333
4. Mobile phase B: Acetonitrile containing 0.1% (v/v) formic
acid.
5. NMR spectrometer for
1
H and
13
C spectroscopy and the corresponding software.
6. DMSO-d 6 (>99.8%) (see Note 3).
3 Methods
1. Dissolve Boc-protected gemcitabine (8 eq) and succinic anhydride (1 eq) in CH 2 Cl 2 .
2. Add DIPEA (10 eq) at 0 °C.
3. Stir the reaction for 12 h at room temperature.
4. Distill the solvent under reduced pressure.
5. Purify the residue by RP-HPLC to receive gemcitabine-linker
(diBoc-Gemcitabine-Hemisuccinate).
6. Characterize the compound with
1
H/
13
C NMR spectroscopy
and mass spectrometry.
1. Dissolve the gemcitabine linker (1 eq), HATU (1 eq) in anhydrous DMF under inert atmosphere (see Note 4).
2. Add DIPEA (2 eq) at 0 °C.
3. After 10 min, add D-Lys
6
-GnRH (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
30 min (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) in anhydrous
acetonitrile under an inert atmosphere.
2. Add DIPEA (40 eq) at 0 °C.
3. After 5 min, add a solution of bis(4-nitrophenyl)carbonate
(8 eq) in anhydrous acetonitrile dropwise and stir at room
temperature for 4 h.
4. Distill the solvent.
3.1 Synthesis of PDCs
3.1.1 Synthesis of PDCs
Using Succinic Acid
as Linker (Ester and Amide
Bonds) (Scheme 1)
Preparation
of Gemcitabine-Linker
(diBoc-GemcitabineHemisuccinate)
Preparation of the Final
Conjugate (GemcitabineHemisuccinate- DLys
6
- GnRH)
3.1.2 Synthesis of PDCs
Using Carbamate Bond
in the Linker (Scheme 2)
Preparation
of Gemcitabine-Linker
(diBoc-Gemcitabine-Bis
(4- Nitrophenyl)Carbonate)
Construction of Peptide-Drug Conjugates for Selective Targeting of Malignant Tumor Cells
4. Mobile phase B: Acetonitrile containing 0.1% (v/v) formic
acid.
5. NMR spectrometer for
1
H and
13
C spectroscopy and the corresponding software.
6. DMSO-d 6 (>99.8%) (see Note 3).
3 Methods
1. Dissolve Boc-protected gemcitabine (8 eq) and succinic anhydride (1 eq) in CH 2 Cl 2 .
2. Add DIPEA (10 eq) at 0 °C.
3. Stir the reaction for 12 h at room temperature.
4. Distill the solvent under reduced pressure.
5. Purify the residue by RP-HPLC to receive gemcitabine-linker
(diBoc-Gemcitabine-Hemisuccinate).
6. Characterize the compound with
1
H/
13
C NMR spectroscopy
and mass spectrometry.
1. Dissolve the gemcitabine linker (1 eq), HATU (1 eq) in anhydrous DMF under inert atmosphere (see Note 4).
2. Add DIPEA (2 eq) at 0 °C.
3. After 10 min, add D-Lys
6
-GnRH (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
30 min (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) in anhydrous
acetonitrile under an inert atmosphere.
2. Add DIPEA (40 eq) at 0 °C.
3. After 5 min, add a solution of bis(4-nitrophenyl)carbonate
(8 eq) in anhydrous acetonitrile dropwise and stir at room
temperature for 4 h.
4. Distill the solvent.
3.1 Synthesis of PDCs
3.1.1 Synthesis of PDCs
Using Succinic Acid
as Linker (Ester and Amide
Bonds) (Scheme 1)
Preparation
of Gemcitabine-Linker
(diBoc-GemcitabineHemisuccinate)
Preparation of the Final
Conjugate (GemcitabineHemisuccinate- DLys
6
- GnRH)
3.1.2 Synthesis of PDCs
Using Carbamate Bond
in the Linker (Scheme 2)
Preparation
of Gemcitabine-Linker
(diBoc-Gemcitabine-Bis
(4- Nitrophenyl)Carbonate)
Construction of Peptide-Drug Conjugates for Selective Targeting of Malignant Tumor Cells