Chemistry Reference
In-Depth Information
reversibility: (i) the exchange reaction should occur at a rate that allows equilibrium to be
reached within an acceptable time-scale; (ii) it should be bioorthogonal with the template
biomolecule, i.e. with reactivity inert to the functional groups of the biomolecule; (iii) it
must operate under conditions that preserve native state (or other biologically relevant
conformations) of the biomolecule - typically aqueous buffer at physiological temper-
ature and pH; (iv) the reaction conditions should not inhibit formation of noncovalent
interactions involved in molecular recognition between target biomolecule and fragments;
and (v) ideally the reactivity of all fragments should be similar to allow access to unbiased
DCLcompositions. The reversible reactions that meet these requirements have been detailed
in a recent review;
[
7
]
here we will present only a specific example, the hydrazone exchange
reaction (Scheme 7.1). This C
N exchange reactio
n
has been widely used in DCC with
multiple examples of drug discovery applications.
[
11 15
]
Hydrazone exchange is the revers-
ible reaction between a hydrazine or hydrazide [R
1
R
2
N-NH
2
or R
1
(C
=
=
O)NH-NH
2
] and an
aldehyde [R
3
(C
N exchange reactions include imine and oxime exchange;
however, to ensure a balanced product distribution, libraries relying on C
=
O)H]. Other C
=
=
N are ideally
prepared from closely related X-NH
2
fragments.
[
7
]
O
O
R
3
NH
2
N
R
1
R
1
N
H
N
H
H
hydrazide
O
OR
OR
+
+H
2
O
R
3
H
R
2
aldehyde
R
2
NH
2
R
1
R
3
N
H
N
R
1
N
H
H
hydrazones
hydrazine
Scheme 7.1
Reversible hydrazone formation and exchange.
4), but
importantly for
in situ
DCC applications
i
t can also occur under mild conditions, such as
in aqueous environments at neutral pH.
[
16 18
]
The amino functional groups on proteins are
predominantly protonated in this pH range so that this reaction is essentially bioorthogonal
and potential imine products, from the reaction of aldehyde fragments with amino groups
on the protein target, are not detected at neutral or acidic pH. If the aldehyde is present as
a hydrate (reversibly formed from the aldehyde), the aldehyde along with the hydrate is
consumed within minutes of initiating the exchange reaction.
[
19
]
The equilibration kinetics
of hydrazone exchange are fairly slow and it may take days to reach equilibrium, whereas
this is not necessarily a barrier for drug discovery applications: an increase in the kinetics of
reaction while preserving the bioorthogonal reaction attributes is indeed desirable. A recent
investigation by Dawson and colleagues demonstrated that the equilibration kinetics of
hydrazone formation from -carbonyl aldehydes and subsequent transimination could be
Hydrazone formation is rapid under acidic conditions (typically fastest at pH
≈
