Chemistry Reference
In-Depth Information
The hit compound detection limit for the HPLC-SIM-MS technique was investigated
and it was shown that triazole molecules could be detected down to 0.4 mol% of the active
AChE concentration. The rate of any background 1,3-DCR between azide and acetylene
fragments was also assessed by monitoring for triazole formation over 2 weeks. A plot of
the integrated second-order kinetic equations appropriate to each tested binary combin-
ation gave the observed rate constants and demonstrated that the background 1,3-DCRs
proceeded to less than 0.5% triazole formation over the 2 weeks. The experimental details
reported in these papers demonstrate that the level of triazole formation with
in situ
chem-
istry is clearly very small, certainly
<<
2%, likely
<
1%, but necessarily greater than the
background reaction if a 'hit' is to be claimed. Ample evidence is included in these proof-
of-concept studies to confirm that the observed triazole formation is not a consequence of
background reaction but is indeed induced by the presence of AChE. More specifically,
this evidence includes a number of results from control experiments, all of which gave
no detectable amounts of triazoles (analysis by either DIOS-MS or HPLC-SIM-MS): (i) a
mixture of azides and acetylenes in the absence of AChE; (ii) a mixture of azides and
acetylenes in the absence of AChE and in the presence of 10 M bovine serum albumin;
(iii) pretreatment of AChE with 100 M tacrine to saturate the AChE active site, followed
by incubation with azides and acetylenes; and (iv) prior inactivation of AChE by covalent
phosphorylation of the active site serine followed by incubation with azides and acetylenes.
In this last experiment, AChE was subsequently reactivated by treatment with prali-
doxime chloride, and the reactivated enzyme then induced formation of the triazole from
fragments.
7.11.2
Integrated Microfluidics
In 2006, Kolb and co-workers reported the application of an integrated microfluidic device
for parallel screening of an
in situ
Click chemistry library.
[
41
]
The intention in this proof-of-
concept study was to make lead discovery through
in situ
Click chemistry more convenient,
more reliable, less expensive and more diverse compared with earlier efforts in which
the Click chemistry was carried out in parallel in 96-well microtitre plates using largely
manual operation. The microfluidics-based study targeted the already successful Click
chemistry systemwith carbonic anhydrase (CA) and utilized 4-ethynylbenzenesulfonamide
as the CA anchor fragment, with 20 complementary azide fragments (Scheme 7.7). The
microfluidic chemical reaction circuit consisted of four major components (Figure 7.17).
These components were utilized as follows:
1. A nanolitre-level rotary mixer - used to mix nanolitre quantities of reagents in a precise
manner; this mixer has a total volume of 250 nL.
2. A microliter-level chaotic mixer - used to combine reagent solutions from the rotary
mixer with microlitre quantities of enzyme solution in buffer to give a homogeneous
reaction mixture (CA in PBS, pH 7.4);
3. A microfluidic multiplexer - used to deliver the reaction mixture to one of the 32
microvessels
4. Microvessels - 32 in total, used to store the reaction mixture for the
in situ
chemistry.
The microvessels are 1.3 mm in diameter and 6 mm in depth, with a volume of
∼
8 L.
