A chemoenzymatic route to diversify aminolgycosides enables a microarray-based method to probe acetyltransferase activity

Article abstract of DOI:10.1002/cbic.201000300

(Figure Presented) Chemoenzymatic synthesis: A chemoenzymatic route for modification of aminoglycosides is disclosed. The significant feature of this approach is the discovery that an aminoglycoside 3-N-acetyltransferase, AAC(3)-IV, from Escherichia coli accepts azidoacetyl coenzyme A (AzAcCoA) as a substrate that can be further chemically diversified. This approach was used to develop a carbohydrate microarray method to study modification of aminoglycosides.

Full text of DOI:10.1002/cbic.201000300

DOI: 10.1002/cbic.201000300
A Chemoenzymatic Route to Diversify Aminolgycosides Enables a
Microarray-Based Method to Probe Acetyltransferase Activity
Pavel B. Tsitovich,[a] Alexei Pushechnikov,[a] Jonathan M. French,[a] and Matthew D. Disney*[a, b]
This worked is dedicated to Professor Peter H. Seeberger in honor of his receiving the Tetrahedron Young Investigator Award.
Specific modification of functional groups in aminoglycosides
poses a significant synthetic challenge. A chemoenzymatic
route for modification of aminoglycosides is disclosed. The crit-
ical feature of this approach is the discovery that the aminogly-
coside 3-N-acetyltransferase, AAC(3)-IV, from Escherichia coli[1]
accepts azidoacetyl coenzyme A (AzAcCoA) as a substrate in a
similar manner as the natural substrate, acetyl coenzyme A
(AcCoA). After enzymatic delivery of an azidoacetyl group, it
can be chemically modified by a Huisgen dipolar cycloaddition
reaction (HDCR),[2] therefore enabling further diversification.
Thus, this method accelerates access to modified compounds
with diversity beyond that which can be installed directly by
AAC(3) and a modified CoA thioester. The approach was fur-
ther developed to study modification of aminoglycosides with
AAC(3), which causes broadscale aminoglycoside inactivation,
by using a fluorescence-based microarray platform. This plat-
form is a useful analytical tool for the facile identification of
both protein and carbohydrate substrates for acetyltransferas-
es, which play critical roles in a multitude of cellular process-
es.[3]
by resistance-causing enzymes or more specific recognition of
target RNAs, is needed. Synthesis of designer aminoglycosides,
however, is difficult because they contain a variety of amino
and hydroxy groups with similar reactivities.
In an effort to develop expedited routes that lead to site-
specifically derivatized aminoglycosides, we studied the ability
of AAC(3) to accept modified acetyl coenzyme A substrate to
install functional groups in their structures that can be further
chemically diversified. Towards this end, AzAcCoA was synthe-
sized, and its ability to acylate the aminoglycoside apramycin
was studied (Figure 1A). AzAcCoA was used because the azido
group can be diversified by reaction with alkynes using a cop-
per(I)-catalyzed HDCR. Modification of apramycin by AzAcCoA
was monitored by UV-vis[1] and mass spectrometry, and each
study unambiguously showed that AzAcCoA is accepted as a
substrate (Figure 1B and C). In fact, AzAcCoA is accepted in a
similar manner as the natural substrate. AzAcCoA has a KM and
a kcat value that are fourfold and threefold lower, respectively,
than AcCoA (Figure 1B). The specificity constants (kcat/KM) are
similar: 5.1ꢀ105 mÀ1 sÀ1 for AcCoA and 6.3 x105 mÀ1 sÀ1 for
AzAcCoA. In contrast, when propionyl CoA, malonyl CoA, and
butyryl CoA substrates were studied, they had kcat/KM values
that are decreased by 7-, 23-, and 1730-fold relative to AcCoA,
respectively.[1] Another recent and extensive study has shown
that a variety of aminoglycosides can be modified by CoA
derivatives to provide new aminoglycosides. The kcat/KM of the
modified CoA substrates in that study are also lower than
AcCoA.[7] Thus, the similarity in specificity constants of AzAc-
CoA and AcCoA as substrates for AAC(3) points to unique fea-
tures for AzAcCoA interacting with the enzyme.
