Deploying Fluorescent Nucleoside Analogues for High-Throughput Inhibitor Screening

Article abstract of DOI:10.1002/cbic.201900671

High-throughput small-molecule screening in drug discovery processes commonly rely on fluorescence-based methods including fluorescent polarization and fluorescence/F?rster resonance energy transfer. These techniques use highly accessible instrumentation; however, they can suffer from high false-negative rates and background signals, or might involve complex schemes for the introduction of fluorophore pairs. Herein we present the synthesis and application of fluorescent nucleoside analogues as the foundation for directed approaches for competitive binding analyses. The general approach describes selective fluorescent environment-sensitive (ES) nucleoside analogues that are adaptable to diverse enzymes that act on nucleoside-based substrates. We demonstrate screening a set of uridine analogues and development of an assay for fragment-based lead discovery with the TcdB glycosyltransferase (GT), an enzyme associated with virulence in Clostridium difficile. The uridine-based probe used for this high-throughput screen has a K_D value of 7.2 μm with the TcdB GT and shows a >30-fold increase in fluorescence intensity upon binding. The ES-based probe assay is benchmarked against two other screening approaches.

Full text of DOI:10.1002/cbic.201900671

Accepted Article
Title: Deploying Fluorescent Nucleoside Analogues for High
Throughput Inhibitor Screening
Authors: Leah Seebald, Amael Madec, and Barbara Imperiali
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201900671
Link to VoR: http://dx.doi.org/10.1002/cbic.201900671
A Journal of
www.chembiochem.org

ChemBioChem
10.1002/cbic.201900671
Deploying Fluorescent Nucleoside Analogues for High Throughput
Inhibitor Screening
Leah Seebald, Amaël Madec, Barbara Imperiali*[a]
[
a]
Dr. L. Seebald, Dr. A. Madec, Prof. B. Imperiali,
Department of Biology and Department of Chemistry Massachusetts Institute of Technology, Cambridge, MA 02139 (USA)
E-mail: imper@mit.edu
[**] This research was supported by the National Institutes of Health GM097241 and GM131627.
Keywords: environment-sensitive fluorophore • glycosyltransferase • high throughput screening • nucleoside analogue
Abstract:
High-throughput small molecule screening in drug discovery processes commonly rely on
fluorescence-based methods including fluorescent polarization and fluorescence/Förster
resonance energy transfer. These techniques use highly accessible instrumentation, however
may suffer from high false negative rates and background signals or, may involve complex
schemes for the introduction of fluorophore pairs. Herein, we present the synthesis and
application of fluorescent nucleoside analogues as the foundation for directed approaches for
competitive binding analyses. The general approach describes selective fluorescent
environment-sensitive (ES) nucleoside analogues that are adaptable to diverse enzymes that
act on nucleoside-based substrates. We demonstrate screening a set of uridine analogues and
development of an assay for fragment-based lead discovery with the TcdB glycosyltransferase
(GT), an enzyme associated with virulence in Clostridium difficile. The uridine-based probe used
for this HTS has a K of 7.2 µM with the TcdB GT and shows a >30-fold increase in
D
fluorescence intensity upon binding. The ES-based probe assay is benchmarked against two
other screening approaches.
1
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900671
ON
wavel
λ
e
/
n
n
g
m
th (nm)
Enzy ml i eg and
OFF
+
ligand
We present the development of nucleoside-based environment sensitive fluorophore
ESF) probes that are applicable to competitive-binding analyses. Use of the ESF probes is
(
illustrated by application to a high throughput fragment-based screen for identification of leads
for inhibitor development against a clinically-relevant glycosyl transferase from the Clostridium
difficile TcdB toxin.
2
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900671
Introduction:
Fluorescence-based analytical approaches are ubiquitous in biochemical and biological
studies and form the mainstay of small-molecule ligand discovery efforts in chemical biology
and medicinal chemistry research. Fluorescence is particularly useful in high throughput
screening applications where alternative approaches, such as those involving radioactivity or
complex instrumentation, are less accessible. Methods including fluorescence polarization (FP)
[
1]
[2]
and differential scanning fluorimetry (DSF) analyses are widely applied for initial steps in
small molecule screening. FP assays are based on changes in the orientation and mobility of
ligands upon binding to macromolecules, thereby providing information on solution state
[
3]
interactions. FP plays an important role in the drug discovery process; when adapted to a
multi-well plate reader format, FP assays provide relatively low cost analyses across a wide
[
4]
range of screening conditions. In DSF, changes in protein stability due to small molecule
binding are quantified by assessing thermal shifts in the presence of a dye, such as SYPRO®
Orange, which becomes more fluorescent when bound to unfolded proteins. Applications of
both FP and DSF in high-throughput screening (HTS) and drug development are well known
[
5]
and have been thoroughly reviewed.
