 mg) and
-S28 -
H2 gas. The solution was stirred at RT for 2 h. The metal catalyst was removed by
filtration through Celite pad, and rinsed with MeOH. The filtrate was concentrated
under reduced pressure and dried in vacuo. The resulting crude product was purified by
column chromatography on silica gel (DCM/MeOH = 9:1, v/v) to afford S30 as a
colorless oil (477 mg, 81% yield). 1H NMR (400 MHz, CDCl3) δ 1.39 (s, 9H), 1.98 (s,
2H), 2.83 (t, J = 4.8 Hz, 2H), 3.26 (q, J = 5.2 Hz, 2H), 3.44-3.51 (m, 4H), 3.4-3.61 (m,
8H), 5.29 (br s, 1H). 13C NMR (100 MHz, CDCl3) δ 28.47, 40.40, 41.61, 70.25, 70.31,
70.55, 70.57, 73.03, 79.10, 156.13.
S31
To a stirred solution of S30 (157 mg, 1.6 mmol) in anhydrous toluene (12 mL) was
added maleic anhydride (486 mg, 1.6 mmol) at RT under nitrogen atmosphere. After
12 h, reaction mixture was concentrated in vacuo to give crude acid that was used for
next cyclization step without further purification. To a mixture of resultant acid in Ac2O
(2 ml) was added sodium acetate (132 mg, 1.6 mmol) at RT under argon atmosphere.
The reaction mixture was heated to 60 C for 16 h and concentrated in vacuo. The
residue was purified by column chromatography on silica gel (PE/EtOAc = 1:1, v/v) to
give S31 as clear oil (391 mg, 66% yield). 1H NMR (400 MHz, CDCl3) δ 1H NMR (400
MHz, CDCl3) δ 1.41 (s, 9H), 3.28 (q, J = 5.5 Hz, 2H), 3.50 (t, J = 5.2 Hz, 2H), 3.54-
3.60 (m, 8H), 3.60-3.64 (m, 2H), 3.70 (t, J = 5.2 Hz, 2H), 5.06 (br s, 1H), 6.68 (s, 2H).
13
C NMR (100 MHz, CDCl3) δ 28.50, 37.20, 40.44, 67.93, 70.13, 70.28, 70.31, 70.63,
79.23, 134.23, 156.11, 170.72.
5
Compound S31 (372 mg, 1.0 mmol) was added to a saturated solution of methanolic
hydrogen chloride (10 mL), and the mixture was stirred at RT for 4 h. The volatiles
-S29 -
were removed by rotary evaporation to give crude ammonium salt for the next step. A
solution of S5 (261 mg, 1.0 mmol) in anhydrous DMF (5 mL) was added dropwise to
the mixture of resulting ammonium salt and DIPEA (340 L, 2.0 mmol) in anhydrous
DMF (5 mL) in an ice bath. The reaction mixture was allowed to warm to RT and stirred
overnight. The solution was evaporated to dryness, and the residue was dissolved in
EtOAc. The solution was washed successively with H2O, saturated aqueous NaHCO3
and brine, dried over anhydrous MgSO4, filtered, and the filtrate was concentrated
under reduced pressure. The residue was purified by column chromatography on silica
gel (PE/EtOAc = 95:5, v/v) to afford 5 as a waxy solid (373 mg, 75% yield). 1H NMR
(400 MHz, CDCl3) δ 3.56-3.73 (m, 16H), 6.66 (s, 2H), 7.25 (s, 1H), 8.05 (d, J = 8.8 Hz,
1H), 8.27 (dd, J = 8.8, 2.0 Hz, 1H), 8.41 (d, J = 2.0 Hz, 1H). 13C NMR (101 MHz,
CDCl3) δ 37.27, 40.30, 67.95, 69.63, 70.20, 70.43, 70.61, 70.72, 126.90, 128.65, 130.15,
134.22, 137.24, 140.08, 141.77, 146.46, 146.93, 165.75, 170.72. HRMS (ESI+): m/z
calcd for C21H23Cl2N4O6 ([M+H]+) 497.0995; found 497.0996.
S32
Compound S31 (298 mg, 0.8 mmol) was added to a saturated solution of methanolic
hydrogen chloride (10 mL), and the mixture was stirred at RT for 4 h. The volatiles
were removed by rotary evaporation to give crude ammonium salt for the next step. S24
(290 mg, 0.8 mmol), HATU (608 mg, 1.6 mmol) and DIPEA (275 L, 1.6 mmol) were
added to the resultant ammonium salt in DMF (5 mL). The reaction mixture was stirred
overnight at RT, and the solution was concentrated under reduced pressure. The residue
was dissolved in EtOAc, then the solution was washed successively with H2O, saturated
aqueous Na2CO3 and brine, dried over anhydrous MgSO4, filtered, and the filtrate was
concentrated under reduced pressure to yield the crude that was purified by column
chromatography on silica gel (PE/EtOAc = 1:1, v/v) to afford S32 as a colorless oil
(283 mg, 56% yield). 1H NMR (400 MHz, CDCl3) δ 1H NMR (400 MHz, CDCl3) δ
-S30 -
1.26 (s, 18H), 3.52-3.66 (m, 16H), 6.63 (s, 2H), 6.88 (t, J = 4.8 Hz, 1H), 7.54 (dd, J =
8.0, 1.2 Hz, 1H), 7.77 (s, J = 8.0 Hz, 1H), 8.13 (d, J = 1.2 Hz, 1H). 13C NMR (101 MHz,
CDCl3) δ 29.84, 29.87, 37.08, 39.83, 49.76, 49.91, 67.84, 69.73, 69.99, 70.23, 70.48,
70.57, 125.46, 127.28, 127.64, 132.95, 134.11, 137.32, 141.96, 166.55, 170.59.2
HRMS (ESI+): m/z calcd for C27H41N2O6S4 ([M+H]+) 617.1847; found 617.1853.
S34
Prepared according to the procedure reported in literature.[11] NaHCO3 (441 mg, 5.25
mmol) in water (10 mL) was added to N-CBZ-L-lysine tert-butyl ester hydrochloride
(S33) (1.86 g, 5.0 mmol) in chloroform (15 mL). The mixture was stirred at RT for 5
min under argon atmosphere, and then (Boc)2O (1.15 g, 5.25 mmol) in chloroform (8
mL) was added. The mixture was refluxed for 1.5 h and allowed to cool to RT. The
organic layer was collected and the aqueous layer was extracted with chloroform. The
combined organic layer was washed with saturated aqueous NaCl, dried over MgSO4,
concentrated under reduced under pressure. The crude product was purified by column
chromatography on silica gel (PE/EtOAc = 4:1, v/v) to afford S34 as a colorless oil
(1.93 g, 88% yield). 1H NMR (400 MHz, CDCl3) δ 1.33-1.78 (m, 6H), 1.43 (s, 9H),
1.45 (s, 9H), 3.19 (q, J = 6.8 Hz, 2H), 4.15 (m, 1H), 4.83 (br s, 1H), 5.08 (m, 3H), 7.29-
7.36 (m, 5H). 13C NMR (400 MHz, CDCl3) δ 22.40, 28.07, 28.40, 29.45, 32.68, 40.80,
53.77, 66.65, 79.72, 81.89, 128.13, 128.18, 128.56, 136.70, 155.57, 156.54, 172.01.
-S31 -
S35
Prepared according to the procedure reported.[12] To a solution of S34 (1.31 g, 3.0 mmol)
in MeOH (20 mL), Pd/C (150 mg, 10%) was added. The reaction mixture was stirred
at RT under a hydrogen atmosphere for 3 h. The reaction mixture was filtered, then the
solid was washed with MeOH, and the combined solution was evaporated under
reduced pressure. The crude product was purified by column chromatography on silica
gel (DCM/MeOH = 98:2, v/v) to afford S35 as a colorless oil (798 mg, 88% yield). 1H
NMR (400 MHz, CDCl3) δ 1.38 (s, 9H), 1.41 (s, 9H), 1.50-1.84 (m, 6H), 2.96 (m, 2H),
4.06 (m, 1H), 5.17 (d, J = 8.4 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 22.62, 27.30,
28.09, 28.42, 32.36, 39.85, 53.93, 79.72, 81.98, 155.61, 171.90.
S36
S24 (544 mg, 1.5 mmol), S35 (454 mg, 1.5 mmol), HATU (1.14 mg, 3.0 mmol) and
DIPEA (252 L, 1.5 mmol) were mixed in DMF (8 mL), and the mixture was stirred
overnight at RT. The resulting solution was concentrated under reduced pressure, and
the residue was dissolved in EtOAc, then the solution was washed successively with
H2O, saturated aqueous Na2CO3 and brine, dried over anhydrous MgSO4, filtered, and
the filtrate was concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel (PE/EtOAc = 3:1, v/v) to afford S36 as a white
solid (738 mg, 76% yield). 1H NMR (400 MHz, CDCl3) δ 1.30 (s, 9H), 1.31 (s, 9H),
1.41 (s, 9H), 1.44 (s, 9H), 1.44-1.83 (m, 6H), 3.44 (m, 2H), 4.16 (m, 1H), 5.08 (d, J =
8.4 Hz, 1H), 6.28 (d, J = 6.0 Hz, 1H), 7.56 (dd, J = 8.4, 1.6 Hz, 1H), 7.82 (d, J = 8.4
Hz, 1H), 8.14 (d, J = 1.6 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 22.67, 27.99, 28.32,
29.06, 29.86, 29.86, 32.63, 39.82, 49.72, 49.87, 53.75, 79.58, 81.81, 125.35, 127.27,
127.67, 132.99, 137.39, 141.94, 155.51, 166.67, 171.85. HRMS (ESI+): m/z calcd for
C30H51N2O5S4 ([M+H]+) 647.2681; found 647.2693.
-S32 -
(49) Synthesis of N6-(3,4-bis(tert-butyldisulfanyl)benzoyl)-L-lysine trifluoroacetate
(S37)
S37
To a cooled (0 ºC) solution of S36 (324 mg, 0.5 mmol) in anhydrous DCM (2 ml) was
added TFA (2 ml). The reaction mixture was allowed to warm to RT and stirred for
additional 6 h. The volatiles were removed by rotary evaporation to dryness, and the
resulting precipitate was rinsed with Et2O three times, dried in vacuo to give S37 as a
waxy solid (233 mg, 77% yield). 1H NMR (400 MHz, CD3OD) δ 1.31 (s, 9H), 1.32 (s,
9H), 1.54 (m, 2H), 1.69 (m, 2H), 1.89-2.05 (m, 2H), 3.41 (m, 2H), 3.98 (t, J = 6.4 Hz,
1H), 7.66 (dd, J = 8.4, 2.0 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 8.26 (d, J = 2.0 Hz, 1H).
13
C NMR (100 MHz, DMSO-d6) δ 22.09, 28.79, 29.56, 29.63, 29.99, 39.20, 49.87,
50.08, 52.23, 112.08, 115.00, 117.92, 120.86, 126.48, 127.32, 128.81, 133.67, 136.51,
141.08, 158.52, 158.86, 159.21, 159.55, 165.25, 171.26. HRMS (ESI+): m/z calcd for
C21H35N2O3S4 ([M+H]+) 491.1531; found 491.1535.
-S33 -
Scheme S7. Synthesis of TP10 analogue (7)
Initially, the MBHA resin (S40) was swelled with DCM/DMF (1:1) in a screw-cap glass
peptide synthesis reaction vessels about 3 h. The Fmoc protecting group was removed
with 20% piperidine in DMF (15 min and 110 min). After that, the resin was washed
with DMF (3 times), DCM (3 times) and DMF (3 times), respectively. Fmoc-Amino
acid residues were coupled by a preactivated solution (4 equiv Fmoc-amino acid, 4
equiv HATU, 4 equiv HOAt, 8 equiv DIPEA). After 2 h, the resin was washed with
DMF (3 times), DCM (3 times) and DMF (3 times), respectively. Couplings were
checked by ninhydrin test. Cleavage reagent: A mixture of 95% TFA, 2.5% water and
2.5% TIPS was added. After 2.5 h, the solution was filtered, and the filtrate was
concentrated under reduced pressure. The crude peptide was obtained by precipitation
in cold diethyl ether, isolated by centrifugation, washed with cold diethyl ether,
dissolved in ACN/H2O (3:7, v/v), purified by preparative HPLC and analyzed by ESI-
MS. The solution was then lyophilized to obtain the tBuS-protected peptide S41 as a
white solid.
-S34 -
Figure S1. RP-HPLC of S41. (The gradient was programmed as follows: 0-25 min:
30%-95% B, 25-30 min: 95%-100% B. 100% Water with 0.06% TFA (solvent A) and
100% ACN with 0.06% TFA (solvent B), were used as the mobile phase at a flow rate
of 1.0 mL min-1. Monitoring wavelength λ = 214 nm)
Figure S3. RP-HPLC of 7. (The gradient was programmed as follows: 0-25 min: 30%-
95% B, 25-30 min: 95%-100% B. Solution of 100% water with 0.06% TFA (solvent A)
and 100% ACN with 0.06% TFA (solvent B) was used as the mobile phase at a flow
rate of 1.0 mL min-1. Monitoring wavelength λ = 214 nm)
-S35 -
Figure S4. ESI-MS spectrum of 7. Theoretical molecular weight: 2350.26. Calculated
molecular weight: 2350.380.2.
S38
EDCI (380 mg, 2.0 mmol) and DIPEA (340 uL, 2.0 mmol) were successively added to
a solution of S24 (600 mg, 1.65 mmol) in anhydrous DCM (80 mL), and the mixture
was stirred at RT under argon atmosphere for 10 min. Following this time, NHS (196
mg, 1.7 mmol) was added and then the mixture was stirred at RT under argon
atmosphere for 6 h. The reaction mixture was purified by column chromatography on
silica gel (PE/EtOAc = 3:1, v/v) to afford S38 as a white solid (493 mg, 65% yield). 1H
NMR (400 MHz, CDCl3) δ 1.33 (s, 18H), 2.91 (s, 4H), 7.91 (dd, J = 8.4, 1.6 Hz, 1H),
7.96 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 1.6 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 25.80,
29.97, 50.10, 50.47, 123.06, 126.89, 128.43, 130.59, 137.88, 147.09, 161.44, 169.33.
S39
To a solution of Fmoc-Lys-OH (405 mg, 1.1 mmol) in anhydrous DMF (15 mL), S38
(460 mg, 1.0 mmol) and DIPEA (340 uL, 2.0 mmol) were added, and the mixture was
stirred overnight at RT under argon atmosphere. The reaction mixture was evaporated
under reduced pressure, and the residue was dissolved in EtOAc, washed successively
with 5% citric acid, H2O and saturated aqueous NaCl, dried over anhydrous MgSO4,
filtered, and the filtrate was concentrated under reduced pressure. The residue was
-S36 -
purified by column chromatography on silica gel (DCM/MeOH/AcOH = 98:2:0.5,
v/v/v) to afford S39 as a white solid (600 mg, 84% yield). 1H NMR (600 MHz, DMSO-
d6) δ 1.26 (s, 18H), 1.34-1.76 (m, 6H), 3.23 (q, J = 6.6 Hz, 2H), 3.86 (s, 1H), 4.18-4.26
(m, 3H), 7.16 (s, 1H), 7.30 (m, 2H), 7.39 (t, J = 6.6 Hz, 2H), 7.69 (d, J = 7.8 Hz, 2H),
7.79 (m, 2H), 7.87 (d, J = 7.8 Hz, 2H), 8.23 (s, 1H), δ 8.61 (t, J = 6.0 Hz, 1H). 13C NMR
(150 MHz, DMSO-d6) δ 23.49, 29.62, 29.91, 29.96, 30.01, 32.87, 47.44, 50.09, 50.33,
55.89, 65.91, 79.75, 120.50, 125.67, 125.76, 126.79, 127.53, 127.70, 128.04, 129.20,
134.00, 134.14, 136.75, 136.87, 141.26, 144.30, 144.54, 156.13, 165.41, 165.46,
177.89. HRMS (ESI+): m/z calcd for C36H45N2O5S4 ([M+H]+) 713.2211; found
713.2216.
