Journal of the American Chemical Society
Article
170.42, 170.06, 169.57, 132.81, 128.92, 128.24, 100.66, 85.70, 76.88,
76.11, 74.01, 73.74, 72.86, 72.00, 70.18, 68.12, 62.41, 62.14, 20.90,
20.87, 20.83. ESI-MS: 662.21 [(M + NH4)+].
100.80, 85.63, 76.22, 73.74, 73.13, 73.08, 71.75, 70.97, 70.19, 69.14,
62.31, 61.39, 20.89, 20.79, 20.69.
Phenyl ((3-18O)-β-D-Galactopyranosyl)-(1→4)-1-thio-β-D-gluco-
pyranoside (4b). A solution of freshly prepared NaOMe (0.17 M,
20 mL) was added to 10 (80.0 mg, 0.12 mmol) and stirred at room
temperature for 5 h. Amberlite IR-120+ resin (H+ form) was added to
neutralize the reaction, and the suspension was stirred for another 15
min before being filtered. The filtrate was concentrated under reduced
pressure to afford the desired product as an off-white solid (46.1 mg,
Phenyl (2,6-Di-O-acetyl-4-O-(18O)acetyl-β-D-galactopyranosyl)-
(1→4)-2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside (8). A flame-
dried flask was charged with 7 (200 mg, 0.31 mmol), dry acetonitrile
(10 mL), triethyl orthoacetate (0.3 mL, 1.64 mmol), and p-
toluenesulfonic acid (10 mg, 0.05 mmol). The resultant solution was
stirred at room temperature and TLC analysis showed the reaction to
be complete after 30 min, Rf = 0.48 (1:4 v/v acetone/toluene).
Following addition of 18O-water (25 μL, 1.24 mmol; 95.1 atom % O-
18) to the reaction mixture, stirring was continued for 1 h. After
addition of EtOH (5 mL), the volatiles were removed under reduced
pressure, and the resulting crude residue was purified by flash
chromatography (3:7 v/v acetone/toluene) to afford the desired
product as a light yellow syrup (187.9 mg, 0.27 mmol, 88% yield). 1H
NMR (600 MHz, CDCl3) δ 7.45−7.36 (m, 2H), 7.28−7.21 (m, 3H),
5.22 (s, 1H), 5.14 (t, J = 8.9, 1H), 4.85 (t, J = 9.5, 1H), 4.78 (s, 1H),
4.61 (m, 1H), 4..47 (m, 1H), 4.35 (m, 1H), 4.09 (m, 1H), 4.06−3.94
(m, 2H), 3.78−3.54 (m, 4H), 2.11 (s, 3H), 2.10 (s, 3H) 2.07−2.00
(6H, 2 × CH3), 1.99 (s, 3H), 1.95 (s, 3H); 13C NMR (151 MHz,
CDCl3) δ 171.13, 170.71, 170.49, 170.44, 169.86, 169.61, 132.87,
131.97, 128.93, 128.29, 100.77, 85.65, 76.77, 76.20, 73.74, 73.02,
71.61, 70.97, 70.19, 69.19, 62.35, 61.44, 30.98, 20.87, 20.79, 20.70.
Phenyl (2,6-Di-O-acetyl-4-O-(18O)acetyl-β-D-gulopyranosyl)-(1→
4)-2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside (9). A solution of 8
(151.6 mg, 0.22 mmol) in dichloromethane (10 mL) was cooled to
−78 °C. Upon the addition of pyridine (68.8 μL, 0.85 mmol) and
trifluoromethanesulfonic anhydride (74 μL, 0.35 mmol), the mixture
was allowed to warm to room temperature. After 30 min, the mixture
was successively washed with 1 N HCl (20 mL), sat. NaHCO3 (20
mL), and brine (20 mL). The organic layer was dried (Na2SO4) and
concentrated under reduced pressure. The resultant crude residue was
dissolved in anhydrous DMF and treated with NaNO2 (71 mg, 1.03
mmol). This reaction mixture was stirred overnight at 50 °C. Upon
removal of the solvent, the crude residue was purified via flash
chromatography (3:7 v/v acetone/toluene) to afford the desired
product as a white solid (113.4 mg, 0.17 mmol). Mp = 91−92 °C; 1H
NMR (600 MHz, CDCl3) δ 7.55−7.48 (m, 2H), 7.38−7.31 (m, 3H),
5.24 (t, J = 9.1, 1H), 4.96 (s, 2H), 4.82 (s, 2H), 4.72 (d, J = 10.0, 1H),
4.50 (d, J = 13.5, 1H), 4.35−4.24 (m, 2H), 4.21 (s, 1H), 4.13 (d, J =
6.4, 2H), 3.79 (t, J = 9.5, 1H), 3.68 (m, 1H), 2.33 (m, 1H), 2.15 (s,
9H), 2.12 (s, 3H), 2.11 (s 3H), 2.09 (s, 3H); 13C NMR (151 MHz,
CDCl3) δ 171.23, 170.48, 170.22, 170.12, 169.76, 169.69, 132.81,
128.95, 128.28, 98.61, 86.37, 85.86, 76.47, 73.85, 70.92, 70.20, 69.69,
69.41, 67.45, 62.46, 61.42, 20.89, 20.84, 20.73.
1
0.11 mmol, 93% yield). The H and 13C NMR spectra were identical
to those for the unlabeled compound (4a) except that the resonance
for C3 in the 13C NMR spectrum was shifted upfield by ∼0.021 ppm.