Despite the advantages of the aforementioned methods, there are notable drawbacks.
[
6]
For example, with FP there may be fluorophore aggregation, unacceptable false-negative
[
7]
rates from high background due to excess fluorophore, small variations in buffer and pH
[
2]
influencing the polarization of free fluorophore, and in particular for fragment-based screening,
competition between the large excess of screening compounds and fluorophores for
[
8]
hydrophobic patches on protein surfaces. In DSF, thermal shifts may be very small (<1 °C)
and information on the site of small-molecule binding is not defined by the assay. In light of
these issues, there remains a need for equally accessible and complementary methods for HTS
screening for small-molecule protein ligands.
As an alternative to FP and DSF, enzyme-targeted ligands that have been elaborated
with environment-sensitive fluorophores (ESFs) can provide advantages for HTS. ESFs have
[
9]
been extensively used as solvent polarity indicators and as probes for investigating protein
[
10]
interactions and dynamics. ESFs are highly sensitive to the polarity of the local
microenvironment due to modulation of the fluorophore quantum yield, which impacts signal
brightness. Some ESFs may also undergo changes in emission wavelengths and thus are
[
11]
designated as solvatochromic. Importantly, for ESFs that show an increased quantum yields
with decreased solvent polarity, an excess of unbound ESF-containing reagents in HTS assays
[
12]
does not significantly contribute to background fluorescence, thereby mitigating the problem
3
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900671
[
13]
of false negatives arising in some FP-based screens. Additionally, ESF probes that are
selective for a specific hydrophobic binding pocket (i.e. ligand targeted) on a protein can
compete for binding against candidate ligands used in HTS, providing an alternative readout to
nonspecific fluorophore-dependent measurements to determine thermal stabilization by ligands
[
2, 14]
in DSF-based assays.
Finally, ligand-targeted ESFs enable validation of competitive binding
at a protein binding site by observed decreases in fluorescence in the presence of the ligand.
Selectivity of the ESF probe for an enzyme active site can be achieved by incorporating
key features of the cognate substrate scaffold. In this report, we describe ESF-derivatized
nucleosides as probes for HTS of enzymes that use sugar nucleotides as substrates including
the plethora of glycosyltransferases (GTs) that are implicated in complex glycan biosynthesis
(Figure 1). First, we present the synthesis and screening of four nucleoside analogs as probes
for HTS. Then, we exemplify the application of ESF-conjugated nucleoside probes in an HTS
assay of the N-terminal GT domain of TcdB. TcdB is a polyprotein produced by Clostridium
difficile, a Gram-positive gastrointestinal pathogen that is currently a leading cause of hospital-
[
15]
acquired infections in developed countries. TcdB is a primary virulence factor for C. difficile,
and is a member of a family of glucosylating toxins that inactivate small GTPases responsible
[
16]
for eukaryotic signaling pathways. TcdB is a potential pharmacological target for inhibition to
[
17]
prevent the host target glucosylation that is associated with bacterial virulence, and because
[
18]
of the current medical relevance of C. difficile.
Figure 1. A) Probes synthesized for this study. Probes are based on a synthetically-accessible
C5' amino-uridine analogue coupled to DMAP and DMN ESFs via varied linkers. B) Application
of tailored uridine-based probes for HTS of enzymes selective for uridine-based enzyme
substrates.
4
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900671
The ESFs employed in the current studies are based on N,N-4-dimethylamino-
[
19]
phthalimide (DMAP) and N,N-4-dimethylamino-naphthalimide (DMN) derivatives. These
imides exhibit minimal fluorescence in polar aqueous environments – thus fluorescence is
“turned off” in an unbound state – and enhanced fluorescence in hydrophobic environments
[
20]
(
e.g. protein binding sites) – thus fluorescence is “turned on” in a bound state. These
[
19]
fluorophores also show a small (40-80 nm) hypsochromic shifts. DMAP and DMN are ideal
fluorophores for substrate analogue preparation due to their relatively small size, which is less
[
10a, 21]
likely to perturb a natural substrate scaffold.