S43
Prepared following the procedure reported.[14] To a solution of D-biotin (S42) (489 mg,
2 mmol) in DMF (15 mL) was added NHS (253 mg, 2.2 mmol) and EDCI (460 mg, 2.4
mmol). After being stirred for 24 h at RT, the reaction solution was concentrated under
reduced pressure to remove DMF. The white solid was washed with methanol three
times, and excess solvent was removed in vacuo to afford biotin-NHS (S43) (572 g, 84%
-S37 -
yield), that was used directly in next step without further purification. 1H NMR (400
MHz, DMSO-d6) δ 1.38-1.68 (m, 6H), 2.58 (d, J = 12.4 Hz, 1H), 2.67 (t, J = 7.6 Hz,
2H), 2.80-2.85 (m, 5H), 3.10 (m, 1H), 4.15 (m, 1H), 4.30 (m, 1H), 6.38 (s, 1H), 6.45 (s,
1H). 13C NMR (150 MHz, DMSO-d6) δ 24.36, 25.50, 27.65, 27.89, 30.04, 39.98, 55.31,
59.23, 61.06, 162.80, 169.01, 170.36.
S45
S24 (362 mg, 1.0 mmol), S44 (174 mg, 1.0 mmol), HATU (760 mg, 2.0 mmol) and
DIPEA (170 L, 1.0 mmol) were mixed in DMF (6 mL), and the mixture was stirred
overnight at RT The resulting solution was concentrated under reduced pressure, and
the residue was dissolved in EtOAc. The solution was washed successively with H2O,
saturated aqueous Na2CO3 and brine, dried over anhydrous MgSO4, filtered, and the
filtrate was concentrated under reduced pressure. The residue was purified by column
chromatography on silica gel (PE/EtOAc = 3:1, v/v) to afford S45 as a white solid (440
mg, 85% yield). 1H NMR (400 MHz, CDCl3) δ 1.30 (s, 18H), 1.44 (s, 9H), 1.69 (m,
2H), 3.23 (q, J = 6.0 Hz, 2H), 3.48 (q, J = 6.0 Hz, 2H), 4.97 (t, J = 6.8 Hz, 1H), 7.43 (t,
J = 6.4 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 8.24 (s, 1H). 13C
NMR (101 MHz, CDCl3) δ 28.53, 29.99, 29.99, 30.35, 36.12, 37.06, 49.82, 50.00,
79.66, 125.60, 127.56, 127.65, 132.95, 137.49, 142.13, 157.14, 166.61. HRMS (ESI+):
m/z calcd for C23H39N2O3S4 ([M+H]+) 519.1844; found 519.1852.
S46
To a cooled (0 ºC) solution of S45 (519 mg, 1.0 mmol) in anhydrous DCM (4 ml) was
added TFA (2 ml). The reaction mixture was allowed to warm to RT and stirred for
additional 4 h. The volatiles were removed by rotary evaporation to dryness. The
-S38 -
residue was partitioned with EtOAc and saturated aqueous Na2CO3. The organic layer
was collected, and the aqueous layer was extracted twice with EtOAc. The combined
organic phase was dried over MgSO4, filtered, and concentrated under reduced
pressure. The crude was purified by flash chromatography on silica gel (DCM/MeOH
= 85:15, v/v) to afford S46 as a white solid (381 mg, 91% yield). 1H NMR (400 MHz,
CDCl3) δ 1.29 (s, 18H), 1.80 (s, 2H), 2.96 (s, 2H), 3.26 (br s, 2H), 3.56 (q, J = 6.0 Hz,
2H), 7.62 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 5.6 Hz, 1H), 8.19
(s, 1H). 13C NMR (101 MHz, CDCl3) δ 29.96, 29.98, 39.26, 39.40, 40.31, 49.83, 50.00,
125.61, 127.46, 127.67, 132.88, 137.39, 142.03, 166.65. HRMS (ESI+): m/z calcd for
C18H31N2OS4 ([M+H]+) 419.1319; found 419.1310.
8
To a mixture of S46 (147 mg, 0.35 mmol) and TEA (41 L, 0.3 mmol) in DMF (10 mL)
was added biotin-NHS (S43) (102 mg, 0.3 mmol). The reaction mixture was stirred at
RT for 4 h, and the solution was concentrated under reduced pressure. The residue was
dissolved in CHCl3, and the solution was washed with H2O and brine, dried by MgSO4,
and evaporated under reduced pressure. The crude product was purified by column
chromatography on silica on gel (DCM/MeOH = 9:1, v/v) to afford 8 as a white solid
(165 mg, 85% yield). 1H NMR (600 MHz, DMSO-d6) δ 1.31 (s, 18H), 1.43 (m, 2H),
1.65-1.75 (m, 6H), 2.24 (t, J = 7.2 Hz, 2H), 2.70 (d, J = 12.6 Hz, 1H), 2.89 (m, 1H),
3.13 (m, 1H), 3.29-3.37 (m, 2H), 3.46 (q, J = 6.0 Hz, 2H), 4.30 (m, 1H), 4.50 (m, 1H),
5.71 (s, 1H), 6.69 (s, 1H), 6.92 (t, J = 6.0 Hz, 1H), 7.53 (t, J = 6.0 Hz, 1H), 7.65 (dd, J
= 8.4, 1.8 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 8.26 (d, J = 1.8 Hz, 1H). 13C NMR (150
MHz, DMSO-d6) δ 25.33, 28.04, 28.23, 29.25, 29.38, 29.44, 35.27, 36.30, 37.11, 39.87,
49.81, 50.03, 55.43, 59.21, 61.04, 126.29, 127.33, 128.37, 133.46, 136.27, 140.76,
162.73, 164.90, 172.11. HRMS (ESI+): m/z calcd for C28H45N4O3S5 ([M+H]+)
645.2095; found 645.2090.
-S39 -
(57) Synthesis of 3,4-bis(tert-butyldisulfanyl)-N-(3-(3-(3',6'-dihydroxy-3-oxo-3H-
spiro[isobenzofuran-1,9'-xanthen]-5-yl)thioureido)propyl)benzamide (9)
9
S46 (84 mg, 0.2 mmol), fluorescein-5-isothiocyanate (5-FITC, 78 mg, 0.1 mmol) and
K2CO3 (56 mg, 0.4 mmol) were dissolved in anhydrous MeOH (6 mL), and the solution
was stirred at RT for 24 h under argon atmosphere and then adjusted pH to 5-6 with
HCl (0.5 M). The resulting mixture was evaporated under reduced pressure, and the
residue was purified by column chromatography on silica gel (DCM/MeOH = 9:1) to
afford 9 as an orange solid (117 mg, 72%). 1H NMR (600 MHz, DMSO-d6) δ 1.28 (s,
9H), 1.28 (s, 9H), 1.83 (m, 2H), 3.31-3.35 (m, 2H), 3.59 (s, 2H), 6.55 (dd, J = 8.4, 2.4
Hz, 2H), 6.61 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 2.4 Hz, 2H), 7.18 (d, J = 8.4 Hz, 1H),
7.74 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.4Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 8.15 (br s,
1H), 8.23 (s, 1H), 8.24 (s, 1H), 8.65 (t, J = 5.4 Hz, 1H), 10.04 (s, 1H), 10.13 (s, 2H).
13
C NMR (150 MHz, DMSO-d6) δ 28.75, 29.12, 29.42, 29.49, 37.03, 41.62, 49.81,
50.02, 83.69, 102.33, 109.85, 112.72, 116.83, 124.17, 126.39, 126.82, 127.33, 128.48,
129.09, 129.60, 133.43, 136.37, 140.95, 141.34, 147.02, 152.02, 159.69, 165.30,
168.59, 180.52. HRMS (ESI+): m/z calcd for C39H42N3O6S5 ([M+H]+) 808.1677; found
808.1675.
-S40 -
Scheme S9. Synthesis of compounds S49 and S52
S47
Compound S47 was prepared following the procedures described[15]. To a suspension
of D-biotin (1.17 g, 4.8 mmol) in a mixture of CH3CN/MeOH (50 mL, 7/3) were added
S38 (1.06 g, 4.8 mmol) and EDCI (1.11 g, 7.2 mmol). After stirring overnight at RT,
the clear solution was evaporated to dryness. The residue was partitioned with DCM
and H2O. The aqueous layer was extracted with extracted with DCM, and the combined
organic phase was washed with brine, dried over anhydrous MgSO4, and filtered. The
filtrate was concentrated under reduced pressure, and the crude product was purified by
column chromatography on silica gel (DCM/MeOH = 95:5, v/v) to afford S47 as a
white solid (1.7 g, 78% yield). 1H NMR (600 MHz, DMSO-d6) δ1.28-1.68 (m, 6H),
2.10 (t, J = 7.2 Hz, 2H), 2.58 (d, J = 12.6 Hz, 1H), 2.82 (q, J = 4.8 Hz, 1H), 3.13 (m,
1H), 3.22 (t, J = 6.0 Hz, 2H), 3.43 (t, J = 5.4 Hz, 4H), 3.53-3.61 (m, 8H), 3.64 (t, J =
5.4 Hz, 2H), 4.17 (m, 1H), 4.34 (m, 1H), 6.39 (s, 1H), 6.45 (s, 1H), 7.86 (t, J = 5.4 Hz,
1H). 13C NMR (100 MHz, CDCl3) δ 25.60, 28.05, 28.31, 35.93, 39.05, 40.44, 50.58,
55.74, 60.19, 61.72, 69.87, 69.92, 69.97, 70.36, 70.36, 70.54, 164.37, 173.39. HRMS
(ESI+): m/z calcd for C18H33N6O5S ([M+H]+) 445.2233; found 445.2228.
-S41 -
(59) Synthesis of N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-5-
((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (S48)
S48
To a solution of S47 (445 mg, 1.0 mmol) in THF/H2O (18 mL, 7/2, v/v) was added
PPh3 (315 mg, 1.2 mmol), and the mixture was stirred at RT for 18 h under argon
atmosphere. The solvent was evaporated under reduced pressure, and the residue was
washed with Et2O (340 mL) and dried in vacuo to give S48 as a pale white solid (388
mg, 87%). Without further purification, the crude product was used directly in the next
step.
S49
To an ice-cold solution of S48 (398 mg, 0.95 mmol) in anhydrous DMF (5 mL) were
added 2,3-dichloro-6-quinoxalinecarbonyl chloride (S5) (248 mg, 0.95 mmol) and
DIPEA (170 L, 1.0 mmol). The reaction mixture was allowed to warm to RT and
stirred for additional 4 h. The solution was evaporated to dryness, and the residue was
partitioned with CHCl3 and H2O. The aqueous layer was extracted with CHCl3, and the
combined organic phase was washed successively saturated aqueous NaHCO3, H2O and
brine, dried over anhydrous MgSO4 and concentrated under reduced pressure The
residue was purified by column chromatography on silica gel (DCM/MeOH = 95:5, v/v)
to afford S49 as a white solid (378 mg, 62% yield). 1H NMR (600 MHz, CDCl3) δ 1.39
(m, 2H), 1.1.59-1.77 (m, 4H), 2.18 (t, J =12.6 Hz, 2H), 2.73 (d, J = 12.6 Hz, 1H), 2.87
(m, 1H), 3.10 (m, 1H), 3.35-3.43 (m, 2H), 3.53 (m, 2H), 3.59-3.75 (m, 12H), 4.27 (m,
1H), 4.48 (m, 1H), 5.68 (s, 1H), 6.67 (s, 1H), 6.72 (t, J = 5.4 Hz, 1H), 7.86 (t, J = 5.4
Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 8.32 (dd J = 8.4, 1.8 Hz, 1H), 8.51 (d, J = 1.8 Hz,
1H). 13C NMR (150 MHz, CDCl3) δ 25.64, 28.10, 28.30, 35.95, 39.13, 40.20, 40.49,
55.74, 60.27, 61.77, 69.65, 69.92, 70.00, 70.22, 70.34, 70.35, 127.07, 128.38, 130.17,
137.07, 139.92, 141.60, 146.19, 146.69, 164.41, 165.82, 173.46. HRMS (ESI+): m/z
-S42 -
calcd for C27H37Cl2N6O6S ([M+H]+) 643.1872; found 643.1868.
S50
To an ice-cold solution of 7 (2.62 g, 10 mmol) in anhydrous THF (50 mL) were added
S44 (1.74 mg, 10 mmol) and DIPEA (340 L, 2.0 mmol). The reaction mixture was
allowed to warm to RT and stirred overnight. The solution was evaporated to dryness,
and the residue was partitioned between saturated aqueous NaHCO3 and EtOAc. After
isolation of the organic layer, the aqueous layer was extracted with two additional
portions of EtOAc. The combined organic phase was then washed with H2O and brine,
dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (PE/EtOAc = 95:5, v/v) to afford S50
as a white solid (3.51 g, 88% yield). 1H NMR (400 MHz, CDCl3) δ 1.46 (s, 9H), 1.76
(m, 2H), 3.29 (q, J = 6.4 Hz, 2H), 3.56 (q, J = 6.4 Hz, 2H), 4.90 (t, J = 6.4 Hz, 1H),
7.87 (s, 1H), 8.07 (d, J = 8.8 Hz, 1H), 8.29 (dd, J = 8.8, 1.6 Hz, 1H), 8.50 (d, J = 1.6
13
Hz, 1H). C NMR (100 MHz, CDCl3) δ 28.47, 30.12, 36.63, 37.18, 79.82, 127.15,
128.50, 129.75, 137.19, 140.02, 141.58, 146.30, 146.77, 157.26, 165.83. HRMS (ESI+):
m/z calcd for C17H20Cl2N4NaO3 ([M+Na]+) 421.0810; found 421.0799.
S51
To a cooled (0 ºC) solution of S50 (599 mg, 1.5 mmol) in anhydrous DCM (4 mL) was
added TFA (2 mL). The reaction mixture was allowed to warm to RT and stirred for
additional 2 h. The volatiles were removed under reduced pressure to give crude
ammonium trifluoroacetate salt, that was partitioned with EtOAc and saturated aqueous
NaHCO3. The organic layer was collected, and the aqueous layer was extracted twice
with EtOAc. The combined organic phase was dried over MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by flash
-S43 -
chromatography on silica gel (DCM/MeOH = 85:15, v/v) to afford S51 as a white solid
(341 mg, 76% yield). 1H NMR (400 MHz, DMSO-d6) δ 1.87 (m, 2H), 2.90 (t, J = 6.8
Hz, 2H), 3.41 (q, J = 6.8 Hz, 2H), 7.87 (s, 2H), 8.16 (d, J = 8.8 Hz, 1H), 8.32 (dd, J =
8.8, 2.0 Hz, 1H), 8.54 (d, J = 2.0 Hz, 1H), 9.07 (t, J = 5.2 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6) δ 27.32, 36.78, 36.97, 126.82, 128.00, 129.98, 136.55, 139.46, 141.06,
145.57, 146.03, 164.87. HRMS (ESI+): m/z calcd for C12H13Cl2N4O ([M+H]+)
299.0466; found 299.0464.
