The Journal of Organic Chemistry
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added. The solution was concentrated in vacuo, and then the residue
was taken up in 100 mL of methanol, concentrated in vacuo two times,
and then dried under high vacuum at ambient temperature for 18 h.
This gave 3.32 g (76%) of 14 as a yellow hygroscopic foam, containing
A 47 g amine-functionalized silica gel column was equilibrated with
0.1 M acetic acid in methanol. The reaction mixture in methanol was
injected onto this column at a rate of 30 mL/min. During the
injection, the product was retained while trimethyl phosphate, sodium
acetate, and any unreacted 14 eluted from the column. The column
was then eluted with 200 mL of 0.1 M acetic acid in methanol (30
mL/min) to remove the remaining methanol-soluble reaction
components. Next, the column was eluted with 200 mL of 0.1 M
aqueous acetic acid (30 mL/min) to recover the product. The column
was washed with 300 mL of 1 M sodium acetate and 1 M acetic acid in
water and then with 200 mL of 0.1 M acetic acid in methanol and
stored in 0.1 M acetic acid in methanol. The product containing
fractions were combined and concentrated in vacuo at 40 °C to give an
oily residue. This was taken up in water and concentrated (2 × 5 mL)
to remove most of the remaining acetic acid. The residue was taken up
in 5 mL of water, frozen, and lyophilized to give 480 mg (76%) of a
pale yellow amorphous solid. This foam was not appreciably
hygroscopic upon handling at ambient temperature and humidity
and could be stored in a capped glass vial for several months. Spectral
data were in agreement with those of Slama and Simmons.12
1
1 wt % methanol by H NMR. The product was kept protected from
atmospheric moisture for use in the subsequent phosphorylation step.
Spectral data were in agreement with those of Slama and Simmons.12
Assignment of some 1H NMR signals was by analogy to Abraham and
Moinuddin.56,57 1H NMR (D2O) δ 9.40 (s, 1H, H2), 9.15 (dt, 1H, J =
6.2, 1.1 Hz, H6), 8.95 (dt, 1H, J = 8.1, 1.5 Hz, H4), 8.25 (dd, 1H, J =
8.0, 6.3 Hz, H5), 5.09 (ddd, 1H, J = 10.8, 9.4, 8.0 Hz, H1′), 4.44 (dd,
1H, J = 9.4, 6.0 Hz, H2′), 4.11 (dd, 1H, J = 5.9, 3.1 Hz, H3′), 3.75 (d,
2H, J = 5.9 Hz, H5′), 2.70 (dd, 1H, J = 13.2, 8.2 Hz, H6α′(cis to
OH)), 2.34 (m, 1H, H4′), 1.99 (ddd, 1H, J = 13.2, 11.0, 8.6 Hz,
H6β′(trans to OH)); 13C NMR (D2O) δ 166.45 (C7), 146.03 (C6-
H), 145.41 (C4-H), 143.77 (C2-H), 138.80 (C3), 129.24 (C5-H),
77.36 (C2′-H), 76.22 (C1′-H), 72.20 (C3′-H), 63.24 (C5′-H), 44.61
(C4′-H), 29.19 (C6′-H2); HR-MS (ESI) m/z ([M + H]+) calcd for
C12H17N2O4 253.1188, found 253.1187.
3-Carbamoyl-1-(2,4-dinitrophenyl)pyridin-1-ium Chloride
(15). A 100 mL round-bottomed flask was charged with 6.07 g
(30.0 mmol) of 2,4-dinitrochlorobenzene and 3.66 g (30.0 mmol) of
nicotinamide (3). The reaction was heated at 100 °C for 20 min with
magnetic stirring. The solids melted to give a homogeneous liquid, and
then the mixture hardened and became difficult to stir. Heating was
removed, and the mixture was cooled to ambient temperature to give a
dark orange glass. This was dissolved in 50 mL of methanol, and then
75 mL of silica gel was added, adding more methanol if necessary to
suspend all of the silica gel. The mixture was concentrated to dryness
in vacuo, using heptanes to chase the last traces of methanol from the
mixture. A 120 g prepacked silica gel column was equilibrated with
20% CH3OH/80% CH2Cl2, the silica gel mixture was fitted to the top
of the column, and the column was eluted with 20% CH3OH/CH2Cl2
for 5 min at a rate of 85 mL/min to remove unreacted
dinitrochlorobenzene. A gradient of 20% CH3OH/80% CH2Cl2 to
40% CH3OH/60% CH2Cl2 over 25 min was run, followed by 40%
CH3OH/60% CH2Cl2 for 5 min. During the gradient, nicotinamide
eluted, followed closely by the product. The product-containing
fractions were concentrated in vacuo to 5.99 g (58%) of a yellow foam,
containing 0.66 mol CH3OH/1 mol product. Attempts to crystallize
this material from various solvents were unsuccessful, but the
amorphous solid could be stored for several months under ambient
conditions. 1H NMR (D2O) δ 9.67 (m, 1H, H2), 9.40 (d, 1H, J = 2.5
Hz, H3′), 9.34 (dt, 1H, J = 6.1, 1.3 Hz, H6), 9.27 (dt, 1H, J = 8.2, 1.3
Hz, H4), 8.95 (dd, 1H, J = 8.6, 2.5 Hz, H5′), 8.49 (ddd, 1H, J = 8.2,
6.1 0.5 Hz, H5), 8.26 (d, 1H, J = 8.6 Hz, H6′). 13C NMR (D2O) δ
165.6 (C7), 150.4 (C2′), 148.1 (C6-H), 147.9 (C4-H), 146.3 (C2-H),
143.4 (C4′), 139.0 (C1′), 134.8 (C3), 131.7 (C6′-H), 131.4 (C5′-H),
129.2 (C5-H), 123.4 (C3′-H). HR-MS (ESI) m/z ([M + H]+) calcd
for C12H9N4O5 289.0573, found 289.0573.