Additionally, synthesis can be accomplished
while simultaneously minimizing the overall effort by utilizing standard coupling methods, such
[
22]
as amide coupling or Cu(I)-catalyzed azide-alkyne cycloaddition (Cu(I)AAC), and a common
nucleoside building block - in this case a uridine analog, with a primary amine handle at the
ribose C-5'.The general approach described herein should be readily adaptable to diverse
enzymes that act on nucleoside-based substrates and should be compatible with HTS discovery
efforts.
Results
Uridine diphosphate glucose (UDP-Glc) is the glucosyl donor substrate for the TcdB GT
[
23]
(K
m
21 µM),
therefore we chose a uridine core as the key structural feature for active-site
[
21a]
[21b]
recognition and conjugated both 4-DMAP
and 4-DMN
ESFs via varied linkers to screen
for binding and fluorescence. The ESFs were appended to the ribose C-5' of uridine, using the
modified amino-uridine derivative (4), by standard amide coupling methods or Cu(I)AAC
(Scheme 1) to afford probes 5 through 8. When conjugated, each of the probes exhibits
comparable fluorescence properties to those of the unmodified dyes in water or buffer (data not
shown).
Scheme 1. Syntheses of ESF probes. Reagents and conditions: (a) Acetone, sulfuric acid; (b)
p-Toluenesulfonyl chloride, 4-(dimethylamino)pyridine, pyridine, dichloromethane; (c) Sodium
azide, 40 ⁰C, DMF; (d) Pd(OH)-C, isopropyl alcohol, H
Diisopropylethylamine, hydroxybenzotriazole, HBTU, DMF, 4-(N,N-dimethylamino)phthalic
anhydride; (f) Trifluoroacetic acid, MeOH; (g) CuSO , tris(3-hydroxypropyltriazolylmethyl)amine,
2
O, formic acid; (e) N,N-
4
sodium ascorbate, 7: compound 10, 8: compound 11; (h) i. Fmoc-Gly-OH, N,N-
diisopropylethylamine, HBTU, DMF, ii. 20% piperidine in DMF; (i) 4-(N,N-
Dimethylamino)phthalic anhydride, dioxane, reflux, 10: Propargyl amine, 11: 1-Amino-but-3-yne.
5
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900671
For the presented assay, the best probe for the TcdB GT was 5; it showed a low
background signal in buffer and the fluorescence signal upon binding to the TcdB GT was linear
across the µM concentration range (Figure S2). As determined by fluorescence titration, probe
5
bound to the TcdB GT with a K of 7.2 ± 1.2 µM and, at saturation showed a >30-fold increase
D
in fluorescence at 512 nm relative to the unbound state (Figure 2). In general, we anticipate that
signal changes with the ESF-based probes above 5-fold would be sufficient for general
adaptation to HTS assays as this would provide statistically robust data for analysis. Addition of
competing UDP-Glc reduced fluorescence to unbound levels showing that binding of probe 5 is
both competitive and reversible (Figure S3). Probe displacement with UDP-Glc also serves as
an ideal positive control for further assay development towards HTS applications.
Figure 2. A) Fluorescence spectra showing titration of probe 5 with 20 µM TcdB GT. B) Binding
of 5 with 20 µM TcdB GT measured at 518 nm.
Probes 6 - 8 did not signal the TcdB GT, however, binding to other select GTs and
phosphoglycosyl transferases (PGTs) was observed (Figure S1). All fluorescence studies
6
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900671
[
6]
included TWEEN-20 (0.09%) to minimize non-specific binding. Full experimental details for
fluorescence studies can be found in the Supplementary Information.
Towards the development of an HTS method, the ESF probe assay was transferred to a
multiwell-plate reader format; volume was scaled to 20 µL and optimal working concentrations
for the TcdB GT and 5 were found to be 10 µM each. The Maybridge Ro3 1000 compound
diversity library, which includes stock solutions of fragments dissolved in DMSO to a final
concentration of 100 mM, was used for the screen. The screen was carried out at 5 mM final
[
24]
fragment concentration, which is typical in fragment screening. The working assay solution,
with the ESF probe and the fragments (at 5 mM), included DMSO at 5.5% of the final volume.
