Peptide-Based Drug Design

232 Cudic and Stawikowski
in α-hydroxy acid (see Note 7) and activation of aminoalkylphosphonate with a
4-nitrophenethyloxycarbonyl (NPEOC) group.
These protocols were adopted from refs. (35–37),(40), and(41):
Fmoc protection of hydroxyl group:
1. Dissolve �-hydroxy acid (1 eq) in anhydrous pyridine (20 mL) at 0 o C.
2. Add Fmoc-chloroformate (3 eq).
3. Stir reaction mixture for 3 h at 0 o C.
4. Concentrate reaction mixture under vacuum and purify by column chromatography
over silica gel (eluent: DCM/CH3OH/AcOH=9/0.9/0.1).
Aminoalkylphosphonate activation:
1. Add 1-(N-benzyloxycarbonyl-amino)-alkylphosphonate (1 eq) to aqueous KOH
(5 eq) at room temperature. Monitor reaction by TLC.
2. Wash reaction mixture with diethyl ether.
3. Acidify water phase with concentrated HCl to pH 2 and then extract with EtOAc
(3x).
4. Dry organic phase over MgSO4, filter, and concentrate under vacuum.
5. Dissolve concentrated residue in EtOH and hydrogenate in the presence of 10%
Pd/C catalyst.
6. Upon completion of reaction, filter the mixture through celite, wash catalyst with
MeOH, and concentrate filtrate under vacuum.
7. Dissolve obtained material in H2O.
8. Add Na2CO3 (1.1 eq) and 4-nitrophenylethyl chloroformate (1.1 eq) dissolved in
dioxane.
9. Monitor reaction by TLC.
10. Upon completion of the reaction wash the mixture with diethyl ether, acidify with
concentrated HCl to pH 2 and extract with EtOAc (3x).
11. Dry organic phase over Mg SO4, filter, and concentrate under vacuum.
This protocol was adopted from refs. (35–37).
1. Place resin in dry reaction vessel.
2. Synthesize the desired peptide sequence using standard Fmoc chemistry.
3. Couple α-O-Fmoc–protected hydroxyl acid using HBTU/HOBt or PyBroP
protocol.
4. Remove the Fmoc-protecting group by agitating the resin with 30% piperidine in
NMP (25 mL/mmol, 3 × 10 min).
5. Wash resin with NMP (25 mL/mmol, 5 × 2 min) and diethyl ether
(25 mL/mmol, 5 × 2 min) and dry overnight under vacuum in the presence of
P2O5.
6. Suspend NPEOC-aa-�(HO-P(=O)-OMe) (1 eq) and NMP (1 eq) in anhydrous
THF, then add tris(4-chlorophanyl)phosphine (1 eq) and DIAD (1 eq).
7. Leave reagent mixture for 5 min and then add it to the resin (see Note 8).