Design, synthesis, and biological evaluation of BODIPY-erythromycin probes for bacterial ribosomes

Article abstract of DOI:10.1016/j.bmcl.2005.11.028

BODIPY-erythromycin probes of bacterial ribosomes were designed and synthesized by attaching a BODIPY fluorophore to the 4″- and 9-positions of the erythromycin structure. The probes exhibited excellent binding affinity to bacterial

Full text of DOI:10.1016/j.bmcl.2005.11.028

Bioorganic & Medicinal Chemistry Letters 16 (2006) 794–797
Design, synthesis, and biological evaluation of
BODIPYÒ–erythromycin probes for bacterial ribosomes
Jing Li,a, In Ho Kim,a Eric D. Roche,b Doug Beeman,b A. Simon Lynch,b
Charles Z. Dinga,* and Zhenkun Maa,à
aDepartment of Medicinal Chemistry, Cumbre Inc., 1502 Viceroy Dr., Dallas, TX 75235, USA
bDepartment of Research, Cumbre Inc., 1502 Viceroy Dr., Dallas, TX 75235, USA
Received 18 August 2005; revised 8 November 2005; accepted 8 November 2005
Available online 23 November 2005
Abstract—BODIPYÒ–erythromycin probes of bacterial ribosomes were designed and synthesized by attaching a BODIPYÒ fluoro-
phore to the 400- and 9-positions of the erythromycin structure. The probes exhibited excellent binding affinity to bacterial ribosomes
and competed with erythromycin and other drugs whose binding sites are in the same vicinity of the 50S subunit. The synthetic
fluorescent probe 5 was successfully adapted in our ultra high-throughput screening (uHTS) to identify novel ribosome inhibitors.
Ó 2005 Elsevier Ltd. All rights reserved.
Bacterial resistance to antibiotics has become a serious
public health issue. Novel classes of antibacterial agents
that have distinct chemical structure and no cross-resis-
tance with currently used agents are urgently needed.
Interruption of bacterial ribosome function represents
a proven way to kill bacteria. There are naturally occur-
ring antibiotics and synthetic antibacterial agents that
target bacterial ribosomes. A prominent group of ribo-
some inhibitors is the macrolide family, which comprises
a large number of naturally occurring and semi-synthet-
ic antibiotic agents, such as erythromycin, clarithromy-
cin, and azithromycin.
by a series of contacts between residues of the domain V
region of the 23S ribosomal RNA and the antibiotic
molecule. Macrolides bound at this site effectively block
the peptide exit tunnel, and erythromycin class macro-
lides do so without directly interfering with the catalytic
activity of the peptidyl transferase reaction center. The
erythromycin co-crystal structure indicates that there
are no productive interactions between the ribosome
and the 400-, 6-, 9-, and 11-positions of the macrolide
molecule. This is consistent with the available struc-
ture–activity relationships as all four positions tolerate
the introduction of large substituent groups without
significantly reducing the binding affinity.4
Macrolides bind to the large subunit (50S) of bacterial
ribosomes and prevent the elongation step of protein
synthesis. The structures of 50S ribosomal subunits
co-crystallized with seven separate macrolides have been
reported.1–3 These crystal structures revealed that all
macrolides bind to the entrance of the peptide exit
tunnel located in the peptidyl transferase reaction center
of the 50S subunit. The macrolide binding site is defined
Fluorescence polarization is a powerful technique appli-
cable to the probing of ribosome–ligand interactions. A
fluorescent probe bound to a ribosome when excited
with polarized light of appropriate wavelength emits
light with enhanced polarization relative to that of an
unbound probe. Hence, when a bound probe is compet-
itively or allosterically displaced by a ligand the polar-
ized light signal diminishes. The ligand thus identified
is likely to interact with the ribosome in a similar
fashion at or near the same binding site as the probe.
Since the binding site is well validated, the ligand there-
fore serves as an attractive start-point for further opti-
mization. An advantage of this screening methodology
is that the approximate binding site of the identified
novel ribosome ligand is immediately apparent, which
can be used for structure-guided drug design and
Keywords: Antibacterial agent; Ribosome inhibitor; Macrolides;
Erythromycin; Fluorescent polarization probes; BODIPY; Synthesis.
*
Corresponding author. Tel.: +1214 631 4700x7670; fax: +1 214 631
4710; e-mail: charles.ding@cumbre.net
Major contributor to this research.
à
Present address: Zhenkun Ma, Global Alliance for TB drug
development, 80 Broad Street, 3rd Floor, New York, NY 10004,
USA.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.028

J. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 794–797
795
chemical optimization. Furthermore, the simple mixing
of inexpensive reagents required for fluorescence polari-
zation displacement assays means that they are readily
adaptable for ultra high-throughput screening, unlike
alternative methods that may require physical separa-
tion steps and/or expensive reagents.
followed by the deprotection of the acetate group to give
the desired BODIPY-fluorescent probe 3.11
Fluorescent probe 5 was prepared by tethering BODIPY
fluorophore to the 9-position of 9-aminoerythromycin 4
(Scheme 2), which in turn was prepared from erythro-
mycin A by a known process described in the litera-
ture.12 Treatment of 4 with BODIPY-FL propionic
acid in the presence of succinimidyl ester produced the
desired probe 5.
Previous reports used fluorescent probes that covalently
link fluorophores to ribosome inhibitors to probe inhib-
itor–ribosome interactions and to estimate the location
of the inhibitor binding sites.5–9 Some of those probes
were also successfully used in uHTS to identify small
molecules that interact with ribosomes. In an ongoing
ribosome program, we desired to synthesize ribosome
probes suitable for our ultra high-throughput screening
needs. We settled on 4,4-difluoro-5,7-dimethyl-4-bora-
3a,4a-diaza,s-indacene (BODIPY-FL) as our fluoro-
phore for two reasons. First, the relatively small size
of BODIPY-FL should minimize the impact on binding
affinity of the resulting macrolide-BODIPY probes to
the ribosome. Second, BODIPY-FL has excitation and
emission wavelengths (504 nm excitation and 513 nm
emission in methanol, respectively) that are distinct
from most compounds in our screening library. The
distinct spectral properties should facilitate fluorescence
anisotropy-based high-throughput screening. We report
herein the synthesis and biological evaluation of three
fluorescent probes (3, 5, and 7) which have BODIPY
fluorophore tethered to the 400- or 9-position of the
erythromycin structure through different linker groups.
BODIPY probe 7 is a version of probe 3 (Scheme 3)
optimized to be more useful in screening due to an in-
crease in the ribosomal off rate as described below.
The synthesis involved protecting the amine group of
compound 4 with benzyl carbamate and otherwise
followed the similar synthesis described for the prepara-
tion of fluorescent probe 3.
Biological evaluation of all three BODIPY probes 3, 5,
and 7 included determination of their binding affinity
to 70S bacterial ribosomes isolated from Escherichia coli
using a method reported by Turconi et al.8 As shown in
Table 1, binding affinities (Kd) to the ribosome vary
from less than 0.5 nM for probe 3 to 30 nM for probe
5. The excellent binding affinity of probe 3 may be
attributable to the BODIPY-fluorophore attachment
point. The 400-position of erythromycin seems to tolerate
large groups without detrimental effect. This finding was
reinforced by probe 7 where position 9 was modified
while maintaining the large BODIPY fluorophore linked
to the 400-position, yet the affinity of probe 7 was only
slightly affected when compared to probe 3. However,
attaching BODIPY fluorophore to the 9-position led
to probe 5 with weaker (but still potent) binding affinity
for the ribosome. Only probes with appropriate binding
affinities and kinetics are useful in a FP displacement
assay. Higher affinity probes allow greater resolution
of ligand potency and require less assay reagents,13 but
they may also result in slower displacement binding
kinetics or in low signal as probe levels should ideally
not be significantly above the probe Kd.
The preparation of probe 3 commenced with 6-O-methyl
-erythromycin (clarithromycin) 1 (Scheme 1).10 The 20-
hydroxy group was selectively protected by an acetate
group. The 400-hydroxy group was selectively reacted
with 1,10-carbonyldiimidazole (CDI), followed by addi-
tion of ethylenediamine in situ to generate intermediate
2. Compound 2 was then coupled with BODIPY-FL
propionic acid in the presence of succinimidyl ester
O
O
9
9
Inhibition of ribosome function mediated by erythromy-
cin and the synthesized probes 3, 5, and 7 was evaluated
in a coupled transcription and translation assay using
E. coli S30 extracts by measuring inhibition of light
N
O
N
O
OH
O
O
OH
O
O
AcO
O
2'
2'
O
HO
O
OH
OH
a, b
O
O
O
O
O
production based on expression of
a
luciferase
O
4"
4"
OH
OMe
NH2
O
N
2
1
OMe
H
O
9
NH2
N
B
N
O
9
O
NH
9
OH
O
O
HO
O
N
2'
F
OH
N
O
OH
HO
HO
F
c, d
N
2'
OH
OH
N
OH
O
2'
N
HO
B
OH
a
F
F
O
O
O
O
O
O
O
O
O
4"
O
O
O
NH
O
O
O
O
N
4"
OH
OMe
3
OMe
H
4"
OH
OMe
5
4
Scheme 1. Preparation of probe 3. Reagents and conditions: (a)
1.0 equiv Ac2O, 3.0 equiv Et3N, CH2Cl2, rt, 16 h; (b) 1.0 equiv CDI,
THF, 35 °C, 12 h, then added 10 equiv NH2CH2CH2NH2, 45 °C, 1 h;
(c) BODIPY-FL propionic acid, succinimidyl ester, DMF, rt, 1 h, 34%
from compound 1; (d) MeOH, 60 °C, 1 h, 33%.
Scheme 2. Preparation of probe 5. Reagents and conditions: (a)
1.0 equiv BODIPY-FL propionic acid, 1.0 equiv succinimidyl ester,
DMF, rt, 1 h, 62%.

