334
5. Purify the residue via column chromatography (eluent:
CH 2 Cl 2 /acetone 9/1) to afford gemcitabine linker (diBocgemcitabine- bis(4-nitrophenyl)carbonate).
6. Characterize with
1
H and
13
C NMR spectroscopy and mass
spectrometry.
1. Dissolve D-Lys
6
-GnRH (1 eq) in anhydrous DMF under inert
atmosphere.
2. Add DIPEA (3 eq) at 0 °C dropwise.
3. After 5 min, add gemcitabine-linker (1 eq) dissolved in anhydrous DMF dropwise and stir the reaction for 12 h at room
temperature.
4. Distill the solvent.
5. Dissolve the residue in TFA-H 2 O-TIS (95:2.5:2.5) and stir for
12 h (see Note 5).
6. Distill the solvent.
7. Purify the residue with RP-HPLC and lyophilize the peak to
receive the final conjugate in pure form.
8. Characterize the compound with mass spectrometry (see
Note 6).
1. Dissolve Boc-protected gemcitabine (1 eq), t-Boc-AminooxyPEG 4 -CH 2 CO 2 H (1.5  eq), DCC (1.5  eq), and DMAP
(0.5  eq) in anhydrous dichloromethane under inert
atmosphere.
2. Add DIPEA (4 eq) at 0 °C and stir the reaction for the required
time at room temperature (monitor the reaction progress with
TLC).
3. Distill the solvent.
4. Purify the residue with column chromatography (eluent:
CH 2 Cl 2 /acetone 9/1) to afford gemcitabine-linker
(diBoc-Gemcitabine-Boc-Aminooxy-PEG 4 -CH 2 CO 2 H).
Preparation of the Final
Conjugate (Gemcitabinecarbamate- D-Lys
6
-GnRH)
3.1.3 Synthesis of PDC
Using Aminooxy-PEG 4 -
CH 2 CO 2 H as Linker (Amide
and Oxime Bond)
(Scheme 3)
Preparation
of Gemcitabine-Linker
(diBoc-Gemcitabine-BocAminooxy-PEG 4 -CH 2 CO 2 H)
Scheme 1 Synthesis of PDCs using carbamate bond as linker. Reagents and conditions: (a) Succinic anhydride, DIPEA, CH 2 Cl 2 , rt., 12 h; (b) D-Lys
6
-GnRH, HATU, DIPEA, DMF, rt., 12 h; (c)TFA-H 2 O-TIS (95:2.5:2.5), rt.,
30 min
Eirinaios I. Vrettos and Andreas G. Tzakos