Development of tag-free photoprobes for studies aimed at identifying the target of novel Group A Streptococcus antivirulence agents

Article abstract of DOI:10.1016/j.bmcl.2014.01.079

We previously reported the identification and development of novel inhibitors of streptokinase (SK) expression by Group A Streptococcus (GAS), originating from a high throughput cell-based phenotypic screen. Although phenotypic screening is well-suited to

Full text of DOI:10.1016/j.bmcl.2014.01.079

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1538–1544
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of tag-free photoprobes for studies aimed
at identifying the target of novel Group A Streptococcus antivirulence
agents
Bryan D. Yestrepsky a, Colin A. Kretz b, Yuanxi Xu c, Autumn Holmes b, Hongmin Sun c, David Ginsburg b,
Scott D. Larsen a,
⇑
a Vahlteich Medicinal Chemistry Core, Department of Medicinal Chemistry, University of Michigan, 428 Church St., Ann Arbor, MI 48109, United States
b Department of Human Genetics, Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109, United States
c Department of Internal Medicine, School of Medicine, University of Missouri—Columbia, 1 Hospital Dr., DC043.00, Columbia, MO 65212, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously reported the identification and development of novel inhibitors of streptokinase (SK)
expression by Group A Streptococcus (GAS), originating from a high throughput cell-based phenotypic
screen. Although phenotypic screening is well-suited to identifying compounds that exert desired biolog-
ical effects in potentially novel ways, it requires follow-up experiments to determine the macromolecular
target(s) of active compounds. We therefore designed and synthesized several classes of chemical probes
for target identification studies, guided by previously established structure–activity relationships. The
probes were designed to first irreversibly photolabel target proteins in the intact bacteria, followed by
cell lysis and click ligation with fluorescent tags to allow for visualization on SDS–PAGE gels. This step-
wise, ‘tag-free’ approach allows for a significant reduction in molecular weight and polar surface area
compared to full-length fluorescent or biotinylated probes, potentially enhancing membrane permeabil-
ity and the maintenance of activity. Of the seven probes produced, the three most biologically active were
employed in preliminary target identification trials. Despite the potent activity of these probes, specific
labeling events were not conclusively observed due to a considerable degree of nonspecific protein bind-
ing. Nevertheless, the successful synthesis of potent biologically active probe molecules will serve as a
starting point for initiating more sensitive methods of probe-based target identification.
Received 22 January 2014
Accepted 27 January 2014
Available online 7 February 2014
Keywords:
Group A Streptococcus
Virulence inhibitor
Streptokinase
Antibiotic
Phenotypic screening
Target identification
Tag-free photoprobes
Photo-crosslinking
Click chemistry
Ó 2014 Elsevier Ltd. All rights reserved.
The growing threat of bacterial resistance to antibiotic treat-
ment necessitates the development of new antibacterial agents
with novel mechanisms of action.1 Bacterial pathogens express
genes known as virulence factors that do not contribute to the
growth and maintenance of the cell, but are critical for infection
in the host. As a result, virulence-attenuating therapeutics have
emerged as a potential mechanism for managing bacterial infec-
tion without selecting for mutants that are resistant to treatment.2
Our group has applied this paradigm toward the development of a
new class of anti-virulence antibiotics that suppress the expression
of streptokinase (SK), a bacterial activator of human plasmin, that
plays a direct role in enhancing Group A Streptococcus (GAS) viru-
lence.3 The lead compound of this series (1, Scheme 1) was identi-
fied via a cell-based high-throughput screen (HTS) for compounds
that reduce ska gene transcription without inhibiting bacterial
growth.4 Optimization of the lead compound through structure-
activity relationship (SAR) studies5 led to a number of incremental
improvements in activity and lipophilicity (2 and 3), eventually
resulting in the discovery of potent analog 4 with a 35-fold greater
potency and 2-log reduction in c LogP compared to lead 1.
Phenotype-based HTS strategies, like the one used to identify 1,
return hit compounds with physicochemical properties sufficient
for activity in whole cells and do not rely on a priori knowledge of
the affected biological pathway, making them useful for discovering
compounds with novel mechanisms of action.6,7 These advantages,
Abbreviations: SK, streptokinase; GAS, Group
A Streptococcus; HTS, high-
throughput screen; SAR, structure–activity relationship; TPSA, topological polar
surface area; cLogP, logarithm of the calculated octanol–water partition coefficient;
LDA, lithium diisopropylamide; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide
gel electrophoresis; SILAC, stable isotope labeling by amino acids in cell culture;
THY, Todd–Hewitt media with 0.2% yeast extract; ESI-MS, electrospray ionization
mass spectrometry.