S52
S51 (60 mg, 0.2 mmol), 5-FITC (78 mg, 0.2 mmol) and K2CO3 (56 mg, 0.4 mmol) was
dissolved in anhydrous MeOH (6 mL), and the solution was stirred at RT for 24 h under
argon atmosphere and adjusted pH to 5-6 with HCl (1N). The resulting solution was
evaporated under reduced pressure, and the residue was purified by column
chromatography on silica gel (DCM/MeOH = 9:1) to afford S52 as an orange solid (89
mg, 65%). 1H NMR (600 MHz, DMSO-d6) δ 1.89 (m, 2H), 3.42 (q, J = 6.6 Hz, 2H),
3.62 (s, 2H), 6.55 (dd, J = 9.0, 2.4 Hz, 2H), 6.60 (d, J =9.0 Hz, 2H), 6.67 (d, J = 2.4 Hz,
2H), 7.74 (d, J = 8.4 Hz, 1H), 8.14 (br s, 1H), 8.16 (d, J = 8.4 Hz, 1H), 8.21 (s, 1H),
8.32 (dd, J = 8.4, 1.8 Hz, 1H), 8.55 (d, J = 1.8 Hz, 1H), 8.98 (t, J = 5.4 Hz, 1H), 10.02
(s, 1H), 10.12 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 29.11, 37.82, 42.15, 83.56,
102.78, 110.25, 113.10, 117.17, 124.63, 127.11, 127.25, 128.58, 129.56, 130.16, 130.52,
137.32, 140.06, 141.63, 141.81, 146.17, 146.59, 147.78, 152.41, 159.99, 165.38, 169.06,
181.02. HRMS (ESI+): m/z calcd for C33H24Cl2N5O6S ([M+H]+) 688.0824; found
688.0823.
-S44 -
Scheme S10. Synthesis of compounds S55 and S58
S54
Prepared according to the procedures reported.[16] β-Alanine (S53) (2.5 g, 28 mmol)
and maleic anhydride (2.8 g, 28 mmol) were dissolved in 50 mL of anhydrous DMF,
and the solution was stirred at RT for 2 h. Following this time, NHS (4.0 g, 35 mmol)
and EDCI (11.6 g, 56 mmol) were added successively. After 36 h stirring at RT, the
reaction mixture was evaporated to dryness. The residue was partitioned with DCM and
H2O. After isolation of the organic layer, the aqueous layer was extracted with two
additional portions of DCM. The combined organic phase was then washed
successively with saturated NaHCO3 and saturated aqueous NaCl. The resulting organic
solution was dried over MgSO4, filtered, and concentrated under reduced pressure.
Purification of the crude by chromatography on silica gel (DCM/acetone = 9/1, v/v)
furnished S54 as a white powder (5.44 mg, 73% yield). 1H NMR (400 MHz, CDCl3) δ
2.81 (s, 4H), 3.01 (t, J = 7.2 Hz, 2H), 3.93 (t, J = 7.2 Hz, 2H), 6.73 (s, 2H). 13C NMR
(100 MHz, CDCl3) δ 25.68, 29.86, 33.12, 134.43, 166.11, 168.85, 170.20.
S55
Activated ester S54 (160 mg, 0.6 mmol) was added to a solution of S51 (180 mg, 0.6
-S45 -
mmol) in DMF (10 mL). The reaction mixture was stirred for overnight at RT under
argon atmosphere. The solvent was concentrated under reduced pressure, and the
residue was partitioned with EtOAc and H2O. The organic layer was collected, and the
aqueous layer was extracted with additional two portions of EtOAc. The combined
organic phase was washed with saturated aqueous NaCl, dried over MgSO4, filtered,
and concentrated. The residue was purified by column chromatography on silica gel
(DCM/MeOH = 95:5 v/v) to afford S55 as a white solid (121 mg, 45% yield). 1H NMR
(400 MHz, CDCl3) δ 1.77 (m, 2H), 2.60 (t, J = 6.8 Hz, 2H), 3.40 (q, J = 6.4 Hz, 2H),
3.50 (q, J = 6.4 Hz, 2H), 3.88 (t, J = 6.8 Hz, 2H), 6.08 (t, J = 6.4 Hz, 1H), 6.74 (s, 2H),
7.77 (t, J = 6.4 Hz, 1H), 8.10 (d, J = 8.8 Hz, 1H), 8.31 (dd, J = 8.8, 2.0 Hz, 1H), 8.52
(d, J = 2.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 28.91, 34.08, 34.11, 36.37, 37.32,
126.62, 128.02, 129.95, 134.55, 136.82, 139.52, 141.06, 145.59, 146.01, 164.54, 169.42,
170.74. HRMS (ESI+): m/z calcd for C19H18Cl2N5O4 ([M+H]+) 450.0736; found
450.0737.
S56
S50 (799 mg, 2.0 mmol), imidazole (136 mg, 2.0 mmol) and K2CO3 (552 mg, 4.0 mmol)
were dissolved in DMF (20 mL), and the solution was stirred at RT for 1 h. The solution
was concentrated under reduced pressure, and the residue was partitioned with EtOAc
and H2O. The organic layer was collected and the aqueous was extracted with two
additional portions of EtOAc. The combined organic phase was washed saturated
aqueous NaCl, dried over MgSO4, filtered, and concentrated. The residue was purified
by column chromatography on silica gel (DCM/MeOH = 95:5, v/v) to afford S56 as a
white solid (487 g, 57% yield). 1H NMR (600 MHz, CDCl3) δ 1.47 (s, 9H), 1.77 (m,
2H), 3.30 (q, J = 6.0 Hz, 2H), 3.58 (q, J = 6.0 Hz, 2H), 4.87 (s, 1H), 7.27 (s, 1H), 7.83
(s, 1H), 7.89 (s, 1H), 8.12 (d, J = 9.0 Hz, 1H), 8.34 (d, J = 9.0 Hz, 1H), 8.46 (s, 1H),
8.56 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 28.44, 30.04, 36.67, 37.21, 79.70, 119.39,
-S46 -
127.04, 128.73, 130.15, 130.20, 137.25, 137.55, 140.20, 140.24, 140.60, 141.88, 157.19,
165.80. HRMS (ESI+): m/z calcd for C20H24ClN6O3 ([M+H]+) 431.1598; found
431.1596.
S57
To a cooled (0 ºC) solution of S56 (431 mg, 1.0 mmol) in DCM (4 mL) was added TFA
(2 mL). The reaction mixture was allowed to warm to RT and stirred for additional 3 h.
The volatiles were removed under reduced pressure to give crude ammonium
trifluoroacetate salt, and the salt was added to saturated aqueous NaHCO3, and the
solution was extracted with three portions of iPrOH/DCM (2/1, v/v). The combined
organic phase was washed saturated aqueous NaCl, dried over MgSO4, filtered, and
concentrated under reduced pressure to to afford crude product (S57) as an off white
solid (265 mg, 80% yield, Rf 0.4 in DCM/MeOH 85:15) that was directly used in the
next step without further purification.
S58
Activated ester S54 (133 mg, 0.5 mmol) was added to a solution of S57 (165 mg, 0.5
mmol) in DMF (6 mL). The reaction mixture was stirred for overnight at room under
argon atmosphere. The solution was concentrated under reduced pressure, and the
residue was partitioned with EtOAc and H2O. The organic layer was collected, and the
aqueous layer was extracted with additional two portions of EtOAc. The combined
organic phase was washed with saturated aqueous NaCl, dried over MgSO4, filtered,
and concentrated. The crude product was purified by column chromatography on silica
gel (DCM/MeOH = 92:8 v/v) to afford S58 as a white solid (123 mg, 51% yield). 1H
-S47 -
NMR (600 MHz, DMSO-d6) δ 1.69 (m, 2H), 2.35 (t, J = 7.2 Hz, 2H), 3.11 (q, J = 6.6
Hz, 2H), 3.31 (m, 2H), 3.62 (t, J = 6.6 Hz, 2H), 7.01 (s, 2H), 7.20 (s, 1H), 7.86 (s, 1H),
8.02 (t, J = 5.4 Hz, 1H), 8.20 (d, J = 9.0 Hz, 1H), 8.33 (dd, J = 9.0, 1.8 Hz, 1H), 8.37
(s, 1H), 8.58 (d, J = 1.8 Hz, 1H), 8.88 (t, J = 5.4 Hz, 1H). 13C NMR (100 MHz, DMSO-
d6) δ 28.95, 34.15, 34.15, 36.46, 37.39, 120.30, 126.50, 128.46, 129.21, 129.96, 134.55,
136.65, 137.94, 139.74, 140.13, 142.07, 142.78, 164.67, 169.54, 170.78. HRMS (ESI+):
m/z calcd for C22H21ClN7O4 ([M+H]+) 482.1344; found 482.1338.
S60
Prepared according to the procedures described in literature.[17] Trityl chloride (2.79 g,
10 mmol) and 4-mercaptobenzoic acid (S59) (1.54 g, 10 mmol) were dissolved in DMF
(30 mL) and stirred overnight at RT under argon atmosphere. After removal of the
solvent, the residue was diluted with CHCl3, washed with H2O, dried with MgSO4, and
concentrated under reduced pressure. The residue was purified by column
chromatography on silica gel (DCM/acetone = 96/4, v/v) to give S60 as a white solid
(3.29 g, 83% yield). 1H NMR (400 MHz, CDCl3) δ 6.94 (d, J = 7.2 Hz, 2H), 7.22 (m,
9H), 7.38 (d, J = 6.8 Hz, 6H), 7.61 (d, J = 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-
d6) δ 70.53, 127.07, 127.99, 128.94, 129.43, 131.37, 140.05, 143.56, 167.15.
S62
Prepared according to similar procedures described in literature.[17] S61 (1.73 mg, 4.0
mmol) was dissolved in a solution of TFA/DCM/TIPS (80 mL, 50/45/5, v/v/v), and the
solution was stirred at RT for 3 h under argon atmosphere. After the completion of the
reaction, the reaction mixture was concentrated under reduced pressure. The residue
was diluted with EtOAc, washed with H2O, dried over MgSO4, filtered, evaporated
under reduced pressure. The residue was purified by column chromatography on silica
gel (DCM/MeOH = 99/1, v/v) to give S62 as a white solid (550 mg, 72% yield). 1H
NMR (400 MHz, CDCl3) δ 2.28 (t, J = 2.8 Hz, 1H), 3.59 (s, 1H), 4.24 (dd, J =5.6, 2.8
Hz, 2H), 6.26 (s, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.4 Hz, 2H). 13C NMR (100
-S49 -
MHz, CDCDl3) δ 29.91, 72.05, 79.57, 127.90, 128.63, 130.73, 136.86, 166.55.
S63
To a solution of S50 (479 mg, 1.2 mmol) in DMF (10 mL) were added S62 (229 mg,
1.2 mmol) and K2CO3 (331 mg, 2.4 mmol). The reaction mixture was stirred at RT for
6 h under argon atmosphere, concentrated under reduced pressure, and the residue was
then partitioned between H2O and EtOAc. The organic layer was collected, and the
aqueous layer was extracted with extracted with two additional portions of EtOAc. The
combined organic phase was washed with H2O and brine, dried over anhydrous MgSO4,
and filtered. The filtrate was concentrated under reduced pressure and the crude product
was purified by column chromatography on silica gel (DCM/MeOH = 97:3, v/v) to
afford S63 as a light white solid (438 mg, 66% yield). 1H NMR (600 MHz, DMSO-d6)
δ 1.36 (s, 9H), 1.66 (m, 2H), 3.00 (q, J = 6.6 Hz, 2H), 3.17 (t, J = 2.4 Hz, 1H), 3.30 (q,
J = 6.6 Hz, 2H), 4.11 (dd, J = 5.4, 2.4 Hz, 2H), 6.84 (t, J = 6.0 Hz, 1H), 7.72 (d, J =8.4
Hz, 1H), 7.78 (d, J = 8.4 Hz, 2H), 8.00 (d, J = 8.4 Hz, 2H), 8.14 (dd, J =8.4, 1.8 Hz,
1H), 8.44 (d, J = 1.8 Hz, 1H), 8.77 (t, J = 5.4 Hz, 1H), 9.14 (t, J = 5.4 Hz, 1H). 13C
NMR (100 MHz, DMSO-d6) δ 28.28, 28.65, 29.44, 37.31, 37.76, 73.05, 77.53, 81.18,
126.76, 127.54, 128.33, 129.37, 131.05, 134.96, 135.00, 138.64, 141.50, 144.39, 155.62,
164.69, 165.26. HRMS (ESI+): m/z calcd for C27H29ClN5O4S ([M+H]+) 554.1629;
found 554.1633.
S64
To a cooled (0 ºC) solution of S63 (305 mg, 0.55 mmol) in DCM (4 mL) was added
TFA (2 mL). The reaction mixture was allowed to warm to RT and stirred for additional
3 h. The volatiles were removed under reduced pressure to give crude ammonium
trifluoroacetate salt that was added saturated aqueous NaHCO3, and the solution was
-S50 -
extracted with three portions of iPrOH/DCM (2/1, v/v). The combined organic phase
was washed saturated aqueous NaCl, dried over MgSO4, filtered, and concentrated
under reduced pressure to to afford crude product (S64) as a faint yellow solid (204 mg,
82% yield, Rf = 0.3 in DCM/MeOH 88:12) that was directly used in the next step
without further purification.
S65
S54 (133 mg, 0.5 mmol) was added to a solution of S64 (204 mg, 0.45 mmol) in DMF
(6 mL). The reaction mixture was stirred for overnight at room under argon atmosphere.
The solvent was concentrated under reduced pressure, and the residue was partitioned
with EtOAc and H2O. The organic layer was collected, and the aqueous layer was
extracted with additional two portions of EtOAc. The combined organic phase was
washed with saturated aqueous NaCl, dried over MgSO4, filtered, and concentrated.
The crude product was purified by column chromatography on silica gel (DCM/MeOH
= 95:5, v/v) to afford S65 as a white solid (118 mg, 43% yield). 1H NMR (400 MHz,
DMSO-d6) δ 1.65 (m, 2H), 2.33 (t, J = 7.2 Hz, 2H), 3.08 (q, J = 6.8 Hz, 2H), 3.17 (t, J
= 2.4 Hz, 1H), 3.29 (q, J = 6.8 Hz, 2H), 3.60 (t, J = 7.2 Hz, 2H), 4.10 (dd, J = 5.6, 2.4
Hz, 2H), 7.00 (s, 2H), 7.74 (d, J = 8.8 Hz, 1H), 7.80 (d, J = 8.4 Hz, 2H), 8.00 (m, 3H),
8.15 (dd, J = 8.8, 2.0 Hz, 1H), 8.45 (d, J = 2.0 Hz, 1H), 8.77 (t, J = 5.6 Hz, 1H), 9.13
(t, J = 5.6 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ 29.16, 29.47, 34.63, 36.94, 37.82,
73.53, 81.68, 127.25, 128.04, 128.82, 129.86, 131.56, 135.06, 135.48, 135.56, 139.14,
142.01, 144.90, 156.09, 165.20, 165.79, 169.96, 171.27. HRMS (ESI+): m/z calcd for
C29H26ClN6O5S ([M+H]+) 605.1374; found 605.1370.