Each microwell plate in the HTS included analyses of the following samples: background
fragment fluorescence, positive controls, negative controls, and maximum expected signal
control. At high concentrations many fragments are fluorescent, so measurement of the
fragment fluorescence in assay buffer with no added enzyme was used as the background. This
background was subtracted from the fluorescence of the working assay, which included the ESF
probe, enzyme, fragment, and assay buffer. Additionally, UDP-Glc (at 1 mM and 100 µM) was
used as a positive control for probe displacement on each microwell plate of the HTS. Blank
wells containing assay buffer and 5.5% DMSO were added to each plate as a negative control.
Lastly, wells with probe 5, the TcdB GT and 5.5% DMSO in the absence of fragment were
included to provide the maximum signal for ratio-based calculations.
Hits were measured as the relative fluorescence ratio of background-corrected
displacement signal to maximum signal (Eq. S1). The distribution of percent probe displaced
(proportional to fragment binding) from the 1000-member library showed 34 fragments with a
signal equal to, or more than 60% of the maximum observable signal (Figure S4). In HTS
compound screening assays, validation of apparent hits is critical because false positives can
arise from experimental artefacts, such as those due to fragment insolubility and fluorescence
[
8]
quenching. In this case, the selection of 34 apparent hits was reduced to 24 hits after 10
fragments were determined to be false positives due to fluorescence quenching. In addition to
the 24 apparent hits, five additional fragments were randomly selected as negative controls for
further validation studies. The 24 apparent hits are numbered 1-24 and the additional five
negative controls are numbered 25-29. The table also includes DMSO at 5.5% as a blank and
additional negative control, numbered 30 (Table 1).
Hits and negative controls (1-30) from the ESF probe assay were tested with a DSF
assay using SYPRO® Orange. In DSF, the protein T
m
is the half maximum fluorescence signal
if stabilized or destabilized
found at complete protein denaturation. Samples exhibit a shifted T
m
7
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900671
[
8]
in the presence of a screening compound, calculated as ΔT
determined in triplicate) of TcdB in the presence of fragments 1-29. The T
equivalent amount of DMSO, but without a fragment, was used as a control. Typically,
fragments that stabilize the native conformation of a protein increase the T . However, some
fragment binders may destabilize the native conformation upon binding to a non-native state of
m
. Table 1 includes the ΔT
m
(
m
of TcdB with an
m
[
25]
the enzyme. Such fragments are still considered apparent hits, and are subject to further
evaluation. DSF analyses may be complicated by interference from highly fluorescent
[
4]
fragments. In this case the ΔT
m
values cannot be determined and are thus recorded as not
compatible (ΔT : NC) with the DSF assay. A total of 11 fragments, of the 29 studied (Table 1),
m
were deemed not compatible with DSF analysis, underscoring the need for alternative
approaches for HTS of fragments and other weak binders. Thus, in each of these cases, the
ESF probe assay was able to provide a read-out for fragments that would have been eliminated
from the HTS based solely on DSF incompatibility.
The 29 fragments were also subjected a biochemical activity assay. For this, we applied
®
the Promega UDP-Glo assay, which detects UDP release over the course of a GT reaction.
Because this is a multienzyme-based detection assay, an equivalent high concentration of
fragment to that used in ESF and DSF analyses was not compatible due to excessive off-target
inhibition of assay enzymes. This is a well-known complication of fragment screening by activity
analysis due to the high concentrations (typically 5 mM) that are necessary for hit identification
[
6]
in initial steps. The activity of TcdB GT was tested in the presence of fragments at 800 µM.
Fragments are typically weak binders, so high GT inhibition is not anticipated (Figure S5). Off-
target inhibition of the Glo® reagent enzymes by the fragments was also tested and corrected
for, however, in several cases we noted off-target inhibition of the Glo® assay at over 50% and
these cases were deemed not compatible (NC) with the fragment assay (for complete error
analysis see SI). Of the 29 fragments, 10 were found to be NC with the biochemical activity
assay. This incompatibility rate is comparable that observed with the DSF assay and again
underscores the value of the ESF probe-based approach. Structures of the fragment hits are
available in the “Supplemental Information” and can now be further elaborated for structure-
activity relationship (SAR) studies towards inhibitor development for TcdB (Figure S6).
In summary, although some of the hits from the ESF probe screen of the TcdB GT can
be further validated using DSF or biochemical assay-based approaches, the significant rates of
assay incompatibility with the high concentrations required for initial fragment-based screening
highlights the challenges. From the 24 ESF-based leads, 30% were validated as hits by DSF
and, an additional 46% could not be analyzed by DSF due to fluorescence interference from the
8
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900671
fragments. The enzyme-based biochemical assay also showed high incompatibility; 42% of the
fragments showed excessive background inhibition of the assay itself (>50% even at 800 µM)
and in this case only 20% of the ESF hits could be supported by activity analysis in the
presence of 800 µM fragment. These percentages serve to highlight the potential of the ESF
probes to address the challenges of low affinity fragments, a challenge that has been
[
6, 26]
documented between various HTS assays.
