Chemistry Reference
In-Depth Information
7.11 Mass Spectrometry and
In Situ
Click Chemistry
The pioneering application of the 1,3-DCR for
in situ
Click chemistry to target a bio-
molecule was described by Sharpless, Finn and co-workers in 2002.
[
37
]
The enzyme
acetylcholinesterase (AChE) was selected as the biological target for this proof-of-concept
investigation and for subsequent optimization work in which the specificity, sensitivity
and general capability of the analysis employed to screen with this target-guided synthetic
strategy was enhanced.
[
37 39
]
Additional biomolecular targets have since been reported and
include the enzymes carbonic anhydrase
[
40, 41, 44
]
and HIV-protease.
[
42
]
More recently,
in
situ
Click chemistry was miniaturized by use of a microfluidic chemical reaction cir-
cuit in which the consumption of biomolecule and fragments alike was significantly
reduced and together with automation this has undoubtedly improved the compatibility
of this approach with drug discovery.
[
41
]
Each of the published examples has utilized
mass spectrometry for the detection of the ligands formed by the
in situ
Click chem-
istry. Here we will describe the AChE proof-of-concept and optimization work and also
the
in situ
microfluidic reactor-based work with a deliberate focus on the attributes of
mass spectrometry for screening to identify lead compounds generated by
in situ
Click
chemistry.
7.11.1 Proof-of-Concept
Acetylcholinesterase (AChE) is an enzyme that catalyses the hydrolysis of the neur-
otransmitter acetylcholine to give acetate and choline. This reaction terminates the
neurotransmission process in both the central and peripheral nervous systems. It is already
known that AChE has two distinct binding sites, a catalytic site and a peripheral site, and
that these sites are in close proximity. The fragment library for the proof-of-concept study
was derived from structural variants of known site-specific AChE inhibitors, namely tac-
rine (an active site ligand with a
K
d
of 18 nM) and phenylphenanthridinium (a peripheral
site ligand with a
K
d
of 1.1 M). These tacrine and phenylphenanthridinium fragments
were thus complementary with respect to reactive functional groups (azide or acetylene)
and binding site recognition (catalytic or peripheral). This investigation screened a total
of 49 binary azide-acetylene fragment combinations with 98 potential triazole products
(including the
syn-
and
anti-
triazole regioisomers) (Scheme 7.6). The
in situ
reactions con-
sisted of solutions of AChE (
1 M active enzyme), ammonium citrate buffer (2 mM, pH
7.3-7.5), tacrine fragment (
TZ2
-
6, TA1-3
;30M) and complementary coupling partner
(either 30 Mof tacrine fragment or 66 Mof phenanthridinium fragment
PZ6-8
,
PA2-6
).
Each reaction mixture was allowed to stand at room temperature with analysis performed
at daily intervals for 7 days.
As for DCC, a robust analytical method to identify fragment combinations that are
assembled in the presence of the biological target is critical for the application of
in situ
Click chemistry to drug discovery. A variant of the MALDI (matrix-assisted laser desorp-
tion/ionization)MS technique was adopted for screening, wherein the desorption/ionization
is performed on a silicon surface, known as desorption/ionization mass spectrometry
(DIOS-MS). The advantage of the DIOS variant of the MALDI-MS technique is that it
is unhampered by interfering background matrix peaks normally associated with MALDI.
DIOS-MS was performed on a MALDI-TOF mass spectrometer, with just 0.25-0.5 Lof
∼