Aminoglycosides represent one of the largest classes of anti-
bacterials with activity against both Gram-negative and Gram-
positive bacteria. Many aminoglycosides exert their antibacteri-
al activity by binding to the decoding site (A-site) in 16S
rRNA.[4] Binding to this site affects recognition of cognate and
noncognate tRNAs by the ribosome. As is common with all
antibiotics, resistance to aminoglycosides has emerged since
their initial introduction as therapeutic agents.[5] One of the
most important resistance-causing mechanisms against amino-
glycosides is enzymatic modification, for example, by acetyl-
transferases (AACs). Aminoglycosides have also been used to
facilitate translational readthrough in diseases caused by non-
sense mutations, including Duchenne’s muscular dystrophy,
cystic fibrosis, and hemophilia.[6] To better exploit these targets,
simple access to modified aminoglycosides with improved bio-
logical activity, such as reduced susceptibility to modification
Encouraged by the results of enzymatic azidoacetyl transfer,
the scope of the 3-N-azidoacetyl-apramycin (3; Figure 1A)
modification by HDCR was further investigated. In initial stud-
ies, dansyl alkyne (4; Figure 1A) was used to modify the azi-
doacetyl aminoglycosides (Figure 2). 3-N-Azidoacetyl-apramy-
cin (3) was partially purified by passing the reaction mixture
over Dowex ion-exchange resin (HO- form) to capture any
CoA-containing materials. This material was then subjected to
HDCR modification with compound 4. Mass spectral analysis
indicated complete conversion to 5. The reaction was purified
by HPLC, and 5 was obtained in 44% isolated yield over two
steps.
[a] Dr. P. B. Tsitovich,+ Dr. A. Pushechnikov,+ J. M. French, Prof. Dr. M. D. Disney
Department of Chemistry & The Center of Excellence in Bioinformatics
and Life Sciences, University at Buffalo, The State University of New York
657 Natural Sciences Complex, Buffalo, NY 14260 (USA)
Fax: (+1)716-645-6963
[b] Prof. Dr. M. D. Disney
Current address:
Armed with these results, a microarray-based method to
study the modification of 2-deoxystreptamine aminoglycosides
by AAC(3) was developed. In these experiments, the extent of
AAC(3) modification on the array surface was monitored by
treating the generated azidoacetyl aminoglycosides with an
alkyne dye through a HDCR (Figure 3 and 4).
The Scripps Research Institute, Department of Chemistry
130 Scripps Way, Jupiter, FL 33458 (USA)
E-mail: disney@scripps.edu
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000300.
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Figure 1. Studying the ability of AAC(3) to accept AzAcCoA as a substrate. A) Reaction of AzAcCoA with apramycin. B) Michaelis–Menten plots for the modifi-
cation of apramycin by AcCoA and AzAcCoA. C) Mass spectral analysis of the reaction of apramycin with AzAcCoA.
In order to site-specifically immobilize the aminoglycosides
onto a microarray, a small library of azide-modified aminogly-
cosides was synthesized (Figure 3). These compounds can be
anchored onto alkyne-functionalized agarose microarrays.[8]
Syntheses of compounds 6–14 were completed according to
modification of previously published procedures in which a pri-
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Figure 2. HDCR modification of 3-N-azidoacetyl-apramycin (3). A) Reaction between compounds 3 and 4. B) Mass spectral analysis of the reaction to form 5.
mary hydroxy group is activated by reaction with 2,4,6-triiso-
propylbenzenesulfonyl chloride. The resulting adduct is dis-
placed with sodium azide.[8b,9] Compound 15 was synthesized
by reductive amination of streptomycin with 3-azidopropyla-
mine. Compound 16 was synthesized by acylation of spermine
with 4-azidobutyric acid. Structures 6–14 are known to bind to
the bacterial rRNA A-site,[10] and most have been studied as
AAC(3) substrates.[1] Structures 15 (streptomycin derivative)
and 16 (spermine derivative) are not known to be specific bac-
terial rRNA A-site binders. Compounds 15 and 16 are not ex-
pected to be substrates for AAC(3) because previous studies
have shown that guanidiniylated aminoglycosides are not
modified by acetyltransferases.[11]
tion-based studies, neomycin B has the highest kcat/KM value
followed by tobramycin, paromomycin, apramycin, ribostamy-
cin, kanamycin, and amikacin. The order of the extent of modi-
fication on the arrays at the highest spot loading is: neomy-
cin B (13)>paromomycin (6 and 7) = amikacin (9)>kanamy-
cin A (12)=neamine (14). Other aminoglycosides are modified
to a lesser extent.
