Chemistry Reference
In-Depth Information
Table 7.2
Molecular weights for CA II-bound ligands identified from
in situ
screening of the
DCL by ESI-FTICR-MS.
Molecular weight observed
for [M - H]
−
(Da)
a
Compound
=
[M]
Molecular weight calculated
for [M - H]
−
(Da)
403.06
403.07
1A
417.07
1B
417.09
445.10
1C
445.12
459.11
1D
459.13
493.09
1E
493.12
a
Observed molecular weights taken from mass spectrum in Figure 7.13c.
for investigating protein-ligand noncovalent complexes compared with standard electros-
pray; the lower sample flow rates (10-200 nL min
−
1
) and smaller sample droplets improve
transfer of specific noncovalent complexes to the gas phase and hence increase the sig-
nal intensity with the added benefit of reducing protein consumption. As nanoESI sources
become more user friendly and commonplace with FTMS, it is expected that more DCL
applications will emerge that utilize MS for screening biomolecular targets to identify bind-
ing partners. The FTMS instrument that permitted these proof-of-concept experiments has
a 4.7 T superconducting magnet and clearly with the higher field magnets now more com-
monly available more demanding DCLs could be analyzed with excellent mass accuracy
results. In addition, coupling nanoESI and higher field magnets with automated sample
handling could significantly reduce both the time and target quantity required to facilit-
ate analysis; this is clearly desirable for drug discovery applications. In-line size-exclusion
chromatography or dialysis techniques can be directly coupled to an ESI-FTMS instrument.
Our preliminary investigations with such a system have permitted us to exchange DCLs
from noncompatible ESI-MS buffers (e.g. phosphate-buffered saline, Tris) into compat-
ible ESI-MS buffers directly prior to analysis (unpublished work); this simple procedure
could undoubtedly further expand the range of biomolecular targets amenable to direct MS
screening by permitting DCL experiments to be carried out in other buffers to fulfil stability
requirements.
The application of mass spectrometry to the direct screening of a DCL has been adopted
also by Schofield and colleagues, the biomolecular target for their study being the therapeut-
ically relevant enzyme metallo--lactamase (BcII) from
Bacillus cereus
.
[
28
]
This enzyme
(molecular weight
25 kDa) catalyses the hydrolysis of a range of clinically used -lactam
antibiotics and is of interest as a medicinal chemistry target owing to its involvement in the
resistance of bacteria to antibiotics.
[
58
]
This DCC investigation stemmed from some earlier
work by the same group in which it was confirmed, also using mass spectrometry, that BcII
contains two active site zinc cations and that simple thiol fragments formed BcII-inhibitor
complexes through a zinc binding interaction.
[
59
]
A thiol fragment identified in this pre-
cursor study was adapted to give five anchor fragments (
F-J
) with suitable functionality
for DCC using thiol-disulfide exchange as the reversible reaction. Each anchor fragment
(
F-J
) had two thiol groups: one to facilitate Zn binding (thiol
1
) and the second to particip-
ate in thiol-disulfide exchange for DCC (thiol
2
). The DCC study examined 19 additional
novel fragments: monothiols
(4-17, 19-22)
and dithiol
(18)
as the coupling partners for
F-J
(Scheme 7.4).
∼
