Chemistry Reference
In-Depth Information
been qualitatively indicated that the quantities of triazole products generated are much
lower than the concentration of the target biomolecule.
[
24, 38
]
In the examples of
in situ
Click chemistry that follow, we will attempt to make more transparent the level of product
formation by scrutinizing the experimental data so far reported.
7.8 Electrospray Ionization Mass Spectrometry (ESI-MS)
The analytical demands of combinatorially generated compound libraries have directed the
attention of the pharmaceutical industry towards mass spectrometry owing to its attributes
of speed, intrinsic sensitivity, specificity, low sample consumption and the capability of
resolving vast numbers of compounds in complex compound mixtures. Mass spectral ana-
lysis requires that the species of interest in solution be transferred to the gas phase as fully
desolvated, charged species, for which the mass spectrometer then determines the mass-to-
charge ratio (
m
/
z
). Using the well-established technique of ESI-MS, charged molecules in
a solution are desolvated by a process that involves the formation of fine droplets generated
by an applied high voltage; this process is usually assisted by pressure or ultrasonic nebuliz-
ation.
[
48
]
The fine droplets successively decrease in size by evaporation of solvent and as the
charge density in the droplet becomes higher, the ejection of yet smaller droplets occurs.
This process continues until a final stage of desolvation by evaporation leaves fully desolv-
ated, charged molecules in the gas phase which are then transferred into the mass analyzer
of the mass spectrometer guided by a series of ion guides and ion optics. The mass ana-
lyzer of the mass spectrometer then measures the
m
/
z
of the ion; this is an information-rich
parameter for the medicinal chemist, as will become evident in the following text.
7.8.1 ESI-MS and Proteins
ESI-MS is generally used to accomplish the transfer of intact biological macromolecules
such as proteins from solution into the gas phase. Many commonly used mass analyzers
such as quadrupoles and ion traps have optimal performance in the
m
/
z
range up to 3000
with a maximum typically of 4000-10 000. Therefore, in order to bring the
m
/
z
of a protein
with a mass of 30 kDa into the optimal
m
/
z
range of a typical mass spectrometer, 10-
30 charges must be incorporated on the protein in the gas phase. When using ESI-MS
for molecular weight determination, samples are typically dissolved in a denaturing 1:1
water-organic solvent (acetonitrile or methanol) buffer system containing a few percent of
acetic or formic acid from which a very stable electrospray can be produced. Under these
conditions, the protein is unfolded, exposing many of the amino acid side-chains to the
bulk solvent (Figure 7.6a). The trace acid ensures that many basic residues on the protein
are protonated, thus maximizing the signal obtained from the protein when running the
mass spectrometer in the positive ion mode. The mass spectrum for denatured carbonic
anhydrase is shown in Figure 7.6b. The spectrum is characterized by a broad envelope
of charge states from
20, corresponding to the family of differently protonated
molecular species present in solution.
If the protein molecular weight is unknown, then the charge states are also not known.
The process of determining the molecular weight for an unknown protein from such a
mass spectrum is known as deconvolution and is based on simple algebra. If
MW
is the
+
35 to
+