796
J. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 794–797
variety of macrolides and ribosome inhibitors with adja-
O
NH2
9
cent binding sites that were present in the screening li-
brary were identified as hits, while known inhibitors
that bind to more distal ribosome sites were silent. These
data further validate the nature and utility of these
BODIPY-FP probes as tools in the discovery of novel
ligands for bacterial ribosomes.
NH
9
O
N
O
OH
HO
N
O
HO
OH
OH
AcO
2'
OH
2'
OH
a, b
O
O
O
O
O
O
O
O
O
O
4"
OH
OMe
4"
OH
OMe
As indicated above, the binding affinity and kinetics are
important for a fluorescent probe. Although probe 3
demonstrated the best binding affinity, the competitive
ligand exchange rate was slow and required up to two
days incubation. Fluorescent probe 7, with an addition-
al group at the 9-position, also showed strong binding
affinity and slow kinetics requiring an overnight incuba-
tion for full displacement under the conditions in which
it was employed in high-throughput screening.
However, fluorescent probe 5, with a moderate binding
affinity and shorter ligand exchange time (about 1 h),
was found to be ideal for use in high-throughput screen-
ing. Probe 5 was successfully used to identify a series of
novel ribosome ligands that competitively or allosteri-
cally displace the fluorescent probe from the bacterial
ribosome. This result will be reported in elsewhere
(Roche et al., manuscript in preparation).
4
6
c, d, e
O
O
OH
NH
9
N
O
OH
HO
2'
OH
N
O
O
N
B
F
F
O
O
O
O
O
4"
NH
O
N
H
OMe
7
Scheme 3. Preparation of probe 7. Reagents and conditions: (a)
1.0 equiv N-(benzyloxycarbonyloxy)succinamide, DMF; (b) 1.2 equiv
Ac2O, 4.0 equiv Et3N, CH2Cl2, rt, 16 h; (c) 2.0 equiv CDI, THF, 35 °C,
12 h, then added 10 equiv NH2CH2CH2NH2, 45 °C, 1 h; (d) 1.0 equiv
BODIPY-FL propionic acid, 1.0 equiv succinimidyl ester, DMF, rt,
1 h, 48% from 1; (e) MeOH, 60 °C, 1 h, 42%.
In summary, macrolides with BODIPY-FP fluorophore
groups attached at either the 400- or 9-positions were de-
signed, synthesized, and evaluated. The binding specific-
ity of the prepared BODIPY-FP probes to the product
exit channel of the peptidyl transferase reaction center
of the ribosome was verified through competitive bind-
ing studies that employed erythromycin and other anti-
biotics known to bind in the same location of the 50S
subunit. Finally, erythromycin-BODIPY-FL probe 5
was found to be ideal for use in an ultra high-through-
put screen to identify novel small molecules that bind
to the bacterial ribosome. These small molecules are
being evaluated as potential start-points for chemistry
optimization efforts toward the development of a novel
antibacterial drug.
Table 1. Binding constants and inhibition IC50s of fluorescent probes
Compound
Ribosome binding
Kd (nM)
Transcription and
translation
IC50 (lM)
Erythromycin
—
0.08
0.16
1.5
3
5
7
<0.5
30
1.5
0.55
reporter.14 As shown in Table 1, probe 3 with tight bind-
ing affinity exhibited a roughly equivalent inhibition
when compared to erythromycin. Similarly, the lower
affinity probes 5 and 7 showed reduced inhibition
accordingly.
References and notes
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We undertook competitive binding studies to demon-
strate that the synthesized probes bind to the bacterial
ribosome in a biologically relevant manner, using
methods similar to those previously described.8 Erythro
mycin competed off all three probes with potent IC50s.
However, in fluorescence polarization competition as-
says, the potency of an inhibitor that binds to ribosomes
more tightly than the assay probe itself cannot be deter-
mined but rather is limited by the affinity of the probe.13
For example, the observed IC50 of erythromycin using
probe 5 was 30 nM, corresponding to the probe affinity.
The binding site for lincosamides such as clindamycin
overlaps with the macrolide binding site.1 Displacement
of probe 5 or 7 by clindamycin yielded identical IC50
values of 6 lM (corresponding to a Ki = 2 lM), in
reasonable agreement with the published literature.15
Finally, in high-throughput screening with probe 5, a

J. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 794–797
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4717.
11. Full
name:
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-
diaza,s-indacene-3-propionic acid, succinimidyl ester, pur-
chased from Molecular Probes.