Molecular Pharmaceutics
Article
labeling precursors 1e and 1f, all intermediates and precursors
for the syntheses of the reference RGD peptides [19F]6Glc-
RGD (4b), [19F]Gal-RGD (4c), [19F]Mlt-RGD (4e), and
[19F]Cel-RGD (4f), and their analytical data are given in the
Supporting Information file (see associated content). The
syntheses of 1a, 2a, 3a, 4a and [18F]2a, [18F]3a, and [18F]4a
were performed as previously described.20,21
2,3,4-Tri-O-acetyl-6-O-p-toluenesulfonyl-β-D-galacto-
pyranosyl Azide (1c). The synthesis was performed according
to the procedure described in the literature.23 To a solution of
p-toluenesulfonyl chloride (465 mg, 2.44 mmol) in anhydrous
pyridine (7 mL) at 0 °C β-galactosyl azide (500 mg, 2.44
mmol; Sigma-Aldrich) was added, and the solution was stirred
overnight at 0 °C. Acetic anhydride (2 mL) was added
dropwise at 0 °C and the solution was stirred for 1 h. The
solution was diluted with dichloromethane, and the organic
phase was washed several times with saturated NaHCO3, 0.2 M
HCl, and water, dried with Na2SO4, and concentrated to afford
the crude product 1c which was crystallized from absolute
2,3,4-Tri-O-acetyl-6-O-p-toluenesulfonyl-β-D-gluco-
pyranosyl Azide (1b). To a solution of p-toluenesulfonyl
chloride (3.2 g, 16.7 mmol) in anhydrous pyridine (45 mL) at 0
°C D-glucose (3 g, 16.7 mmol) was added. The solution was
stirred overnight at 0 °C. Subsequently, acetic anhydride (13.2
mL) was added dropwise. The solution was allowed to warm to
room temperature, and after 1 h the solvent was removed in
vacuo. The residue was dissolved in dichloromethane, and the
solution was washed several times with saturated NaHCO3, 0.2
M HCl, and water, dried with Na2SO4, and concentrated again
to afford the crude 1,2,3,4-tetra-O-acetyl-6-O-p-toluenesulfonyl-
β-D-glucopyranose 7b which was crystallized from ethanol: 3 g,
1
EtOH: 350 mg, 730 μmol, 30% yield. H NMR (600 MHz,
CDCl3): δ ppm 1.97 (s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 2.46
(s, 3H), 4.03 (m, 2H, H-5, H-6a), 4.13 (dd, J = 9.42, 5.73 Hz,
1H, H-6b), 4.55 (d, J = 8.77 Hz, 1H, H-1), 5.00 (dd, J = 10.37,
3.37 Hz, 1H, H-3), 5.10 (dd, J = 10.37, 8.77 Hz, 1H, H-2), 5.40
(dd, J = 3.33, 0.71 Hz, 1H, H-4), 7.38−7.35 (m, 2H), 7.79−
7.76 (m, 2H). 13C NMR (151 MHz, CDCl3): δ ppm 169.83,
169.80, 169.32, 145.38, 132.20, 130.02, 128.07, 88.24, 72.73,
70.49, 67.84, 66.66, 65.98, 21.69, 20.65, 20.48.
1
6.0 mmol, 36% yield. H NMR (600 MHz, CHCl3): δ ppm
1.99 (s, 3H, OAc), 2.00 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.09
(s, 3H, OAc), 2.46 (s, 3H, CH3-Ts), 3.84 (ddd, J = 10.07, 4.55,
2.87 Hz, 1H, H-5), 4.15 (dd, J = 11.17, 2.89 Hz, 1H, H-6b),
4.11 (dd, J = 11.21, 4.59 Hz, 1H, H-6a), 5.04 (dd (t), J = 9.5
Hz, 1H, H-4), 5.05 (dd, J = 9.41, 8.23 Hz, 1H, H-2), 5.20 (t, J =
9.37 Hz, 1H, H-3), 5.65 (d, J = 8.19 Hz, 1H, H-1), 7.35 (d, J =
7.97 Hz, 2H), 7.77 (d, J = 8.29 Hz, 2H). 13C NMR (151 MHz,
CHCl3): δ ppm 170.09, 169.27, 169.12, 168.75 (4x CO,
OAc), 145.14 (Ts), 132.43 (Ts), 129.86 (2C, Ts), 128.17 (2C,
Ts), 91.53 (C-1), 72.60, 72.17, 70.05, 67.95, 66.74 (C-2 − C-
6), 21.68 (CH3, Ts), 20.74, 20.55, 20.52, 20.49 (4x CH3, OAc).
To a solution of 1,2,3,4-tetra-O-acetyl-6-O-p-toluenesulfonyl-
β-D-glucopyranose 7b (3 g, 6 mmol) in dichloromethane (20
mL) HBr (33% in acetic acid, 30 mL) was added dropwise at 0
°C, and the solution was stirred for 3 h at room temperature.
The mixture was diluted with dichloromethane (150 mL) and
washed with saturated NaHCO3 (3 × 50 mL), water (2 × 50
mL), dried with Na2SO4 and concentrated in vacuo. The crude
product was purified by column chromatography (EtOAc-n-
hexan 1:1) to afford 2,3,4-tri-O-acetyl-6-O-p-toluenesulfonyl-β-
D-glucopyranosyl bromide as a yellow oil (2.8 g, 5.3 mmol, 88%
yield) that was dissolved in in EtOAc (30 mL) and
tetrabutylammonium hydrogen sulfate (1.8 g, 5.3 mmol) and
sodium azide (1.4 g, 21.2 mmol), and saturated NaHCO3 (30
mL) was added. The two-phase mixture was vigorously stirred
for 2 h at room temperature; EtOAc (70 mL) was added, and
the organic phase was washed with saturated NaHCO3 (2 × 50
mL) and brine (50 mL). After drying with Na2SO4 and
evaporation of the solvent, the crude product 1b was
recrystallized twice from EtOH: 1.8 g, 3.8 mmol, yield 70%.