⇑
Corresponding author at present address: College of Pharmacy, 428 Church St.,
Ann Arbor, MI 48109-1065, United States. Tel.: +1 734 615 0454.
E-mail address: sdlarsen@umich.edu (S.D. Larsen).
http://dx.doi.org/10.1016/j.bmcl.2014.01.079
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

B. D. Yestrepsky et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1538–1544
1539
regulation of GAS virulence mechanisms. Several proteins
governing GAS virulence have been studied in detail,9 including
Mga,10 Rgg,11,12 and CovR/CovS,13 but the genomic sequencing of
several clinically relevant GAS serotypes has revealed multiple
well-conserved virulence control elements that remain uncharac-
terized.14 Thus, identifying the target of this compound series has
the potential to ascertain novel control mechanisms and further
elucidate the complex nature of GAS virulence.
N
O
S
N
S
N
N
O
1
2
IC50 >50 μM
cLogP = 7.1
IC50 = 19 6 μM
cLogP = 5.5
The use of chemical probes is a proven strategy for successfully
establishing the protein targets of small molecules.15–18 We chose
to pursue
a tandem photolabeling-bioorthogonal conjugation
strategy that has become widespread since the development of
click chemistry.19–21 In this approach, a small-molecule analog of
a potent compound possessing a photoreactive group and a termi-
nal alkyne is covalently crosslinked to target proteins in the intact
cellular milieu with UV light. After cell lysis, an azide-modified
fluorescent or biotin-derived moiety can then be appended to the
alkyne-functionalized protein(s) via copper(I)-mediated click
chemistry, resulting in target proteins with covalently attached
tags for visualization or selective purification. The lower molecular
weight and topological polar surface area (TPSA) of these ‘tag-
free’20 compounds compared to traditional biotinylated probes in-
creases their likelihood of being cell-permeable, allowing them to
be used in whole-cell systems rather than lysates. Cell-permeable
affinity probes are advantageous in that their biological activity
can be confirmed in phenotypic assays before beginning target
identification studies. The probes also have access to all proteins
in their native cellular conformations.
We envisioned the design of several tag-free photoprobes based
on structural insights gained from our SAR studies on this scaffold.5
Although the maintenance of a high level of potency was a primary
concern, the nature and positioning of the UV-active and terminal
alkyne groups were equally important to us as they are crucial for
ensuring a compatible orientation for labeling.22,23 A number of
different photolabile groups have been successfully employed in
the literature, varying in their stability, reactivity, and preference
for carbon–carbon or carbon–heteroatom bond formation.24 The
unknown nature of the binding site ultimately determines the
structural features necessary for a functional probe, so we set out
O
N
S
N
O
S
O
N
N
O
4
3
IC50 = 1.3 0.6 μM
cLogP = 5.1
IC50 = 3.2 0.6 μM
cLogP = 4.3
Scheme 1. Selected compounds from the SAR effort leading to potent analogs of
screening hit 1.
taken together with the lack of antibiotic leads discovered via bac-
terial target-based screening,8 suggest phenotypic screening may
be a potentially more fruitful tool for identifying novel antibacterial
agents. However, phenotypic screening does not explicitly identify
the molecular target of individual hit compounds, and therefore
they must be established through subsequent studies. In the context
of our project, identifying the target of this compound series would
be important for (a) helping to establish a biochemical assay with
which to improve the potency and specificity of the series, and (b)
elucidating potentially novel virulence control pathways.