-S51 -
S66
CuI (4.0 mg, 0.02 mmol) and TEA (6.0 L, 0.04 mmol) were added to a solution of
S65 (60.5 mg, 0.1 mmol) and S47 (44.5 mg, 0.1 mmol) in DMF (5 mL), and the mixture
was stirred at RT for overnight under argon atmosphere. The reaction was concentrated
under reduced pressure, and the residue was purified by purified by column
chromatography on silica gel (DCM/MeOH = 88:12, v/v) to afford S66 as a white solid
(70 mg, 67% yield). 1H NMR (600 MHz, DMSO-d6) δ 1.23-1.60 (m, 6H), 1.65 (m, 2H),
2.04 (t, J = 7.2 Hz, 2H), 2.33 (t, J = 7.2 Hz, 2H), 2.56 (d, J = 12.6 Hz, 1H), 2.80 (dd, J
= 12.6, 5.4 Hz, 1H), 3.08 (m, 3H), 3.16 (m, 2H), 3.29 (q, J = 6.6 Hz, 2H), 3.36 (m, 2H),
3.45-3.47 (m, 6H), 3.52 (m, 2H), 3.60 (t, J = 7.2 Hz, 2H), 3.81 (t, J = 5.4 Hz, 2H), 4.13
(m, 1H), 4.29 (m, 1H), 4.51 (t, J = 5.4 Hz, 2H), 4.55 (d, J = 6.0 Hz, 2H), 6.36 (s, 1H),
6.42 (s, 1H), 7.00 (s, 2H), 7.73 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 7.8 Hz, 2H), 7.82 (t, J
= 5.4 Hz, 1H), 8.00 (m, 2H), 8.02 (d, J =7.8 Hz, 2H), 8.15 (d, J = 8.4 Hz, 1H), 8.45 (s,
1H), 8.77 (t, J = 5.4 Hz, 1H), 9.23 (t, J = 5.4 Hz, 1H). 13C NMR (150 MHz, DMSO-d6)
δ 25.80, 28.57, 28.73, 29.47, 34.64, 35.49, 35.64, 36.95, 37.83, 38.99, 49.87, 55.97,
59.77, 61.57, 61.62, 69.30, 69.68, 70.08, 70.19, 70.24, 123.95, 127.28, 128.02, 128.85,
129.89, 131.36, 135.06, 135.41, 135.58, 135.83, 139.15, 142.03, 144.93, 145.31, 156.13,
163.31, 165.24, 165.97, 170.01, 171.28, 172.73. HRMS (ESI+): m/z calcd for
C47H58ClN12O10S2 ([M+H]+) 1049.3529; found 1049.3532.
-S52 -
(76) Synthesis of tert-butyl (3-(3-chloro-2-(4-
(hydroxymethyl)phenoxy)quinoxaline-6-carboxamido)propyl)carbamate (S67)
S67
S50 (1.6 g, 4.0 mmol), p-hydroxybenzyl alcohol (497 mg, 4.0 mmol) and K2CO3 (1.1
g, 8.0 mmol) were dissolved in DMF (20 mL), and the solution was stirred for overnight
at RT under argon. The solution was concentrated under reduced pressure, and the
residue was partitioned with EtOAc and H2O. The organic layer was collected, and the
aqueous was extracted with two additional portions of EtOAc. The combined organic
phase was washed saturated aqueous NaCl, dried over MgSO4, filtered, and
concentrated. The residue was purified by column chromatography on silica gel
(DCM/MeOH = 95:5, v/v) to afford S67 as a white solid (1.27 g, 65% yield, mixture of
regio-isomers). 1H NMR (600 MHz, CD3OD, 0.85/0.15 = regioisomeric equivalents) δ
1.44/1.41 (s, 9H), 1.79/1.75 (m, 2H), 3.16/3.12 (t, J = 6.6 Hz, 2H), 3.46/3.42 (t, J = 6.6
Hz, 2H), 4.67 (s, 2H), 7.28/7.27 (d, J = 8.4 Hz, 2H), 7.49/7.48 (d, J = 8.4 Hz, 2H),
7.70/7.69 (d, J = 9.0 Hz, 1H), 8.09/8.05 (dd, J = 9.0, 1.8 Hz, 1H), 8.38/8.11 (d, J = 1.8
Hz, 1H). 13C NMR (150 MHz, CD3OD, 0.85/0.15 = regioisomeric equivalents) δ 28.78,
30.72/30.61, 38.58/38.53, 38.97/38.85, 64.59, 80.02, 122.64, 128.02/127.18,
128.17/127.61, 129.35/128.96, 129.89/129.26, 135.13/137.26, 139.42/139.77,
140.74/140.68, 141.62/141.35, 141.93/142.11, 152.86152.92, 155.22154.95,
158.62/158.57, 168.45. HRMS (ESI+): m/z calcd for C24H28ClN4O5 ([M+H]+) 487.1748;
found 487.1746.
S68
To a cooled (0 C) solution of S67 (877 mg, 1.8 mmol) and pyridine (160 L, 2.0 mmol)
in anhydrous THF (10 mL) was added dropwise a solution of p-nitrophenyl
chloroformate (373 mg, 1.85 mmol) in anhydrous THF (10 mL). The reaction mixture
-S53 -
was then allowed to warm to RT and stirred for overnight. The solution was evaporated
to dryness, and the residue was partitioned between EtOAc and H2O. The organic layer
was collected, and the aqueous layer was extracted with EtOAc. The combined organic
phase was washed successively H2O and brine, dried over anhydrous MgSO4 and
concentrated under reduced pressure The residue was purified by column
chromatography on silica gel (PE/EtOAc = 1:1, v/v) to afford S68 as a white solid (763
mg, 65% yield). 1H NMR (400 MHz, CDCl3) δ 1.46 (s, 9H), 1.76 (m, 2H), 3.28 (q, J =
6.0 Hz, 2H), 3.55 (q, J = 6.0 Hz, 2H), 4.89 (br s, 1H), 5.36 (s, 2H), 7.36 (d, J = 8.8 Hz,
2H), 7.41 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.8 Hz, 2H), 7.61 (s, 1H), 7.76 (d, J = 8.8 Hz,
1H), 8.16 (dd, J = 8.8, 2.0 Hz, 1H), 8.29 (d, J = 8.8 Hz, 1H), 8.45 (s, 1H). 13C NMR
(100 MHz, CDCl3) δ 28.40, 30.09, 36.75, 37.32, 70.23, 79.47, 121.71, 121.94, 125.24,
126.81, 127.32, 129.00, 130.10, 131.98, 134.54, 138.50, 140.13, 140.43, 145.40, 152.37,
152.78, 153.21, 155.49, 156.95, 166.30. HRMS (ESI+): m/z calcd for C31H31ClN5O9
([M+H]+) 652.1810; found 652.1813.
S69
S68 (587 mg, 0.9 mmol) and propargylamine (68.5 L, 1.0 mmol) were dissolved in
anhydrous THF (10 mL), and the solution was stirred for overnight at RT under argon
atmosphere. The reaction solution was removed by rotary evaporation under reduced
pressure, and the residue was purified by column chromatography on silica gel
(DCM/MeOH = 98:2, v/v) to afford S69 as a white solid (429 mg, 84% yield). 1H NMR
(400 MHz, DMSO-d6) δ 1.37 (s, 9H), 1.67 (m, 2H), 3.00 (q, J = 6.4 Hz, 2H), 3.13 (t, J
= 2.4 Hz, 1H), 3.30 (q, J = 6.4 Hz, 2H), 3.82 (dd, J = 5.6, 2.4 Hz, 2H), 5.11 (s, 2H),
6.84 (t, J = 5.6 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.79 (m, 2H),
8.15 (dd, J = 8.8, 2.0 Hz, 1H), 8.47 (d, J = 2.0 Hz, 1H), 8.74 (t, J = 5.6 Hz, 1H). 13C
NMR (150 MHz, DMSO-d6) δ 28.25, 29.44, 29.87, 37.25, 37.75, 65.09, 73.10, 77.50,
81.33, 121.67, 126.45, 126.84, 129.21, 129.46, 133.99, 134.75, 137.88, 139.91, 140.04,
-S54 -
151.82, 153.48, 155.61, 155.92, 164.83. HRMS (ESI+): m/z calcd for C28H31ClN5O6
([M+H]+) 568.1963; found 568.1963.
S70
Prepared according to the method for synthesis of S64, with S69 (398 mg, 0.7 mmol),
TFA (2 mL), and DCM (4 mL) to afford crude product (S70) as a faint yellow solid
(265 mg, 81% yield, Rf 0.3 in DCM/MeOH 88:12) that was directly used in the next
step without further purification.
S71
Prepared according to the method for synthesis of S65, with S54 (106 mg, 0.4 mmol),
S70 (187 mg, 0.4 mmol) and DMF (5 mL). Purification by column chromatography on
silica gel (DCM/MeOH = 96:4, v/v) afforded S71 as a white solid (102 mg, 41% yield).
1
H NMR (400 MHz, DMSO-d6) δ 1.66 (m, J = 6.8 Hz, 2H), 2.34 (t, J = 7.2 Hz, 2H),
3.09 (q, J = 6.8 Hz, 2H), 3.13 (d, J = 2.0 Hz, 1H), 3.27-3.31 (m, 2H), 3.61 (t, J = 7.2
Hz, 2H), 3.82 (dd, J = 5.6, 2.0 Hz, 2H), 5.11 (s, 2H), 7.00 (s, 2H), 7.39 (d, J = 8.4 Hz,
2H), 7.50 (d, J = 8.4 Hz, 2H), 7.79 (m, 2H), 7.99 (t, J = 5.6 Hz, 1H), 8.15 (dd, J = 8.8,
1.6 Hz, 1H), 8.47 (d, J = 1.6 Hz, 1H), 8.74 (t, J = 5.6 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6) δ 28.87, 29.82, 33.99, 34.03, 36.33, 37.17, 64.98, 72.88, 81.23, 121.48,
126.35, 126.74, 129.10, 129.26, 134.03, 134.40, 134.65, 137.84, 139.78, 139.95,
151.74, 153.30, 155.79, 164.77, 169.33, 170.58. HRMS (ESI+): m/z calcd for
C30H28ClN6O7 ([M+H]+) 619.1708; found 619.1708.
-S55 -
((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-12-
azaheptadecyl)-1H-1,2,3-triazol-4-yl)methyl)carbamate (S72)
S72
Prepared according to the method for synthesis of S66, with S71 (62 mg, 0.1 mmol),
S47 (44.5 mg, 0.1 mmol), CuI (4.0 mg, 0.02 mmol), TEA (6.0 l, 0.04 mmol) and DMF
(5 ml). Purification by column chromatography on silica gel (DCM/MeOH = 88:12,
v/v) afforded S72 as a white solid (67 mg, 63% yield). 1H NMR (600 MHz, DMSO-d6)
δ 1.25-1.60 (m, 6H), 1.66 (m, 2H), 2.05 (t, J = 7.2 Hz, 2H), 2.34 (t, J = 7.2 Hz, 2H),
2.57 (d, J = 12.6 Hz, 1H), 2.80 (dd, J = 12.6, 5.4Hz, 1H), 3.08 (m, 3H), 3.17 (q, J = 6.0
Hz, 2H), 3.30 (q, J = 6.0 Hz, 2H), 3.38 (m, 2H), 3.45-3.52 (s, 8H), 3.61 (t, J = 7.2 Hz,
2H), 3.80 (t, J = 5.4 Hz, 2H), 4.12 (m, 1H), 4.28 (m, 3H), 4.50 (t, J = 5.4 Hz, 2H), 5.10
(s, 2H), 6.35 (s, 1H), 6.41 (s, 1H), 7.00 (s, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.49 (d, J =
8.4 Hz, 2H), 7.78 (d, J = 8.4 Hz, 1H), 7.83 (m, 2H), 7.91 (s, 1H), 8.00 (t, J = 5.4 Hz,
13
1H), 8.15 (d, J = 8.4 Hz, 1H), 8.47 (s, 1H), 8.74 (t, J = 5.4 Hz, 1H). C NMR (150
MHz, DMSO-d6) δ 25.31, 28.09, 28.25, 29.01, 34.17, 35.16, 36.09, 36.48, 37.32, 38.52,
49.38, 55.50, 59.29, 61.10, 61.15, 64.99, 68.83, 69.20, 69.60, 69.69, 69.76, 121.72,
123.12, 126.53, 126.92, 129.27, 129.44, 134.05, 134.58, 134.97, 137.95, 140.00,
140.12, 145.09, 151.82, 153.55, 156.26, 162.84, 164.94, 169.56, 170.81, 172.29.
HRMS (ESI+): m/z calcd for C48H60ClN12O12S ([M+H]+) 1063.3863; found 1063.3865.
-S56 -
(82) Synthesis of tert-butyl (3-(3-chloro-2-((2-hydroxyethyl)thio)quinoxaline-6-
carboxamido)propyl)carbamate (S73)
S73
Prepared according to the method for synthesis of S67, with S50 (1.6 g, 4.0 mmol), 2-
mercaptoethanol (293 L, 4.0 mmol), K2CO3 (1.1 g, 4.0 mmol) and DMF (20 mL).
Purification by column chromatography on silica gel (DCM/MeOH = 96:4, v/v)
afforded S73 as a white solid (1.3 g, 74% yield, mixture of regio-isomers). 1H NMR
(400 MHz, CDCl3, 0.8/0.2 = regioisomeric equivalents) δ 1.45 (s, 9H), 1.75 (m, 2H),
3.08 (t, J = 6.0 Hz, 1H), 3.27 (q, J = 6.0 Hz, 2H), 3.51 (t, J = 6.0 Hz, 2H), 3.54 (q, J =
6.0 Hz, 2H), 4.01 (q, J = 6.0 Hz, 2H), 4.95 (t, J = 6.0 Hz, 1H), 7.76 (t, J = 6.0 Hz,
1H), 7.87/7.92 (d, J = 8.4 Hz, 1H), 8.15/8.08 (d, J = 8.4 Hz, 1H), 8.33/8.36 (s, 1H). 13C
NMR (100 MHz, CDCl3, 0.8/0.2 = regioisomeric equivalents) δ 28.51, 30.05,
33.94/33.73, 36.87, 37.43, 60.73, 79.78, 127.17/126.37, 127.38/127.23, 128.87/128.31,
134.58/136.10, 138.15/140.02, 141.76,/140.08, 145.85/146.43, 156.97156.24, 157.17,
166.62. HRMS (ESI+): m/z calcd for C19H26ClN4O4S ([M+H]+) 441.1363; found
441.1364.
