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crc press - E-Lib FK UWKS

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330 Cell-Penetrating Peptides: Processes and Applications<br />

Two methods, Fmoc (9-fluorenylmethyloxycarbonyl) and Boc (tert-butyloxycarbonyl)<br />

chemistry, are typically used in peptide synthesis. In Fmoc chemistry, the N<br />

terminus and side chains of the amino acids are protected by Fmoc and t-butyl<br />

groups, respectively. On each cycle, the Fmoc group is removed with 20% piperidine.<br />

After synthesis, the peptide is cleaved from the support and side-chain deprotected<br />

with 90% trifluoroacetic acid (TFA). Boc chemistry needs stronger acids, i.e., TFA<br />

and hydrogen fluoride. TFA was used to remove the N-terminal t-Boc protectinggroup<br />

on each cycle; hydrogen fluoride was needed to remove the side chain benzylprotecting<br />

group and to cleave peptide off solid support. Thus the stability of the<br />

cargo molecules in all these chemical conditions is critical for conjugate preparation.<br />

The common functional groups on peptide for cargo molecule attachment are<br />

N-terminal amino, side-chain amino, sulfhydryls, and carboxylic groups. These<br />

groups can be conveniently introduced by adding additional amino acids, such as<br />

lysine, cysteine, glutamic acid, or aspartic acid. The reactive functional groups can<br />

also come from non-nature amino acids or other synthetic linkers. The cargo molecules<br />

then can be anchored to the peptide through these functional groups.<br />

The biggest advantage of performing conjugation on the solid support is that<br />

the structure of the conjugates is well defined. Using orthogonal deprotecting strategies,<br />

the cargo molecules can be attached to a specific position; even the peptide<br />

contains several identical functional groups. CPPs are good examples because most<br />

of them have several lysine residues. Applying cargo molecule to a specific lysine<br />

side chain can be done by selecting various protected lysine residues during the<br />

synthesis. The example shown below is to prepare a Tat peptide and metal chelator<br />

conjugate used for cell imaging. 33<br />

EXAMPLE 15.1<br />

SYNTHESIS OF TAT-MACROCYCLIC CHELATOR CONJUGATE<br />

Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Tyr-Lys(DOTA)-NH 2 (Figure 15.2)<br />

1. Reagents:<br />

Amino acids: Boc-Gly, Fmoc-Arg(Pbf), Fmoc-Lys(Boc), Fmoc-Gln(Trt),<br />

Fmoc-Gly, Fmoc-Tyr(Bu) and Fmoc-Lys(Dde) (NovaBiochem, San<br />

Diego, CA).<br />

Resin: Rink amide MBHA resin (NovaBiochem, San Diego, CA).<br />

Coupling agents: 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium<br />

hexafluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBt) (Nova-<br />

Biochem, San Diego, CA).<br />

Chelator: 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid t-butyl ester)-10-acetic<br />

acid (DOTA-3tBu) (Macrocyclics, Richardson, TX).<br />

2. The peptide was synthesized on an automatic peptide synthesizer (PS3,<br />

Rainin, Woburn, MA) by Fmoc chemistry at 0.1 mmol scale. To each<br />

cycle, four-fold excess on amino acid and coupling agents was used. The<br />

amino acid sequence and protecting groups are shown in Figure 15.2.<br />

Since the subsequent hydrazine treatment is able to remove the Fmoc

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