The potent activity of our compounds against SK expression,
combined with RNA microarray data indicating the down-
regulation of other important GAS virulence factors,4 suggests that
their macromolecular target(s) are involved in the upstream
O
R2
Br
H
a
NH2
N
S
d
CN
N
CO2Et
O
5
2
6:
7:
8:
R
R
R
= Br
b
c
2 = SnBu3
2 = COPh
9
O
R2O
TsO
g
O
O
e
N
S
TBS
R1
11:
12:
3
R = H
f
13
3
N
R = Ts
O
1
10a:
R
= propargyl
10b: R1 =
O
Scheme 2. Synthesis of benzophenone-functionalized tag-free probes 10a–b. Reagents and conditions: (a) LDA, 1 h; then ethyl 3,3-dimethyl acrylate, ZnI2, diglyme, À78 °C to
rt, 2 h, 50%; (b) (Bu3Sn)2, Pd(PPh3)4, toluene, reflux, 16 h, 37%; (c) 1 atm CO, PdCl2(PPh3)2, iodobenzene, DMF, 60 °C, 4 h, 58%; (d) allyl isothiocyanate, AcOH, EtOH, 70 °C, 16 h,
27%; (e) propargyl bromide or 12, Cs2CO3, 2-butanone, 70 °C, 16 h, 69–70%; (f) Ts–Cl, pyridine, 0–4 °C, 24 h, 72%; (g) nBuLi, À78 °C, THF, 2 h, then TBS-OTf, À78 °C to rt, 1 h,
63%.

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B. D. Yestrepsky et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1538–1544
to synthesize a diverse series of compounds with one of three
photolabile groups (benzophenone, diazirine, aryl azide) at differ-
ent points on the scaffold to maximize the probability of identify-
ing the target.
I
N3
NH2
a,b
d,e
c
NH2
NH2
CN
The chemistry to install benzophenone, diazirine, and aryl azide
functionality onto the core scaffold of this class of compounds
required three distinct synthetic routes. Synthesis of the
benzophenone-based probes (10a–b, Scheme 2) began with the
LDA-promoted Michael addition of 4-bromo-2-methylbenzonitrile
(5) to ethyl 3,3-dimethyl acrylate and subsequent ZnI2-mediated
cyclization to afford b-aminoester 6.25 Stannylation via halogen–
metal exchange26 followed by carbonylative Stille coupling27
successfully delivered benzophenone 8. Acid-catalyzed addition
of the amine to allyl isothiocyanate and subsequent cyclization
generated 2-thioxopyrimidinone 9 that underwent S-alkylation
with propargyl bromide or 12 (generated via tosylation of
commercially available propynol ethoxylate 11) under mildly basic
conditions to generate probes 10a and 10b.
Diazirine probes 19a–d were synthesized via an analogous
route beginning from aryl methyl ether 14 (Scheme 3). Demethyl-
ation and protection of the resulting phenol as a silyl ether resulted
in 15, which could be employed in the synthesis of b-aminoester
16 in a manner similar to the one used to generate 6 in Scheme 3.
In the course of our investigation, we found that cyclization with
allyl isothiocyanate in the presence of cesium carbonate installed
the desired 2-thioxopyrimidinone ring system while simulta-
neously cleaving the silyl ether protecting group, affording com-
mon intermediate 17. Successive chemoselective alkylations at
sulfur and oxygen were exploited to install the diazirine and
alkyne linker moieties in four different arrangements, yielding
probes 19a–d. Diazirine-containing alkylating agent 21 was syn-
thesized from 4-hydroxy-2-butanone (20) via diaziridination with
7N methanolic ammonia and hydroxylamine O-sulfonic acid,28
followed by oxidation and tosylation.29
CO2Et
CO2Et
22
24
23
N3
N3
H
f,g
N
S
N
O
S
O
R
NH
N
O
26: R = TBS
27: R = H
25
h
Scheme 4. Synthesis of aryl azide-functionalized tag-free probe 27. Reagents and
conditions: (a) NaNO2, HCl, then KI, H2O, 0 °C; (b) LDA, then ethyl 3,3-dimethyl
acrylate, ZnI2, diglyme, À78 °C to rt, 3 h, 16% over 2 steps; (c) NaN3, CuI, N,N-
dimethyl ethylene diamine, Na-ascorbate, DMSO/H2O (5:1), rt, 3 h, 85%; (d) benzoyl
isothiocyanate, EtOH, 70 °C, 3 h; (e) KOH, EtOH/H2O, 2:1, 70 °C, 3 h, 54% over 2
steps; (f) 13 (Scheme 2), NaHCO3, 70 °C, DMF, 16 h; (g) NaOMe, allyl bromide, EtOH,
70 °C, 3 h, 42% over 2 steps; (h) TBAF, THF, 0 °C, 1 h, 65%.
