The Journal of Organic Chemistry
Article
[1,3-15N2]3 (56 mg, 0.43 mmol), and 0.3 mL of AcOH were
combined to give 76.8 mg of a crude yellow solid (86% pure as
determined by HPLC); HRMS (ESI+) [M + H]+ calculated for
C17H21N215N2O6 379.1396, observed 379.1401.
5-Deazariboflavin (8). 5-Deazariboflavin (88% pure as determined
by HPLC) was synthesized as previously described28 and was a
generous gift from Dr. Seoung Ryoung-Choi. HRMS (ESI+) [M + H]+
calculated for C18H22N3O6 376.1503, observed 376.1505.
yellow solid; 1H NMR (500 MHz, D2O) δ ppm 7.76 (s, 1H), 7.60 (s,
1H), 5.03 (t, J = 10 Hz, 1H), 4.68 (d, J = 15 Hz, 1H), 4.38−4.35 (m,
1H), 4.15−3.97 (m, 4H), 2.49 (s, 3H), 2.37 (s, 3H); 13C NMR (125
1
2
MHz, D2O) δ ppm 163.6 (dd, JC−C = 76.1 Hz, JC−N = 7.56 Hz),
160.3, 153.2, 152.4 (d, 1JC−C = 55.44 Hz), 142, 136.8, 136.4 (d, JC−N
=
3
3
3.78 Hz), 134.2 (d, JC−C = 6.17 Hz), 132.9 (t, JC−C = 6.3 Hz), 129,
119.5, 75, 73.8 (d, JC−P = 7.56 Hz), 71.8, 68.8, 50, 23.3, 21.2; 31P NMR
(121 MHz, D2O) δ ppm 1.95; HRMS (ESI−) calculated for
C1613CH2014N315NO9P (M − H)− m/z = 457.0972, found 457.0971.
[4a-13C]FMN. Following the procedure described for FMN; crude
[4a-13C]7 (10 mg, 0.0265 mmol, 0.53 mM) and ATP (137.785 mg,
0.25 mmol, 5 mM) were incubated with riboflavin kinase (9.423 mg,
0.5 μmol, 10 μM) in 50 mL of buffer containing 5 mM MgCl2 and
Expression and Purification of Homo sapiens (hs) Riboflavin
Kinase (RFK). E. coli BL21 (DE3) cells were transformed with Homo
sapiens (hs) riboflavin kinase plasmid (HS_RFK_EC_1_pQE-T7)
containing the gene for RFK with a N-terminal His6 affinity tag
(Qiagen). Starting from a single colony, the cells were grown in 1 L of
MDG [Luria Broth34 + 1% glucose] containing 34 μg/mL kanamycin
at 37 °C, 250 rpm to OD600 ≈ 0.6. Expression of the protein was
induced by addition of isopropyl-β-D-thiogalactoside (IPTG, final
concentration of 1 mM). Incubation was continued for 4 h at 37 °C.
The cells were harvested by centrifugation (6000g, 25 min, 4 °C) and
stored at −80 °C until used. A frozen cell pellet was suspended in lysis
buffer (50 mM sodium phosphate, pH 8.0, containing 300 mM NaCl
and 10 mM imidazole), and the cells were lysed by incubation with
lysozyme, one protease inhibitor tablet (Roche), and DNase I (2 mg)
on ice for 30 min followed by sonication (6 cycles of 30 s, 1 min
cooling on ice). The cell lysate was centrifuged (12 000 rpm, 25 min, 4
°C), and the resulting supernatant was mixed with 10 mL of Ni-NTA
Agarose resin (Qiagen) and loaded onto a column. The resin was
washed with 80 mL of 50 mM sodium phosphate buffer, pH 8.0,
containing 300 mM NaCl and 20 mM imidazole and then eluted with
50 mM sodium phosphate buffer, pH 8.0, containing 300 mM NaCl
and 500 mM imidazole. Fractions were analyzed by SDS gel
electrophoresis, and those containing RFK were pooled, concentrated
by centrifugation with a 10 kDa MWCO filter (Centriprep, Millipore),
and dialyzed against 10 mM Tris-HCl buffer, pH 8, containing 10%
glycerol. Protein concentration was determined by the BCA assay
(Pierce).
