Biology Reference
In-Depth Information
4.1.1 Amine-Selecive Chemistries
Amines are by far the most commonly used targets for bioconjugation
chemistry. Primary amines in proteins are derived from the N-terminus (the
terminal
-amine that is not engaged in a peptide bond) and the amine group
of Lys side chains (Fig. 4.1), termed the
α
ε
-amine.
-amine is a highly reactive nucleophile. A nucleophile is any atom
containing an unshared pair of electrons or an excess of electrons able to
participate in covalent bond formation. The reactivity stands in relation to the
relative nucleophilicity and is dependent on whether the amine is protonated
or unprotonated. An uncharged, unprotonated amine is more reactive
compared with the protonated form. In its unprotonated form, the amine
contains an unshared pair of electrons and is thus a good nucleophile. The Lys
side chain is a strong base with a p
The
ε
is the acid dissociation
constant and a measure of the strength of an acid in solution; the lower the
value, the higher the extent of dissociation and strength of the acid). Based on
the Henderson-Hasselbalch equation (Eq. 4.1), at a pH close to its p
K
of 9.3-9.5 (p
K
a
a
K
50%
a
of the
of a specific
amino acid side chain in a protein is dependent on the microenvironment and
is typically lower than the theoretical value; Lys modification can be carried
out at pH values as low as pH 7.0 with negligible loss of reactivity, although
of course this is dependent on the individual protein (Aslam & Dent, 1999;
Hermanson, 1996).
ε
-amines are unprotonated and reactive. The realistic p
K
a
pH = p
K
+ log{[base]/[acid]}
(4.1)
a
Amine-selective chemistries are a popular choice, and numerous
reagents for amine modification are commercially available. For examples,
browse the catalogs from Pierce (http://www.piercenet.com), Invitrogen
(http://www.invitrogen.com), and Solulink (http://www.solulink.com).
Reagents used for amine modification typically contain isothiocyanates or
-
hydroxysuccinimide (NHS) esters as the functional group. Derivatization with
carboxylate containing compounds can also be achieved using a carbodiimide
as a coupling reagent (Aslam & Dent, 1999; Hermanson, 1996).
N
.1.1.1
Isothiocyanate-mediated coupling
The coupling reaction of an isothiocyanate with an amine is outlined in
Fig. 4.2 (panel A); coupling results in the formation of a stable thiourea bond.
The most commonly used isothiocyanate-containing label is fluorescein-
isothiocyanate (FITC), which has been conjugated to a large variety of VNPs.
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