S74
Prepared according to the method for synthesis of S68, with S73 (794 mg, 1.8 mmol)
and pyridine (160 L, 2.0 mmol), p-nitrophenyl chloroformate (373 mg, 1.85 mmol)
and anhydrous THF (20 mL). Purification by column chromatography on silica gel
(PE/EtOAc = 1:1, v/v) afforded S74 as a white solid (731 mg, 67% yield, mixture of
regio-isomers). 1H NMR (400 MHz, CDCl3, 0.8/0.2 = regioisomeric equivalents) δ 1.46
(s, 9H), 1.76 (m, 2H), 3.28 (q, J = 6.4 Hz, 2H), 3.56 (q, J = 6.4 Hz, 2H), 3.71 (t, J = 6.4
Hz, 2H), 4.64 (t, J = 6.4 Hz, 2H), 4.93 (t, J = 6.4 Hz, 1H), 7.37 (d, J = 9.2 Hz, 2H), 7.73
-S57 -
(t, J = 6.4 Hz, 1H), 7.98/7.99 (d, J = 8.8 Hz, 1H), 8.20/8.12 (d, J = 8.8 Hz, 1H), 8.27
13
(d, J = 9.2 Hz, 2H), 8.41/8.46 (s, 1H). C NMR (100 MHz, CDCl3, 0.8/0.2 =
regioisomeric equivalents) δ 28.49, 29.07, 30.17/30.11, 36.57, 37.19, 66.63, 79.72,
121.81, 125.39, 127.28/126.83, 127.76/127.41, 129.08/128.54, 135.20/136.42,
138.70/140.40, 142.03/140.46, 145.45, 145.76/146.31, 152.33, 155.43, 155.66, 157.19,
166.21. HRMS (ESI+): m/z calcd for C26H29ClN5O8S ([M+H]+) 606.1425; found
606.1417.
S75
Prepared according to the method for synthesis of S69, with using S74 (545 mg, 0.9
mmol), propargylamine (68.5 L, 1.0 mmol) and THF (10 mL). Purification by column
chromatography on silica gel (PE/EtOAc = 3:1 to 1:1, v/v) afforded S75 as a white solid
(373 mg, 75% yield, mixture of regio-isomers). 1H NMR (600 MHz, CDCl3, 0.85/0.15
= regioisomeric equivalents) δ 1.46 (s, 9H), 1.76 (m, 2H), 2.25 (t, J = 2.4 Hz, 1H), 3.28
(q, J = 6.0 Hz, 2H), 3.56 (m, 4H), 3.99 (dd, J = 6.0, 2.4 Hz, 2H), 4.43 (t, J = 6.0 Hz,
2H), 4.98 (t, J = 6.0 Hz, 1H), 5.12/5.19 (s, 1H), 7.71 (t, J = 6.0 Hz, 1H), 7.95 (d, J =8.4
Hz, 1H), 8.17/8.09 (d, J = 8.4 Hz, 1H), 8.37/8.40 (s, 1H). 13C NMR (150 MHz, CDCl3)
δ 28.49, 29.99/29.93, 30.19/30.13, 30.90, 36.80, 37.39, 62.86, 71.70, 79.64, 79.69,
127.19/126.55, 127.68/127.34, 128.90/128.43, 134.96/136.37, 138.53/140.34,
142.07/140.40, 145.83/146.37, 155.77/155.66, 156.25, 157.03, 166.38. HRMS (ESI+):
m/z calcd for C23H29ClN5O5S ([M+H]+) 522.1578; found 522.1586.
S76
Prepared according to the method for synthesis of S64, with S75 (331 mg, 0.6 mmol),
-S58 -
TFA (2 mL) and DCM (4 mL) to afford crude product (S76) as a faint yellow solid (233
mg, 92% yield, Rf 0.3 in DCM/MeOH 9:1) which was directly used in the next step
without further purification.
S77
Prepared according to the method for synthesis of S65, with S54 (133 mg, 0.5 mmol),
S76 (211 mg, 0.5 mmol) and DMF (5 mL). Purification by column chromatography on
silica gel (DCM/MeOH = 96:4, v/v) afforded S77 as a white solid (109 mg, 38% yield,
mixture of regio-isomers). 1H NMR (400 MHz, CD3OD, 0.9/0.1 = regioisomeric
equivalents) δ 1.81 (m, 2H), 2.49 (t, J = 6.8 Hz, 2H), 2.55 (t, J = 2.8 Hz, 1H), 3.26 (t, J
= 6.8 Hz, 2H), 3.45 (t, J = 6.8 Hz, 2H), 3.63 (t, J = 6.0 Hz, 2H), 3.79 (t, J = 6.8 Hz, 2H),
3.85 (d, J = 2.8 Hz, 2H), 4.41 (t, J = 6.0 Hz, 2H), 6.82 (s, 2H), 8.03/7.97 (d, J = 8.8 Hz,
1H), 8.18/8.12 (dd, J = 8.8, 2.0 Hz, 1H), 8.37/8.43 (d, J = 2.0 Hz, 1H). 13C NMR (100
MHz, DMSO-d6) δ 29.02, 29.67, 29.89, 34.19, 36.51, 37.36, 61.92, 73.10, 81.34,
126.83, 127.33, 129.38, 134.59, 134.72, 137.98, 141.54, 145.15, 155.78, 164.90,
169.62, 170.82. HRMS (ESI+): m/z calcd for C25H26ClN6O6S ([M+H]+) 573.1323;
found 573.1323.
S78
Prepared according to the method for synthesis of S66, with S77 (57.3 mg, 0.1 mmol),
S47 (44.5 mg, 0.1 mmol), CuI (4.0 mg, 0.02 mmol), TEA (6.0 L, 0.04 mmol) and
DMF (5 mL). Purification by column chromatography on silica gel (DCM/MeOH =
88:12, v/v) afforded S78 as a white solid (69 mg, 68% yield). 1H NMR (600 MHz,
-S59 -
DMSO-d6) δ 1.28-1.60 (m, 6H), 1.66 (m, 2H), 2.05 (t, J = 7.2 Hz, 2H), 2.34 (t, J = 7.2
Hz, 2H), 2.57 (d, J = 12.6 Hz, 1H), 2.80 (dd, J = 12.6, 5.4 Hz, 1H), 3.09 (m, 3H), 3.16
(q, J = 5.4 Hz, 2H), 3.30 (m, 2H), 3.37 (m, 2H), 3.3.51 (s, 8H), 3.60 (m, 4H), 3.79 (t, J
= 5.4 Hz, 2H), 4.11 (m, 1H), 4.21 (d, J = 5.4 Hz, 2H), 4.31 (m, 3H), 4.48 (t, J = 5.4 Hz,
2H), 6.35 (s, 1H), 6.41 (s, 1H), 7.00 (s, 2H), 7.75 (t, J = 5.4 Hz, 1H), 7.81 (t, J = 5.4
Hz, 1H), 7.87 (s, 1H), 8.00 (t, J = 5.4 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 8.24 (dd, J =
8.4, 1.2 Hz, 1H), 8.45 (d, J = 1.2 Hz, 1H), 8.77 (t, J = 5.4 Hz, 1H). 13C NMR (150 MHz,
DMSO-d6) δ 25.30, 28.07, 28.24, 29.00, 29.73, 34.15, 35.14, 36.00, 36.47, 37.33, 38.50,
49.35, 55.49, 59.28, 61.08, 61.13, 61.71, 68.80, 69.18, 69.58, 69.66, 69.73, 123.08,
126.81, 127.31, 129.38, 134.57, 134.73, 138.00, 141.56, 145.02, 145.16, 155.83,
156.00, 162.81, 164.85, 169.54, 170.79, 172.25. HRMS (ESI+): m/z calcd for
C43H58ClN12O11S2 ([M+H]+) 1017.3478; found 1017.3476.
S80
S79 (164 mg, 1.0 mmol) and DIPEA (170 uL, 1.0 mmol) were added to a solution of 1t
(287 mg, 1.0 mmol) in anhydrous DMF (15 ml), and the mixture was stirred overnight
at RT under argon atmosphere. The reaction mixture was evaporated under reduced
pressure, and the residue was purified by column chromatography on silica gel
(PE/EtOAc = 3:1, v/v) to afford S80 as a white solid (182 mg, 40% yield). 1H NMR
(600 MHz, DMSO-d6) δ 5.27 (s, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H),
7.77-7.70 (m, 5H), 8.01 (d, J = 7.8 Hz, 1H), 8.22 (d, J = 9.0 Hz, 2H), 10.58 (s, 1H). 13C
NMR (150 MHz, DMSO-d6) δ 65.67, 117.58, 121.30, 124.59, 126.55, 127.35, 128.31,
-S60 -
129.52, 130.58, 133.42, 138.35, 138.45, 138.52, 141.69, 145.28, 152.13, 152.20,
152.85. HRMS (ESI+): m/z calcd for C22H16ClN4O5 ([M+H]+) 451.0809; found
451.0805.
S81
Prepared according to the method for synthesis of S80, with 1q (241 mg, 1.0 mmol),
S79 (164 mg, 1.0 mmol), DIPEA (170 uL, 1.0 mmol) and DMF (15 mL). Purification
by column chromatography on silica gel (PE/EtOAc = 4:1, v/v) afforded S81 as a white
solid (251 mg, 62% yield). 1H NMR (600 MHz, DMSO-d6) δ 3.68 (t, J = 6.0 Hz, 2H),
4.49 (t, J = 6.1 Hz, 2H), 7.60 (d, J = 9.0 Hz, 2H), 7.73-7.68 (m, 2H), 7.85 (d, J = 8.4
Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 9.0 Hz, 2H), 10.35 (s, 1H). 13C NMR
(150 MHz, DMSO-d6) δ 28.99, 62.54, 117.51, 124.44, 126.87, 127.48, 129.11, 130.42,
138.38, 140.16, 141.60, 143.93, 145.21, 152.64, 153.61. HRMS (ESI+): m/z calcd for
C17H14ClN4O4S ([M+H]+) 405.2464; found 405.2465.
-S61 -
3. Optimization of conditions
-S62 -
Table S2: Investigations on couplings of o-dichloroquinoxaline (1) with various
substrates (2a-2j) containing double functional groups[a]
1 94[c]
2a 3a
2 95[c]
2b 3b
3[b] 97[c]
2c 3c
4[b] 97[c]
2d 3d
14[d]
3e
5
2e
42[d]
3'e
2[d]
3f
6
2f
61[d]
3'f
0[d]
3g
7
2g
0[d]
3'g
6[d]
3h
8
2h
9[d]
3'h
0[d]
3i
9
2i
15[d]
1q
-S63 -
0[d]
3j
10
2j
16[d]
1i
[a] Reaction conditions: 1a (0.5 mmol, 50 mM), 2a-2j (0.5 mmol, 50 mM), K2HPO4
(1.0 mmol, 100 mM), H2O (2.0 mL) , DMF (8.0 mL) at RT for 10 min. [b] K2HPO4 (1.5
mmol, 150 mM). [c] Yield of isolated product. [d] Yield determined by HPLC.
-S64 -
Table S3: Investigations on couplings of 4-methylbenzene-1,2-dithiol (2b) with
several o-dihalo substrates (1a-1e)[a]
1 95
1a 3b
2 95
1b 3b
3 3k 78[d]
1c
4 96
1d 3l
5[b] 98
1e
[a] Reaction conditions: 1a-1e (0.5 mmol, 50 mM), 2b (0.5 mmol, 50 mM), K2HPO4
(1.0 mmol, 100 mM), H2O (2.0 mL), DMF (8.0 mL) at RT for 10 min. [b] K2HPO4 (1.5
mmol, 150 mM). [c] Yield of isolated product. [d] Reaction time 30 min.
-S65 -
Table S4: Investigations on couplings of o-dichloroquinoxalines (1a, 1e) with
different ratios of o-dithiophenols (2a-2d) in mixed solvent of DMF and PBS (pH
7.4)[a]
Yield of 3 (%)[b]
Entry 1 (1 equiv) 2a-d
1 equiv of 2 5 equiv of 2 10 equiv of 2
[a] Reaction conditions: 1a or 1e (50 nmol, 0.5 mM), 2a-2d (50-500 nmol, 0.5-5.0 mM),
PBS (20 mM, pH 7.4) (50 L), DMF (50 L) at RT for 60 min. [b] Yield determined
by HPLC. Results represent three replicate experiments.
-S66 -
containing functionalized molecules were attempted, and they provided satisfactory
results (entries 4-9). Substrate (1x) with 1-hydroxypyrrolidine-2,5-dione also was
suitable bioorthogonal reagent (entry 10). Therefore, the present bioorthogonal
cleavage methods should provide a novel strategy for deprotection of biologically
active molecules.
1 10 min 94
1f
2 10 min 97
1g
3 30 min 71
1h
4 30 min 67
1i
5 30 min 85
1j
6 30 min 78
1k
7 4h 11
1l
8 4h NR
1m
9 4h NR
1n
[a] Reaction conditions: 1f-1n (0.5 mmol, 50 mM), 2b (0.5 mmol, 50 mM), K2HPO4
(1.0 mmol, 100 mM), H2O (2.0 mL), DMF (8.0 mL) at RT for 10 min-4 h. [b] Yield of
isolated product. NR = No reaction.
-S67 -
Table S6: Investigations on couplings of 4-methylbenzene-1,2-dithiol with other
chloroquinoxalines[a]
1 30 92
1o
2 30 91
1p
3 60 73
1q
4 30 62
1r
5 60 72
1s
6 60 86
1t
7 60 94
1u
8 60 93
1v
9 10 88
1w
10 30 85
1x
[a] Reaction conditions: 1o-1x (0.5 mmol, 50 mM), 2b (0.5 mmol, 50 mM), K2HPO4
(1.0 mmol, 100 mM), H2O (2.0 mL), DMF (8.0 mL) at RT for 10-60 min. [b] Yield of
isolated product.
Based on the results above, we synthesized three chloroquinoxalines (1g, 1i and 1k)
containing amino acid units and surveyed their couplings of with different ratios of 2-
(3,4-dimercaptophenyl)acetic acid (2d) in mixed solvent of PBS (pH 7.4) and DMF
(v/v, 1:1). As shown in Table S7, order of reactivity on substrates (1g, 1i and 1k)
containing three amino acid units are as follows: His > Tyr > Cys, and yields obviously
increased with ratio rise of 2d from one equivalent to ten equivalents.
-S68 -
Table S7: Investigations on couplings of three chloroquinoxalines (1g, 1i and 1k)
containing amino acid units with different ratios of 2-(3,4-
dimercaptophenyl)acetic acid (2d)[a]
Yield of 3d (%)[b]
Entry 1g, 1i, 1k
1 equiv of 2d 5 equiv of 2d 10 equiv of 2d
-S69 -
Table S8: Investigations on reactivity of o-dichloroquinoxaline (1a) with
compounds (4a-4h) containing common functional groups[a]
1 92
4a 1h
2 4b 1o 88
3 1j NR
4c
4 NR
4d 1l
5 NR
4e 1f
6 4f NR
1m
7 4g NR
1n
8 NaOH 4h NR
1y
[a] Reaction conditions: 1a (0.5 mmol, 50 mM), 4a-4h (0.5 mmol, 50 mM), K2HPO4
(1.0 mmol, 100 mM), DMF (8.0 mL), H2O (2.0 mL) at RT for 2 h. [b] Yield of isolated
product. NR = No reaction.
