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330 Cell-Penetrating Peptides: Processes and Applications
Two methods, Fmoc (9-fluorenylmethyloxycarbonyl) and Boc (tert-butyloxycarbonyl)
chemistry, are typically used in peptide synthesis. In Fmoc chemistry, the N
terminus and side chains of the amino acids are protected by Fmoc and t-butyl
groups, respectively. On each cycle, the Fmoc group is removed with 20% piperidine.
After synthesis, the peptide is cleaved from the support and side-chain deprotected
with 90% trifluoroacetic acid (TFA). Boc chemistry needs stronger acids, i.e., TFA
and hydrogen fluoride. TFA was used to remove the N-terminal t-Boc protectinggroup
on each cycle; hydrogen fluoride was needed to remove the side chain benzylprotecting
group and to cleave peptide off solid support. Thus the stability of the
cargo molecules in all these chemical conditions is critical for conjugate preparation.
The common functional groups on peptide for cargo molecule attachment are
N-terminal amino, side-chain amino, sulfhydryls, and carboxylic groups. These
groups can be conveniently introduced by adding additional amino acids, such as
lysine, cysteine, glutamic acid, or aspartic acid. The reactive functional groups can
also come from non-nature amino acids or other synthetic linkers. The cargo molecules
then can be anchored to the peptide through these functional groups.
The biggest advantage of performing conjugation on the solid support is that
the structure of the conjugates is well defined. Using orthogonal deprotecting strategies,
the cargo molecules can be attached to a specific position; even the peptide
contains several identical functional groups. CPPs are good examples because most
of them have several lysine residues. Applying cargo molecule to a specific lysine
side chain can be done by selecting various protected lysine residues during the
synthesis. The example shown below is to prepare a Tat peptide and metal chelator
conjugate used for cell imaging. 33
EXAMPLE 15.1
SYNTHESIS OF TAT-MACROCYCLIC CHELATOR CONJUGATE
Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Tyr-Lys(DOTA)-NH 2 (Figure 15.2)
1. Reagents:
Amino acids: Boc-Gly, Fmoc-Arg(Pbf), Fmoc-Lys(Boc), Fmoc-Gln(Trt),
Fmoc-Gly, Fmoc-Tyr(Bu) and Fmoc-Lys(Dde) (NovaBiochem, San
Diego, CA).
Resin: Rink amide MBHA resin (NovaBiochem, San Diego, CA).
Coupling agents: 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBt) (Nova-
Biochem, San Diego, CA).
Chelator: 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid t-butyl ester)-10-acetic
acid (DOTA-3tBu) (Macrocyclics, Richardson, TX).
2. The peptide was synthesized on an automatic peptide synthesizer (PS3,
Rainin, Woburn, MA) by Fmoc chemistry at 0.1 mmol scale. To each
cycle, four-fold excess on amino acid and coupling agents was used. The
amino acid sequence and protecting groups are shown in Figure 15.2.
Since the subsequent hydrazine treatment is able to remove the Fmoc