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Cell-Penetrating Peptide Conjugations and Magnetic Cell Labels 331
Pbf Boc Boc Pbf Pbf Trt Pbf Pbf Pbf tBu Dde
Boc-Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Tyr Lys
Pbf Boc Boc Pbf Pbf Trt Pbf Pbf Pbf tBu NH2
Boc-Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Tyr Lys
Pbf Boc Boc Pbf Pbf Trt Pbf Pbf Pbf tBu NH
Boc-Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Tyr Lys
tBu-COO
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Tyr Lys NH 2
tBu-COO
O
N
DOTA-3tBu = HO
C N N COO-tBu
N
COO-tBu
2% Hydrazine
DOTA-3tBu/HBTU/HOBT
TFA
N N O
C N
N
COO-tBu
COO-tBu
N N O
C N
N
COOH
FIGURE 15.2 Synthetic scheme of Tat-DOTA. (From Bhorade, R. et al., Bioconjug. Chem.,
11, 301, 2000. With permission.)
group, Fmoc-Gly cannot be used for the last cycle. Instead, a t-Boc-Gly
was used to cap the N-terminal amino group.
3. The Dde protecting group on the C- terminal lysine residue was selectively
removed with 10 ml of 2% hydrazine in DMF (2 × 3 min). The other
lysine residues were still protected by t-Boc groups.
4. The deprotected lysine residue was then reacted with four-fold excess of
DOTA-3tBu with HOBT/HBTU in 5 mL of DMSO/diisopropylethylamine
(20% v/v). The coupling efficiency was monitored by Ninhydrin reaction.
5. After synthesis the peptide was cleaved by 5 ml of TFA/thioanisole/ethandithiol/anisole
(90/5/3/2) for 3 h, precipitated by methyl-t-butyl
ether and purified by C18 reverse phase high pressure liquid chromatography.
6. Finally, the purified peptide was characterized by mass spectrum, MALDI-
MS; (M + H) + : 2131.4 (calc.), 2131.3 (found).
NH
HOOC
COOH