1H NMR (600 MHz, CDCl3): δ ppm 1.99 (s, 3H, OAc), 2.02
(s, 3H, OAc), 2.06 (s, 3H, OAc), 2.46 (s, 3H, CH3-Ts), 3.83
(ddd, J = 10.10, 5.70, 2.66 Hz, 1H, H-5), 4.09 (dd, J = 11.24,
5.72 Hz, 1H, H-6a), 4.15 (dd, J = 11.24, 2.65 Hz, 1H, H-6b),
4.56 (d, J = 8.87 Hz, 1H, H-1), 4.85 (t, J = 9.26 Hz, 1H, H-2),
4.95 (t, J = 9.77 Hz, 1H, H-4), 5.18 (t, J = 9.50 Hz, 1H, H-3),
7.37 (d, J = 8.08 Hz, 2H, Ts), 7.79 (d, J = 8.29 Hz, 2H, Ts). 13C
NMR (151 MHz, CDCl3): δ ppm 170.04, 169.32, 169.11 (3×
CO, OAc), 145.33 (Ts), 132.25 (Ts), 129.94 (2C, Ts),
128.13 (2C, Ts), 87.69 (C-1), 73.65, 72.34, 70.40, 68.14, 67.21
(C-2−C-6), 21.69 (CH3, Ts), 20.53, 20.51, 20.50 (3× CH3,
OAc). ESI-MS: m/z = 508.1 [M + Na]+.
2,3,4-Tri-O-acetyl-6-O-p-nitrobenzenesulfonyl-β-D-
galactopyranosyl Azide (1d). 1d was synthesized starting
from β-galactosyl azide (500 mg, 2.44 mmol) following the
protocol described for 1c but using p-nitrobenzenesulfonyl
chloride (540 mg, 2.44 mmol) instead of p-toluenesulfonyl
chloride. 1d was obtained after column chromatography
(toluene−EtOAc 1:1) as an off-white solid in a yield of 40%
1
(500 mg, 970 μmol). H NMR (360 MHz, CDCl3): δ ppm
1.98 (s, 3H), 2.08 (s, 3H), 2.12 (s, 3H), 4.07 (ddd, J = 6.77,
5.40, 1.20 Hz, 1H, H-5), 4.20 (dd, J = 10.53, 5.39 Hz, 1H, H-6),
4.22 (dd, J = 10.52, 6.88 Hz, 1H, H-6), 4.56 (d, J = 8.68 Hz,
1H, H-1), 5.01 (dd, J = 10.37, 3.32 Hz, 1H, H-3), 5.11 (dd, J =
10.46, 8.63 Hz, 1H, H-2), 5.41 (dd, J = 3.31, 1.11 Hz, 1H, H-4),
8.44−8.40 (m, 2H), 8.13−8.09 (m, 2H). 13C NMR (90.56
MHz, CDCl3): δ ppm 169.91, 169.84, 169.26, 151.08, 141.08,
129.40, 124.58, 88.26, 72.67, 70.42, 67.72, 67.24, 66.68, 20.62,
20.53, 20.46. ESI-MS (m/z): = 539.0 [M + Na]+, m/z = 555.0
[M + K]+.
Syntheses of Reference Compounds 4b, 4c, 4e, or 4f.
To a solution of 6-deoxy-6-fluoro-D-glucopyranosyl azide (3b)
(1.86 mg, 2.4 μmol), 6-deoxy-6-fluoro-D-galactopyranosyl azide
(3c) (1.86 mg, 2.4 μmol), 6′-deoxy-6′-fluoro-β-maltosyl azide
(3e) (2.25 mg, 2.4 μmol), or 6′-deoxy-6′-fluoro- β-cellobiosyl
azide (3f) (2.25 mg, 2.4 μmol) and c(RGDfPra)21 (1.37 mg,
2.4 μmol) in a solution of phosphate-buffered saline (PBS; 10%
ethanol) (0.5 mL), a solution of copper(II)sulfate pentahydrate
(0.2 M, 10 μL) and a solution of sodium ascorbate (0.6 M, 10
μL) were added. The mixture was stirred at room temperature
for 30 min, then diluted with water/acetonitrile (9:1, 0.5 mL),
and subjected to semipreparative RP-HPLC for purification of
the desired product (Kromasil C8, 125 × 8 mm, 10−40%
acetonitrile (0.1% trifluoroacetic acid, TFA) in water (0.1%
TFA) in a linear gradient over 30 min, 4 mL/min, tR(4b) =
11.0 min, tR(4c) = 9.3 min, tR(4e) = 7.5 min, tR(4f) = 6.4 min).
After lyophilization of the product fraction, the glycopeptides
were obtained as white solids in yields of 70−80%. ESI-MS: 4b:
m/z = 778.4 [M + H]+; 4c: m/z = 778.4 [M + H]+; 4e: m/z =
940.6 [M + H]+; 4f: m/z = 940.4 [M + H]+, 481.7 [M + H +
Na]2+.
Synthesis of c(RGDfPra). The peptide was synthesized
using Fmoc-protocols as described previously.21
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dx.doi.org/10.1021/mp4004817 | Mol. Pharmaceutics 2014, 11, 505−515