Fragment Conjugation

An alternative strategy for peptide-PNA conjugate synthesis is a solution-phase fragment coupling between a peptide and a PNA, each containing a specific functionality and each synthesized separately on solid phase and purified prior to the conjugation step. This route is more popular for studies that involve synthesis of a range of peptides attached to the same PNA. Although theoretically only a stoichiometric amount of each reagent is needed to obtain the desired conjugate, often the less expensive reagent, usually the peptide, is used in excess to drive the conjugation reaction to completion. In recent years, a number of stable or labile linkage chemistries have been developed to conjugate a peptide to a PNA moiety, and described below are some of the most well-known methods used for gene-modulation applications. Among stably linked conjugates, the thiol-maleimide conjugation method requires addition of a cysteine residue to the peptide and preparation of an N-maleimide derivative of the PNA65 (Figure 4.2a). The latter can be obtained by reacting the terminal primary amino group of purified PNA with the hetero-bifunctional cross-linker succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate. The solution-phase coupling is very fast when carried out in potassium phosphate buffer (pH 7.0). Similarly, ester-containing conjugates (Figure 4.2b) can be prepared from PNAs bearing a glycyl(hydroxy-methyl)benzoic acid residue at the N-terminus, and this linkage is potentially labile to esterases within cells.46

We have recently described the formation of stably linked thioether peptide-PNAs by conjugation of a peptide containing a cysteine at the C-terminus with N-terminally bromoacetylated PNA in BisTris. HBr buffer [pH 7.5; Figure 4.3].47 This work is based on original thioether conjugate formation between bromoacetyl oligodeoxynucleotides and cysteine-containing peptides.66 By far the most popular linkage is one based on a disulfide bond between the peptide and the PNA residue. Such a linkage is expected to be cleaved within the reducing environment of the cell and was first used by Langel and colleagues for biological studies that involved Transportan-PNA and Pene-tratin-PNA.67 To obtain a disulfide linkage, both the peptide and the PNA should contain an additional cysteine residue, since the disulfide bond is formed between the cysteine thiol groups. The asymmetric disulfide conjugation reaction requires a pre-activation of one of the thiols, which is achieved

Peptide SH

Peptide S

Peptide SH

N PNA H

N PNA H

Peptide S

N PNA H

N PNA H

Peptide S

Peptide S

H PNA

Figure 4.2 Formation of (a) thiol-maleimide and (b) ester-linked peptide-PNA conjugates.

H PNA

Figure 4.2 Formation of (a) thiol-maleimide and (b) ester-linked peptide-PNA conjugates.

Figure 4.3 Formation of thioether-linked peptide-PNA conjugates.
Figure 4.4 Formation of disulfide-linked peptide-PNA conjugates.

typically by use of either the pyridylsulfenyl (pys) or 3-nitropyridylsulfenyl (Npys) group (Figure 4.4). We have used the disulfide linkage extensively in studies of peptide-PNA conjugates targeted to the HIV-1 TAR RNA58 and very recently in the luciferase splice correction model.47

A convergent synthetic route for PNA-N-to-C-peptide conjugate construction involves regioselective chemical ligation of a thioester peptide fragment to an N-terminal cysteine-modified PNA under denaturing conditions (Figure 4.5a).68 Furthermore, a native ligation approach, but using a bifunc-tional PNA derivative with a C-terminal thioester and an N-terminal thiaproline (Figure 4.5b), can be applied to the synthesis of trimeric peptide-PNA-peptide conjugates.69,70

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