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126 Cell-Penetrating Peptides: Processes and Applications
On the other hand, the vector and cargo may be synthesized separately and
coupled together using a ligation strategy. One of the most commonly used ligation
techniques in biological chemistry is the formation of a disulphide bond between
peptides and proteins containing activated thiol groups. Disulphide bond formation
has been used to link both peptide 108-110 and oligonucleotide 111,112 cargo to translocating
peptide vectors. The nature of the disulphide bond requires that separation
procedures used to isolate and characterize the desired product must not include any
method that may reduce the disulphide bond, e.g., SDS-PAGE with reducing buffers.
Another drawback associated with the formation of disulphide bonds is that a large
quantity of byproducts usually results due to the nonspecific nature of the interaction,
leading to low reaction efficiency. Formation of this type of bond has the advantage,
however, of releasing the cargo as the disulphide bond breaks in the reducing
environment of the cytoplasm, thereby minimizing interactions between the translocating
peptide and the biologically active cargo in vivo.
The formation of other nonamide linkages between translocating peptide and
cargo has also been investigated. 77,83,113 Independently prepared translocating peptide
and cargo peptides have been linked by a single-step ligation through a nonamide
thiazolidine linkage (Figure 6.2c) and the biological activity of the resulting peptides
compared to counterparts synthesized by the conventional tandem synthesis
method. 113 Combinations of two different MTSs and two different cargo peptides
were used in this study: the h-region of kFGF and that of human integrin β 3
33,88 as
MTSs, and peptides derived from the human integrin β 3 cytoplasmic tail and the
NF-κB p50 NLS as cargo. A general and mild method for the chemoselective ligation
of an aldehyde on one reactant with a 1,2-amino thiol moiety on a second
reactant 114,115 was used to assemble the MTS-cargo peptides. An aldehyde functionality
was generated at the C terminus of each MTS peptide by oxidation of an
aminopropanediol moiety or a 1,2-amino alcohol moiety present at the C terminal
following stepwise solid-phase peptide synthesis and cleavage from the solid support.
Preparation of the cargo peptides involved addition of a cysteine residue to the
N terminus of the sequence. The ligations, performed under various conditions to
optimize solubility of hydrophobic and functional peptide modules, were complete
within 6 h and standard procedures used to purify the desired products. The results
of the biological assays indicate that the hybrid peptides are functionally equipotent
with those containing an amide linkage synthesized by the conventional method.
The thiazolidine ring formation protocol was employed by Chang et al. 77 to
generate a series of peptides comprising the kFGF MTS and cargo peptides designed
to examine the intracellular signaling mechanism of the 5-HT 2C receptor. In this case
lysine and serine residues were added at the C terminus of the MTS peptide to serve
as a linker and a masked aldehyde, respectively. The lysine was attached to the
C terminus of the MTS by its primary amine, and then serine was attached at the
side chain ε-amine. The serine residue was treated with an oxidizing agent to generate
the reactive aldehyde moiety subsequently reacted with effector peptides containing
N-terminal cysteines to form the vector–cargo product.
This method for the rapid preparation of functional cell-permeant peptides may
be applied to virtually any peptide or protein containing an N-terminal cysteine and
allows bulk preparation of translocating peptide that may be used in ligation to any