J. Feng et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
C16H16Cl2N6O2: C, 48.62; H, 4.08; N, 21.26. Found: C, 48.55; H, 4.22;
N, 21.40.
131.3, 136.2, 139.2, 142.5, 157.2, 161.4, 166.0, 168.2; HRMS (ESI):
calcd. for C16H14ClIN6O2 [M + 1]+ 484.99842, found: 484.99794;
Elemental Anal. Calcd for C16H14ClN6O2(483.99): C, 39.65; H, 2.91;
N, 17.34. Found: C, 39.38; H, 2.73; N, 17.23.
4.1.3.6. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl-4-methylbenzoate hydrochloride 6g. Yellow solid; Yield 89%;
m.p 132–134 °C; 1H NMR (400 MHz, DMSO‑d6) δ(ppm): 2.50 (s, 3H,
CH3), 2.65 (s, 3H, CH3), 5.50 (s, 2H, CH2), 5.75 (s, 2H, OCH2), 7.44 (s,
2H, NH2), 7.44 (s, 2H, Ar-H), 7.96 (s, 2H, Ar-H), 7.96 (s, 1H, 1,2,3-
triazol-4-yl-H), 8.44 (s, 1H, pyrimidin-6-yl-H); 13C NMR (100 MHz,
DMSO‑d6) δ(ppm): 22.6, 25.0, 47.3, 58.2, 108.2, 125.5, 127.5, 129.2,
131.8, 138.7, 141.8, 156.7, 161.6, 164.8, 167.5; ESI-MS m/z: 339.2
[M + 1]+; HRMS (ESI): calcd. for C17H18N6O2 [M + H]+ 339.15640,
found: 339.15706; Elemental Anal. Calcd for C17H19ClN6O2: C, 54.47;
H, 5.11; N, 22.42. Found: C, 54.66; H, 5.34; N, 22.56.
4.1.3.12. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)methyl-3-chlorobenzoate 8f. Celadon solid; Yield 88%; m.p.
185–186 °C; 1H NMR(400 MHz, DMSO‑d6) δ(ppm): 2.50(s, 3H, CH3),
5.39(s, 2H, CH2), 5.51(s, 2H, -CH2O), 6.95(s, 2H, NH2), 7.57(s, 1H, Ar-
H), 7.75(s, 1H, Ar-H), 7.90(s, 2H, Ar-H), 13C NMR(100 MHz, DMSO‑d6)
δ(ppm): 25.7, 47.0, 58.1, 108.5, 124.7, 127.9, 128.8, 131.1, 138.3,
142.3, 143.0, 156.4, 161.9, 165.6, 167.7; HRMS (ESI): calcd. for
C
16H14ClIN6O2 [M + 1]+ 484.99842, found: 484.99568; Elemental
Anal. Calcd for C16H14ClN6O2 (483.99): C, 39.65; H, 2.91; N, 17.34.
Found: C, 39.74; H, 2.86; N, 17.15.
4.1.3.7. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)-methylbenzoate 8a. Green solid; Yield 91%; m.p.
198–199 °C; 1H NMR (400 MHz, DMSO‑d6) δ (ppm): 2.33 (s, 3H,
CH3), 5.36 (s, 2H, CH2), 5.48 (s, 2H, -CH2O), 6.95 (s, 2H, NH2), 7.52
4.1.3.13. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)methyl-4-methoxybenzoate 8g. Green solid; Yield 75%; m.p.
200-201 °C ; 1H NMR(400 MHz, DMSO‑d6) δ(ppm): 2.29(s, 3H, CH3),
2.36(s, 3H, -CH3), 5.35(s, 2H, CH2), 5.44(s, 2H, -CH2O), 6.95(s, 2H,
NH2), 7.32(d, 2H, J = 8.0 Hz, Ar-H), 7.83(d, 2H, J = 8.0 Hz, Ar-H),
8.01(s, 1H, pyrimidin-5-yl-H); 13C NMR(100 MHz, DMSO‑d6) δ(ppm):
25.7, 29.7, 46.9, 59.2, 109.1, 124.8, 127.6, 129.0, 131.1, 139.0, 143.3,
155.8, 163.3, 165.1, 167.5; HRMS (ESI): calcd. for C17H17IN6O2 [M
+1]+ 465.05304, found: 465.05161; Elemental Anal. Calcd for
C17H17IN6O3(464.05): C, 42.51; H, 3.57; N, 17.50. Found: C, 42.44;
H, 3.58; N, 17.67.
