10.1002/cbic.201800665
ChemBioChem
FULL PAPER
70.57, 61.62, 55.15, 53.15. ESI-HRMS calculated for C13H17N3O6
[M+Na]+ 334.1010, found 334.1007.16
Experimental Section
Materials and Methods: Reagents were purchased from Sigma-Aldrich,
TCI, and Acros and were used without further purification unless
otherwise specified. Solvents were purchased from Sigma-Aldrich and
Fisher Scientific, and dried by standard techniques. NMR solvents were
purchased from Cambridge Isotope Laboratories (Andover, MA). All
reactions were monitored with analytical TLC (Merck Kieselgel 60 F254).
Column chromatography was carried out with Teledyne ISCO Combiflash
Rf with silica gel particle size 40–63 um. NMR spectra were obtained on
Varian Mercury 300 MHz and Varian VX 500 MHz. Mass spectra were
obtained on an Agilent 6230 HR-ESI-TOF MS at the Mass Spectrometry
Facility at the UCSD Chemistry and Biochemistry Department.
5-chloro-7-benzylamino-1-deaza-purine riboside (11): Solid 7 (7.9 mg,
0.025 mmol) was placed in a pressure vial and flushed with argon. n-
Butanol (2 mL) and benzylamine (0.11 mL, 1.0 mmol, 40 eq.) were added
to the mixture and then stirred at 90⁰C for 24 hours. The mixture was
cooled and the excess solvent removed under vacuum. The resulting
residue was purified with column chromatography with a gradient of 0 to
5% MeOH in DCM (Rf: 0.3 in 5% MeOH in DCM) yielding 11 as a glassy
yellow solid (8.5 mg, 87%). 1H NMR (300MHz, CD3OD): δ 8.25 (s, 1H),
7.45–7.25 (m, 5H), 6.42 (s, 1H), 5.94 (d, J = 6.4 Hz, 1H), 4.77 (t, J = 6.0
Hz, 1H), 4.62 (s, 2H), 4.33 (dd, J = 5.1, 2.6 Hz, 1H), 4.18 (q, J = 2.6 Hz,
1H), 3.92, 3.76 (ABq, J = 12.5 Hz, 2H). 13C NMR (125MHz, CD3OD): δ
149.10, 147.15, 144.10, 139.96, 122.75, 97.50, 89.92, 86.57, 73.52,
71.23, 62.19, 20.65. ESI-HRMS calculated for C18H19ClN4O4 [M–H]–
389.1022, found 389.1019.16
Abbreviations: DCM: dichloromethane; DMF: N,N-dimethylformamide;
MeOH: methanol; TFA: Trifluoroacetic acid; DMSO: Dimethyl sulfoxide;
ACN: Acetonitrile; TAR: Tetra-O-acetyl-β-D-ribofuranose
5-chloro-7-butylamino-1-deaza-purine riboside (12): Solid 7 (8.2 mg,
0.026 mmol) was placed in a pressure vial and flushed with argon. n-
Butanol (2 mL) and n-butylamine (0.1 mL, 1.0 mmol, 38 eq.) were added
to the mixture and then stirred at 90⁰C overnight. The mixture was
cooled and the solvent evaporated off with a rotary evaporator. The
Key Intermediate 7: Compounds 2–7 were synthesized based on
previously published procedures19,20 and 1 was purchased from Sigma-
Aldrich.
5-chloro-7-methylamino-1-deaza-purine riboside (8): Solid 7 (10 mg,
0.031 mmol) was placed in a pressure vial (8 mL) and then dissolved in
methylamine in ethanol (33wt%, 2 mL, 16 mmol, 520 eq.) while stirring.
The vial was capped and the mixture brought to 80⁰C and stirred for 48
hours. The mixture was allowed to cool and the solvent evaporated off.
