Journal of the American Chemical Society
Article
reaction on His-tagged protein 100 μM NiCl2(100× stock in water)
was also added. The reaction was mixed thoroughly and placed in the
dark at room temperature. After 1 h the reaction was quenched by
adding 500 μM EDTA.
For click reaction with Cy3-N3, proteins were precipitated with
acetone prior to click reaction to remove excess TPAC. The pellet was
dissolved in PBS containing 0.1% SDS for click reaction, and
precipitated again to remove unreacted dye. The pellet was then
dissolved in running buffer and analyzed by SDS-PAGE.
For click reaction on TPAC-labeled, His-tagged proteins, proteins
were buffer-exchanged into 25 mM HEPES, pH 7.5 by extensive
ultrafiltration (Amnicon 10K, EMD Millipore, Hayward CA) prior to
click reaction. After the click reaction, proteins were buffer-exchanged
into PBS by extensive ultrafiltration to remove unwanted chemicals.
Cell Culture and Imaging. Cells were grown in the Cell Culture
Facility at the University of California, Berkeley. HeLa cells were
cultured in DMEM supplemented with 10% FBS and glutamine (2
mM). One day before imaging, cells were passed and plated on eight-
well chamber slides (Lab-Tek, Thermo Fisher).
For imaging, cells were grown on 8-well chamber slides (LabTek,
Thermo Fisher) to desired confluency, washed with PBS and
incubated with 0.1 mg/mL (Arg)9-labeled GFP or mCherry in PBS
for 30 min at 37 °C. The cells were then washed with PBS and stained
with 1 μM Hoechst 33342 for 15 min, washed again and imaged on a
Zeiss LSM710 laser-scanning microscope with a 20× objective lens
for quantification and a 63× oil-immersion objective lens for images.
Excitation was provided at 405 for Hoechst 33 342, 488 nm for GFP
and 543 nm for mCherry.
N2. Triethylamine (1.5 mL, 11 mmol) was dissolved in Et2O (20 mL)
and added dropwise via addition funnel to form a white suspension.
The reaction mixture was warmed to room temperature and was
furthered stirred at room temperature for 2 h. Trimethylamine
hydrochloride was removed by filtration, and the solution was
concentrated to give product 5 as a light yellow oil with a pungent
smell (1.8 g, 99%). The product was used without further purification.
1H NMR (400 MHz, CDCl3) δ 4.92 (dd, J = 14.5, 1.0 Hz, 2H), 2.76
(t, J = 1.0 Hz 1H). 13C NMR (101 MHz, CDCl3) δ 78.93, 74.91 (d, J
= 9.6 Hz), 58.53 (d, J = 7.2 Hz). 31P NMR (162 MHz, CDCl3) δ 8.70.
LRMS (EI+) m/z calcd 137.0, found 136.9 for C3H3ClO2P+ (M−
Cl−).
Synthesis of Methyl Propargyl Phosphorochloridate (3).
Crude 5 with trimethylamine hydrochloride in Et2O was prepared as
described above. The mixture was cooled again in dry ice/acetone
bath. Dry methanol (0.43 mL, 11 mmol) and trimethylamine (1.5
mL, 11 mmol) in Et2O (20 mL) was added dropwise via addition
funnel. The slurry was further stirred at room temperature for 3 h,
filtered and concentrated to give product 3 as light yellow oil with a
pungent smell (1.7 g, 93%). 1H NMR (400 MHz, CDCl3) δ 4.79 (dd,
J = 11.5, 2.5 Hz, 2H), 3.92 (d, J = 13.8 Hz, 3H), 2.66 (t, J = 2.5 Hz,
1H). 13C NMR (101 MHz, CDCl3) δ 78.92, 76.04 (d, J = 9.0 Hz),
56.66 (d, J = 5.3 Hz), 55.97 (d, J = 7.0 Hz). 31P NMR (162 MHz,
CDCl3) δ 6.74. HRMS (APCI+) m/z calcd 168.9816, found 168.9855
for C4H7ClO3P+ (M+H+).