1
purified by HPLC to give 9.38 mg (97% yield) of a yellow solid; H
NMR (500 MHz, D2O) δ ppm 7.74 (s, 1H), 7.56 (s, 1H), 5.01 (t, J =
10 Hz, 1H), 4.65 (d, J = 15 Hz, 1H), 4.38−4.34 (m, 1H), 4.13−3.98
(m, 4H), 2.48 (s, 3H), 2.35 (s, 3H); 13C NMR (125 MHz, D2O) δ
ppm 163.4 (d, 1JC−C = 76.86 Hz), 160.3, 153.2, 152.3 (d, 1JC−C = 55.44
3
3
Hz), 142, 136.7, 136.3, 134.1 (d, JC−C = 6.04 Hz), 132.8 (d, JC−C
=
6.1 Hz), 128.8, 119.5, 75, 73.8 (d, JC−P = 7.2 Hz), 71.8, 68.6, 50, 23.3,
21.2; 31P NMR (121 MHz, D2O) δ ppm 1.85; HRMS (ESI−)
calculated for C1613CH20N4O9P (M − H)− m/z = 456.1001, found
456.0995.
[1,3-15N2]FMN. Following the procedure described for FMN; crude
[1,3-15N2]7 (10 mg, 0.0265 mmol, 0.53 mM) and ATP (137.785 mg,
0.25 mmol, 5 mM) were incubated with riboflavin kinase (9.423 mg,
0.5 μmol, 10 μM) in 50 mL of buffer containing 5 mM MgCl2 and
purified by HPLC to give 10.66 mg (>98% yield) of a yellow solid; 1H
NMR (500 MHz, D2O) δ ppm 7.75 (s, 1H), 7.57 (s, 1H), 5.02 (t, J =
10 Hz, 1H), 4.67 (d, J = 15 Hz, 1H), 4.39−4.36 (m, 1H), 4.17−3.99
(m, 4H), 2.50 (s, 3H), 2.36 (s, 3H); 13C NMR (125 MHz, D2O) δ
ppm 163.4 (d, JC−N = 13.86 Hz), 160.3 (t, JC−N = 11.0 Hz), 153.3,
152.3 (d, JC−N = 7.93 Hz), 142, 136.8, 136.3, 134.1, 132.9, 119.5, 75,
73.8 (d, JC−P = 7.56 Hz), 71.8, 68.8, 50.1, 23.5, 21.2; 31P NMR (121
MHz, D2O)
δ
ppm 1.9; HRMS (ESI−) calculated for
FMN. In a 100 mL round bottomed flask, crude riboflavin 7 (25 mg,
0.066 mmol, 0.66 mM) and ATP (275.57 mg, 0.5 mmol, 5 mM) were
incubated with riboflavin kinase (18.84 mg, 1 μmol, 10 μM) in 100 mL
of buffer (100 mM 3-(N-morpholino)propanesulfonic acid (MOPS),
pH 7.9) containing 5 mM MgCl2 for 12 h at 30 °C. After incubation,
the enzyme was removed by filtration (Millipore Centricon, 10 000
molecular weight cutoff (MWCO)). The solvent was then removed
under lyophilization, and the solid was purified using high performance
liquid chromatography equipped with an RP-C18 column to give 19.2
mg of a yellow solid; 1H NMR (300 MHz, D2O) δ ppm 7.69 (s, 1H),
7.48 (s, 1H), 4.97 (t, J = 11.7 Hz, 1H), 4.6 (d, J = 13.8 Hz, 1H), 4.34−
4.31 (m, 1H), 4.11−3.97 (m, 4H), 2.45 (s, 3H), 2.31 (s, 3H); 13C
NMR (75 MHz, D2O) δ ppm 160.8, 157.8, 150.8, 149.8, 139.6, 134.2,
133.7, 131.6, 130.3, 117.1, 72.6, 71.4 (d, JC−P = 7.95 Hz), 69.3, 66,
47.6, 20.9, 18.7; 31P NMR (121 MHz, D2O) δ ppm 2.72; HRMS
(ESI−) calculated for C17H20N4O9P (M − H)− m/z = 455.0973, found
455.0971.