Arrays were also probed for binding to a fluorescently-la-
beled mimic of the bacterial rRNA A-site (Figure 4D). The data
are in good agreement with a study of aminoglycoside binding
to the A-site using surface plasmon resonance (SPR).[10] For ex-
ample, neomycin B (13-like) gives the highest signal for bind-
ing to the A-site and is the highest affinity binder in the series
(Figure 4F). The second and the third highest binding signals
are observed for the paromomycin derivatives (7 and 6), and
the apramycin (10) and tobramycin (11) derivatives, respective-
ly. The other aminoglycosides that were expected to bind the
A-site gave lower but measurable signals. The lowest binding
signals are observed for the nonspecific binders, streptomycin
(15) and spermine (16) derivatives, as expected.
Our fluorescence-based approach is advantageous over
other microarray approaches that have been developed to
probe aminoglycoside modification by resistance-causing en-
zymes that use radioisotopes. For example, detection of 14C-la-
beled acetylated aminoglycosides requires several days of ex-
posure in a phosphorimager cassette.[13] Furthermore, the reso-
The azido-aminoglycosides and spermine derivatives were
arrayed onto alkyne-agarose microarrays in the presence of
copper(I). Serial dilutions from 500 to 62 pmol of compound
were delivered to the surface through a pin-transfer replicator.
After the reaction to conjugate the ligands onto an array, the
arrays were quenched[12] and then modified with AzAcCoA by
AAC(3). After washing the arrays, they were treated with an
alkyne-functionalized BODIPY dye via a HDCR (Figure 4A). As
expected, scans of modified arrays showed clear spots of
modification for 6–14, whereas 15 and 16 were not labeled
(Figure 4C and 4E). Array data for modification by AAC(3) were
then compared to previous solution-based studies of modifica-
tion.[1] In general, the two studies are in agreement. In solu-
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Figure 3. The compounds used to construct microarrays and the microarray immobilization chemistry used in this study. A) Structures of azide-functionalized
aminoglycosides 6–15, and spermine 16. B) The HDCR used to anchor azide-containing small molecules onto alkyne-functionalized agarose microarray surfa-
ces. (N3-R represents azide-functionalized compounds 6–16).
lution of a phosphorimager is significantly diminished relative
to that of a fluorescent microarray scanner. Enhanced resolu-
tion allows for a significant increase in the number of features
(spots) that can be analyzed on a microarray.
aminoglycosides and in developing microarray-based platforms
to probe modification of biomolecules by acetyltransferases,
including proteins and carbohydrates for which activities are
modulated by acetyltransferases.[3]
During the course of this work, a series of modified amino-
glycosides were synthesized using modified coenzyme A deriv-
atives.[7] Interestingly, these modified antibiotics displayed anti-
bacterial activity despite the fact that they were modified by
resistance-causing enzymes.[7] The results herein expand that
work, demonstrating that aminoglycosides can be modified by
coenzyme As that install reactive groups that can be easily
chemically diversified. Thus, the diversity of modifications in-
troduced into aminoglycosides is not solely limited by the abil-
ity of an AAC to accept a modified coenzyme A as a substrate.
Rather, reactive tags can be installed on an aminoglycoside by
a CoA derivative followed by subsequent chemical modifica-
tion to provide access to novel compounds.
Acknowledgements
We thank Prof. Jessica Childs-Disney for critical review of the
manuscript and Prof. John Blanchard for the gift of the plasmid
encoding AAC(3). This work was supported by the National Insti-
tutes of Health (RO1-GM079235). M.D.D. is a Cottrell Scholar
from the Research Corporation, a J.D. Watson Young Investigator
from NYSTAR, and a Camille and Henry Dreyfus New Faculty
Awardee.
In summary, a chemoenzymatic route toward the synthesis
of aminoglycosides is reported. This method was used to
develop a high-throughput and sensitive microarray-based
method to study the modification of aminoglycosides by ace-
tyltransferases that confer resistance. These developments
have broad applications in both the synthesis of diversified
Keywords: antibiotics · bioorganic chemistry · carbohydrates ·
glycoconjugates · RNA recognition
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Figure 4. Probing aminoglycoside recognition by microarray. A)Labeling aminoglycosides by enzymatic delivery of
an azidoacetyl group followed by installation of a dye by a HDCR. B) Secondary structure of the bacterial rRNA A-
site mimic (boxed nucleotides represent those derived from the native sequence). C) Image of a microarray to
study AAC(3) modification of aminoglycosides. D) Image of a microarray to study binding of an oligonucleotide
mimic of the bacterial rRNA A-site. E) Representative plot of data for AAC(3) modification. F) Representative plot
of data for binding of the rRNA A-site mimic. Each group of bars in the plots from left to right corresponds to de-
livery of 500, 250, 125, 62, and 0 pmol of material to the array surface.
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Received: May 22, 2010
Published online on July 13, 2010
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