(s, 3H, Ar-H), 7.94(s, 2H, Ar-H), 7.66 (s, H, pyrimidin-5-yl-H Ar-H); 13
C
NMR (100 MHz, DMSO‑d6) δ(ppm): 25.9, 46.9, 57.7, 108.8, 128.5,
129.4, 131.5, 133.5, 138.5, 142.6, 156.2, 160.2, 164.8, 167.6; HRMS
(ESI): calcd. for C16H15IN6O2 [M + 1]+ 451.03739, found: 451.03703;
Elemental Anal. Calcd for C16H15IN6O2(450.03): C, 42.68; H, 3.36; N,
18.61. Found: C, 42.64; H, 3.43; N, 19.67.
4.1.3.8. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)methyl-4- nitrobenzoate 8b. Yellow solid; Yield 86%; m.p.
192–193 °C; 1H NMR(400 MHz, DMSO‑d6) δ(ppm): 2.39(s, 3H, CH3),
5.36(s, 2H, CH2), 5.55(s, 2H, -CH2O), 6.98(s, 2H, NH2), 7.36(s, 2H, Ar-
H), 8.01(s, 2H, Ar-H), 8.01(s, 1H, pyrimidin-5-yl-H); 13C NMR
(100 MHz, DMSO -d6) δ(ppm): 25.7, 47.9, 57.6, 108.4, 124.9, 127.9,
129.2, 131.3, 138.7, 141.8, 156.6, 161.6, 165.6, 166.7; HRMS (ESI):
4.2. Expression and purification of Cy-PDHc E1 and its activity
measurements
calcd. for
C
16H14IN7O4 [M + 1]+ 496.02247, found: 496.02199;
The genes encoding Cy-PDHc-E1 (Gene Bank Number: BA000022.2)
were directly amplified from genomic DNA of Synechocystis PCC6803
and then were inserted into the pETDuet-1 vector (Novagen). Positive
transformants were verified by sequencing and then they were co-
transformed into Escherichia coli BL-21 (DE3) together with plasmid
pGro7 for over-expressing chaperonins GroEL and GroES. For expres-
sion of the enzyme, the recombination stain was inoculated in 2xYT
medium with 100 µg/ml ampicillin and 20 µg/ml chloramphenicol at
37 °C until reaching a cell density to A600 of 0.5–0.6. The culture was
induced with a final concentration of 0.5 mM IPTG and 0.5 mg/mL L-
Arabinose for 24 h at 22 °C before harvesting. The harvested pellets
were resuspended in buffer (300 mM NaCl, 50 mM potassium phos-
phate buffer, 1% glycerol and 1 mM DTT, 1 mM MgCl2, pH 7.2) and
sonicated, subsequently the lysate supernatant containing soluble cel-
lular materials was loaded on a Ni2+-NTA resin (Novagen) in a stan-
dard procedure. Proteins were eluted in two steps with 300 mM NaCl,
50 mM potassium phosphate buffer, and 1% glycerol and 1 mM DTT,
1 mM MgCl2, pH 7.2, 75 mM imidazole and 250 mM imidazole. The
isolated proteins were dialyzed against 300 mM NaCl, 50 mM potassium
phosphate buffer, and 1% glycerol and 1 mM DTT, 1 mM MgCl2, pH 7.2.
