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ACS Medicinal Chemistry Letters
putatively bind the cell surface receptor TrkC.14-19 Thus conjugates
1
2
7 and 8 were formed to test this hypothesis via photoaffinity label-
ing.
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7
8
9
Figure 3a shows in-gel fluorescence after a photoaffinity labeling,
click, and SDS-PAGE sequence for solubilized TrkC receptor and
the conjugate 7. A band at around 100 kDa was observed (lanes 1
and 3) but not when 80x of the blocking ligand 8 was added (lanes
2 and 4), or if the illumination step was excluded (lane 5). Figure
3b shows a Western blot after illumination of 7 with NIH3T3 cells
stably transfected with TrkC, “click” with biotin azide, capture onto
NeutrAvidin agarose beads, then SDS-PAGE. Two TrkC-Ab
bands were observed (whereas the parent NIH3T3 cells give none,
see supporting); these are at similar, but not the same, molecular
mass as the solubilized extracellular domain of the TrkC receptor in
Figure 3a.
Other PAL ligands functionalized with alkynes have been reported
prior to our work,20,21 but the synthesis of cassette 1 appears to be
more convenient than most, and provides another type of system.
This is because, unlike some other probes, synthesis of cassette 1
does not require reactive organometallics, except in the first step
wherein the starting material was made on scale. Cassette 1 has a
secondary amine that can be easily coupled to activated carboxylic
acids; this feature makes the probe more portable. Both the small-
molecule conjugates prepared in this work labeled their anticipated
targets with sufficient efficiencies for observation in SDS-PAGE
experiments.
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a
ASSOCIATED CONTENT
Supporting Information. The Supporting Information is available
free of charge on the ACS Publications website at DOI: XXX.
synthetic procedures for compound 1-8, procedures for photoaffinity
labeling, full gel images
b
AUTHOR INFORMATION
Corresponding Author
* FAX: +1 979 845 8839. E-mail: burgess@tamu.edu.
ACKNOWLEDGMENT
We thank DoD BCRP Breakthrough Award (BC141561), CPRIT
(RP150559 and RP170144), The Robert A. Welch Foundation (A-
1121). The NMR instrumentation at Texas A&M University was
supported by a grant from the National Science Foundation (DBI-
9970232) and the Texas A&M University System.
Figure 3. a Experiments with solubilized TrkC extracellular domain.
Solubilized TrkC extracellular domain was incubated with 7 for 1 h,
illuminated for 30 min at 365 nm, clicked with azide-fluor-488,
then subjected to SDS-PAGE. Top gel: fluorescence at 488 nm.
Bottom gel: protein staining with CBB G250. Competition with 8,
which lacks a diazirine, suppresses labeling with 7 (lanes 2 and 4).
b Experiments with NIH3T3 TrkC+ cell lysates. Cell lysate was
treated with PAL probe 7 under 365 nm illumination for 30 min,
then clicked with biotin azide. NeutrAvidin agarose was used to
pull down the biotinylated proteins, and the material captured was
run on a SDS-PAGE gel. Lane 1 shows staining of TrkC on the
Western blot image with anti-TrkC mAb, lane 2 shows sample
pretreated with blocking ligand 8.
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