2 Materials
2.1 NMR
Spectrometer
and Accessories
Although the exact materials required for FBS by NMR will vary
dramatically depending on the target, certain features will remain
constant. The most central of these is, obviously, the NMR spectrometer. Any modern NMR spectrometer should be capable of
running the experiments described in Subheading 1, although the
exact implementation will vary between vendors and local installations. Ideally, the NMR spectrometer should be of 500 MHz or
higher field strength, with a cryogenic probe to enhance sensitivity
and shorten the time for each experiment and automated sample
handling. If
19
F-NMR is to be used, a probe capable of
19
F acquisition with
1
H decoupling is strongly recommended.
A hand centrifuge is strongly recommended in order to allow
spinning of the NMR sample tubes. These are widely available from
laboratory equipment suppliers. Numerical labeling of the centrifuge rotor buckets is advised in order to allow ready identification
of samples after spinning.
2.2 Fragment Library
The second key requirement is a suitable fragment library. A number of publications describe the generation of such libraries [34–
36]; additionally, many chemical vendors sell preselected fragment
libraries. Some libraries are also available from academic groups or
institutions. A recent survey of commercial fragment libraries found
that a relatively small number of vendors were commonly used to
obtain fragment libraries [38]. Regardless of the source of the
library, it is important to consider the suitability of the fragment
library for the NMR screening technique to be used.
For LO-NMR, buffer components may overlap extensively
with resonances (particularly aliphatic resonances) from the fragments, confounding analysis of the LO-NMR experiments. If the
protein is stable in a phosphate buffer, this can be used to reduce
buffer overlap issues; alternatively, deuterated buffers can be used
to good effect. If possible, it is advantageous to have one or more
aromatic protons on the fragments since these are rarely overlapped
with buffer resonances. Compounds with no observable protons
(typically no C–H bonds) should be excluded from a fragment
library intended for use with
1 H LO-NMR. Although probably
unavoidable, it is important to be aware that compounds with
aliphatic methyl groups may cause issues with artifactual direct
irradiation in the STD experiment, while compounds with
exchangeable protons will give rise to artifactual positive signals in
the water-LOGSY experiment. All compounds should also be soluble in water to at least 250–500 μM.
PO-NMR is less sensitive to the chemical nature of the fragment than is LO-NMR. However, the solubility requirement is
rather more stringent since it is advisable to titrate any putative
Fragment Screening by NMR
253
2.1 NMR
Spectrometer
and Accessories
Although the exact materials required for FBS by NMR will vary
dramatically depending on the target, certain features will remain
constant. The most central of these is, obviously, the NMR spectrometer. Any modern NMR spectrometer should be capable of
running the experiments described in Subheading 1, although the
exact implementation will vary between vendors and local installations. Ideally, the NMR spectrometer should be of 500 MHz or
higher field strength, with a cryogenic probe to enhance sensitivity
and shorten the time for each experiment and automated sample
handling. If
19
F-NMR is to be used, a probe capable of
19
F acquisition with
1
H decoupling is strongly recommended.
A hand centrifuge is strongly recommended in order to allow
spinning of the NMR sample tubes. These are widely available from
laboratory equipment suppliers. Numerical labeling of the centrifuge rotor buckets is advised in order to allow ready identification
of samples after spinning.
2.2 Fragment Library
The second key requirement is a suitable fragment library. A number of publications describe the generation of such libraries [34–
36]; additionally, many chemical vendors sell preselected fragment
libraries. Some libraries are also available from academic groups or
institutions. A recent survey of commercial fragment libraries found
that a relatively small number of vendors were commonly used to
obtain fragment libraries [38]. Regardless of the source of the
library, it is important to consider the suitability of the fragment
library for the NMR screening technique to be used.
For LO-NMR, buffer components may overlap extensively
with resonances (particularly aliphatic resonances) from the fragments, confounding analysis of the LO-NMR experiments. If the
protein is stable in a phosphate buffer, this can be used to reduce
buffer overlap issues; alternatively, deuterated buffers can be used
to good effect. If possible, it is advantageous to have one or more
aromatic protons on the fragments since these are rarely overlapped
with buffer resonances. Compounds with no observable protons
(typically no C–H bonds) should be excluded from a fragment
library intended for use with
1 H LO-NMR. Although probably
unavoidable, it is important to be aware that compounds with
aliphatic methyl groups may cause issues with artifactual direct
irradiation in the STD experiment, while compounds with
exchangeable protons will give rise to artifactual positive signals in
the water-LOGSY experiment. All compounds should also be soluble in water to at least 250–500 μM.
PO-NMR is less sensitive to the chemical nature of the fragment than is LO-NMR. However, the solubility requirement is
rather more stringent since it is advisable to titrate any putative
Fragment Screening by NMR
253