The resulting residue was purified using column chromatography with a
gradient of 0 to 10% MeOH in DCM (Rf: 0.3 in 10% MeOH in DCM)
yielding 8 as a glassy, clear solid (8.2 mg, 84%). 1H NMR (300 MHz,
CD3OD): δ 8.22 (s, 1H), 6.43 (s, 1H), 5.94 (d, J = 6.4 Hz, 1H), 4.78 (t, J =
6.0 Hz, 1H), 4.34 (dd, J = 5.0, 2.6 Hz, 1H), 4.18 (q, J = 2.4 Hz, 1H), 3.91,
3.77 (ABq, J = 12.6 Hz, 2H), 3.01 (s, 3H). 13C NMR (125 MHz, CD3OD):
δ 149.10, 147.15, 144.10, 139.96, 139.88, 122.75, 97.50, 89.92, 86.57,
73.52, 71.23, 62.19. ESI-HRMS calculated for C12H15ClN4O4 [M+H]+
315.0855, found 315.0854.16,18a
resulting residue was purified with column chromatography with
a
gradient of 0 to 10% MeOH in DCM (Rf: 0.4 in 10% MeOH in DCM)
yielding 12 as a glassy solid (4.1 mg, 44%). 1H NMR (300MHz, CD3OD):
δ 8.23 (s, 1H), 6.45 (s, 1H), 5.93 (d, J = 6.4 Hz, 1H), 4.77 (t, J = 6.0 Hz,
1H), 4.34 (dd, J = 5.0, 2.6 Hz, 1H), 4.18 (q, J = 2.5 Hz, 1H), 3.92, 3.76
(ABq, J = 12.5 Hz, 2H), 3.38 (t, J = 6.9 Hz, 2H), 1.79 – 1.63 (m, 2H), 1.51
(dq, J = 14.3, 7.2 Hz 2H), 1.02 (t, J = 7.3 Hz, 3H). 13C NMR (125MHz,
CD3OD): δ 149.10, 147.15, 144.10, 139.96, 122.75, 97.50, 89.92, 86.57,
73.52, 71.23, 62.19, 20.65. ESI-HRMS calculated for C15H21ClN4O4
[M+H]+ 357.1324, found 357.1320.16
Henri-Michaelis-Menten Kinetics General Methods: Bovine spleen
ADA was obtained from Sigma Aldrich (EC Number 232-817-5). The
commercial solution [1150 U mL–1 in 3.2 M (NH4)2SO4, 0.01 M potassium
phosphate, pH 6.0] was diluted to 1.15 U mL–1 by dissolving an aliquot (1
μL) in phosphate buffer (999 μL, 50 mM, pH 7.4). The enzyme stock
solution was freshly prepared and kept on ice prior to use. The enzyme
stock solution was diluted to concentrations of 4.5, 5.6, 6.7, 7.8, and 8.9
mU mL–1 with phosphate buffer. A total of 20 μL enzyme solution was
mixed with 200 μL of substrate solution in the stopped flow apparatus
chamber yielding final enzyme concentrations of 4.1, 5.1, 6.1, 7.1, and
5-chloro-7-methylthio-1-deaza-purine riboside (9): Solid 7 (7.6 mg,
0.024 mmol) was placed in a pressure vial and flushed with argon.
Sodium methanethiolate in MeOH (0.5 M, 2 mL, 1.0 mmol, 42 eq.) was
added to the mixture and then stirred at 105⁰C for 24 hours. The mixture
was cooled and the solvent evaporated off. The resulting residue was
purified with column chromatography with a gradient of 0 to 10% MeOH
in DCM (Rf: 0.5 in 10% MeOH in DCM) yielding 9 as a pale yellow solid
(6.7 mg, 84%). 1H NMR (300MHz, CD3OD): δ 8.57 (s, 1H), 7.19 (s, 1H),
6.07 (d, J = 5.7 Hz, 1H), 4.73 (t, J = 5.4 Hz, 1H), 4.37 (t, J = 4.5 Hz, 1H),
4.18 (q, J = 3.1 Hz, 1H), 3.92, 3.80 (ABq, J = 12.3 Hz, 2H), 2.67 (s, 3H).
13C NMR (125MHz, CD3OD): δ 146.20, 146.02, 143.17, 142.75, 131.52,
112.37, 89.43, 86.07, 73.99, 70.75, 61.69, 11.98. ESI-HRMS calculated
for C12H14ClN3O4S [M–H]– 330.0321, found 330.0320.16
8.1 mU mL–1
.
Concentrated stock solutions (3.7 mM, 4.1 mM) in DMSO were prepared
for tzA and tz2-AA. A 1.0 mM solution was prepared for tzA and tz2-AA
from the concentrated stock solutions and autoclaved water. A 1.0 mM
solution of adenosine was prepared in autoclaved water from solid
adenosine.
5,7-dimethoxy-1-deaza-purine riboside (10): Solid 7 (7.8 mg, 0.025
mmol) was placed in a pressure vial and flushed with argon. Sodium
methoxide in MeOH (2 mL, 0.5M, 40 eq.) was added to the mixture and
then stirred at 105⁰C for 24 hours. The mixture was cooled and then
placed under vacuum to remove excess solvent. The resulting residue
was purified with column chromatography with a gradient of 0 to 10%
MeOH in DCM (Rf: 0.4 in 10% MeOH in DCM) yielding 10 as a pale
yellow solid (2.0 mg, 26%). 1H NMR (300MHz, CD3OD): δ 8.26 (s, 1H),
6.30 (s, 1H), 6.06 (d, J = 5.3 Hz, 1H), 4.81 (t, J = 5.3 Hz, 1H), 4.42 (t, J =
4.5 Hz, 1H), 4.11 (dd, J = 7.9, 3.9 Hz, 1H), 4.03 (s, 3H), 3.98 (s, 3H),
3.88, 3.78 (ABq, J = 12.0 Hz, 2H). 13C NMR (125MHz, CD3OD): δ
163.71, 159.66, 144.59, 139.05, 120.86, 88.67, 88.02, 85.14, 73.75,
The ADA-mediated enzymatic conversion of adenosine (and its analogs)
was followed by absorbance and emission (for the emissive analogs tzA
and tz2-AA) spectroscopy by monitoring the intensity variation for
absorbance and voltage output for emission as a function of time. The
real-time conversion of adenosine (and its analogs) to inosine (and its
analogs) were performed on an Applied Photophysics Pistar Stop Flow
Apparatus.
Stopped Flow Fluorescence Measurements and HMM Kinetic
Analysis of Enzymatic Conversion of tzA to tzI: Substrate
concentrations ranging from 3.9 to 34.5 μM were used keeping the
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