-S70 -
treatments of chloroquinoxaline 1f containing imidazolyl with 1 equiv, 5 equiv and 10
equiv of 4a were investigated, and we found that 1f displayed slightly higher reactivity
than 1h and 1j and showed the same reaction activity as 1a (Figure S5d). According to
the results above, mono-substituted chloroquinoxalines containing thioether or phenol
ether should be more suitable as the partners of o-dithiophenols in bioorthogonal
chemistry.
Figure S5. HPLC of the resulting solution from reactions of 1a, 1h, 1j or 1f (100 μM)
with different ratios of N-acetyl cysteine methyl ester (4a) (1 equiv, 5 equiv and 10
equiv) in 1.0 mL PBS (20 mM, pH =7.4)/ACN (1:1) solution. a) Reaction of 1a with
4a. b) Reaction of 1h with 4a. c) Reaction of 1j with 4a. d) Reaction of 1f with 4a. ES
= external standard (100 μM).
-S71 -
5. Competitive effect of glutathione on conjugations
The competitive reaction of 1e (100 M) with 2b (500 M) in the presence of
glutathione (GSH, 0-20 mM) (0-200 equiv relative to 1e and 0-40 equiv relative to 2b)
containing a cysteine residue was investigated in PBS (20 mM, pH 7.4)/ACN (Figure
S6). The resulting solutions were directly analyzed by HPLC using external standard
method (standard curve method) for quantification determination (Figure S7). Injection
volume 15 L. The data are average from three replicate experiments.
Figure S6. HPLC traces of products 3m and 3′m. Competitive reaction of 1e with 2b in the presence
of GSH (0-200 equiv relative to 1e and 0-40 equiv relative to 2b). The gradient was programmed as
follows: 80% B. Water with 0.06% TFA (solvent A) and 80% ACN/20% water with 0.06% TFA
(solvent B), were used as the mobile phase at a flow rate of 1.0 mL min-1, monitoring wavelength λ
= 398 nm.
-S72 -
Figure S7. a) HPLC traces of standards 3m and 3′m under the indicated concentractions. b)
Standard curve with products 3m and 3′m as the standards for HPLC quantification analysis.
Injection volume 15 L. The data are average from three replicate experiments. The gradient was
programmed as follows: 80% B. Water with 0.06% TFA (solvent A) and 80% ACN/20% water with
0.06% TFA (solvent B), were used as the mobile phase at a flow rate of 1.0 mL min-1, monitoring
Figure S8. Comparison of CQ-DT reactions in PBS buffer and cell medium. Yields
were determined by HPLC. The data are average from three replicate experiments.
Figure S9. Stability analysis of 4-methylbenzene-1,2-dithiol (2b) in cell medium. Conversion rates
of 1e were determined by HPLC. The data are average from three replicate experiments. The
gradient was programmed as follows: 80% B. Water with 0.06% TFA (solvent A) and 80% ACN/20%
-S74 -
water with 0.06% TFA (solvent B), were used as the mobile phase at a flow rate of 1.0 mL min -1,
Figure S10. Stability analysis of conjugates 3m and 3′m in cell medium. Monitored by
UV-Vis spectrophotometer at a concentration of 50 M in cell medium.
7. Kinetic measurements
-S75 -
regioisomeric products 3m and 3'm as determined by HPLC at 398 nm. Each data point
is the average of three independent trials. As shown in Figure S11b, the second order
rate constant k2 of 1.30.1 M-1s-1 was calculated from the slope of the graph using the
relationship: kobs = [2b]k2.
Figure S11. a) HPLC trace spectra of products 3m and 3'm as change of time from
reaction of 2,3-dichloroquinoxaline-6-carboxylic acid (1e) (5.010-5 M) and 4-
methylbenzene-1,2-dithiol (2b) (5.010-4 M). The gradient was programmed as follows:
80% B. Water with 0.06% TFA (solvent A) and 80% ACN/20% water with 0.06% TFA
(solvent B), were used as the mobile phase at a flow rate of 1.0 mL min-1, monitoring
wavelength λ = 398 nm. b) Plot of an observed first-order rate constant (kobs) vs. the
concentration of 4-methylbenzene-1,2-dithiol (2b) for the reactions of 2,3-
dichloroquinoxaline-6-carboxylic acid (1e) (5.010-5 M) with increasing concentration
of 4-methylbenzene-1,2-dithiol (2b) (4.010-4 ~ 1.010-3 M) in ACN and PBS (20 mM,
pH 7.4) (v/v, 1:1) at 25.00.1 C. Percent conversion was monitored by appearance of
products (isomers 3m and 3'm) as determined by HPLC at 398 nm. Each data point is
the average of three independent trials. The second order rate constant k2 of 1.30.1 M-
1 -1
s is calculated from the slope of the graph using the relationship: kobs = [2b]k2. Error
bars represent standard deviations from three replicate experiments.
-S76 -
8. Fluorescence investigations of the conjugates
UV-Vis spectra of reactants 1e, 2b and products 3m, 3'm were recorded, and the
tetracyclic heteroacenes 3m and 3'm showed strong absorption peak at 398 nm (Figure
S12a). Importantly, the conjugates 3a, 3c, 3d, 3m and 3'm all exhibit varying degrees
photoluminescence (PL) in the visible light spectral region (Figure S12b).[18]
Figure S12. a) UV-Vis spectra of reactants 1e, 2b and the corresponding products 3m
and 3'm at a concentration of 0.5 M in PBS (10 mM, pH 7.4). b) Emission spectra of
the compound 3a, 3c, 3d and 3m at the concentrations of 2.0 M in PBS (10 mM, pH
7.4) excited at 398 nm. The inset shows photoluminescence of the products at the
concentrations of 20 M in PBS (10 mM, pH 7.4) under irradiation of 365 nm UV.
-S77 -
fluorescence group (9) and all provided the corresponding conjugates in excellent yields.
Alternatively, we first prepared tBuS-protected o-dithiophenol (S90) containing GSH
from coupling of S32 with GSH, and then conjugations of S90 with different o-
dichloroquinoxalines (1e, S49, S52) were performed in the presence of 20 equiv of
TCEP in PBS with 6% DMF at RT. As shown in Figure S14, the bioorthogonal ligations
provided S91-S93 in almost complete conversion rate (HPLC determination). (The
gradient was programmed as follows: 0-25 min: 28%-72% B, 25-28 min: 72%-100%
B, 28-32 min: 100% B, 32-35 min, 100%-28%, B. Water with 0.06% TFA (solvent A)
and 80% ACN/20% water with 0.06% TFA (solvent B), were used as the mobile phase
at a flow rate of 1.0 mL min-1. Monitoring wavelength λ = 254 nm)
-S78 -
Figure S14. Bioorthogonal ligations of o-dichloroquinoxalines with various o-
dithiophenols containing GSH. Reaction conditions: S32 (100 nmol, 500 μM), GSH
(100 nmol, 500 μM), 1e, S49 or S52 (100 nmol, 500 μM), TCEP (2.0 mol, 10 Mm, 20
equiv), PBS (20 mM, pH 7.4) (200 L) at RT for 60 min. (Note: A pair of regioisomers
were observed by HPLC after formation of two C-S bonds.)
To a solution of GSH (2.0 mL, 1.0 mol, 500 M) in PBS buffer (20 mM, pH 7.4) was
added a solution of 5 (20 μL of a 50 mM stock solution in DMF, 1.0 mol, 500 M
final concentration, 1 equiv) and the resulting mixture was vortexed for 30 seconds.
After 60 min of additional shaking at RT, the solution was analyzed by RP-HPLC
(Figure S16) and LC-MS (Figure S17), and a complete conversion to S82 was observed.
-S79 -
Figure S15. RP-HPLC of 5.
AV: 0.21-0.52 m in (18) NL: 2.60E5 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [200.00-1200.00]
104
% 804.3
[M+H]+
88
75
63
50
38
25
13 [M+Na]+
826.4
m /z
0
400 500 600 700 800 900 1,000 1,100 1,200
-S80 -
(2) Reaction of S82 with 2b
A 200 μL of the crude S82 (100 nmol, 500 μM) in PBS buffer (20 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. 4-Methylbenzene-1,2-dithiol (2b) (10 μL of a
10 mM stock solution in DMF, 100 nmol, 500 μM final concentration, 1 equiv) was
added, and the resulting mixture was vortexed for 30 seconds. After 60 min of
additional shaking at RT, the reaction mixture was analyzed by RP-HPLC (Figure S18)
and LC-MS (Figure S19) and a complete conversion to S83 was observed.
AV: 0.25-0.28 m in (3) NL: 5.60E5 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [200.00-2000.00]
104
% 888.6
[M+H]+
88
75
63
50
38
25
13
415.4 463.8
m /z
0
400 500 600 700 800 900 1,000 1,100 1,200
-S81 -
(3) Reaction of S82 with 2c
A 200 μL of the crude S82 (100 nmol, 500 μM) in PBS buffer (20 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. 3,4-Dimercaptobenzoic acid (2c) (10 μL of a
10 mM stock solution in DMF, 100 nmol, 500 μM final concentration, 1 equiv) was
added and the resulting mixture was vortexed for 30 seconds. After 60 min of additional
shaking at RT, the reaction mixture was analyzed by RP-HPLC (Figure S20) and LC-
MS (Figure S21) and a complete conversion to S84 was observed.
AV: 0.19-0.81 m in (36) NL: 1.63E5 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [400.00-2000.00]
104
% 918.6
[M+H]+
88
75
63
50
38
25
13
432.4
476.4 520.2 564.5
m /z
0
400 500 600 700 800 900 1,000 1,100 1,200
-S82 -
(4) Reaction of S82 with 2d
A 200 μL of the crude S82 (100 nmol, 500 μM) in PBS buffer (20 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. 2-(3,4-Dimercaptophenyl)acetic acid (2d) (10
μL of a 10 mM stock solution in DMF, 100 nmol, 500 μM final concentration, 1 equiv)
was added and the resulting mixture was vortexed for 30 seconds. After 60 min of
additional shaking at RT, the reaction mixture was analyzed by RP-HPLC (Figure S22)
and LC-MS (Figure S23) and a complete conversion to S85 was observed.
AV: 0.18-0.79 m in (36) NL: 2.97E5 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [400.00-2000.00]
104
% 932.6
[M+H]+
88
75
63
50
38
25
13
415.2 485.8
m /z
0
400 500 600 700 800 900 1,000 1,100 1,200
-S83 -
(5) Reaction of S82 with S37
A 200 μL of the crude S82 (100 nmol, 500 μM) in PBS buffer (20 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. S37 (10 μL of a 10 mM stock solution in DMF,
100 nmol, 500 μM final concentration, 1 equiv) and tris(2-carboxyethyl)phosphine
hydrochloride (TCEPHCl) (14.3 μL of a stock solution of 4 mg TCEPHCl in 100 μL
0.5 M NaOH aqueous solution, 2.0 mol, 10 mM final concentration, 20 equiv) were
added. The resulting mixture was vortexed for 30 seconds and then shaking additional
60 min at RT. The reaction mixture was analyzed by RP-HPLC (Figure S24) and LC-
MS (Figure S25) and a complete conversion to S86 was observed.
[M+2H]2+
88
75
63
50
1046.8
38 [M+H]+
25
13
1396.0
432.4
m /z
0
400 500 600 700 800 900 1,000 1,100 1,200 1,300 1,400 1,500
-S84 -
(6) Reaction of S82 with 7
A 200 μL of the crude S82 (100 nmol, 500 μM) in PBS buffer (20 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. 7 (10 μL of a 10 mM stock solution in DMF,
100 nmol, 500 μM final concentration, 1 equiv) was added and the resulting mixture
was vortexed for 30 seconds. After 60 min of additional shaking at RT, the reaction
mixture was analyzed by RP-HPLC (Figure S26) and LC-MS (Figure S27) and
complete conversion to S87 was observed.
[M+4H]4+ [M+3H]3+
88 771.4
75
63
50
38
25 [M+2H]2+
[M+5H]5+ 1542.0
617.3
13
1233.9
m /z
0
400 500 625 750 875 1,000 1,125 1,250 1,375 1,500 1,625 1,750 1,875 2,000
A 200 μL of the crude S82 (100 nmol, 500 μM) in PBS buffer (20 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. 8 (10 μL of a 10 mM stock solution in DMF,
100 nmol, 500 μM final concentration, 1 equiv) and TCEPHCl (14.3 μL of a stock
solution of 4 mg TCEPHCl in 100 μL 0.5 M NaOH aqueous solution, 2.0 mol, 10
mM final concentration, 20 equiv) were added. The resulting mixture was vortexed for
30 seconds and then shaking additional 60 min at RT. The reaction mixture was
analyzed by RP-HPLC (Figure S28) and LC-MS (Figure S29) and a complete
conversion to S88 was observed.
[M+2H]2+
88
75
63
50 1200.8
[M+H]+
38
25
13
432.4
m /z
0
400 500 600 700 800 900 1,000 1,100 1,200 1,300 1,400 1,500
A 200 μL of the crude S82 (100 nmol, 500 μM) in PBS buffer (20 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. 9 (10 μL of a 10 mM stock solution in DMF,
100 nmol, 500 μM final concentration, 1 equiv) and TCEPHCl (14.3 μL of a stock
solution of 4 mg TCEPHCl in 100 μL 0.5 M NaOH aqueous solution, 2.0 mol, 10
mM final concentration, 20 equiv) were added. The resulting mixture was vortexed for
30 seconds and then shaking additional 60 min at RT. The reaction mixture was
analyzed by RP-HPLC (Figure S30) and LC-MS (Figure S31) and a complete
conversion to S89 was observed.
-S87 -
AV: 0.21-0.30 m in (6) NL: 1.26E5 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [200.00-2000.00]
104
% 682.4
[M+2H]2+
88
75
63
50
38
25
[M+H]+
1364.8
13
432.3
582.7
m /z
0
400 500 625 750 875 1,000 1,125 1,250 1,375 1,500 1,625 1,750 1,875 2,000
To a solution of GSH (1.0 mL, 0.5 mol, 500 M) in PBS buffer (20 mM, pH 7.4) was
added a solution of S32 (10 μL of a 50 mM stock solution in DMF, 0.5 mol, 500 M
final concentration, 1 equiv) and the resulting mixture was vortexed for 30 seconds.
After 60 min of additional shaking at RT, the solution was analyzed by RP-HPLC
(Figure S33) and LC-MS (Figure S34) and a complete conversion to S90 was observed.
-S88 -
Figure S33. RP-HPLC of S90.
AV: 0.19-0.79 m in (35) NL: 4.78E5 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [400.00-2000.00]
104
%
[M+H]+
924.6
88
75
63
50
38
25
13
m /z
0
400 500 600 700 800 900 1,000 1,100 1,200
A 200 μL of the crude S90 (100 nmol, 500 μM) in PBS buffer (20 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. 1e (10 μL of a 10 mM stock solution in DMF,
100 nmol, 500 μM final concentration, 1 equiv) and TCEPHCl (14.3 μL of a stock
solution of 4 mg TCEPHCl in 100 μL 0.5 M NaOH aqueous solution, 2.0 mol, 10
mM final concentration, 20 equiv) were added. The resulting mixture was vortexed for
30 seconds and then shaking additional 60 min at RT. The reaction mixture was
analyzed by RP-HPLC (Figure S35) and LC-MS (Figure S36) and a complete
-S89 -
conversion to S91 was observed.