Synthesis of N-Propargyl Thiophosphoramidic Dichloride
(6). To a solution of PSCl3 (0.30 mL, 2.9 mmol) in CH2Cl2 (5 mL)
was added propargyl amine (0.19 mL, 2.9 mmol) and K2CO3 (0.41 g,
2.9 mmol). After 2 h stirring at room temperature, the mixture was
filtered and purified by column chromatography (30:1 hexanes/
EtOAc) to give product 6 as a yellow oil with a pungent smell (0.25 g,
Synthesis of Methyl Propargyl Thiophosphorochloridate
(2). To a flask containing PSCl3 (2 mL, 20 mmol) cooled in ice/water
bath was added dry methanol (2.0 mL, 49 mmol) dropwise. The
mixture was stirred for 15 min on ice and excess methanol was
distilled under vacuum at the same temperature to give crude methyl
thiophosphorodichloridate. Sodium (0.453 g, 19.7 mmol) was
dissolved in cooled propargyl alcohol (6.0 mL, 104 mmol) to form
an orange, thick solution, which was added dropwise to methyl
thiophosphorodichloridate cooled in ice/water bath. The suspension
was further stirred for 2 h at room temperature, diluted with CH2Cl2,
filtered, and purified by column chromatography (30:1 hexanes/
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49%). H NMR (400 MHz, Acetone) δ 4.09 (dd, J = 20.7, 2.5 Hz,
2H), 2.89 (t, J = 2.5 Hz, 1H). 13C NMR (101 MHz, Acetone) δ79.90
(d, J = 8.8 Hz), 74.13, 33.42. 31P NMR (162 MHz, Acetone) δ 57.59.
LRMS (EI+) m/z calcd 151.9, found 152.0 for C3H4ClNPS+ (M−
Cl−).
Synthesis of Propargyl Dithiophosphorodichloridate (7). S-
Propargyl thioacetate (0.35 mg, 3.1 mmol) was stirred with NaOH
(0.16 g, 4.0 mmol) in MeOH (10 mL) under N2 for 30 min. The
mixture was then diluted with CH2Cl2 (30 mL), washed with H2O
(×4) and dried (Na2SO4). To this solution was added PSCl3 (0.37
mL, 3.7 mmol) and K2CO3 (0.51g, 3.7 mmol) and the mixture was
stirred overnight. The mixture was filtered and purified by column
chromatography (100:1 hexanes/EtOAc) to give product 7 as a light
yellow oil, which degenerates into a solid mixture within minutes after
concentration. We were able to obtain a crude 1H NMR before
degeneration and also confirmed its negligible reactivity in aqueous
1
EtOAc) to give product 2 as a colorless oil (2.4 g, 67%). H NMR
(400 MHz, CDCl3) δ 4.85−4.80 (m, 2H), 3.91 (d, J = 16.1 Hz, 3H),
2.63 (t, J = 2.5 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 77.16 (s),
76.39 (d, J = 10.5 Hz), 56.90 (d, J = 4.4 Hz), 55.98 (d, J = 7.1 Hz).
31P NMR (162 MHz, CDCl3) δ 71.91. HRMS (APCI+) m/z calcd
184.9587, found 184.9586 for C4H7ClO2PS+ (M+H+).
Synthesis of Methyl Propargyl Thiophosphoramidate (1).
Crude 2 in propargyl alcohol and CH2Cl2 was prepared as described
above. Excess ammonia was led through this mixture to form NH4Cl
as a precipitate. The mixture was filtered, concentrated, and purified
by column chromatography (2:1 hexanes/EtOAc) to give product 1
1
buffer with histidine when generated in situ. H NMR (600 MHz,
CDCl3) δ 3.75 (dd, J = 15.6, 2.7 Hz, 2H), 2.35 (t, J = 2.7 Hz, 1H).
