Biology Reference
In-Depth Information
.1.1.  N -hydroxysuccinimide esters 
Coupling of an NHS ester-activated compound to an amine results in the
formation of a stable amide bond with the NHS as a leaving group (Fig. 4.2,
panel B).
.1.1. 
Carboxylates 
Carboxylates can be covalently attached to amines via the formation
of a peptide bond. To achieve chemical conjugation, the carboxylate-
selective coupling agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) is typically used. EDC activates carboxylates by the formation of an
O
-acylisourea ester intermediate. This unstable intermediate reacts readily
with primary amines, which results in the formation of a stable covalent
amide bond (Fig. 4.2, panel C). NHS is often used in addition to EDC, where
the NHS group reacts with the activated
O
-acylisourea and generates an NHS
ester
, which readily reacts with primary amines (see above). This
coupling technique can be applied toward coupling any molecule containing
a carboxylate to a VNP with addressable Lys side chains.
in situ
4.1.1.4  Transaminaion 
Covalent modification of the N-terminus can be achieved by site-specific
transamination reactions mediated by the biological cofactor pyridoxal
5
-phosphate and has been applied to a genetically engineered version of
the
., 2008). In brief,
the transamination allows the introduction of a ketone or aldehyde, both
of which provide
Tobacco mosaic virus
(TMV) coat protein (Scheck
et al
ligation handles for further reaction such
as oxime ligation (see Section 4.2.2). The word
orthogonal
comes from
the Greek language and means “straight angle”. In chemistry,
orthogonal
orthogonal
reactions are defined as strategies that allow the reaction of functional
groups independently of each other. Orthogonal reactions are in general
highly selective and reactive.
4.1.2 Carboxylate-Selecive Chemistries
Carboxylate groups derived from the acids Asp and Glu (Fig. 4.1) can be used
as targets for bioconjugation chemistry. The p
K
values of Asp and Glu side
a
chains lie at around
4.5. The C-terminus of proteins (that is the terminal
α
-
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