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332 Cell-Penetrating Peptides: Processes and Applications
A
B
a)
b)
c)
a)
b)
c)
R 1
R 1
O
O N
FIGURE 15.3 Common reactions of amino and sulfhydryl groups. (A) Reaction of amino
groups with a) activated esters, b) isothiocyanates, and c) aldehydes. (B) Reaction of sulfydryl
groups with a) haloacetyls, b) pyridyl disulfides, and c) maleimides.
15.2.2 SOLUTION PHASE CONJUGATION
A number of proteins, peptides, oligonucleotides, oligonucleotide analogs, and particles
have been linked to CPPs using solution phase conjugation. Cargo molecules
and CPPs were linked through two reactive ends, usually amino or sulfhydryl groups,
by bifunctional linkers (Figure 15.3). To prevent dimer formation, hetero-bifunctional
linkers are preferable. The most commonly used bridges between CPPs and
cargo molecules are disulfide, maleimide, thioether, and amide linkage. The reactives
for amino and sulfydryl are described in the following section.
15.2.2.1 Amine-Reactive Reagents
O
O
H 2N R 2 R1
Aliphatic primary amine is commonly found at the N terminal of proteins or lysine
side chain and has excellent reactivity, so it is widely applied in conjugation. Lysine
ε-amine has good nucleophilic activity about pH 8.0, while the α-amino group is
more basic than lysine and reactive at around pH 7.0. The optimal pH for amine
reaction is between pH 8 and 9. At this pH range, other amine-containing groups,
such as guanidinium group of arginine, amide group of glutamine and asparagine,
imidazolium group of histidine, and aromatic amine of nucleic acids, are virtually
unreactive. Buffers that contain free amine, e.g., Tris and glycine, should be avoided
in the reaction.
Preactivated esters, isothiocynates and aldehyde are three often used reagents
for amine conjugation (Figure 15.3A). Preactivated N-hydroxysuccinimide (NHS)
O
N
H R 2
R 1 N C S H 2N R 2 R1 N H C N H
R 1
O
R 1
O
C
H 2
R 1 S S
N
O
O
H H 2N R 2 R1 C H
X
N
+
+
+
+
+
+
HS R 2
HS R 2
HS R 2
R 1
R 1
O
N R 2
C
H 2
R 1 S S R 2
N
O
O
O
R 2
S R 2
S R 2
R 1
C H2
N
H R 2