Chemistry Reference
In-Depth Information
biaryls are produced by means of Suzuki couplings. If one of the biaryls is a hit, one of the
aryls can then easily be exchanged with another aryl. The iNovacia fragment library also
contains molecular scaffolds that occur frequently in marketed drug molecules.
4.4.2 NMR Instrumentation
For adequate resolution and sensitivity, the NMR spectrometer used should operate at a
1
H
frequency of 500 MHz or higher and preferably be equipped with a cryogenically cooled
probe that reduces the thermal noise in the receiver circuitry ('cryoprobe'). Automated
sample changing is convenient and it should be possible to keep the samples cold while
they are waiting to be measured. At iNovacia, we are using a 600 MHz spectrometer
equipped with a cryoprobe with a flow cell insert. The samples are loaded into 96-well
plates placed on a Peltier cooling device on a Gilson robot. It should be noted that the
freezing point of pure D
2
O is 3.82
◦
C.
4.4.3 Selection of Buffer
Most important is that the target protein is soluble, stable and monodisperse in the selected
buffer. This will be checked in the limited protein characterization (see below). Phosphate
buffer is very convenient since there are no
1
H signals, and is often the first choice for
ligand-detected NMR techniques. However, there are several proteins that bind phosphate
(notably phosphatases) for which phosphate buffer is not a good choice. Fortunately, there
is now a large selection of commercially available buffers in deuterated form.
4.4.4 Protein Characterization
It is very important to performat least a limited protein characterization study before starting
the NMR screening. Here it is assumed that the identity and purity of the target protein
produced has been checked by methods such as MALDI-MS and N-terminal sequencing.
Protein stability.
It should be clear before starting the screen that the target protein stays
folded in the selected screening buffer during the measurement time in the spectrometer
and while in the queue waiting to be measured. Probably the simplest way to check the
stability is to observe the protein methyl signals during e.g. 24 h, by repeatedly acquiring
1
H 1D spectra. By the appearance of the methyl signals it will be clear if the protein
precipitates or unfolds during this time. If a protein-detected NMR technique is to be used
in the primary screening and a stable isotope-labeled target protein is available, repeatedly
acquired
1
H-
15
N (or
1
H-
13
C) correlation spectra should be collected instead.
Protein homogeneity.
The target protein should be monodisperse in the selected buffer and
at the protein concentration to be used in the screening. The presence of large, soluble
aggregates may cause the hit rate to be very high due to nonspecific interactions with
aggregates. The oligomeric state of the target protein in a given buffer can be checked
by dynamic light scattering (DLS) or analytical ultracentrifugation. DLS is a very useful
technique for buffer optimization.
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