Journal of the American Chemical Society
Page 8 of 13
Synthesis of 2(5(tert-butoxycarbonyl)thiazol-2-yl)-6-amino-5-
A sample of ester 6 (2.13 mg, 0.0040 mmol) in methanol (40 μL) was
hydroxy-benzofuran-N,N,O-triacetic acid trimethyl ester, 3
treated with aqueous potassium hydroxide (40 μL, 1.2 M, 0.048 mmol)
and stirred at room temperature for 24 h. The solution was quantita-
tively transferred to a volumetric flask and diluted with buffer at pH
7.0 (50 mM PIPES, 100 mM KCl) to a final volume of 2 mL. Quantita-
tive hydrolysis was verified by HPLC. The stock solution was divided
into small aliquots, flash frozen in liquid nitrogen, and stored below -
20 °C.
1
2
3
4
5
6
7
8
A solution of salicylaldehyde 2 (100 mg, 0.27 mmol), tert-butyl 2-
(bromomethyl)thiazole-5-carboxylate 1 (113 mg, 0.41 mmol), and
K2CO3 (187 mg, 1.35 mmol) in dry dimethylformamide (2.0 mL) was
heated to 100 °C under inert atmosphere for 1.5 h. After allowing to
cool to room temperature, the reaction was poured into water (30 mL)
and extracted with EtOAc (3×30 mL). The combined extracts were
dried over Na2SO4 and concentrated in vacuo. The residue was puri-
fied by column chromatography on silica gel (1:1 EtOAc:hexanes) to
give product 3 as a yellow solid (110 mg, 74%, Rf = 0.38 in 1:1 hex-
anes:EtOAc). M.p. 129-130 °C. 1H NMR (400 MHz, CDCl3, δ) 8.32 (s,
1H), 7.31 (s, 1H), 7.10 (s, 1H), 7.04 (s, 1H), 4.70 (s, 2H), 4.27 (s, 4H),
3.80 (s, 3H), 3.75 (s, 6H), 1.60 (s, 9H). 13C{1H} NMR (100 MHz,
CDCl3, δ) 171.5, 169.4, 162.3, 160.7, 151.9, 149.5, 149.1, 148.2, 140.5,
130.7, 122.4, 107.1, 107.0, 102.9, 83.0, 67.1, 54.0, 52.3, 52.1, 28.4. ESI-
MS (m/z): [M+H]+ calcd for C25H28N2O10S, 549.2; found 549.2.
Synthesis of Mag-S-Tz
A mixture of succinimidyl ester 5 (30 mg, 0.051 mmol) and tert-butyl
(4-aminobutyl)carbamate 8 (15 mg, 0.076 mmol) in acetonitrile (1.5
mL) was treated with two drops of triethylamine and stirred at room
temperature for 30 min. The white precipitate was filtered off and the
filtrate was concentrated in vacuo and redissolved in dichloromethane
(0.8 mL). The resulting solution was treated with trifluoroacetic acid
(0.2 mL) and stirred at room temperature for 1 hour. The volatiles
were evaporated and the residue was redissolved in dichloromethane
(20 mL). The solution was washed with aqueous K2CO3 (20 mL, 1 M),
dried over Na2SO4 and concentrated in vacuo. To the residue was
added methanol (600 μL) and aqueous KOH (300 μL, 1.2 M), and the
mixture was stirred at room temperature overnight. The reaction
solution was neutralized with aqueous HCl and lyophilized. The resi-
due was dissolved in MeOH (3 mL) and triethylamine (10 μL) and
tetrazine 9 (20 mg, 0.064 mmol) were added. The reaction mixture
was stirred at room temperature for 30 min and concentrated in vac-
uo. The residue was purified by reverse phase semi-prep HPLC to give
Mag-S-Tz as a red solid (10 mg, 27%). M.p. 230-231 °C. 1H NMR (400
MHz, DMSO-d6, δ) 12.5 (br, 3H), 8.76 (t, J = 5.6 Hz, 1H), 8.72 (t, J =
5.6 Hz, 1H), 8.54 (d, J = 8.6 Hz, 2H), 8.45 (s, 1H), 8.10 (d, J = 8.6 Hz,
2H), 7.49 (d, J = 0.6 Hz, 1H), 7.16 (s, 1H), 7.07 (s, 1H), 4.72 (s, 2H),
4.19 (s, 4H), 3.33 (overlapping solvent), 3.01 (s, 3H), 1.62 (s, 4H).