conditions to transform the aniline to the aryl iodide30 permitted
the formation of b-aminoester 23 in sufficient yields. Conversion
to the corresponding aryl azide 24 via Cu(I)-mediated SNAr pro-
ceeded in excellent yields.31 Stepwise generation of the unsubsti-
tuted 2-thioxopyrimidinone with benzoyl isothiocyanate and
potassium hydroxide afforded 25,32 which was then selectively
S-alkylated with TBS-protected alkyne-containing unit 13. Alkyne
protection was necessary to minimize cross-reactivity during the
subsequent N-allylation under basic conditions to furnish 26.
Finally, fluoride ion-promoted removal of the silyl group yielded
the desired aryl azide probe 27.
Synthesis of the aryl azide probe 27 began from commercially
available 2-cyano-3-methylaniline 22 (Scheme 4). Masking the
aniline functionality of 22 as a mono- or bis-Boc carbamate, diphe-
nyl imine, or azide proved incompatible with the LDA/ZnI2 cycliza-
tion conditions. Finally, it was found that modified Sandmeyer
All completed tag-free affinity probes were assessed for their
inhibitory effects on streptokinase expression using our previously
described assay.4 Briefly, GAS culture supernatant was mixed with
HO
TBSO
O
TBSO
H
N
S
d
a,b
c
NH2
CN
CN
N
CO2Et
O
17
14
15
16
O
HO
O
R2
OH
N
S
N
O
S
R1
R1
e
f
20
N
N
g,h
O
1
19a-d (See Table 1)
18a:
R = propargyl
N
N
OTs
18b: R1
=
O
21
18c: R1 =
N
N
Scheme 3. Synthesis of diazirine-functionalized tag-free probes 19a–d. Reagents and conditions: (a) BBr3, DCM, 0 °C–rt, 16 h; (b) TBS-Cl, imidazole, DCM, 0 °C–rt, 16 h, 96%
over 2 steps; (c) LDA, then ethyl 3,3-dimethyl acrylate, ZnI2, diglyme, À78 °C to rt, 3 h, 63%; (d) allyl isothiocyanate, Cs2CO3, EtOH, 70 °C, 16 h, 57%; (e) 12, 21, or propargyl
bromide, NaHCO3, 70 °C, DMF, 3–6 h, 45–71%; (f) 12, 21, or propargyl bromide, Cs2CO3, 70 °C, DMF, 3 h, 16–64%; (g) 7 N NH3:MeOH, hydroxylamine-O-sulfonic acid, 0 °C–rt,
16 h; then I2, 0 °C, 30 min; (h) Ts–Cl, pyridine, 0–4 °C, 24 h, 15% over 3 steps.

B. D. Yestrepsky et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1538–1544
1541
Table 1
Activity data for tag-free photoprobes 10, 19, and 27
R2
9
8
N
O
S
R1
N
c
Compd
R1
R2
T/C
(5
Growth T/C
(50
M)b
IC50
nd
(lM)
l
M)a
l
10a
10b
Propargyl
8-Bz
8-Bz
0.77 0.34
0.51 0.07
1.09 0.04
0.94 0.18
3.8 0.6
19a
19b
Propargyl
1.07 0.16
0.49 0.34
0.96 0.09
1.07 0.02
nd
0.33 0.70
nd
19c
19d
0.71 0.40
0.89 0.16
1.01 0.09
1.02 0.01
8-O-Propargyl
nd
27
4
9-N3
0.30 0.08
0.10 0.06
0.98 0.04
0.73 0.07
0.19 0.26
1.3 0.6
Et
9-OMe
a
Amount of SK activity in GAS culture measured via colorimetric assay (A405) after incubation with 5
lM test compound for 24 h divided by SK activity measured in GAS
culture 24 h after treatment with DMSO control, +/À standard deviation.
b
Growth inhibition as measured by OD600 of GAS culture 24 h after treatment with 50
lM test compound divided by OD600 of control GAS culture treated with DMSO
control after 24 h, +/À standard deviation.
c
Concentration at which 50% maximal inhibitory effect against SK expression was achieved +/À standard deviation. Each data point is derived from the statistical
treatment of at least 3 replicates. nd = not determined.