((1R,2R,3S,4R)-4-(3-Carbamoylpyridin-1-ium-1-yl)-2,3-
dihydroxycyclopentyl)methyl Hydrogen Phosphate (16). Im-
mediately prior to use, 500 mg (1.64 mmol) of 14 was dissolved in 5
mL of methanol and then concentrated in vacuo to give a pale yellow
foam. The foam was dissolved in 5 mL of methanol and concentrated
in vacuo a second time, and then the resulting foam was dried under
high vacuum (<1 Torr) and at ambient temperature for 30 min to give
anhydrous 14, containing approximately 0.6 equiv of residual methanol
as determined by 1H NMR integration. This was suspended in 4 mL of
freshly distilled trimethyl phosphate, and the suspension was stirred for
1 min under high vacuum at ambient temperature to remove a small
amount of volatile material. The reaction flask was backfilled with N2
and then cooled with an ice bath. Next, 450 μL (4.92 mmol) of POCl3
was added via syringe. The starting material dissolved over 5 min after
the POCl3 had been added. The reaction was stirred with ice cooling
for 1 h, and then 2 mL of water was added. This was stirred for 40 min
while cooling with an ice bath, and then the reaction was diluted into a
solution of 2.00 g (24.4 mmol) of sodium acetate in 125 mL of
methanol at ambient temperature.
1
Assignment of some H NMR signals was by analogy to Abraham
and Moinuddin.56,57 1H NMR (D2O) δ 9.39 (t, 1H, J = 1.4 Hz, H2),
9.18 (dd, 1H, J = 6.3, 1.2 Hz, H6), 8.94 (dd, 1H, J = 8.1, 1.4 Hz, H4),
8.24 (dd, 1H, J = 8.0, 6.3 Hz, H5), 5.10 (td, 1H, J = 9.8, 8.7 Hz, H1′),
4.49 (dd, 1H, J = 9.6, 5.7 Hz, H2′), 4.19 (dd, 1H, J = 5.7, 2.4 Hz, H3′),
4.06 (ddd, 1H, J = 10.2, 5.0, 4.7 Hz, H5′a), 3.96 (ddd, 1H, J = 10.2,
5.0, 4.7 Hz, H5′b), 2.74 (dt, 1H, J = 13.6, 8.6 Hz, H6′α(cis to OH)),
2.45 (m, 1H, H4′), 2.13 (ddd, 1H, J = 13.4, 10.6, 7.8 Hz, H6′β(trans
to OH)); 13C NMR (D2O) δ 166.36 (C7), 146.07 (C6-H), 145.58
(C4-H), 143.78 (C2-H), 134.68 (C3), 129.32 (C5-H), 77.83 (C2′-H),
76.56 (C1′-H), 73.07 (C3′-H), 65.89 (d, JC−P = 5.2 Hz, C5′-H), 43.15
(d, JC−P = 8.1 Hz, C4′-H), 29.70 (C6′-H2); HR-MS (ESI) m/z ([M +
H]+) calcd for C12H18N2O7P 333.0852, found 333.0853.
Crystallization and Structural Determination of SIRT3
Complex. SIRT3/ASC2 crystals were obtained by the hanging drop
vapor diffusion method at 18 °C. The drop was composed of 1 μL of
the protein/peptide mixture and 1 μL of the crystallization buffer
containing 0.2 M Li2SO4, 15% (w/v) polyethylene glycol (PEG)
12000, and 0.1 M Bis-Tris, pH 5.3. The SIRT3/ACS2 crystals were
then cross-linked by glutaraldehyde and soaked for 1 h in 10 mM
carba-NAD in crystallization buffer.
The SIRT3/ASC2 crystals soaked with carba-NAD (9) were
cryoprotected in mother liquor containing 20% glycerol and 10 mM
carba-NAD (9) before being flash-frozen in liquid nitrogen. Diffraction
data were collected at APS 21D and processed using the HKL2000
program.58 The structure was solved by molecular replacement using
the structure of SIRT3/ACS2 complex (PDB code 3GLR) as a search
model. Detailed information regarding the diffraction data, refinement,
and structure statistics is listed in the Supporting Information. All the
parameters for each diffraction data set are from that reprocessed by
Mosflm59 and Scala,60 and refinement statistics are from Refmac61 in
the CCP462 suite for consistency.
Crystallization and Structural Determination of SIRT5
Complexes. SIRT5/Succ-IDH2 crystals were obtained by the
hanging drop vapor diffusion method at 18 °C. The drop was
composed of 1 μL of the protein/peptide mixture and 1 μL of a
crystallization solution containing 0.15 M magnesium formate and
20% (w/v) PEG 3350. The SIRT5/Succ-IDH2 crystals were then
cross-linked by glutaraldehyde and soaked in 5 mM carbaNAD (9) in
crystallization solution overnight.
Crystals of SIRT5/Succ-IDH2 cross-linked by glutaraldehyde were
soaked in 5 mM carba-NAD (9) overnight. The data set was collected
at beamline SSRF BL17U. HKL2000 was used to process the data set
to 1.95 Å in space group P212121 with two monomers in one
asymmetric unit. The molecular replacement software Phaser63 was
used to solve the structure with the search model generated from the
monomer structure of the published PDB entry 2NYR. A 2-fold
symmetry related dimer solution was successfully obtained, and then
iterative structure refinement and model building were performed
between Phenix.refine64 and Coot.65 In the final refined structure, only
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dx.doi.org/10.1021/jo301067e | J. Org. Chem. XXXX, XXX, XXX−XXX