13C{1H} NMR (100 MHz, DMSO-d6, δ) 172.1, 170.2, 167.3, 165.4,
162.9, 159.6, 159.5, 150.7, 148.3, 147.5, 144.1, 140.0, 137.9, 135.2,
134.1, 128.2, 127.3, 120.6, 106.8, 105.5, 101.4, 65.6, 53.5, 39.1, 26.6,
20.9. ESI-MS (m/z): [M+H]+ calcd for C32H30N8O10S, 719.2; found
719.1.
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10
11
12
13
14
15
16
17
18
19
20
21
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23
24
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26
27
28
29
30
31
32
33
34
35
36
37
38
39
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41
42
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51
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53
54
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56
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60
Synthesis
of
2(5-carboxythiazol-2-yl)-6-amino-5-hydroxy-
benzofuran-N,N,O-triacetic acid trimethyl ester, 4
A solution of compound 3 (38 mg, 0.069 mmol) in dichloromethane
(0.8 mL) was treated with trifluoroacetic acid (0.2 mL) and stirred at
room temperature overnight. The volatiles were evaporated and the
residue was recrystallized in ethyl acetate/hexanes to give compound 4
as a yellow solid (30 mg, 88%, Rf = 0.37 in 10:1 CH2Cl2/MeOH). M.p.
145-146 °C (decomp.). 1H NMR (400 MHz, DMSO-d6, δ) 8.34 (s, 1H),
7.52 (s, 1H), 7.20 (s, 1H), 7.14 (s, 1H), 4.82 (s, 2H), 4.26 (s, 4H), 3.71
(s, 3H), 3.64 (s, 6H). 13C{1H} NMR (100 MHz, DMSO-d6, δ) 170.9,
169.1, 162.1, 160.6, 150.8, 148.5, 148.4, 147.2, 139.9, 131.8, 120.8,
107.2, 105.9, 101.5, 65.7, 53.4, 51.9, 51.5. ESI-MS (m/z): [M+H]+ calcd
for C21H20N2O10S, 493.1; found 493.1.
Synthesis of 2(5(succinimidylcarboxy)thiazol-2-yl)-6-amino-5-
hydroxy-benzofuran-N,N,O-triacetic acid trimethyl ester, 5
A
solution of compound 4 (30 mg, 0.061 mmol) and N-
hydroxysuccinimide (8 mg, 0.067 mmol) in dry dimethylformamide
(1 mL) was stirred at room temperature for 5 min. Then a solution of
DCC (15 mg, 0.073 mmol) in dry dimethylformamide (0.3 mL) was
added. The reaction mixture was stirred at room temperature over-
night. The white precipitate was filtered off and the filtrate was con-
centrated in vacuo. The residue was purified by column chromatog-
raphy on silica gel (1:1 EtOAc:hexanes) to give product 5 as a yellow
Synthesis of Mag-S-Tz acetoxymethyl ester, Mag-S-Tz-AM
A mixture of compound Mag-S-Tz (0.21 mg, 0.3 μmol) in dimethyl-
formamide (300 μL) was treated with diisopropylethylamine (10 μL,
100 μmol) and bromomethylacetate (5 μL, 50 μmol) at room tempera-
ture for 1 h, protected from light. The mixture was purified by reverse
phase semi-prep HPLC to give Mag-S-Tz-AM as a red solid (0.11 mg,
40%). HR-TOF-MS (m/z): [M+H]+ calcd for C41H42N8O16S, 935.25112;
found 935.25166.