C17H2014N215N2O9P (M − H)− m/z = 457.0909, found 455.0913.
5-deazaFMN (9). Following the procedure described for FMN;
crude 8 (12 mg, 0.032 mmol, 0.53 mM) and ATP (137.785 mg, 0.25
mmol, 5 mM) were incubated with riboflavin kinase (9.423 mg, 0.5
μmol, 10 μM) in 50 mL of buffer containing 5 mM MgCl2 and purified
by HPLC to give 13 mg (>98% yield) of a yellow solid;35 1H NMR
(500 MHz, D2O) δ ppm 7.78 (s, 1H), 7.45 (s, 1H), 7.03 (s, 1H),
4.27−3.97 (m, 7H), 2.3 (s, 3H), 2.12 (s, 3H); 13C NMR (125 MHz,
D2O) δ ppm 164.7, 161, 158.7, 152.1, 143.6, 141.2, 138.9, 132.6,
121.6, 119.5, 113.3, 74.9, 74 (d, JC−P = 7.3 Hz), 72.3, 68.8, 49.3, 23.2,
20.8; 31P NMR (121 MHz, D2O) δ ppm 2.5; HRMS (ESI−) calculated
for C18H21N3O9P (M − H)− m/z = 454.1015, found 454.1016.
ASSOCIATED CONTENT
■
S
* Supporting Information
[5-15N]FMN. Following the procedure described for FMN; crude
[5-15N]7 (10 mg, 0.0265 mmol, 0.53 mM) and ATP (137.785 mg,
0.25 mmol, 5 mM) were incubated with riboflavin kinase (9.423 mg,
0.5 μmol, 10 μM) in 50 mL of buffer containing 5 mM MgCl2 and
The Supporting Information is available free of charge on the
1H, 13C, 31P NMR spectral data of the unlabeled, labeled
intermediates, unlabeled FMN, and labeled FMNs
[5-15N]FMN, [4a-13C]FMN, [5-15N,4a-13C]FMN,
[1,3-15N2]FMN, and 5-deazaFMN (Figures S3−S31);
analytical and prep HPLC chromatograms (Figures
1
purified by HPLC to give 8.94 mg (98%) of a yellow solid; H NMR
(500 MHz, D2O) δ ppm 7.77 (s, 1H), 7.61 (s, 1H), 5.04 (t, J = 10 Hz,
1H), 4.69 (d, J = 15 Hz, 1H), 4.39−4.36 (m, 1H), 4.16−3.99 (m, 4H),
2.51 (s, 3H), 2.38 (s, 3H); 13C NMR (125 MHz, D2O) δ ppm 163.5
2
(d, JC−N = 6.3 Hz), 160.3, 153.3, 152.4, 142, 136.8, 136.4 (d, JC−N
=
=
4.28 Hz), 134.2, 132.9 (d, 2JC−N = 7.56 Hz), 119.5, 75, 73.8 (d, JC−P
7.56 Hz), 71.8, 50.1, 23.4, 21.2; 31P NMR (121 MHz, D2O) δ ppm
1.92; HRMS (ESI−) calculated for C17H2014N315NO9P (M − H)− m/z
= 456.0938, found 456.0950.
AUTHOR INFORMATION
■
[5-15N, 4a-13C]FMN. Following the procedure described for FMN;
crude [5-15N, 4a-13C]7 (10 mg, 0.0264 mmol, 0.53 mM) and ATP
(137.785 mg, 0.25 mmol, 5 mM) were incubated with riboflavin kinase
(9.423 mg, 0.5 μmol, 10 μM) in 50 mL of MOPS buffer containing 5
mM MgCl2 and purified by HPLC to give 7.19 mg (98% yield) of a
Corresponding Author
Notes
The authors declare no competing financial interest.
E
J. Org. Chem. XXXX, XXX, XXX−XXX