O
O
λν, 365 nm
N
N
N
S
N
O
S
O
O
30 min, RT
MeOH
N
N
O
19b
O-H insertion
1,2 hydride shift,
olefination
O
O
R
N
O
S
O
N
S
O
O
N
N
O
28
29a,
R =
29b, R =
Scheme 5. UV-induced reactivity of probe compound 19b in methanol.
human plasma, and the resulting plasmin activity was assayed
by turnover of plasmin-specific chromogenic substrate by
monitoring absorbance at 405 nm. Activity data are reported in
Table 1 as the ratio of plasmin activity in test cultures divided by
plasmin activity in a control culture treated with DMSO alone.
Since retaining high potency is important for target identification,
cutoff value for further evaluating probe compounds. Compounds
meeting this threshold were more completely characterized with
full IC50 determinations. Results are summarized in Table 1.
Benzophenone 10b, with its S-propargyloxyethyl click ligation
sidechain, was found to be more potent than the corresponding
S-propargyl analog 10a. This trend was also evident in diazirine
probes 19a and 19b. Disappointingly, reversing the positioning of
a
50% streptokinase expression inhibition at 5 lM was used as the

1542
B. D. Yestrepsky et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1538–1544
solution of 19b to 365 nm UV light led to conversion of the parent
compound into new isolable compounds within 30 min
2
(Scheme 5). NMR and ESI-MS analysis of the purified products
identified the major product as 28, consistent with carbene inser-
tion into the OAH bond of methanol (43% yield). The most promi-
nent minor product consisted of alkene regioisomers that resulted
from the two possible 1,2 hydride shifts (29, 27% combined yield)
common for carbenes adjacent to saturated carbons.33 A previously
reported study involving methanol capture by aliphatic diazirines
observed analogous insertion and rearrangement products in sim-
ilar ratios.34 Importantly, this study established that the remainder
of the template is stable to degradation by UV irradiation.
Having confirmed the photostability of our probe scaffold, we
began photo-crosslinking35 and fluorescent tagging assays. Briefly,
GAS cultures were incubated in the presence of probe compounds
for 30 min, then exposed to long-wave UV radiation for 5 (19b, 27)
or 10 (10b) min to promote crosslinking. The cells were lysed and
pelleted, and the resulting lysates were subjected to click chemis-
try conditions with Alexa Fluor 488 azide. Proteins were then pre-
cipitated via methanol/chloroform/water solution, separated via
SDS–PAGE gel, and fluorescently visualized. Total protein levels
were subsequently visualized by staining with Sypro Ruby (see
Supplemental data for more detailed protocol).
Benzophenone probe 10b appeared to crosslink to a distinct
subpopulation of proteins when separated on a polyacrylamide
gel and fluorescently imaged (Fig. 1). Tagging was found to be
dose-dependent, evidenced by lower fluorescent signal in protein
bands from samples tagged with lower probe concentrations. An
identically treated control omitting the UV crosslinking step (Lane
6) did not fluorescently tag any proteins. Staining the gel to visual-
ize total protein content with Sypro Ruby stain confirmed an abun-
dance of protein in the sample, indicating that only a small subset
of proteins were crosslinked to 10b and tagged with Alexa Fluor
488. Although the intensity of several protein bands appears to
have been diminished by the introduction of soluble competitor
3, these findings could not be reliably reproduced in biotin pull-
down experiments (data not shown). The presence of several other
protein bands on the gel that did not lose intensity in the presence
of 3 implies some degree of nonspecific protein binding by 10b.
Crosslinking experiments undertaken with diazirine 19b and
azide 27 (Figs. 2 and 3) showed similar trends to 10b. In each case
Figure 1. Crosslinking trials using probe 10b. GAS cultures were grown in the
presence of 10b with or without competitor 3, then subjected to UV crosslinking
conditions. The cells were lysed and a fluorescent Alexa Fluor 488 moiety was
appended to probe-protein adducts via click chemistry, then proteins were isolated
and separated via SDS–PAGE. (A) Fluorescent visualization of proteins from
crosslinking and competition experiments with 10b on PAGE gel. (B) Total protein
stain of gel with Sypro Ruby stain. Lane 1: 100
10b + 500 M 3; Lane 3: 100 M 10b + 1 mM 3; Lane 4: 10
M 10b; Lane 6: 100 M 10b, no UV crosslinking.
l
M
10b; Lane 2: 100
lM
l
l
lM 10b; Lane 5:
1
l
l
the alkyne and diazirine in probes 19c and 19d resulted in com-
plete loss of activity. Azide probe 27, on the other hand, possessed
potency slightly greater than lead compound 4, the most potent
compound derived from our prior SAR studies. Fortuitously, we
were able to identify a sufficiently active probe containing each
of the selected photolabile groups (10b, 19b, and 27) for use in tar-
get identification studies.