AV: 0.18-0.78 m in (35) NL: 2.07E5 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [400.00-2000.00]
104
% 918.6
[M+H]+
88
75
63
50
38
25
13
432.4 459.9
m /z
0
400 500 600 700 800 900 1,000 1,100 1,200
A 200 μL of the crude S90 (100 nmol, 500 μM) in PBS buffer (20 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. S49 (10 μL of a 10 mM stock solution in DMF,
100 nmol, 500 μM final concentration, 1 equiv) and TCEPHCl (14.3 μL of a stock
solution of 4 mg TCEPHCl in 100 μL 0.5 M NaOH aqueous solution, 2.0 mol, 10
mM final concentration, 20 equiv) were added. The resulting mixture was vortexed for
30 seconds and then shaking additional 60 min at RT. The reaction mixture was
-S90 -
analyzed by RP-HPLC (Figure S37) and LC-MS (Figure S38) and a complete
conversion to S92 was observed.
AV: 0.19-0.72 m in (31) NL: 4.99E5 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [400.00-2000.00]
104
% 660.0
[M+2H]2+
88
75
63
50
[M+H]+
38
1319.0
25
13
583.1
453.1 526.0 728.0
m /z
0
400 500 625 750 875 1,000 1,125 1,250 1,375 1,500 1,625 1,750 1,875 2,000
A 200 μL of the crude S90 (100 nmol, 500 μM) in PBS buffer (20 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. S52 (10 μL of a 10 mM stock solution in DMF,
100 nmol, 500 μM final concentration, 1 equiv) and TCEPHCl (14.3 μL of a stock
solution of 4 mg TCEPHCl in 100 μL 0.5 M NaOH aqueous solution, 2.0 mol, 10
-S91 -
mM final concentration, 20 equiv) were added. The resulting mixture was vortexed for
30 seconds and then shaking additional 60 min at RT. The reaction mixture was
analyzed by RP-HPLC (Figure S39) and LC-MS (Figure S40) and a complete
conversion to S93 was observed.
[M+H]+
88
75
63
50
38
25
[M+H]+
13 1364.8
432.2
520.2
m /z
0
400 500 625 750 875 1,000 1,125 1,250 1,375 1,500 1,625 1,750 1,875 2,000
-S92 -
9.2. Bioorthogonal ligations with model protein
9.2.1. Preparation of recombinant protein C2A(S83C)[19]
Sequence of His-C2A domain of Synaptotagmin-I [His-C2A(S105C), modified residue
in bold and underlined] is shown as follow:
SYYHHHHHHDYDIPTTENLYFQGAMGSEKLGKLQYSLDYDFQNNQLLVGIIQAAELPA
LDMGGTSDPYVKVFLLPDKKKKFETKVHRKTLNPVFNEQFTFKVPYCELGGKTLVMAV
YDFDRFSKHDIIGEFKVPMNTVDFGHVTEEWRDLQSAEK
ETKVHRKTLNPVFNEQFTFKVPYCELGGKTLVMAVYDFDRFSKHDIIGEFKVPMNTVDFG
HVTEEWRDLQSAEK
The synthesized C2A(S83C) gene sequence cloned into the bacterial expression vector
pProEx-HTb using the restriction sites BamHI and Xhol. The pProEx-HTb vector
contains an ampicillin resistance cassette and encodes an N-terminal poly-histidine tag
to facilitate purification of proteins expressed with this epitope tag. His-C2A(S83C)
was expressed in BL21 (DE3) and purified as previously described.[20] The His tag was
removed from the fusion protein (at a protein concentration of approx. 2 mg/mL) by
incubation in Tris-HCl buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM DTT, 1 mM
EDTA, pH 7.4), with 10 U of TEV protease per milligram of His-C2A(S83C), for 6 h
at 16°C. The cleaved C2Am-S83C further exchanged assay buffer (10 mM PBS, 150
mM NaCl, pH 7.4) using desalting column (GE Healthcare) after the cleaved His tag
removed using Ni-NTA Resin. The purity and size of the protein were accessed by
HPLC, ESI-MS (Figures S41 and S42) and SDS-PAGE. The purified protein was stored
in assay buffer, flash-frozen with liquid nitrogen and kept at -80 C.
-S93 -
AV: 0.18-0.93 m in (43) NL: 5.20E4 T: {0,0} + c ESI !corona s id=60.00 det=1024.00 Full m s [600.00-1600.00]
104
% 1503.0
12H+
88
75
63
13H+
16H+ 1387.3
50 1127.3
15H+
17H+ 1202.5 14H+
1288.2
1061.1
18H+
38
1002.3
25 19H+
20H+
+ 902.1
949.6
13 22H+21H
820.2
859.1
740.3 784.4
653.1 696.8
m /z
0
600 700 800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600
10H+
88
75 11H+
1381.4
63
50
9H+
12H+
1688.4
38
1266.7
25 13H+
1169.2
15H+
+11 14H+ 8H+
13
17H+16H 1013.5 1085.7
1713.7
1898.9
844.6 950.3
894.6
m /z
0
800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000
-S94 -
Figure S43. Bioorthogonal ligations of o-dichloroquinoxalines protein C2A(S83C)
with various o-dithiophenols. Reaction conditions: C2A(S83C) (2.6 nmol, 13 μM), 5
(13 nmol, 65 μM), 2b-2d or 7-9 (26 nmol, 130 μM), TCEP (520 nmol, 2.6 mM), PBS
(20 mM, pH 7.4) (200 L) at RT for 60 or 90 min. (Note: A pair of regioisomers were
observed by HPLC after formation of two C-S bonds.)
To a solution of C2A(S83C) (1.5 mL, 19.5 nmol, 13 M) in PBS buffer (10 mM, pH
7.4) was added a solution of 5 (10 μL of a 10 mM stock solution in DMF, 97.5 nmol,
-S95 -
65 M final concentration, 5 equiv) and the resulting mixture was vortexed for 30
seconds. After 60 min of additional shaking at RT, the solution was analyzed by LC-
MS (Figure S44) and a complete conversion to 6 (Theoretical molecular weight:
15680.75. Calculated molecular weight: 15685.1) was observed.
AV: 0.22-0.55 m in (19) NL: 1.52E5 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [600.00-2000.00]
104
% 1569.6
11H+ 10H+
1426.9
88
75
63
50
12H+
38 1308.1
25
+ 9H+
14H+ 13H 1743.7
16H+ 15H+
1207.3
8H+
1121.3
17H+
13
1781.8
981.5 1046.6 1961.4
923.9
874.6
m /z
0
800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000
A 200 μL of the crude 6 (2.6 nmol, 13 μM) in PBS buffer (10 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. 2b (2.5 μL of a 10 mM stock solution in DMF,
26 nmol, 130 μM final concentration, 10 equiv) was added and the resulting mixture
was vortexed for 30 seconds. After 60 min of additional shaking at RT, the reaction
mixture was analyzed by LC-MS (Figure S45) and a complete conversion to
S94(Theoretical molecular weight: 15764.09. Calculated molecular weight: 15767.7)
was observed.
-S96 -
AV: 0.18-1.00 m in (46) NL: 7.36E4 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [600.00-2000.00]
104
% 1434.4
11H+ 10H+
1578.0
88
75
63
50
38 12H+
1314.9
9H+
25
13H+ 1753.2
14H+ 1213.8
+ 15H+
17H+16H 1052.2
1127.4
8H+
13
986.6
876.9 928.5 1971.2
m /z
0
800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000
A 200 μL of the crude 6 (2.6 nmol, 13 μM) in PBS buffer (10 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. 2c (2.5 μL of a 10 mM stock solution in DMF,
26 nmol, 130 μM final concentration, 10 equiv) was added and the resulting mixture
was vortexed for 30 seconds. After 90 min of additional shaking at RT, the reaction
mixture was analyzed by LC-MS (Figure 46) and a complete conversion to S95
(Theoretical molecular weight: 15794.08. Calculated molecular weight: 15798.4) was
observed.
-S97 -
AV: 0.18-0.94 m in (43) NL: 8.71E4 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [800.00-2000.00]
104
% 1437.2
11H+
88
10H+
1580.9
75
63
50
12H+
38 1317.5
9H+
1756.3
25
A 200 μL of the crude 6 (2.6 nmol, 13 μM) in PBS buffer (10 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. 2d (2.5 μL of a 10 mM stock solution in DMF,
26 nmol, 130 μM final concentration, 10 equiv) was added and the resulting mixture
was vortexed for 30 seconds. After 90 min of additional shaking at RT, the reaction
mixture was analyzed by LC-MS (Figure 47) and a complete conversion to 10
(Theoretical molecular weight: 15808.10. Calculated molecular weight: 15811.1) was
observed.
AV: 0.18-0.97 m in (45) NL: 8.55E4 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [800.00-2000.00]
104
% 1582.2
11H+ 10H+
1438.2
88
75
63
50
12H+
1318.5
38
9H+
1757.7
13H+
25
15H+ 14H+
1217.3
17H+ 16H+
13
1055.1 1130.2 8H+
931.3 989.2 1977.4
879.0
m /z
0
800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000
-S98 -
(5) Reaction of 6 with 7
A 200 μL of the crude 6 (2.6 nmol, 13 μM) in PBS buffer (10 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. 7 (2.5 μL of a 10 mM stock solution in DMF,
26 nmol, 130 μM final concentration, 10 equiv) was added and the resulting mixture
was vortexed for 30 seconds. After 90 min of additional shaking at RT, the reaction
mixture was analyzed by LC-MS (Figure 48) and a complete conversion to 11
(Theoretical molecular weight: 17958.09. Calculated molecular weight: 17961.9) was
observed.
AV: 0.20-0.92 m in (41) NL: 3.36E4 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [800.00-2000.00]
104
% 1634.1
11H+
88
12H+
75 1284.2
14H+
13H+ 1497.9
63
1382.4 10H+
1796.9
50
38
15H+
16H+ 1198.3
17H+
1123.9 1842.8
1654.6
25
1057.5
13
1001.2
948.8
817.9 899.0
m /z
0
800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000
-S99 -
(6) Reaction of 6 with 8
A 200 μL of the crude 6 (2.6 nmol, 13 μM) in PBS buffer (10 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. 8 (2.5 μL of a 10 mM stock solution in DMF,
26 nmol, 130 μM final concentration, 10 equiv) and TCEPHCl (3.7 μL of a stock
solution of 4 mg TCEPHCl in 100 μL 0.5 M NaOH aqueous solution, 520 nmol, 2.6
mM final concentration, 200 equiv) were added. The resulting mixture was vortexed
for 30 seconds and then shaking additional 90 min at RT. The reaction mixture was
analyzed by LC-MS (Figure 49) and a complete conversion to 12 (Theoretical
molecular weight: 16076.48. Calculated molecular weight: 16080.7) was observed.
AV: 0.18-0.86 m in (39) NL: 6.83E4 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [800.00-2000.00]
104
% 1462.7
11H+
88
75 10H+
1609.2
63
12H+
50
1340.9
38
25
14H+ 13H+
1238.0 9H+
15H+
1149.7 1632.1
1787.9
13
17H+ 16H+ 1072.9
1006.0
947.1
897.2
m /z
0
800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000
-S100 -
(7) Reaction of 26 with 14
A 200 μL of the crude 6 (2.6 nmol, 13 μM) in PBS buffer (10 mM, pH 7.4) was
transferred to a 0.5 mL eppendorf tube. 9 (2.5 μL of a 10 mM stock solution in DMF,
26 nmol, 130 μM final concentration, 10 equiv) and TCEPHCl (3.7 μL of a stock
solution of 4 mg TCEPHCl in 100 μL 0.5 M NaOH aqueous solution, 520 nmol, 2.6
mM final concentration, 200 equiv) were added. The resulting mixture vortexed for 30
seconds and then shaking additional 90 min at RT. The reaction mixture was analyzed
by LC-MS (Theoretical molecular weight: 16239.57, calculated molecular weight:
16244.4) (Figure 50) and Gel analysis (Figure 51), and a complete conversion to 13
was observed.
AV: 0.18-0.90 m in (41) NL: 3.06E4 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [800.00-2000.00]
104
% 1477.8
11H+
88
10H+
1625.7
75
63
12H+
1354.8
50
9H+
38
13H+ 1805.5
1250.7
25
+
16H+ 15H+ 14H
13 17H+ 1016.3 1083.9
1161.3
956.5
903.3
1957.8
m /z
0
800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000
To a solution of His-C2A(S105C) (1.0 mL, 55 M) in PBS buffer (10 mM, pH 7.4) was
added a solution of 5 (14 μL of a 20 mM stock solution in DMF, 275 M final
concentration, 5 equiv) and the resulting mixture was vortexed for 30 seconds. After 60
min of additional shaking at RT, the solution was analyzed by HPLC, and a complete
conversion to 14 was observed. Then the solution was purified by preparative HPLC
and lyophilized to obtain 14 for further conjugations. Dichloroquinoxaline-
functionalized protein 14 was redissolved in 1.0 mL PBS (10 mM, pH 7.4) buffer. The
protein solution was divided into ten parts to 0.5 mL eppendorf tubes. Equal volume
PBS, cell lysate or cell growth medium was added to the protein solutions, respectively.
Dichloroquinoxaline-functionalized His-C2A(S105C) 14 was incubated in cell lysate
-S102 -
or cell growth medium for 0 min, 10 min or 60 min, and then o-dithiophenol derivative
9 (5 equiv 9 + 100 equiv TCEP) or 7 (5 equiv) was added, and the resulting mixtures
were vortexed for 30 seconds. After 90 min of additional shaking at RT, the reaction
mixtures were analyzed by SDS-PAGE (Figure S52).
Figure S52. Efficiency in a crude cell lysate or cell medium on the CQ-DT reactions. a)
Reaction routes. b) Gel analysis of the CQ-DT conjugates in the cell lysate. c) o-
Dichloroquinoxaline-functionalized His-C2A(S105C) 14 was incubated in cell lysate
or cell medium for the indicated time, then treated with o-dithiophenol 7, and the
conjugates were determined by Gel analysis. Fluorescence signals were acquired using
EB channel.
-S104 -
Figure S53. Bioorthogonal ligations and cleavages via reactions of
chloroquinoxalines containing protein C2A(S83C) with 2d or 7. Reaction conditions:
(a-e), S96, S97 or 17-19 (2.6 nmol, 13 μM), 2d (26 nmol, 130 μM), PBS (10 mM, pH
= 7.4) (200 L) at RT for 90 min. f), S96, S97 or 17-19 (2.6 nmol, 13 μM), 7 (26 nmol,
130 μM), PBS (10 mM, pH 7.4) (200 L) at RT for 90 min. (Note: The regioselectivity
is ignored during formation of two C-S bonds).
According to the results above, we thought that the present method could be used in
release of biologically active molecules and drugs. First, chloroquinoxaline derivative
S80 containing 4-nitroaniline through linkage of 4-(hydroxymethyl)phenol was used as
the model to investigated a decaged process of active molecule. Reaction of S80 with
2b in PBS (pH 7.4)/acetonitrile (v/v, 1:1) at RT provided ligation product 3b releasing
S98 via HPLC analysis (see Figure S54a). Next, Reaction of S81 with 2b in PBS (pH
-S105 -
7.4)/acetonitrile (v/v, 1:1) at RT afforded ligation product 3b releasing S98 via HPLC
analysis (see Figure S54b).
