Biomedical Engineering Reference
In-Depth Information
peptidylphosphonate diphenyl esters derived from substitution of fluoride
with the phenoxy group.
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Another difference between diphenyl esters and
fluoridates is their differential stability, with the latter being unstable after
prolonged incubation in aqueous solutions.
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Two of the most important
examples of phosphonates include the FP-biotin (9.9)
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and FP-fluorescein
(9.10)
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used as activity-based probes in chemical proteomics to tag serine
proteases. In the case of 9.9, because of the long spacer between the elec-
trophilic phosphorus atom and the biotin group, biotin that extends out of
the active site is readily available to bind to streptavidin that can be exploited
for detection. A type of phosphonate diphenyl ester was described and its
ability to detect active KLK6 was demonstrated in an immunofluorimetric
assay for KLK6 used as a prototype assay for quantification of active KLK6
in biological samples.
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O
O
P
F
N
H
O
S
H
N
HN
O
NH
9.9
O
O
H
N
H
N
N
P
O
O
F
F
O
O
O
O
O
O
S
OH
O
9.10
O
O
HO
A modern approach exploits click chemistry to probe protease activity
(Scheme 9.1). In this case, a fluorophosphonate alkyne (FP-alkyne, com-
pound 9.11) was used to probe KLK7 activity in vitro. The conjugate was
allowed to react with an azide derivative of fluorescein (9.12) in the presence
of copper ions, and the activity was reported by fluorescence.
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Addition of
tris(triazolyl)amine is necessary in order to stabilize the Cu(I) formed by
the reaction of Cu(II) and the reducing agent TCEP [tris(carboxyethyl)
phosphine]. Cu(I) acts as a catalyst for this reaction.
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Although this is a
two-step procedure comparedtoreactionwith9.10, the overall labeling
eciency is significantly higher. Further, the advantage of 9.11 is that a lack
of the large hydrophobic fluorescein moiety significantly reduces background
caused by unspecific hydrophobic protein binding. In the future, more