Representative probe 19b was subjected to crosslinking condi-
tions in methanol to assess whether the core scaffold would exhi-
bit undesired photo-induced reactivity. Exposure of a methanolic
Figure 2. Crosslinking trials using diazirine probe 19b. GAS cultures were grown in the presence of 19b with or without competitor 3, then subjected to UV crosslinking
conditions. The cells were lysed and a fluorescent Alexa Fluor 488 moiety was appended to probe-protein adducts via click chemistry, then proteins were isolated and
separated via SDS–PAGE. (A) Fluorescent visualization of proteins from crosslinking and competition experiments with 19b on PAGE gel. (B) Total protein stain of gel with
Sypro Ruby stain. Lane 1: 100
lM 19b; Lane 2: 10
l
M 19b; Lane 3: 10
lM 19b + 100 lM 3; Lane 4: 1 lM 19b; Lane 5: 1 lM 19b + 100 lM 3; Lane 6: 100 lM 19b, no UV
crosslinking; Lane 7: 10 M 19b, no UV crosslinking; Lane 8: 1
l
lM 19b, no UV crosslinking; Lane 9: no probe, no UV crosslinking.

B. D. Yestrepsky et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1538–1544
1543
(SILAC)36 and GAS genome phage display library screening.38 These
techniques solve the issue of low abundance by vastly expanding
the detection limit for specific interactions and expressing the en-
tire proteome at equal levels, respectively. The development of
more potent probes is also underway. Using probes with greater
potency should enhance labeling efficiency and allow for the use
of lower probe concentrations, reducing the likelihood of wide-
spread nonspecific labeling.
Acknowledgments
This work was supported by a University of Michigan Life Sci-
ences Institute Innovation Partnership grant to S.D.L., and an NIH
Pharmacological Sciences Training Program fellowship (Grant T32
GM007767) to B.D.Y.
Supplementary data
Supplementary data (methods for activity determination, target
identification, and solvent capture studies, as well as detailed syn-
thetic procedures and characterization data for all synthesized
compounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmcl.2014.01.079.
Figure 3. Crosslinking trials using azide probe 27. GAS cultures were grown in the
presence of 27 with or without competitor 3, then subjected to UV crosslinking
conditions. The cells were lysed and a fluorescent Alexa Fluor 488 moiety was
appended to probe-protein adducts via click chemistry, then proteins were isolated
and separated via SDS–PAGE. (A) Fluorescent visualization of proteins from
crosslinking and competition experiments with 27 on PAGE gel. (B) Total protein
References and notes
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stain of gel with Sypro Ruby stain. Lane 1: 100
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l
l
l
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the intensity of fluorescently-tagged proteins was dependent on
both the concentration of the probe compound and the exposure
to UV light. Qualitative comparison of the fluorescent and total
protein stains suggests that the fluorescent intensity was generally
proportional to protein abundance, with a few exceptions. Bands at
approximately 30 K and 60 K (probe 19b, Fig. 2) and 18 K and
110 K (probe 27, Fig. 3), for example, appear to be disproportion-
ately labeled compared to their relative abundance, but the lack
of competition-induced signal reduction suggests these are due
to nonspecific interactions as well.
In summary, we report the synthesis of 7 tag-free photoaffinity
probe compounds designed to determine the target of a novel class
of Group A Streptococcal virulence inhibitors. Three probes, each
with a different photolabile moiety, were found to be sufficiently
active for use in target identification studies. After confirming ade-
quate photoactivation of representative probe 19b without
destruction of the overall template, we performed preliminary tar-
get identification studies. Crosslinking in whole cells, followed by
cell lysis and covalent attachment of a fluorescent tag, led to rela-
tively widespread fluorescent labeling that appears consistent with
nonspecific labeling of a subset of total GAS proteins.
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