Figure S54. Trace HPLC of formed products 3b and S98 via reactions of S80 or
S81 with 2b. a) Incubation of S80 (100 nmol, 100 μM) and 2b (1.0 mmol, 1.0 mM)
in 1.0 mL of ACN/PBS (20 mM, pH 7.4) (v/v, 1:1) at RT for indicated time. b)
Incubation of S81 (100 nmol, 100 μM) and 2b (1.0 mmol, 1.0 mM) in 1.0 mL of
ACN/PBS (20 mM, pH 7.4) (v/v, 1:1) at RT for indicated time. Release of 4-nitroaniline
(S98) was monitored by HPLC analysis. (The gradient was programmed as follows: 0-
2 min: 50%-60% B, 2-6 min: 60%-90% B, 6-10 min: 90% B. Water (solvent A) and
MeOH (solvent B) were used as the mobile phase at a flow rate of 1.0 mL min-1.
Monitoring wavelength λ = 405 nm). ES = external standard.
To a solution of C2A(S83C) (200 L, 4.0 nmol, 20 M) in PBS buffer (10 mM, pH 7.4)
was added a solution of S55 (2 μL of a 10 mM stock solution in DMF, 20 nmol, 100
M final concentration, 5 equiv), and the resulting mixture was vortexed for 30 seconds.
After 60 min of additional shaking at RT, the solution was analyzed by LC-MS (Figure
-S106 -
S55) and a complete conversion to S96 (Theoretical molecular weight: 15633.70.
Calculated molecular weight: 15635.0) was observed. Small molecules were removed
from the reaction mixture by using a SpinOUT GT-600 desalting column (G-
Biosciences) equilibrated with PBS buffer (10 mM, pH 7.4). The protein sample
(approximately 0.2 mg/mL) was stored at -20 ºC.
AV: 0.20-0.83 m in (36) NL: 1.33E4 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [800.00-2000.00]
104
% 1564.6
10H+
88
75
11H+
1422.2
63
50
12H+
1303.9
38
13H+
14H+
1203.8
1776.3
1117.7
25
16H+ 15H+
978.1
1043.5
872.3 923.3
13
To a solution of C2A(S83C) (200 L, 4.0 nmol, 20 M) in PBS buffer (10 mM, pH 7.4)
was added a solution of S58 (2 μL of a 10 mM stock solution in DMF, 20 nmol, 100
M final concentration, 5 equiv), and the resulting mixture was vortexed for 30 seconds.
After 60 min of additional shaking at RT, the solution was analyzed by LC-MS (Figure
S56) and a complete conversion to S97 (Theoretical molecular weight: 15665.32.
Calculated molecular weight: 15671.6) was observed. Small molecules were removed
from the reaction mixture by using a SpinOUT GT-600 desalting column (G-
Biosciences) equilibrated with PBS buffer (10 mM, pH 7.4). The protein sample
(approximately 0.2 mg/mL) was stored at -20 ºC.
-S107 -
AV: 0.13-0.83 m in (40) NL: 3.41E3 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [800.00-2000.00]
104
% 980.4 1045.8
16H+ 15H+
88
75
1085.9
25
1381.7
1267.0
13 1960.2
1470.5 1822.7
1531.7
1687.3
m /z
0
800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000
To a solution of C2A(S83C) (200 L, 4.0 nmol, 20 M) in PBS buffer (10 mM, pH 7.4)
was added a solution of S66 (2 μL of a 10 mM stock solution in DMF, 20 nmol, 100
M final concentration, 5 equiv), and the resulting mixture was vortexed for 30 seconds.
After 60 min of additional shaking at RT, the solution was analyzed by LC-MS (Figure
S57) and a complete conversion to 17 (Theoretical molecular weight: 16233.04.
Calculated molecular weight: 16236.7) was observed. Small molecules were removed
from the reaction mixture by using a SpinOUT GT-600 desalting column (G-
Biosciences) equilibrated with PBS buffer (10 mM, pH 7.4). The protein sample
(approximately 0.2 mg/mL) was stored at -20 ºC.
-S108 -
AV: 0.18-0.96 m in (44) NL: 1.62E4 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [600.00-2000.00]
104
% 1624.9
11H+ 10H+
1477.1
88
75
63
50
12H+
1354.0
38
13H+ 9H+
25 15H+ 14H+ 1250.0 1804.7
1083.4
16H+
1160.9
17H+
1878.8
13 1514.9 1666.4
847.7 903.4 956.3 1016.2
1851.5
1191.7 1315.1 1748.8 1957.2
m /z
0
800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000
To a solution of C2A(S83C) (200 L, 4.0 nmol, 20 M) in PBS buffer (10 mM, pH 7.4)
was added a solution of S72 (2 μL of a 10 mM stock solution in DMF, 20 nmol, 100
M final concentration, 5 equiv), and the resulting mixture was vortexed for 30 seconds.
After 60 min of additional shaking at RT, the solution was analyzed by LC-MS (Figure
S58) and a complete conversion to 18 (Theoretical molecular weight: 16247.00.
Calculated molecular weight: 16251.7) was observed. Small molecules were removed
from the reaction mixture by using a SpinOUT GT-600 desalting column (G-
Biosciences) equilibrated with PBS buffer (10 mM, pH 7.4). The protein sample
(approximately 0.2 mg/mL) was stored at -20 ºC.
-S109 -
AV: 0.18-0.95 m in (43) NL: 3.28E4 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [600.00-2000.00]
104
% 1626.3
1478.3
10H+
88
11H+
75
63
50
38
12H+ 9H+
1806.6
1355.2
25
15H+ +
1084.5 14H+ 13H
13 16H+ 1162.0
1251.4 1831.7
841.5
1016.7
903.8 959.5 1282.9
1588.9 1748.0 1942.3
m /z
0
800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000
To a solution of C2A(S83C) (200 L, 4.0 nmol, 20 M) in PBS buffer (10 mM, pH 7.4)
was added a solution of S78 (2 μL of a 10 mM stock solution in DMF, 20 nmol, 100
M final concentration, 5 equiv), and the resulting mixture was vortexed for 30 seconds.
After 60 min of additional shaking at RT, the solution was analyzed by LC-MS (Figure
S59) and a complete conversion to 19 (Theoretical molecular weight: 16200.99.
Calculated molecular weight: 16204.41.9) was observed. Small molecules were
removed from the reaction mixture by using a SpinOUT GT-600 desalting column (G-
Biosciences) equilibrated with PBS buffer (10 mM, pH 7.4). The protein sample
(approximately 0.2 mg/mL) was stored at -20 ºC.
-S110 -
AV: 0.18-1.22 m in (58) NL: 2.92E4 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [600.00-2000.00]
104
% 1621.8
10H+
88
75
63
11H+
1474.2
50
38
9H+
25
13H+ 12H+ 1801.4
14H+
1351.3
16H+ 15H+
1247.7
13
17H+ 1013.9 1081.2
1158.1
819.7 954.3
901.4 1727.1 1893.4
1317.7
m /z
0
800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000
To a 200 μL of S96, S97, 17-19 (2.6 nmol, 13 M) in PBS buffer (10 mM, pH 7.4) was
added a solution of 2d (2.6 μL of a 10 mM stock solution in DMF, 26 nmol, 130 M
final concentration, 10 equiv), and the resulting mixture was vortexed for 30 seconds.
After 90 min of additional shaking at RT, the reaction mixture was analyzed by LC-MS
(Figure S60) and a complete conversion to 20 (Theoretical molecular weight: 15761.05.
Calculated molecular weight: 15762.3) was observed releasing HCl, imidazole, 21 or
22.
AV: 0.22-0.29 m in (5) NL: 4.63E4 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [800.00-2000.00]
104
% 1434.1
11H+
88
75
12H+
63
1314.7
50
13H+
10H+
14H+
38
1213.4
1577.1
15H+ 1126.8
9H+
25
1054.5
1752.3
13
928.0 995.6 1617.2
861.3 1357.7
1695.3
1815.1
1541.3 1875.9 1924.2
m /z
0
800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000
-S111 -
(7) Reaction of 7 with S96, S97, 17-19
To a 200 μL of S96, S97 or 17-19 (2.6 nmol, 13 M) in PBS buffer (10 mM, pH 7.4)
was added a solution of 7 (2.6 μL of a 10 mM stock solution in DMF, 26 nmol, 130 M
final concentration, 10 equiv), and the resulting mixture was vortexed for 30 seconds.
After 90 min of additional shaking at RT, the reaction mixture was analyzed by LC-MS
(Figure S61) and gel analysis (Figure S62), and a complete conversion to 23 releasing
HCl, imidazole, 21 or 22 (Theoretical molecular weight: 17910.68. Calculated
molecular weight: 17919.6) was observed.
AV: 0.22-0.31 m in (6) NL: 2.00E4 T: {0,0} + c ESI !corona s id=45.00 det=1024.00 Full m s [800.00-2000.00]
104
% 1120.9
16H+
88
75
15H+
1195.6
63
+
+17H 14H+
50
18H 1055.1
996.4 1281.1
38
855.6
25
13H+
1223.3 1379.6
944.1
896.8
13 1304.6 1629.8
1494.6 1726.2
1548.6 1678.7 1853.4
1938.3
m /z
0
800 900 1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,700 1,800 1,900 2,000
-S112 -
Figure S62. a) Gel analysis of C2A(S83C) (lane 1) and 20 (lanes 2-6 from reactions of
17-19 or S96, S97 with 2d, respectively). Reaction conditions: 17-19 or S96, S97 (2.6
nmol, 13 μM), 2d (26 nmol, 130 μM), PBS (10 mM, pH 7.4) (200 L) at RT for 90 min.
b) Gel analysis of C2A(S83C) (lane 1) and 23 (lanes 2-6 from reactions of 17-19 or
S96, S97 with 7, respectively). Reaction conditions: 17-19 or S96, S97 (2.6 nmol, 13
μM), 7 (26 nmol, 130 μM), PBS (10 mM, pH 7.4) (200 L) at RT for 90 min.
Fluorescence signals were acquired using EB channel (upper panel). Coomassie
staining was used to assess protein loading (lower panel).
-S113 -
9. References
[1] D. R. Romer, J. Heterocycl. Chem. 2009, 46, 317-319.
[2] D. J. Cram, H. J. Choi, J. A. Bryant, C. B. Knobler, J. Am. Chem. Soc. 1992, 114, 7748-7765.
[3] A. J. Thompson, M. H. Verheij, J. E. van Muijlwijk-Koezen, S. C. Lummis, R. Leurs, I. J. de
Esch, Chemmedchem 2013, 8, 946-955.
[4] A. Conchillo, F. Camps, A. Messeguer, J. Org. Chem. 1990, 55, 1728-1735.
[5] A. Keivanloo, S. S. Kazemi, H. Nasr-Isfahani, A. Bamoniri, Tetrahedron 2016, 72, 6536-6542.
[6] A. Keivanloo, M. Bakherad, A. Rahimi, Synthesis 2010, 2010, 1599-1602.
[7] L. H. Klemm, Y. N. Hwang, J. N. Louris, J. Org. Chem. 1983, 48, 1451-1454.
[8] A. Mahendran, A. Vuong, D. Aebisher, Y. Gong, R. Bittman, G. Arthur, A. Kawamura, A. Greer,
J. Org. Chem. 2010, 75, 5549-5557.
[9] F. Yang, W. Wang, K. Li, W. Zhao, X. Dong, Tetrahedron Lett. 2017, 58, 218-222.
[10] C.-P. Zhang, D. A. Vicic, J. Am. Chem. Soc. 2012, 134, 183-185.
[11] R. J. Bergeron, W. R. Weimar, R. Müller, C. O. Zimmerman, B. H. McCosar, H. Yao, R. E.
Smith, J. Med. Chem. 1998, 41, 3888-3900.
[12] D. Shetty, J. M. Jeong, C. H. Ju, Y. J. Kim, J. Y. Lee, Y. S. Lee, D. S. Lee, J. K. Chung, M. C.
Lee, Bioorg. Med. Chem. 2010, 18, 7338-7347.
[13] U. Soomets, M. Lindgren, X. Gallet, M. Hällbrink, A. Elmquist, L. Balaspiri, M. Zorko, M.
Pooga, R. Brasseur, Ü. Langel, Biochim. Biophys. Acta. 2000, 1467, 165-176.
[14] K. Chenoweth, D. Chenoweth, W. A. Goddard, 3rd, Org. Biomol. Chem. 2009, 7, 5255-5258.
[15] U. Pradere, A. Brunschweiger, L. F. Gebert, M. Lucic, M. Roos, J. Hall, Angew. Chem. Int. Ed.
2013, 52, 12028-12032; Angew. Chem. 2013, 125, 12250-12254.
[16] W. H. Parsons, J. Du Bois, J. Am. Chem. Soc. 2013, 135, 10582-10585.
[17] Y. Hori, T. Norinobu, M. Sato, K. Arita, M. Shirakawa, K. Kikuchi, J. Am. Chem. Soc. 2013,
135, 12360-12365.
[18] J. Nafe, S. Herbert, F. Auras, K. Karaghiosoff, T. Bein, P. Knochel, Chem. Eur. J. 2015, 21,
1102-1107.
[19] U. Soomets, M. Lindgren, X. Gallet, M. Hällbrink, A. Elmquist, L. Balaspiri, M. Zorko, M.
Pooga, R. Brasseur, Ü. Langel, Biochim. Biophys. Acta. 2000, 1467, 165-176.
[20] I. S. Alam, A. A. Neves, T. H. Witney, J. Boren, K. M. Brindle, Bioconjug. Chem. 2010, 21,
884-891.
-S114 -
10. 1H, 13C NMR spectra of the synthesized small molecules
-S115 -
-S116 -
-S117 -
-S118 -
-S119 -
-S120 -
-S121 -
-S122 -
-S123 -
-S124 -
-S125 -
-S126 -
-S127 -
-S128 -
-S129 -
-S130 -
-S131 -
-S132 -
-S133 -
-S134 -
-S135 -
-S136 -
-S137 -
-S138 -
-S139 -
-S140 -
-S141 -
-S142 -
-S143 -
-S144 -
-S145 -
-S146 -
-S147 -
-S148 -
-S149 -
-S150 -
-S151 -
-S152 -
-S153 -
-S154 -
-S155 -
-S156 -
-S157 -
-S158 -
-S159 -
-S160 -
-S161 -
-S162 -
-S163 -
-S164 -
-S165 -
-S166 -
-S167 -
-S168 -
-S169 -
-S170 -
-S171 -
-S172 -
-S173 -
-S174 -
-S175 -
-S176 -
-S177 -
-S178 -
-S179 -
-S180 -
-S181 -
-S182 -
-S183 -
-S184 -
-S185 -
-S186 -
-S187 -
-S188 -
-S189 -
-S190 -
-S191 -
-S192 -
-S193 -
-S194 -
-S195 -
-S196 -
-S197 -
-S198 -
-S199 -
-S200 -
-S201 -
-S202 -
-S203 -
-S204 -
-S205 -
-S206 -
-S207 -
-S208 -
-S209 -
-S210 -