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4.2.1 Cu(I)-Catalyzed Azide-Alkyne Cycloaddiion (CuAAC, a.k.a.
Click Chemistry)
Bio-orthogonal reactions are those that involve functional groups that are
inert to most biological molecules, and are thus of great interest for
bioconjugation chemistry. The bio-orthogonal functional groups such as
azide and alkyne have been very successfully applied. The reaction between
an azide and an alkyne is catalyzed by Cu(I) and termed as Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC), also known as click chemistry. Click
reactions have been widely used for bioconjugation and are a popular
strategy because of their specificity, high yield, and wide range of solvents
and pH stabilities (Kolb
, 2001).
The research team led by Finn (The Scripps Research Institute, La Jolla,
CA, USA) has contributed enormously to the development of click reactions
utilizing VNPs and virus-like particles (VLPs) as templates. To facilitate
click conjugations on a VNP, an azide or alkyne functional group needs
to be installed on the VNP scaffold. This can be achieved using a chemical
or genetic approach. For most applications, the azide or alkyne functional
group is covalently introduced by coupling a Lys-, Cys-, or Tyr-reactive azide
or alkyne derivative to solvent-exposed amino acids on the particle surface
(Fig. 4.7, panel A). Alternatively, azide- or alkyne-containing unnatural
amino acids can be introduced into the capsid proteins under genetic control;
this has been demonstrated using Q
et al.
b
and
Hepatitis B virus
(HBV) VLPs
(Strable
, 2008), as discussed in Chapter 3, Section 3.2.1.
The reaction between an azide and an alkyne is outlined in Fig. 4.7
(panel B). A copper-binding ligand is required to accelerate the reaction
and prevent protein aggregation or degradation (Wang
et al.
, 2003a). The
ligands, tris(triazolylmethyl)amine and sulfonated bathophenanthroline
(Fig. 4.7, panel C) have been used for conjugation of a variety of molecules
to
et al.
VNPs.
A
new
improved
protocol
has
recently
been
established
(Hong
, 2009). The new protocol offers the most convenient and
efficient way to perform CuAAC by employing tris(3-hydroxypropyltriazoly-
lmethyl) amine (THPTA), a water-soluble member of the tris(triazolylmethyl)
amine family, which, in excess, intercepts reactive oxygen species generated
in the coordination sphere of the metal. In addition, aminoguanidine
is recommended to capture dehydroascorbate and its decomposition
products before they can react with protein side chains. Click chemistry
protocols have been established for CPMV, Q
et al.
b
Potato virus X
(PVX), and
TMV, and broad classes of molecules ranging from small chemical modifiers
to intact proteins have been covalently attached (Bruckman
,
et al.
, 2008;
Hong
et al.
, 2009; Kaltgrad
et al.
, 2008; Prasuhn
et al.
, 2007, 2008; Sen Gupta
et al.
, 2005a,b; Steinmetz
et al.
, 2009a,c; Udit
et al.
, 2008; Wang
et al.
, 2003a).
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