Conclusions
We have described a robust ESF-based assay for enzymes that act on nucleoside-
based substrates such as the ubiquitous NDP-sugars that are extensively exploited as glycosyl
donor substrates in the assembly of complex glycan structures and for post-translational protein
modification. The exemplar assay uses a readily-accessible nucleoside analogue, which is
modified with the environment-sensitive DMAP and shows a K of 7.2 ± 1.2 µM with a >30-fold
D
increase in fluorescence signal upon binding to the active site of the TcdB GT domain from the
C. difficile TcdB toxin. These properties make this probe highly amenable to HTS. The use of
the probe has been demonstrated in a fragment-based lead discovery screen using the 1000-
member Ro3 Maybridge library; the ESF probe assay revealed 24 leads for further
development.
ESF nucleoside analogues, such as those presented in this report, can be applied to a
diverse range of enzymes that act on nucleoside-based substrates. A subset of these enzymes
includes glycosyltransferases and phosphoglycosyl transferases involved in the glycosylation
[
27]
pathways of bacterial pathogens that use NDP-sugars as substrates. Many of these
glycosylation-related enzymes are potential antibiotic drug targets due to the key roles played in
bacterial glycoconjugate biosynthesis. The strategy for nucleoside-based ESF probe
development provides valuable reagents for HTS applied to inhibitor development towards
these nucleoside-accepting enzymes.
9
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900671
Table 1. Results of ESF probe (5) binding, DSF analysis and biochemical assay determination
for 29 fragments
%
probe
probe
Inhibition
% probe
probe
Inhibition
#
Δ T
m
T
m
hits
#
Δ T
m
m
T hits
displaced
displaced
at 800 µM
displaced
displaced
at 800 µM
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
61
97
90
60
81
82
90
66
61
93
87
61
83
74
95
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
NC
NC
NC
NC
no
yes
NC
yes
NC
no
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30[c]
78
98
80
88
79
76
95
94
68
10
85[b]
10
9
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
-3.5
NC
NC
NC
-3.8
0.3
yes
NC
NC
NC
yes
no
no
NC
NC
yes
no
-0.2
NC
1.0
NC
0.3
-2.5
NC
1.8
NC
NC
NC
0.8
-0.7
NC
yes
NC
no
NC
NC
no
no
1.2
yes
no
no
yes
NC
yes
NC
NC
NC
no
0.5
NC
NC
no
yes
NC
yes
no
1.2
yes
no
0
1
2
3
4
5
0.2
no
-2.4
0.1
yes
no
no
no
no
no
no
0.3
no
no
no
14
0
no
0.7
no
no
no
NC
no
40.5[c]
no
no
[
a] NC: not compatible with DSF or biochemical assay at concentration of probe employed. [b] Concentration dependence validation
showed this fragment to be a false positive as a fluorescence quencher in the ESF assay. [c] Control with equivalent volume of
DMSO. [d] T for TcdB.
m
Acknowledgements
Financial support from the National Institutes of Health (GM097241 and GM131627
to B.I.) is gratefully acknowledged. We also acknowledge Dr. Nathaniel Shocker for expression
and purification of TcdB, Dr. Debasis Das for initial fluorescence titration experiments and Dr.
Christine Arbour for assistance with probe synthesis. We also acknowledge the MIT DCIF for
NMR and HR-MS analyses. We thank Prof. Mathew Pratt and Narek Darabedian providing a
sample of recombinant OGT.
10
This article is protected by copyright. All rights reserved.

ChemBioChem
10.1002/cbic.201900671
[1]
[2]
[3]
[4]
[5]
M. G. Lagorio, G. B. Cordon, A. Iriel, Photochem Photobiol 2015, 14, 1538-1559.
K. Huynh, C. L. Partch, Curr Protoc Protein Sci 2016, 1-19.
D. M. Jameson, J. A. Ross, Chem. Rev. 2010, 110, 2685-2708.
W. A. Lea, A. Simeonov, Expert Opin. Drug Discov. 2011, 6, 17-32.
M. D. Hall, A. Yasgar, T. Peryea, J. C. Braisted, A. Jadhav, A. Simeonov, N. P. Coussens, Methods
Appl Fluoresc. 2016, 4.