Purified protein was stored in 50% (v/v) glycerol at −20 °C. The con-
centrations of purified proteins were determined by the Bradford
Elemental Anal. Calcd for C16H14IN7O4(495.02): C, 38.80; H, 2.85; N,
19.80. Found: C, 38.88; H, 3.01; N, 20.05.
4.1.3.9. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)methyl-4-fluorobenzoate 8c. Yellow solid; Yield 73%; m.p.
207–209 °C ; 1H NMR(400 MHz, DMSO‑d6) δ(ppm): 2.30(s, 3H, CH3),
4.50(s, 2H, CH2), 5.42(s, 2H, -CH2O), 6.91(s, 2H, NH2), 7.53(d, 2H,
J = 7.4 Hz, Ar-H), 7.88(d, 2H, J = 7.5 Hz, Ar-H), 8.45(s, 1H, pyrimidin-
5-yl-H); 13C NMR(100 MHz, DMSO‑d6) δ(ppm): 24.7, 47.9, 57.6, 108.6,
115.1, 124.2, 127.5, 131.6, 142.5, 157.4, 161.6, 164.2, 165.3, 166.9,
167.9; HRMS (ESI): calcd. for C16H14FIN6O2 [M + 1]+ 469.02797,
found: 469.02847; Elemental Anal. Calcd for C16H14FIN6O2 (468.02):
C, 41.04; H, 3.01; N, 17.95. Found: C, 40.87; H, 3.42; N, 18.01.
4.1.3.10. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)methyl-2-chlorobenzoate 8d. Celadon solid; Yield 86%; m.p.
185–187 °C; 1H NMR(400 MHz, DMSO‑d6) δ(ppm): 2.36(s, 3H, CH3),
4.94(s, 2H, CH2), 5.50(s, 2H, -CH2O), 7.00(s, 2H, NH2), 7.55–7.59(m,
2H, Ar-H), 7.76(d, 2H, J = 4.0 Hz, Ar-H), 8.18 (s, 1H, pyrimidin-5-yl-
H); 13C NMR(100 MHz, DMSO‑d6) δ(ppm): 26.1, 48.1, 58.3, 108.6,
125.0, 128.3, 129.0, 130.8, 132.0, 138.7, 141.7, 142.6, 155.8, 161.2,
164.0, 167.5; HRMS (ESI): calcd. for
C
16H14ClIN6O2 [M + 1]+
.
484.99842, found: 484.99809; Elemental Anal. Calcd for
Cy-PDHc E1 activity was determined by monitoring the reduction of
2, 6 – DiChlorophenol IndoPhenol (2,6-DCIP) at 600 nm using a mi-
croplate reader (Bioteck Synergy2, USA)20. Cofactor ThDP and sub-
strate pyruvate were purchased from Sigma. A standard reaction mix-
ture containing 50 mM K3PO4 (pH 7.2), 0.375 mM 2, 6-
DiChlorophenolIndoPhenol (DCIP), 0.8 mM sodium pyruvate as sub-
strate, 0.1 mg/ml purified Cy-PDHc E1 enzyme. The reaction mixtures
were incubated for 3 min at 37 °C, then added different concentrations
of ThDP (ranging from 0 to 200 µM). One unit of activity is defined as
the amount of 2, 6-DCIP reduced.
C
16H14ClN6O2(483.99): C, 39.65; H, 2.91; N, 17.34. Found: C, 39.28;
H, 3.16; N, 17.79.
4.1.3.11. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)methyl-4-chlorobenzoate 8e. Grass green solid; Yield 84%;
m.p. 192–193 °C; 1H NMR(400 MHz, DMSO‑d6) δ(ppm): 2.33(s, 3H,
CH3), 5.36(s, 2H, CH2), 5.49(s, 2H, CH2O), 6.94(s, 2H, NH2), 7.59(s,
2H, Ar-H), 7.94(s, 2H, Ar-H), 7.59(s, 1H, pyrimidin-5-yl-H); 13C NMR
(100 MHz, DMSO‑d6) δ(ppm): 25.9, 46.7, 58.1, 108.2, 124.7, 128.4,
6