Kinetic Isotope Effect Measurements. The NMR spectroscopic
measurements of 13C- and 18O-KIEs, at 25 °C, followed the published
protocols16 except that: (i) deuterium oxide (5 μL, < 1.0% of the final
reaction volume) was added directly into the NMR tube containing
the labeled substrates (∼1.5−2.0 mg) and buffer (645 μL, 50 mM
NaOAc pH 5.5) for signal locking; (ii) automated gradient shimming
2
of the magnetic field using the H lock signal was initially performed
and this operation was followed by manual shimming, which involved
adjusting the various shim currents in order to ensure optimal
(symmetric) line shapes in the 1H NMR spectrum (spectra were
automatically Fourier transformed in real-time every 3 s during this
procedure); and (iii) the amount of VcNA was adjusted such that 80%
of the catalyzed reaction was complete within approximately 8 h. To
measure β-secondary deuterium KIEs, a simultaneous inverse-gated 1H
and 2H decoupling sequence was used during 13C NMR spectral
acquisition. Specifically, pulse lengths were measured on the sample
and the powers used to calculate pulse power levels for simultaneous
1H and 2H Globally optimized Alternating phase Rectangular Pulse
(GARP)24,25 composite pulse decoupling (CPD). GARP-4 was used to
reduce amount of power required for efficient decoupling and to
minimize sample heating.24,25 The spectral width was 240 ppm (36
058 Hz), 102 400 complex points acquired (acquisition time 1.42 s),
and a recycle delay 2 s between transients. The dead time after the 13C
pulse was set to 20 μs to minimize pulse breakthrough. The 13C
transmitter was set to 50 ppm; 12 μs π/2 pulse at 29.6 W was used.
The 1H transmitter was set to 4 ppm, GARP-4 CPD used 80 μs pulses
2
2
at 0.21 W, and the H transmitter was set on resonance of the H
signal (1.76 ppm). GARP-4 CPD used 300 μs pulses at 3.40 W and 2H
locking was gated on during the recycle delays but off during FID
acquisition. Under these conditions, 13C{1H, H} resonances had line
2
widths at half-height of 0.01 ppm or less. Of note, an inverse gated
decoupling pulse sequence was used in all KIE experiments to
minimize NOE enhancements. In addition, we checked that transient
NOE build-up from previous scans did not contribute to the signal
intensities by increasing the relaxation delay to >15 × T1, and showed
that under these conditions the measured signal intensities were found
to be identical to those measured using our published experimental
protocols in which the shorter relaxation delays were utilized.16
For all β-secondary 2H-KIEs experiments on the 2,3-diastereomer 2,
a filtered solution of E. coli Neu5Ac aldolase was added into the NMR
tube in order to reduce the signal intensity of β-Neu5Ac by
equilibrating the Neu5Ac generated during the hydrolysis reaction
with N-acetylmannosamine (ManNAc) and pyruvate.26 Experimental
spectra were fit using the ‘deconvolution’ function from the Bruker
Topspin 2.1 program (fits are provided in Supporting Information).
Peaks were fit to a Lorentzian shape, detection sensitivity = 0.5, and
peak overlapping factor = 0.5 ppm.
Computational Modeling. Calculations for the VcNA-catalyzed
hydrolyses of α-sialosides were determined with Gaussian 0927 using
the B3LYP method with a 6-31G* basis set. The 4-hydroxyl and the 5-
N-acetamido functional groups as well as the 6-glycerol side chain were
removed from the Neu5Ac core in order to generate a truncated
substrate. In addition, a methoxy leaving group was used in place of
the natural 3- and 6- hydroxyl groups of the terminal galactose residue
found in many natural substrates.28 From several starting geometries,
calculations were performed to locate the ground state structure by
minimizing its energy in vacuo. Subsequent frequency calculations
Phenyl (2,3,6-Tri-O-acetyl-β-D-(3-18O)galactopyranosyl)-(1→4)-
2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside (10)). A solution of 9
(95 mg, 0.14 mmol) in dichloromethane (15 mL) was treated with
pyridine (150 μL, 1.85 mmol) and cooled to 0 °C. After addition of
trifluoromethanesulfonic anhydride (144 μL, 0.675 mmol), the
resultant mixture was warmed to room temperature, and after 4 h,
the mixture was washed successively with 1 N HCl (20 mL), sat.
NaHCO3 (20 mL), and brine (20 mL). After the organic layer was
dried (Na2SO4) and concentrated under reduced pressure, the crude
residue was dissolved in THF (10 mL) and treated with H2O (100 μL,
5.5 mmol) and 2,6-lutidine (150 μL, 1.3 mmol). The resultant mixture
was and stirred at 40 °C for 1 h, at which time the volatiles were
removed under reduced pressure. Purification of the resultant crude
residue was accomplished using flash chromatography (3:7 v/v
acetone/toluene) to afford the desired product as a white solid
(87.4 mg, 0.13 mmol). Mp = 90−92 °C; 1H NMR (600 MHz, CDCl3)
δ 7.54−7.48 (m, 2H), 7.37−7.31 (m, 3H), 5.32 (s, 1H), 5.24 (t, J =
8.5, 1H), 4.95 (t, J = 9.7, 1H), 4.87 (m, 1H), 4.71 (d, J = 10.1, 1H),
4.57 (d, J = 11.9, 1H), 4.44 (d, J = 7.8, 1H), 4.19 (dd, J = 5.8, 11.9,
1H), 4.16−4.05 (m, 2H), 3.85−3.73 (m, 3H), −3.69 (m, 1H), 2.54 (d,
J = 6.3, 1H), 2.20 (s, 3H), 2.15 (s, 3H), 2.14 (s, 3H), 2.12 (s, 3H),
2.09 (s, 3H), 2.06 (s, 3H); 13C NMR (151 MHz, CDCl3) δ 171.44,
170.51, 170.32, 170.22, 169.85, 169.63, 132.90, 131.99, 128.94, 128.28,
3750
dx.doi.org/10.1021/ja208564y | J. Am. Chem. Soc. 2012, 134, 3748−3757