[
[
[
[
[
6]
7]
8]
9]
B. J. Davis, D. A. Erlanson, Bioorg Med Chem Lett 2013, 23, 2844-2852.
N. Malo, J. A. Hanley, S. Cerquozzi, J. Pelletier, R. Nadon, Nat. Biotechnol. 2006, 24, 167-175.
B. J. Davis, S. D. Roughley, Fragment-Based Lead Discovery, Vol. 50, 1st ed., Elsevier Inc., 2017.
C. Reichardt, Chem. Rev. 1994, 94, 2319-2358.
a) G. S. Loving, M. Sainlos, B. Imperiali, Trends Biotechnol. 2010, 28, 73-83; b) W. L. Goh, M. Y.
Lee, T. L. Joseph, S. T. Quah, C. J. Brown, C. Verma, S. Brenner, F. J. Ghadessy, Y. N. Teo, J. Am.
Chem. Soc. 2014, 136, 6159-6162.
10]
[
[
11]
12]
A. Marini, A. Muñoz-Losa, A. Biancardi, B. Mennucci, J. Phys. Chem. B 2010, 114, 17128-17135.
C. A. Telmer, R. Verma, H. Teng, S. Andreko, L. Law, M. P. Bruchez, ACS Chem. Biol. 2015, 10,
1239-1246.
[
13]
14]
I. A. Karpenko, M. Collot, L. Richert, C. Valencia, P. Villa, Y. Mély, M. Hibert, D. Bonnet, A. S.
Klymchenko, J. Am. Chem. Soc. 2015, 137, 405-412.
H. L. Silvestre, T. L. Blundell, C. Abell, A. Ciulli, Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 12984-
[
12989.
[15]
[16]
[17]
J. A. Karas, D. A. Enoch, S. H. Aliyu, J. Infect. 2010, 61, 1-8.
D. T. Denhardt, Biochem. J. 1996, 318, 729.
J. Tam, G. L. Beilhartz, A. Auger, P. Gupta, A. G. Therien, R. A. Melnyk, Chem. Biol. 2015, 22, 175-
185.
[
[
[
[
18]
19]
20]
21]
S. S. Ghantoji, K. Sail, D. R. Lairson, H. L. DuPont, K. W. Garey, J Hosp Infect 2010, 74, 309-318.
G. S. Loving, B. Imperiali, J. Am. Chem. Soc. 2009, 130, 13630-13638.
S. Fery-Forgues, J. P. Fayet, A. Lopez, Photochem. Photobiol. 1993, 70, 229-243.
a) P. Venkatraman, T. T. Nguyen, M. Sainlos, O. Bilsel, S. Chitta, B. Imperiali, L. J. Stern, Nat.
Chem. Biol. 2007, 3, 222; b) G. Loving, B. Imperiali, J. Am. Chem. Soc. 2008, 130, 13630-13638.
H. Li, R. Aneja, I. Chaiken, Molecules 2013, 18, 9797-9817.
[22]
[23]
[24]
[25]
Z. L. Wu, C. M. Ethen, B. Prather, M. MacHacek, W. Jiang, Glycobiology 2011, 21, 727-733.
B. Lamoree, R. E. Hubbard, Essays in biochemistry 2017, 61, 453-464.
a) P. Cimmperman, L. Baranauskiene, S. Jachimovičiute, J. Jachno, J. Torresan, V. Michailoviene,
J. Matuliene, J. Sereikaite, V. Bumelis, D. Matulis, Biophys. J. 2008, 95, 3222-3231; bA. Kabir, R.
P. Honda, Y. O. Kamatari, S. Endo, M. Fukuoka, K. Kuwata, Protein Sci. 2016, 25, 2132-2141.
A. Boettcher, S. Ruedisser, P. Erbel, D. Vinzenz, N. Schiering, U. Hassiepen, P. Rigollier, L. M.
Mayr, J. Woelcke, J Biomol Screen 2010, 15, 1029-1041.
[
26]
27]
[
a) L. L. Lairson, B. Henrissat, G. J. Davies, S. G. Withers, Annu. Rev. Biochem. 2008, 77, 521-555;
b) V. Lukose, M. T. C. Walvoort, B. Imperiali, Glycobiology 2017, 9, 820-833.
11
This article is protected by copyright. All rights reserved.