Shake for 4 h, drain and flow-wash consecutively with THF,
MeOH, DCM, and THF (see Fig. 3, step 2.2).
4. Treat the resin (2 mmol) with triphenylphosphine (PPh 3 ,
10 mmol) in THF (5 mL) and HO-PEG 2 -CH 2 CH 2 COOtBu
(10 mmol) in THF (5 mL) under N 2 atmosphere. Add dropwise diisopropyl azodicarboxylate (DIAD, 10 mmol) and shake
for 1 h (see Fig. 3, step 2.3).
5. Wash the resin with THF and DCM, dry under vacuum and
treat with cleavage cocktail for 2.5 h. Collect the TFA-mixture
and wash the resin with TFA and DCM. Evaporate the combined TFA/DCM fractions and coevaporate with diethyl ether
(2 Â 30 mL) (see Fig. 3, step 3).
6. Dissolve the final product in water–MeCN (75/25, 100 mL)
and lyophilize to provide Ns-NPEG4 linker.
3.2.3 Synthesis
of TAT-N-Dimeric Peptide
1. Preactivate the Ns-NPEG4 linker (0.1 eq) with HBTU (0.2 eq)
and DIPEA (0.4 eq) in DMF. Subsequently, add the preactivated solution to the Fmoc-deprotected Wang-resin-bound
IETDV (1 eq). Shake the mixture for 45 min, drain the preactivated solution and wash with DMF (5 times, each of
10 mL). Repeat this step 5 times (see Fig. 4, step 1).
Fig. 4 SPPS synthesis scheme of the Tat-N-Dimer using the Ns-NPEG4 linker. (1) SPPS synthesis of the PDZ
binding peptide and addition of the Ns-NPEG4 linker on-resin. (2) Treatment with DBU and mercaptoethanol
and addition of Fmoc-Arg(Pbf)-OH. (3) Synthesis of the remaining Tat sequence and cleavage from the resin.
(Figure adapted from Bach et al. (2012))
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