332
2. Gemcitabine base (gemcitabine is Boc-protected on the –NH 2
and the primary –OH prior to usage, as previously described
[13]) (see Note 1).
3. 1-hydroxybenzotriazole (HOBt).
4. O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HATU).
5. t-Boc-Aminooxy-PEG 4 -CH 2 CO 2 H.
6. 4-dimethylaminopyridine (DMAP).
7. Triisopropylsilane (TIS).
8. N,N-diisopropylethylamine (DIPEA).
9. Trifluoroacetic acid (TFA).
10. Fmoc-Ser(tBu)-OH.
11. Piperidine.
12. Imidazole.
13. Sodium periodate (NaIO 4 ).
14. Ethylene glycol.
15. Anhydrous N,N-dimethylformamide (DMF).
16. Anhydrous acetonitrile.
1. Column chromatography performed using SiO 2 .
2. Thin-layer chromatography (TLC) plates, silica gel coated
with fluorescent indicator F254 (see Note 2).
3. Dichloromethane, analytical grade.
4. Methanol, analytical grade.
5. Acetone, analytical grade.
1. HPLC solvent delivery system with binary gradient capability
and a UV detector.
2. Reversed-phase Jupiter
®
C18 column (25 cm × 21.2 mm) at a
flow rate of 20 mL/min and 10 μm particle size as stationary
phase.
3. Sample filters, 0.2 μm pore size.
4. Acetonitrile, water, and trifluoroacetic acid (TFA), HPLC
grade.
5. Mobile phase A: TFA 0.1% (v/v) in water.
6. Mobile phase B: TFA 0.1% (v/v) in acetonitrile.
1. Acetonitrile, water, and formic acid (LC-MS grade).
2. Mass spectrometer instrumentation with C18 column
100 mm × 2.1 mm, 2.6 μm, with ProGuard column 2.1 mm.
3. Mobile phase A: Water containing 0.1% (v/v) formic acid.
2.2 Purification
and Characterization
of Intermediates
and Final Conjugates
2.2.1 Column
Purification
2.2.2 RP-HPLC
Purification
2.2.3 Characterization
with Mass Spectrometry
and/or NMR Spectroscopy
Eirinaios I. Vrettos and Andreas G. Tzakos
2. Gemcitabine base (gemcitabine is Boc-protected on the –NH 2
and the primary –OH prior to usage, as previously described
[13]) (see Note 1).
3. 1-hydroxybenzotriazole (HOBt).
4. O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HATU).
5. t-Boc-Aminooxy-PEG 4 -CH 2 CO 2 H.
6. 4-dimethylaminopyridine (DMAP).
7. Triisopropylsilane (TIS).
8. N,N-diisopropylethylamine (DIPEA).
9. Trifluoroacetic acid (TFA).
10. Fmoc-Ser(tBu)-OH.
11. Piperidine.
12. Imidazole.
13. Sodium periodate (NaIO 4 ).
14. Ethylene glycol.
15. Anhydrous N,N-dimethylformamide (DMF).
16. Anhydrous acetonitrile.
1. Column chromatography performed using SiO 2 .
2. Thin-layer chromatography (TLC) plates, silica gel coated
with fluorescent indicator F254 (see Note 2).
3. Dichloromethane, analytical grade.
4. Methanol, analytical grade.
5. Acetone, analytical grade.
1. HPLC solvent delivery system with binary gradient capability
and a UV detector.
2. Reversed-phase Jupiter
®
C18 column (25 cm × 21.2 mm) at a
flow rate of 20 mL/min and 10 μm particle size as stationary
phase.
3. Sample filters, 0.2 μm pore size.
4. Acetonitrile, water, and trifluoroacetic acid (TFA), HPLC
grade.
5. Mobile phase A: TFA 0.1% (v/v) in water.
6. Mobile phase B: TFA 0.1% (v/v) in acetonitrile.
1. Acetonitrile, water, and formic acid (LC-MS grade).
2. Mass spectrometer instrumentation with C18 column
100 mm × 2.1 mm, 2.6 μm, with ProGuard column 2.1 mm.
3. Mobile phase A: Water containing 0.1% (v/v) formic acid.
2.2 Purification
and Characterization
of Intermediates
and Final Conjugates
2.2.1 Column
Purification
2.2.2 RP-HPLC
Purification
2.2.3 Characterization
with Mass Spectrometry
and/or NMR Spectroscopy
Eirinaios I. Vrettos and Andreas G. Tzakos