1
solid (30 mg, 84%, Rf = 0.58 in EtOAc). M.p. 164-165 °C. H NMR
(600 MHz, DMSO-d6, δ) 8.89 (s, 1H), 7.73 (d, J = 0.7 Hz, 1H), 7.24 (s,
1H), 7.14 (s, 1H), 4.84 (s, 2H), 4.28 (s, 4H), 3.72 (s, 3H), 3.64 (s, 6H),
2.90 (s, 4H). 13C{1H} NMR (150 MHz, DMSO-d6, δ) 170.9, 170.2,
169.1, 164.2, 156.7, 153.4, 151.5, 147.5, 147.3, 140.8, 120.6, 109.7,
105.8, 101.2, 65.6, 53.5, 51.9, 51.6, 25.6. ESI-MS (m/z): [M+H]+ calcd
for C25H23N3O12S, 590.1; found 590.5.
General Spectroscopic Methods
All aqueous solutions were prepared using de-ionized water having a
resistivity of 18 MΩ/cm. Other solvents were obtained from commer-
cial vendors and used as received. Piperazine-N,N- bis(2-
ethanesulfonic acid) (PIPES), 99.999% KCl, 99.999% MgCl2, 99.999%
ZnCl2, 99.99% CaCl2, and high-purity 25% HCl, 45% KOH, and 50%
NaOH were purchased from Sigma Aldrich. Stock solutions of the
sensors in their acid form were stored at -20 °C in 100-200 μL ali-
quots, and thawed immediately before each experiment. Spectroscopic
measurements at pH 7.0 were conducted in aqueous buffer containing
50 mM PIPES and 100 mM KCl. Measurements containing purified
HaloTag protein were conducted at in 50 mM PIPES buffer at pH 7.
Buffers were treated with Chelex resin (Bio-Rad) to remove adventi-
tious metal ions according to the manufacturer’s protocol, unless
otherwise noted. Measurements of pH were conducted using a Mettler
Toledo FE20 with glass electrode. UV-visible spectra were acquired on
a Cary 100 spectrophotometer using 1 cm quartz cuvettes. Fluores-
cence spectra were acquired on a QuantaMaster 40 Photon Technolo-
gy International spectrofluorometer equipped with xenon lamp
source, emission and excitation monochromators, excitation correc-
tion unit, and PMT detector. Emission spectra were corrected for the
detector wavelength-dependent response. The excitation spectra were
corrected for the wavelength-dependent lamp intensity. Measure-
Synthesis of 2(5(propylaminocarbonyl)thiazol-2-yl)-6-amino-5-
hydroxy-benzofuran-N,N,O-triacetic acid trimethyl ester, 6
To a solution of compound 5 (7.0 mg, 12 μmol) and propylamine (1.5
μL, 18 μmol) in MeCN (1.0 mL) was added Et3N (20 μL) and the reac-
tion mixture was stirred at room temperature for 1 h. The solvent was
evaporated and the residue was redissolved in EtOAc (5.0 mL). The
resulting solution was washed with water (3×10 mL), dried over
Na2SO4 and concentrated in vacuo. The residue was purified by col-
umn chromatography on silica gel (40:1 CH2Cl2/MeOH) to give com-
pound 6 as a yellow solid (5.2 mg, 80%, Rf = 0.56 in 19:1
1
CH2Cl2/MeOH). M.p. 169-170 °C. H NMR (400 MHz, CD3CN, δ):
8.20 (s, 1H), 7.34 (d, J = 0.8 Hz, 1H), 7.15 (t, J = 5.3 Hz, 1H), 7.11 (d, J
= 0.8 Hz, 1H), 7.10 (s, 1H), 4.73 (s, 2H), 4.23 (s, 4H), 3.75 (s, 3H), 3.67
(s, 6H), 3.32-3.27 (m, 2H), 1.65-1.55 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H).
13C{1H} NMR (100 MHz, CD3CN, δ):172.2, 170.3, 161.2, 161.0, 152.2,
150.4, 148.8, 144.8, 141.1, 136.1, 122.7, 107.2, 107.0, 103.1, 67.0, 54.5,
52.7, 52.3, 42.3, 23.4, 11.7. HR-TOF-MS (m/z): [M+H]+ calcd for
C24H27N3O9S, 534.15408; found 534.15647.
Preparation of compound 7 (quantitative stock solution for fluo-
rescence spectroscopy)
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