Chemistry Reference
In-Depth Information
The individual reagent quantities per microvessel were acetylene, 80 nL of 30 mM stock
=
=
0.514 g or 2.4 nmol; azide, 120 nL of 30 mM stock
3.6 nmol; CA, 3.8 Lof
5mgmL
−
1
=
in PBS stock
19 g or 0.65 nmol; and CA inhibitor, 40 nL of 11 mM
stock
4 nmol. The interested reader is directed to the original paper for specific pro-
tocol details.
[
41
]
Significant for drug discovery applications is that the 32
in situ
reactions
were prepared in
=
57 s per reaction cycle); this is a remarkably short opera-
tion time compared with the microtitre plate reaction format and also uses substantially
less protein (3.8 Lof5mgmL
−
1
protein compared with
∼
30 min (
∼
94 Lof1mgmL
−
1
pro-
tein for microtitre plate). The reaction circuit was incubated at 37
◦
C for 40 h prior to
analysis by HPLC-SIM-MS using electrospray ionization as described above for AChE
(Figure 7.18). The major determinant of the reagent and target quantities for these micro-
fluidics experiments was the need to detect the triazole product formed, and in principle
a more sensitive MS instrument than that used could facilitate even smaller reagent
quantities.
∼
7.12 Summary and Outlook
The
in situ
chemistry examples presented here are testament to a synthetic achievement
that, although a niche component of medicinal chemistry, has the potential to impact on
and influence the direction of future drug discovery campaigns. Typically, the reported
in situ
medicinal chemistry successes have stemmed from prior knowledge of a reli-
able target recognition fragment or 'anchoring' fragment. This anchor fragment is then
furnished with the necessary functional group(s) to participate in either DCC or Click
chemistry, typically with a much larger panel of complementary functionalized fragments.
The
in situ
chemistry is free to scan the active site architecture of the target to link
those fragments that can best exploit additional molecular recognition interactions with
the target. As these approaches continue to develop, the field should aim to deliver res-
ults driven entirely from novel anchor fragments identified independently and without
this prior knowledge. Thus fragments discovered through fragment-based screening are
well poised to lead into either
in situ
DCC or Click chemistry campaigns to underpin
target-guided fragment optimization, and it is expected that the currently small overlap-
ping footprint of fragment-based drug discovery with these synthetic methodologies will
expand.
In situ
DCC and Click chemistry can in principle be applied to a multitude of
biomolecular targets for which small-molecules drug are sought. So far enzymes pre-
dominate in published examples, but receptor proteins, carbohydrate-binding proteins,
DNA, RNA and whole cells have been targeted successfully. The real challenge that now
presents itself is to demonstrate practicality of these synthetic methods in a drug discovery
setting.
This research has progressed only because the science was willing to allow Nature's
biomolecules to intervene earlier than is usual for medicinal chemistry protocols. The role
of biomolecules has been elevated from spectator to key player, to guide and instruct the
synthesis of potent inhibitor molecules, while the medicinal chemist acts to facilitate this
process. The
in situ
selection and assembly of fragments could in principle be screened by an
activity assay or by any number of analytical techniques; for drug discovery, however, it is a
