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Cell-Penetrating Peptide Conjugations and Magnetic Cell Labels 333
esters react with amine efficiently, and the amide linkages formed are stable in most
conditions. Some linkers with NHS ester may have poor water solubility; in these
cases, NHS can be replaced by sulfonate-NHS. Isothiocyanates like NHS esters are
amine-reactive reagents: a stable thiourea bond was formed after the reaction. A
number of isothiocyanates of fluorescence dye have been widely used in labeling
biomolecules.
The third type of amine-reactive group is aldehyde, which will form an imine
linkage. The aldehyde first reacts with amine to form an intermediate Schiff base,
which can be selectively reduced by mild reducing agent sodium cyanoborohydride
to give a stable alkylamine bond. This type of linkage is used often to react with
sugar because the sugar molecule can be readily oxidized into aldehyde by sodium
periodate. However, the reaction is not limited to aliphatic amines; it can also react
with aromatic amines. CPPs such as Tat, antennpedia, transportan, and others have
multiple lysine residues. Any modification on these lysine side chains may abolish
their penetrating capability; therefore, instead of amino groups, a sulfhydryl group
is usually introduced for CPP attachment.
15.2.2.2 Sulfhydryl-Reactive Reagents
Sulfhydryl-specific reactions include α-haloacetyls, pyridyl disulfides, and maleimides,
(Figure 15.3B). Reacting with maleimide and haloacetyl groups will form thiol
ether bonds, and with thiol–pyridyl will form a disulfide linkage that is cleavable.
All these thiol-specific reactions happen rapidly in the physiological pH range, pH
6.5 to 8.0, at or below room temperature. Since thiol and amino groups have different
optimum pH, the selectivity of the reactions can be controlled well.
Although the thiol–ether bond created from haloacetyl group is a stable bond,
the ones from maleimide or pyridyle disulfide are not. The disulfide linkage can be
readily cleaved under reducing conditions. If a reversible linkage on the CPP conjugate
were preferred, the disulfide bond would be an ideal choice. The maleimide
linkage is stable at neutral pH; once the pH is above 8, side reactions may occur. 37
Compared to amino groups, thiol groups are less commonly found in most
proteins; thus the sulfhydryl reactive group can provide means of selective modification
at a defined site. For synthetic compounds, a sulfhydryl group can be conveniently
generated during synthesis. Peptides can be functionalized by adding a
cysteine residue at the N terminus, C terminus, or even at an internal position. If an
oligonucleotide is involved in conjugation, many ready-to-use thiol linkers for oligonucleotide
3′ or 5′-terminal modification are also commercially available.
15.2.2.3 Heterobifunctional Conjugation
Checking the amino acid sequences of CPPs, it is found that the positively charged
arginine and lysine residues are the two most dominant amino acids. Compared to
the amino group of lysine, the guanidinium group of arginine is inert at the optimal
pH range for amine reaction. Arginine residue would cause no trouble during conjugation,
but the lysine residues might, especially multiple lysine residues. Heterobifunctional
linkers usually have two distinct reactive groups that allow for sequential