K. Sakurai et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Fig. 1. Photoaffinity labeling scheme to capture and enrich binding proteins using small molecule-conjugated photoaffinity nanoprobes.
6
nanoprobe 4 was prepared with a mixed presentation of the hCAII li-
gand (1, SBz) and diazirine group (2a, DA) at a ratio of 2:1
(Fig. 1), photoaffinity nanoprobes were incubated with proteins to bind at
4 °C for 1 h then were subjected to photoirradiation condition
(λ = 365 nm) at 0 °C for varied duration of time. After the photoaffinity
labeling step, the unreacted proteins were removed by centrifugation and
by repeated washing with 3 M guanidinium hydrochloride (Gdn-HCl)
buffer, which is a strong protein denaturing agent. Only the photo-
crosslinked proteins having covalent bonds could be enriched on AuNPs,
which were then cleaved by 2-mercaptoethanol at the S-Au bond to be
eluted from AuNPs. The eluted proteins were analyzed by SDS-PAGE and
fluorescence imaging. Among the three diazirine nanoprobes 4, 8–9, na-
noprobe 8 with a 1:1:1 ratio of SBz, DA and a spacer group displayed the
highest crosslinking efficiency (Fig. 2a, lane 5, Fig. S3, lane 7). These data
showed that the spacer density affected the outcomes of photoaffinity
labeling. While a judicious density of the spacer group is necessary to
provide water dispersive AuNPs, sufficiently high local concentration of
SBz and the photoreactive group on AuNPs is apparently important to
promote an efficient photocrosslinking reaction.
To compare the photocrosslinking efficiency and selectivity of dif-
ferent photoreactive groups (DA, AA, BP), we next employed probes 8,
10, 11 for photoaffinity labeling reactions with hCAII in HeLa cell ly-
sate. The ligand-dependent reactivity of nanoprobes was assessed by
performing a photoaffinity labeling reaction in the presence of excess
concentration of sulfamoylbenzoic acid as a competitor (10 μM), which
is a known hCAII inhibitor. All three nanoprobes crosslinked hCAII with
similar efficiency (Fig. 2b, lane 3, 6, 9), although significant levels of
nonspecific bands were observed under all conditions with diazirine
probe 8 showing the highest level of background reactivity (Fig. S4,
lane 9). The results from the competitive photoaffinity labeling condi-
tions showed different outcomes for the three photoreactive groups in
terms of selectivity of the photoaffinity labeling reaction. As verified by
the absence of the hCAII band under the competitive photoaffinity la-
beling condition in the presence of a competitor, arylazide (10) and
benzophenone nanoprobes (11) enabled ligand-dependent crosslinking
(Fig. 2b, lane 6, 7 and 3, 4). On the other hand, the hCAII bands were
detected by the diazirine nanoprobe 8 under both reaction conditions
with or without a competitor. It suggested that photocrosslinking of
hCAII by diazirine nanoprobe 8 was not solely dependent on ligand
binding and therefore nonselective (Fig. 2b, lane 9, and 10). These re-
sults are in contrast to our previous findings that when the reactivity of
the three major photoreactive groups (DA, AA, BP) were compared in
the context of conventional photoaffinity probes, diazirine group was
(
Scheme 1b). As a reference, nanoprobe 5 was also prepared, which
only presents the hCAII ligand (1). Initial attempts to functionalize
6
,11
AuNPs by the ligand exchange method in aqueous buffer
proved
problematic because of severe flocculation resulting in precipitation. As
it was likely due to poor miscibility between the hydrophobic ligands
and anionic AuNPs, citrate-coated AuNPs were transferred from the
citrate solution to DMF by centrifugation to remove the aqueous su-
pernatant solution and the ligand exchange reaction was performed in
DMF. The PEG-lipoic acid based building block presenting SBz (1) and a
photoreactive group 2a mixed at a desired ratio were assembled on
AuNPs by treating with citrate-stabilized AuNPs to give nanoprobe 4–5.
It was found that the functionalized AuNPs tend to stick to micro-
centrifuge tubes when they are transferred to an aqueous buffer solu-
tion. We thought this was due to the hydrophobic surface formed by
densely functionalized hydrophobic ligands on AuNPs. We therefore
decided to incorporate a hydrophilic spacer bearing a hydroxyl-termi-
nated PEG linker (3) into the new design of nanoprobes to confer col-
1
2
loidal stability (Scheme 1b). Control nanoprobes presenting the hCAII
ligand (SBz, 1) and a spacer group 3 were initially prepared with two
different molar ratios (6, SBz: spacer = 1:1 or 7, SBz: spacer = 1:2) to
evaluate the effect of spacer density to their dispersibility in water. Both
nanoprobes were found to be water dispersible as judged by UV–VIS
analysis and no precipitates were observed. We then prepared photo-
affinity nanoprobes with different composition of SBz, a photoreactive
group and a spacer (8–11, Scheme 2). Similar to probe 6–7, nanoprobes
8
–11 showed no apparent precipitation or color changes, showing the
desired water dispersibility conferred by the hydrophilic PEG spacer.
The functionalization of AuNPs 4–11 was characterized by UV–VIS,
6
,13
SDS-PAGE and MALDI-TOF MS analysis (Figs. S1 and S2).
A slight
red-shift to 527 nm were observed for the absorbance maxima in the
UV–VIS spectra of nanoprobes, indicating an increase in the particle
1
3
sizes by functionalization. SDS-PAGE analysis showed that nanop-
robes were on average uniformly functionalized as compared to un-
functionalized AuNPs, which do not electrophorese readily under the
present experimental condition (Fig. S1). MALDI-TOF MS analysis de-
monstrated the desired ligands were attached on the AuNP scaffold.
We next evaluated the photocrosslinking efficiency toward hCAII by
different design of nanoprobes 4–5, 8–9. Based on our previously estab-
lished one-pot method for photoaffinity labeling reactions and enrichment
2
Sakurai, Kaori