Synthesis of 2-Azidoethyl Thiophosphorodichloridate (8).
2-Azidoethanol (0.43 g, 4.9 mmol) was dissolved in dry THF (15
mL) under N2 and cooled in dry ice/acetone bath. To this solution
was added dropwise nBuLi (2.0 mL, 2.5 M in hexanes) and stirred for
20 min at room temperature to form the lithium salt. To another flask
cooled in dry ice/acetone bath was added THF (15 mL) and PSCl3
(1.0 mL, 4.9 mmol) under N2. The lithium salt solution was then
added dropwise at this temperature and the mixture was then stirred
for 1 h at room temperature. The mixture was concentrated, diluted in
CH2Cl2, filtered to remove lithium chloride and purified by column
chromatography (50:1 hexanes/EtOAc) to give product 8 as a light
1
as a light yellow oil (2.1 g, 65% overall yield). H NMR (400 MHz,
MeOD) δ 4.62 (dd, J = 10.5, 2.5 Hz, 2H), 3.68 (d, J = 13.8 Hz, 3H),
2.94 (t, J = 2.5 Hz, 1H). 13C NMR (101 MHz, MeOD) δ 79.47 (d, J
= 10.9 Hz), 76.35, 55.04 (d, J = 2.8 Hz), 53.72 (d, J = 5.3 Hz). 31P
NMR (162 MHz, MeOD) δ 78.25. HRMS (APCI+) m/z calcd
166.0086, found 166.0097 for C4H9NO2PS+ (M+H+).
Synthesis of Propargyl Thiophosphorodichloridate (4,
TPAC). To a solution of PSCl3 (1.0 mL, 9.8 mmol) in CH2Cl2 (10
mL) were added propargyl alcohol (0.57 mL, 9.8 mmol) and K2CO3
(1.36 g, 9.8 mmol). After overnight stirring at room temperature, the
mixture was filtered and purified by column chromatography (50:1
hexanes/EtOAc) to give product TPAC as a colorless oil with a
pungent smell (1.1 g, 55%). 1H NMR (400 MHz, CDCl3) δ 4.94 (dd,
J = 15.9, 2.5 Hz, 2H), 2.72 (t, J = 2.5 Hz, 1H). 13C NMR (101 MHz,
CDCl3) δ 78.37, 75.48 (d, J = 11.1 Hz), 58.52 (d, J = 7.4 Hz). 31P
NMR (162 MHz, CDCl3) δ 59.68. HRMS (APCI+) m/z calcd
188.9092, found 188.9115 for C3H4Cl2OPS+ (M+H+).
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yellow oil with a pungent smell (0.60 g, 56%). H NMR (400 MHz,
CDCl3) δ 4.45 (dt, J = 11.0, 5.1 Hz, 2H), 3.63 (t, J = 4.4 Hz, 2H). 13
C
NMR (101 MHz, CDCl3) δ 69.57 (d, J = 9.9 Hz), 50.11 (d, J = 10.6
Hz). 31P NMR (162 MHz, CDCl3) δ 59.05. HRMS (APCI+) m/z
calcd 191.9202, found 191.9219 for C2H5Cl2NOPS+ (M−N2+H+).
Synthesis of ( )-3-Butyn-2-yl Thiophosphorodichloridate
(9). ( )-3-Butyn-2-ol (0.77 mL, 9.8 mmol) was dissolved in dry THF
(10 mL) under N2 and cooled in dry ice/acetone bath. To this
solution was added dropwise nBuLi (3.9 mL, 2.5 M in hexanes) with
stirring for 20 min at room temperature to form the lithium salt. To
Synthesis of Propargyl Phosphorodichloridate (5). POCl3
(1.0 mL, 11 mmol) and propargyl alcohol (0.62 mL, 11 mmol) were
dissolved in Et2O (20 mL) and cooled in dry ice/acetone bath under
F
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX