Selectively Guanidinylated Aminoglycosides as Antibiotics

Article abstract of DOI:10.1002/cmdc.201200150

The emergence of virulent, drug-resistant bacterial strains coupled with a minimal output of new pharmaceutical agents to combat them makes this a critical time for antibacterial research. Aminoglycosides are a well-studied, highly potent class of naturally occurring antibiotics with scaffolds amenable to modification, and therefore, they provide an excellent starting point for the development of semisynthetic, next-generation compounds. To explore the potential of this approach, we synthesized a small library of aminoglycoside derivatives selectively and minimally modified at one or two positions with a guanidine group replacing the corresponding amine or hydroxy functionality. Most guanidino-aminoglycosides showed increased affinity for the ribosomal decoding rRNA site, the cognate biological target of the natural products, when compared with their parent antibiotics, as measured by an in vitro fluorescence resonance energy transfer (FRET) A-site binding assay. Additionally, certain analogues showed improved minimum inhibitory concentration (MIC) values against resistant bacterial strains, including methicillin-resistant Staphylococcus aureus (MRSA). An amikacin derivative holds particular promise with activity greater than or equal to the parent antibiotic in the majority of bacterial strains tested.

Full text of DOI:10.1002/cmdc.201200150

MED
DOI: 10.1002/cmdc.201200150
Selectively Guanidinylated Aminoglycosides as Antibiotics
Richard J. Fair,[a] Mary E. Hensler,[b] Wdee Thienphrapa,[b] Quang N. Dam,[b] Victor Nizet,*[b]
and Yitzhak Tor*[a]
The emergence of virulent, drug-resistant bacterial strains cou-
pled with a minimal output of new pharmaceutical agents to
combat them makes this a critical time for antibacterial re-
search. Aminoglycosides are a well-studied, highly potent class
of naturally occurring antibiotics with scaffolds amenable to
modification, and therefore, they provide an excellent starting
point for the development of semisynthetic, next-generation
compounds. To explore the potential of this approach, we syn-
thesized a small library of aminoglycoside derivatives selective-
ly and minimally modified at one or two positions with a guani-
dine group replacing the corresponding amine or hydroxy
functionality. Most guanidino-aminoglycosides showed in-
creased affinity for the ribosomal decoding rRNA site, the cog-
nate biological target of the natural products, when compared
with their parent antibiotics, as measured by an in vitro fluo-
rescence resonance energy transfer (FRET) A-site binding assay.
Additionally, certain analogues showed improved minimum in-
hibitory concentration (MIC) values against resistant bacterial
strains, including methicillin-resistant Staphylococcus aureus
(MRSA). An amikacin derivative holds particular promise with
activity greater than or equal to the parent antibiotic in the
majority of bacterial strains tested.
Introduction
The discovery of antibiotics—small molecules of natural or syn-
thetic origin that specifically interfere with vital processes in
bacteria—can be viewed as one of the major medical break-
throughs of the 20th century. Indeed, antibiotics facilitated the
cure of previously untreatable life-threatening infectious dis-
eases. However, many of the originally identified antibiotics are
no longer clinically useful as they are compromised by bacteri-
al resistance mechanisms, which include modification of the
drug molecules, mutation of the molecular drug targets, or in-
creased cellular drug efflux by small-molecule transporters and
biofilm formation.[1] The emergence of pathogens resistant to
nearly all antibiotics in current use is of particular concern to
clinicians. While infections caused by Gram-positive organisms,
such as methicillin-resistant Staphylococcus aureus (MRSA),
remain a major problem worldwide, the emergence within the
last decade of multiple-drug-resistant Gram-negative organ-
isms, such as Acinetobacter baumannii, Pseudomonas aerugino-
sa, and Klebsiella pneumonia, is equally worrisome.[2] In fact,
Gram-negative resistance to drugs of last resort, such as coli-
stin, has become alarmingly more commonplace in the clinical
setting.[3] The widespread and frequently indiscriminate use of
antibiotics in human and veterinary medicine has further accel-
erated the emergence of these resistant, highly pathogenic
bacteria that can cause life-threatening infections.[4] Unfortu-
nately, the surge seen in the appearance of resistant bacteria
has not been met by a parallel development of new effective,
broad-spectrum antibiotics, and only two novel classes of anti-
bacterial agents, fluoroquinolones and oxazolidinones, have
been identified over the past few decades.[5] It is apparent,
therefore, that new potent antibiotics are required to comple-
ment or even replace currently used drugs, whose utility is
ever increasingly compromised by bacterial resistance.
ucts and synthetic libraries can, in principle, lead to the iden-
tification of potent novel agents.[6] Such screening strategies
can involve empirical, whole-cell testing or target-based in
vitro assays for known or underutilized, genomically identified
targets with the former approach having the potential to iden-
tify antibacterial agents with distinct modes of action.[7] Un-
fortunately, such efforts have resulted in minimal output and
lead compound discovery. Alternatively, modifications of exist-
ing and perhaps retired antibiotics can revive some of their
utility through systematic structural modifications.[8] While in-
cremental, such efforts rely on established bioactive scaffolds
targeting known bacterial pathways, with the prospective of
generating potent agents, which can potentially evade deacti-
vation by prevailing resistance mechanisms. Here, we address
the selective modification of aminoglycoside antibiotics,
a large family of natural products active primarily against
Gram-negative bacteria.[9]
Aminoglycosides are polycationic antibiotics, and most bind
to the 16S ribosomal A-site RNA, leading to diminished transla-
tional fidelity.[10] Being a particularly well-studied antibacterial
class, a wealth of information exists regarding their interactions
with their intracellular target and bacterial resistance mecha-
nisms.[1a,b,d,11] This vast knowledge makes the aminoglycoside
[a] R. J. Fair, Prof. Dr. Y. Tor
Department of Chemistry & Biochemistry
University of California, San Diego
9500 Gilman Drive, La Jolla, CA 92093 (USA)
E-mail: ytor@ucsd.edu
[b] Dr. M. E. Hensler, W. Thienphrapa, Q. N. Dam, Prof. Dr. V. Nizet
Department of Pediatrics
University of California, San Diego
9500 Gilman Drive, La Jolla, CA 92093 (USA)
E-mail: vnizet@ucsd.edu
Diverse approaches to the discovery of new antibiotics exist.
High-throughput screening campaigns of novel natural prod-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200150.
ChemMedChem 2012, 7, 1237 – 1244
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1237

V. Nizet, Y. Tor et al.
MED
scaffold an ideal starting point for the development of new,
potentially more effective antibiotics.[12] Indeed, semisynthetic,
second-generation aminoglycosides such as amikacin have
proven very successful in the clinic.[9] With this in mind, we set
out to make minor structural modifications to selected mem-
bers of these known antibiotics in an effort to retain or even
improve upon their affinity for the 16S A-site, while potentially
decreasing their susceptibility to the most prevalent modes of
bacterial deactivation.
using di-tert-butyl dicarbonate. The single primary alcohol of
(Boc)5tobramycin (17) was then selectively converted to a steri-
cally demanding sulfonate by treatment with 2,4,6-triisopropyl-
benzenesulfonyl chloride (TPSCl) in pyridine.[12f] Reflux in meth-
anolic ammonia then afforded 6’’-deoxy-6’’-amino(Boc)5tobra-
mycin (19). This three-step process, converting primary alco-
hols into amines, has been previously used in our laboratory
for the synthesis of other modified aminoglycosides.[14] Treat-
ment of the single free amine with 1,3-di-Boc-2-(trifluorome-
thylsulfonyl)guanidine in the presence of triethylamine gave
fully Boc-protected guanidino-aminoglycoside, 6’’-deoxy-6’’-
guanidino(Boc)7tobramycin (20). Acidic deprotection of all Boc
groups using a one to one mixture of trifluoroacetic acid (TFA)
and tri-iso-propyl silane (TIPS) in dichloromethane, followed by
HPLC purification, afforded the analytically pure 6’’-deoxy-6’’-
guanidinotobramycin (2).
A slightly different approach was employed for the synthesis
of 6’’-deoxy-6’’-guanidinoapramycin (16). Care had to be taken
in preparing the sulfonate to prevent activation of multiple hy-
droxy groups. This was achieved by using fewer equivalents of
the sulfonyl chloride (Step b, Scheme 2), in comparison with
the other aminoglycosides. In addition, unlike other aminogly-
coside derivatives, 6’’-deoxy-6’’-triisopropylbenzylsulfonyl(Boc)5-
apramycin (22) was found to degrade in refluxing methanolic
ammonia. Instead, it was converted to the amino intermediate
via a two-step process wherein the sulfonyl functionality was
first substituted for an azide using sodium azide and then sub-
sequently reduced in a palladium-catalyzed hydrogenation to
give 6’’-deoxy-6’’-guanidino(Boc)7apramycin (23).
Here we disclose the synthesis of a small focused library of
aminoglycoside derivatives selectively modified at one or two
positions. We strategically replace amine or hydroxy functional-
ities with a guanidine group in tobramycin, amikacin, kanamy-
cin A, neomycin B neamine, paromomycin, and apramycin (Fig-
ure 1).[12a,c] Most of the newly synthesized guanidino-aminogly-
cosides displayed enhanced affinity for the ribosomal A-site,
the biological target of the parent derivatives, as determined
by an in vitro fluorescence resonance energy transfer (FRET)-
based binding assay. The results of antibacterial tests on a di-
verse collection of regular and resistant strains illustrate that
certain analogues exhibit improved potency against resistant
strains, including MRSA. An amikacin analogue shows particu-
lar promise with activities greater or equal to those of the
parent antibiotic in the majority of strains tested.
Results
Design strategy
The bacterial A-site is a highly discriminating RNA target that is
not tolerant of major structural changes to its cognate li-
gands.[12n,13] Therefore, we decided to selectively and strategi-
cally functionalize aminoglycosides at positions that are less
likely to perturb binding and, at the same time, are synthetical-
ly accessible.[12a,c] A relatively small modification, which would
retain or enhance the overall charge of the RNA-targeting anti-
biotic, could be achieved by replacing a hydroxy or amine
group with a guanidine functionality. In contrast to amines,
the planar guanidine functional group is highly basic and can
participate in well-defined directional hydrogen bonds. To
probe this strategy, we derivatized several aminoglycoside anti-
biotics, including neamine, kanamycin A, tobramycin, paromo-
mycin, neomycin B, amikacin, and apramycin, by converting se-
lected primary alcohols into guanidine groups, or turning an
existing aminomethyl group into the corresponding guanidine
derivative. We hypothesized that beyond yielding a greater af-
finity for the A-site, functional group changes at some sites
could potentially lead to decreased recognition by aminoglyco-
side-modifying enzymes, one of the major bacterial resistance
mechanisms.
A third protocol was used for selectively converting amino-
methyl groups in aminoglycosides to the corresponding guani-
dine derivatives, relying on their higher nucleophilicity com-
pared with the other more sterically hindered amines. Treat-
ment of unprotected tobramycin with sub-stoichiometric
quantities of 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine
followed by deprotection in a one to one mixture of TFA in di-
chloromethane provided desired derivative 3 (Scheme 3). Simi-
lar protocols were applied to amikacin and neamine. Note, all
guanidino-aminoglycoside derivatives were first converted to
their free-base form by exposure to a strong basic anion (OHÀ)
exchange resin (Monosphere 550A, Dowex) prior to their eval-
uation in any A-site binding assays or antibacterial experi-
ments.
Affinity for the bacterial 16S A-site RNA construct
To determine the affinity of all derivatives to the bacterial 16S
A-site, we used a modified version of a FRET-based assay that
was previously developed in our lab.[15] In this assay, a coumar-
in–aminoglycoside conjugate placeholder binds to a Dy-547-la-
beled 16S A-site construct. Coumarin acts as a FRET donor to
its matched Dy-547 acceptor. The affinity of unlabeled ligands
for the A-site can be measured in a competition experiment,
where the compound of interest is titrated in and displaces
the coumarin–aminoglycoside placeholder, resulting in a de-
creased sensitized acceptor emission. Different coumarin–ami-
Synthesis
A general synthetic approach for the conversion of aminogly-
coside primary alcohols to guanidinium groups is illustrated
using tobramycin (1) as an example (Scheme 1). First, all
amines were globally tert-butyloxycarbonyl (Boc)-protected
1238
www.chemmedchem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 1237 – 1244

Antibiotic Guanidinylated Aminoglycosides
MED
Figure 1. Parent aminoglycosides and guanidino-aminoglycosides. The guanidine and 2-deoxystreptamin (2-DOS) moieties are highlighted in bold.
noglycoside conjugates can be used to cover distinct affinity
ranges of putative competitor antibiotics.
Initial titrations were performed with a coumarin–kanamycin
derivative, the lowest affinity placeholder aminoglycoside con-
ChemMedChem 2012, 7, 1237 – 1244
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1239

V. Nizet, Y. Tor et al.
MED
improvements compared with their parent com-
pounds, which were the weakest binders tested. 5’’-
Deoxy-5’’-guanidinoneomycin (10) and 5’’,6’-dideoxy-
5’’,6’-diguanidinoparomomycin (14) both showed
higher affinities for the A-site than neomycin (9),
which was the highest affinity binder among the nat-
ural aminoglycosides. The only exception to this
trend was 6’’-deoxy-6’’-guanidinoapramycin (16),
which has a similar affinity to the unmodified antibi-
otic. Most of the aminoglycoside derivatives, where
amines at primary carbon centers are replaced with
guanidinium groups, show comparable affinity to
their parent antibiotics. This is not surprising since
the overall positive charge is unlikely to drastically
change; this is in contrast to derivatives where pri-
mary alcohols were converted to a guanidine group,
which would have increased positive character.
Antibacterial activities
While the structure–activity relationship (SAR) data
generated is intriguing, the ultimate test is the actual
efficacy against bacteria. To assess the relative anti-
bacterial activities of the synthetic derivatives, mini-
mum inhibitory concentration (MIC) values of both
the modified and parent antibiotics were determined
against a wide variety of bacterial strains (Table 3).
The compounds were first tested against the antibac-
terial-agent-susceptible
control
E. coli
strain
ATCC25922. We also used the clinically relevant
Gram-positive MRSA strain ATCC33591. Most of the
guanidino-aminoglycosides showed improved effica-
cy over their respective parent aminoglycosides
against MRSA. The two compounds showing the
greatest improvements were 6’’-deoxy-6’’-guanidino-
kanamycin A (8) with an MIC value dropping to
3.125–6.25 mgmLÀ1 from 25 mgmLÀ1 for the parent
compound, and 6’’-deoxy-6’’-guanidinoamikacin (5),
which improved to 3.125 mgmLÀ1 from a parent MIC
value of 12.5–25 mgmLÀ1.
Scheme 1. Primary alcohol to guanidinium conversions. Reagents and conditions:
a) Boc2O, Et3N, H2O, DMF, 558C, 4–6 h, 91–97%; b) TPSCl, pyridine, RT, 2 days, 51–66%;
c) NH3, MeOH, 808C, 2–2.5 days, 57–93%; d) 1,3-di-Boc-2-(trifluoromethylsulfonyl)guani-
dine, Et3N, CH2Cl2, MeOH, RT, 3 days, 71–85%; e) TFA, TIPS, CH2Cl2, RT, 2.5–3 h, 79–96%.
All synthetic derivatives were also tested against
a variety of antibacterial-drug-resistant, Gram-nega-
tive, clinical isolates including strains of P. aeruginosa,
K. pneumoniae, and A. baumannii. Compound
5
showed the broadest spectrum activity, with potency
jugate (Table 1). Potent A-site binders, such as neomycin and greater or equal to its parent compound in six out of the eight
paromomycin derivatives 10 and 14, respectively, were titrated strains tested. For example, the MIC values of compound 5
against a coumarin–neomycin derivative (Table 2). In all cases, against the two P. aeruginosa strains were 1.56 mgmLÀ1 and
titration curves were generated by plotting the fractional fluo- 3.125 mgmLÀ1, respectively, compared with 3.125 mgmLÀ1 and
rescence saturation of the acceptor against the concentration 6.25 mgmLÀ1 for the parent compound—a twofold increase in
of the molecule of interest (for a representative example, see potency in both cases. With the exception of the aforemen-
Figure 2 and the Supporting Information).
tioned derivative, the guanidino-aminoglycosides did not per-
In general, derivatives with primary alcohols converted to form as well in general against these strains. Surprisingly, 5’’,6’-
guanidinium groups were found to have significantly higher dideoxy-5’’,6’-diguanidinoparomomycin (14) showed a vast im-
affinities for the A-site in comparison with their parent amino- provement against the A. baumannii strain GNR1753 compared
glycosides. In particular, 6’’-deoxy-6’’-guanidinoamikacin (5) with paromomycin (13), dropping from an MIC value of greater
and 6’’-deoxy-6’’-guanidinokanamycin A (8) showed marked than 50 mgmLÀ1 to 6.25–12.5 mgmLÀ1
.
1240
www.chemmedchem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 1237 – 1244

Antibiotic Guanidinylated Aminoglycosides
MED
Scheme 3. Amine to guanidinium conversions. Reagents and conditions:
a) 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine, Et3N, CH2Cl2, MeOH, RT,
5 days; b) TFA, TIPS, CH2Cl2, RT, 2 h, 12–22% (two steps).
Table 1. Initial evaluation of inhibitory concentrations determined by ti-
tration against the coumarin–kanamycin derivative.[a]
Compd
IC50 [mm]
Tobramycin (1)
1.6Æ0.2
1.0Æ0.1
1.6Æ0.1
6.7Æ0.7
1.4Æ0.04
3.2Æ0.2
7.0Æ0.7
1.8Æ0.1
4.5Æ0.4
4.4Æ0.5
1.7Æ0.1
1.9Æ0.2
6’’-Deoxy-6’’-guanidinotobramycin (2)
6’-Guanidinotobramycin (3)
Amikacin (4)
6’’-Deoxy-6’’-guanidinoamikacin (5)
6’,g-Diguanidinoamikacin (6)
Kanamycin A (7)
6’’-Deoxy-6’’-guanidinokanamycin A (8)
Neamine (11)
6’-Guanidinoneamine (12)
Apramycin (15)
6’’-Deoxy-6’’-guanidinoapramycin (16)
[a] Conditions: A-site RNA (1 mm), kanamycin–coumarin (0.53 mm), cacody-
late buffer pH 7.0 (20 mm), NaCl (100 mm), EDTA (0.5 mm). Values repre-
sent the mean ÆSD of n=3–5 experiments.
Table 2. Inhibitory concentrations of potent A-site binders determined
by titration against the coumarin–neomycin derivative.[a]
Compd
IC50 [mm]
Neomycin (9)
5’’-Deoxy-5’’-guanidinoneomycin (10)
Paromomycin (13)
4.4Æ0.3
1.5Æ0.1
19.6Æ2.0
1.8Æ0.1
Scheme 2. 6’’-Deoxy-6’’guanidinoapramycin synthesis. Reagents and condi-
tions: a) Boc2O, Et3N, H2O, DMF, 558C, 8 h, 92%; b) TPSCl, pyridine, RT,
1.5 days, 31%; c) NaN3, DMF, 558C, 2 days; d) Pd/C, H2, MeOH, RT, overnight,
80% (two steps); e) 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine, Et3N,
CH2Cl2, MeOH, RT, 3 days, 74%; f) TFA, TIPS, CH2Cl2, RT, 2 h, 78%.
5’’,6’-Dideoxy-5’’,6’-diguanidinoparomomycin (14)
[a] Conditions: A-site RNA (1 mm), neomycin–coumarin (0.53 mm), cacody-
late buffer pH 7.0 (20 mm), NaCl (100 mm), EDTA (0.5 mm). Values repre-
sent the mean ÆSD? of n=3 experiments.
provement over their parent compounds when tested for anti-
bacterial activity. In contrast, analogues with primary alcohols
converted to guanidinium groups, particularly those of the ka-
namycin class, consistently showed marked increases in A-site
affinity, which was coupled, in most cases, with improved anti-
bacterial activity.
Discussion
Aminoglycoside derivatives with guanidinylated aminomethyl
groups or primary alcohols selectively converted to guanidini-
um groups were synthesized. Analogues with guanidinylated
amines showed modest, if any, improvement in affinity for the
bacterial A-site RNA. These analogues did not show any im-
ChemMedChem 2012, 7, 1237 – 1244
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1241

V. Nizet, Y. Tor et al.
MED
Table 3. Inhibitory activities of both the modified and parent compounds against a panel of bacterial strains.[a]
Compd
E. coli
S. aureus
P. aeruginosa
P. aeruginosa
K. pneumoniae
K. pneumoniae
A. baumannii
A. baumannii
(ATCC25922)
(ATCC33591)
(PA01)
(GNR0697)
(GNR0713)
(GNR1100)
(GNR0717)
(GNR1753)
1
2
3
4
5
6
7
8
0.78–1.56
3.125–6.25
25–50
1.56–3.125
1.56–3.125
>50
6.25
6.25
0.78
1.56
6.25
12.5
6.25
12.5–25
3.125
1.56
25
12.5–25
3.125
>50
0.39
6.25
50
3.125
1.56
>50
0.78
25
>50
6.25
3.125
>50
>50
>50
12.5–25
>50
>50
>50
12.5
>50
>50
>50
25
>50
>50
>50
25
6.25–12.5
0.78
1.56
25
0.39
0.39
>50
12.5
1.56
3.125
6.25
12.5
12.5
0.39–0.78
3.125–6.25
0.39
6.25
6.25
25
25–50
>50
>50
>50
>50
>50
>50
>50
>50
>50
25
>50
>50
>50
6.25
6.25
3.125
>50
>50
>50
6.25
25
>50
>50
>50
>50
6.25
3.125–6.25
1.56–3.125
0.78–1.56
12.5–25
6.25–12.5
25–50
9
3.125–6.25
6.25–12.5
>50
25–50
6.25
10
13
14
15
16
6.25
>50
6.25–12.5
0.39
>50
50
>50
0.39
[a] Minimum inhibitory concentration (MIC) values [mgmLÀ1]. MIC value equal to parent compound (italics); MIC value lower than parent compound (bold).
Compounds 11 and 12 showed no activity.
ly rationalized by examining the crystal structure of the parent
aminoglycoside. A crystal structure of apramycin (15) with the
16S A-site shows that the 6’’-hydroxy group forms a unique hy-
drogen-bonding interaction in which it functions as both
a donor and an acceptor along the edge of the G1491–C1409
base pair.[11a] Disrupting these interactions could explain why
an alteration at that site was not as well tolerated (Figure 3).
The lack of improvement in binding seen for derivatives
with modifications to the 6’ amine is not entirely surprising.
We recognize that the guanidine groups, being highly basic,
can also somewhat attenuate the pKa of neighboring ammoni-
um groups, leading to derivatives with similar overall char-
ge.[12a] Additionally, all of these derivatives are modified at the
6’ amine, which is known to make critical contacts with A1408
within the A-site binding pocket.[11b,e] 6’,g-Diguanidinoamikacin
(6) was the only derivative of this class to show statistically sig-
nificant improvements in affinity compared with the parent
compound. However, 6’,g-diguanidinoamikacin (6) still showed
weaker affinity than 6’’-deoxy-6’’-guanidinoamikacin (5), the
Figure 2. Representative A-site competitive displacement curve.
corresponding derivative with a guanidinium group replacing
The marked increase in the affinity of the kanamycin class of
derivatives upon replacing the 6’’ hydroxy with a guanidinium
group is interesting since the 6’’ hydroxy group in the parent
compounds in not involved in hydrogen bonding with the A-
site, at least not in the published co-crystal structures, but the
6’’ hydroxy group is in close proximity to U1406 and C1407.
This could suggest that the new guanidine group in these de-
rivatives, in addition to its overall electrostatic contribution,
might be extended far enough to make new contacts with
these RNA nucleobases (Figure 3).[11b,c,e] The increased binding
affinity of 5’’,6’-dideoxy-5’’,6’-diguanidinoparomomycin (14) is
most likely due to the increased overall charge and perhaps
also replacement of the hydroxy-based hydrogen bonds ob-
served for the parent molecule paromomycin (13), which is
known to make A-site contacts at both the 5’’ and 6’ posi-
tions,[9d] with stronger (charged) hydrogen bonds. The affinity
of 6’’-deoxy-6’’-guanidinoapramycin (16) can also be potential-
an alcohol.
When analyzing the potency and MIC values, it is important
to remember that affinities to the A-site do not necessarily cor-
relate with antibacterial potency.[16] It is interesting to note
that all but two of the synthesized compounds, 6’’-deoxy-6’’-
guanidinoamikacin (5) and 6’’-deoxy-6’’-guanidinokanamycin A
(8), showed inferior antibacterial activity against the control
E. coli strain ATCC25922, suggesting that improvement in activ-
ity against resistant strains is at least partially due to overcom-
ing bacterial resistance mechanisms. This makes the broad-
spectrum improvement of 6’’-deoxy-6’’-guanidinoamikacin (5)
a particularly intriguing observation given that amikacin is
a semi-synthetic aminoglycoside structurally derived from ka-
namycin A with an amino 2-hydroxybutyryl (AHB) side chain,
which lowers its susceptibility to aminoglycoside-modifying en-
zymes. It is possible that the AHB and guanidinium modifica-
tions operate synergistically to further decrease its affinity for
1242
www.chemmedchem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 1237 – 1244

Antibiotic Guanidinylated Aminoglycosides
MED
deoxy-6’’-guanidinoamikacin (5) proved to be partic-
ularly promising; showing equal or better activity
than amikacin (4) against almost all of the bacterial
strains tested, including several clinical isolates.
Experimental Section
Materials: Unless otherwise specified, materials pur-
chased from commercial suppliers were used without
further purification. All aminoglycosides were obtained
from Sigma–Aldrich as their sulfate salts. Tobramycin sul-
fate was converted to the trifluoroacetic acid (TFA) salt
by first passing it over an anion (OHÀ) exchange resin
(Monosphere 550A, Dowex) to get the free base, then
stirring in 0.1% TFA/H2O. Neamine hydrochloride was
made by methanolysis of commercially available neomy-
cin sulfate.[17] 1,3-Di-Boc-2-(trifluoromethylsulfonyl)guani-
dine was synthesized according to an established proce-
dure.[18] Anhydrous NH3 was purchased from Airgas. All
other anhydrous solvents and reagents, and ion ex-
change resins were purchased from Sigma–Aldrich. NMR
solvents were purchased from Cambridge Isotope Labo-
ratories (Andover, MA, USA). The Dy-547-labeled A-site
construct was purchased from Thermo Scientific and pu-
rified by gel electrophoresis. Kanamycin–coumarin and
neomycin–courmarin conjugates were synthesized and
purified according to established procedures.[15] Chemi-
cals for preparing buffer solutions (enzyme grade) were
purchased from Fisher Biotech. Autoclaved water was
used in all fluorescence titrations. Mueller–Hinton broth
used for sensitivity testing was obtained from Hardy Di-
agnostics (Santa Maria, CA, USA). Polystyrene 96-well mi-
croplates for MIC testing were purchased from Corning
Inc. (Corning, NY, USA). Bacterial strains for sensitivity
testing included two reference strains from the American
Type Culture Collection (Manassas, VA, USA): hospital-as-
sociated MRSA strain 33591 rendered resistant to rifami-
cin by serial passage and E. coli strain 25922. P. aerugino-
sa strain PA01 was used as a general antibiotic-sensitive
P. aeruginosa strain.[19] Other Gram-negative strains used
were clinical isolates obtained from a tertiary academic
Figure 3. Proposed interactions between guanidinium groups of guanidino-aminoglyco-
sides and A-site RNA bases. a) 6’’-Deoxy-6’’-guanidinokanamycin A (8) interactions with
U1406 and C1407 and 6’’-deoxy-6’’-guanidinoapramycin (16) interactions with C1409 and
G1491 (in bold). b) Kanamycin A (7) crystal structure with 6’’ OH modification site and
potential new contacts of 6’’-deoxy-6’’-guanidinokanamycin A (8) highlighted.
modifying enzymes. Derivatives where amines were guanidiny-
hospital in the New York metropolitan area; these were: P. aerugi-
nosa strain GNR0697 (blood isolate), K. pneumoniae strain GNR0713
(blood isolate), K. pneumoniae strain GNR1100 (respiratory isolate),
A. baumannii strain GNR0717 (urine isolate), and A. baumannii
strain GNR1753 (wound isolate).
lated generally show poor antibacterial activity, in all cases in-
ferior to the parent aminoglycosides, which could suggest that
the amine at position 6’ plays an important role in antibacterial
activity. This is somewhat surprising, since the majority of ami-
noglycoside-modifying enzymes make alterations to ring I in-
cluding the AAC(6’) enzyme which directly modifies 6’ amine-
s.[1a] In contrast, 5’’,6’-dideoxy-5’’,6’-diguanidino-paromomycin
(14), which contains a 6’ hydroxy functionality, exhibits several
improved antibacterial activities.
Instrumentation: NMR spectra were recorded on Varian Mercury
300 and 400 MHz, Varian VX 500 MHz, and Jeol ECA 500 MHz spec-
trometers. All two-dimensional NMR spectra were recorded on
a Jeol ECA 500 MHz spectrometer and processed using the Delta
NMR Processing and Control Software (version 4.3.6). Mass spectra
(MS) were recorded at the University of California, San Diego
Chemistry and Biochemistry Mass Spectrometry Facility, utilizing an
Agilent 6230 HR-ESI-TOF mass spectrometer. Reverse-phase HPLC
(Vydac C18 column) purification and analysis were carried out
using an Agilent 1200 series instrument. Products were lyophilized
utilizing a Labconco FreeZone 2.5 freeze drier. Steady-state fluores-
cence experiments were carried out in a microfluorescence cell
with a path length of 1.0 cm (Hellma GmH & Co KG, Mullenheim,
Germany) on a Jobin Yvon Horiba FluoroMax-3 luminescence spec-
trometer. A background spectrum (buffer) was subtracted from
each sample. A VersaMax plate reader (Molecular Devices, Moun-
Conclusions
A series of guanidino-aminoglycosides, selectively modified
aminoglycosides, was synthesized. In almost all cases, these
derivatives have proven to be superior binders of the bacterial
A-site compared with their parent antibiotics when tested in
an in vitro FRET-based assay. Some of the compounds showed
potent antibacterial activity, frequently performing as well or
even better than the parent aminoglycosides. In particular, 6’’-
ChemMedChem 2012, 7, 1237 – 1244
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1243

V. Nizet, Y. Tor et al.
MED
[10] a) J. Davies, L. Gorini, B. D. Davis, Mol. Pharmacol. 1965, 1, 93–106; b) J.
Davies, B. D. Davis, J. Biol. Chem. 1968, 243, 3312–3316; c) D. Moazed,
H. F. Noller, Nature 1987, 327, 389–394; d) A. P. Carter, W. M. Clemons,
D. E. Brodersen, R. J. Morgan-Warren, B. T. Wimberly, V. Ramakrishnan,
Nature 2000, 407, 340–348; e) J. M. Ogle, D. E. Brodersen, W. M. Clem-
ons, M. J. Tarry, A. P. Carter, V. Ramakrishnan, Science 2001, 292, 897–
902.
[11] a) Q. Vicens, E. Westhof, Structure 2001, 9, 647–668; b) Q. Vicens, E.
Westhof, Chem. Biol. 2002, 9, 747–755; c) B. FranÅois, R. J. M. Russell,
J. B. Murray, F. Aboul-ela, B. Masquida, Q. Vicens, E. Westhof, Nucleic
Acids Res. 2005, 33, 5677–5690; d) Q. Han, Q. Zhao, S. Fish, K. B. Simon-
sen, D. Vourloumis, J. M. Froelich, D. Wall, T. Hermann, Angew. Chem.
2005, 117, 2754–2760; Angew. Chem. Int. Ed. 2005, 44, 2694–2700; e) J.
Kondo, B. FranÅois, R. J. M. Russell, J. B. Murray, E. Westhof, Biochimie
2006, 88, 1027–1031.
[12] For examples of synthetic and semi-synthetic aminoglycoside ana-
logues see: a) H. Wang, Y. Tor, J. Am. Chem. Soc. 1997, 119, 8734–8735;
b) H. Wang, Y. Tor, Bioorg. Med. Chem. Lett. 1997, 7, 1951–1956; c) H.
Wang, Y. Tor, Angew. Chem. 1998, 110, 117–120; Angew. Chem. Int. Ed.
1998, 37, 109–111; d) K. Michael, Y. Tor, Chem. Eur. J. 1998, 4, 2091–
2098; e) Y. Tor, T. Hermann, E. Westhof, Chem. Biol. 1998, 5, R277–R283;
f) K. Michael, H. Wang, Y. Tor, Bioorg. Med. Chem. 1999, 7, 1361–1371;
g) K. Michael, Y. Tor, RNA-Binding Antibiotics (Eds.: R. Schroeder, M.
Wallis), Landes Bioscience, Austin, 2001, pp. 130–145; h) J. Haddad, L. P.
Kotra, B. Llano-Sotelo, C. Kim, E. F. Azucena, Jr., M. Liu, S. B. Vakulenko,
C. S. Chow, S. Mobashery, J. Am. Chem. Soc. 2002, 124, 3229–3237; i) Y.
Tor, ChemBioChem 2003, 4, 998–1007; j) T. Hermann, Y. Tor, Expert Opin.
Ther. Pat. 2005, 15, 49–62; k) F. Zhao, Q. Zhao, K. F. Blount, Q. Han, Y.
Tor, T. Hermann, Angew. Chem. 2005, 117, 5463–5468; Angew. Chem. Int.
Ed. 2005, 44, 5329–5334; l) M. Hainrichson, V. Pokrovskaya, D. Shallom-
Shezifi, M. Fridman, V. Belakhov, D. Shachar, S. Yaron, T. Bassov, Bioorg.
Med. Chem. 2005, 13, 5797–5807; m) K. Blount, Y. Tor, ChemBioChem
2006, 7, 1612–1621; n) Y. Tor, Biochimie 2006, 88, 1045–1051; o) V. Pok-
rovskaya, V. Belakhov, M. Hainrichson, S. Yaron, T. Baasov, J. Med. Chem.
2009, 52, 2243–2254; p) S. Bera, G. G. Zhanel, F. Schweizer, Bioorg. Med.
Chem. Lett. 2010, 20, 3031–3035; q) J. Revuelta, T. Vacas, F. Corzana, C.
Gonzales, A. Bastida, J. L. Asensio, Chem. Eur. J. 2010, 16, 2986–2991;
r) V. Pokrovskaya, I. Nudelman, J. Kandasamy, T. Baasov, Methods Enzy-
mol. 2010, 478, 437–462; s) L. Yang, X. S. Ye, Curr. Top. Med. Chem.
2010, 10, 1898–1926; t) R. B. Yan, M. Yuan, Y. Wu, X. You, X. S. Ye,
Bioorg. Med. Chem. 2011, 19, 30–40; u) S. Bera, G. G. Zhanel, F. Schweiz-
er, Carbohydr. Res. 2011, 346, 560–568; v) L. McCoy, Y. Xie, Y. Tor, WIREs
RNA 2011, 2, 209–232; w) T. Cottin, C. Pyrkotis, C. I. Stathakis, I. Mavri-
dis, I. A. Katsoulis, P. Anastasopoulou, G. Kythreoti, A. L. Zografos, V. R.
Nahmias, A. Papakyriakou, D. Vourloumis, ChemBioChem 2011, 12, 71–
87; x) V. Udumula, M. Chittapragada, J. B. Marble, D. L. Dayton, Y. W.
Ham, Bioorg. Med. Chem. Lett. 2011, 21, 4713–4715; y) J. Szychowski, J.
Kondo, O. Zahr, K. Auclair, E. Westhof, S. Hanessian, J. W. Keillor, Chem-
MedChem 2011, 6, 1961–1966; z) L. Guan, M. D. Disney, ACS Chem. Biol.
2012, 7, 73–86.
tain View, CA, USA) set at 600 nm wavelength was used for MIC
assays.
Synthesis: Full synthetic procedures and characterization of all
compounds can be found in the Supporting Information.
Desalting: Guanidino-aminoglycoside·TFA (up to 40 mg) was dis-
solved in autoclaved H2O (0.6 mL) in a sterile eppendorf tube.
Monosphere 550 A (75 mg) was added, and the suspension was
shaken lightly on a Fisher Vortex Genie 2 overnight. The resin was
removed by centrifugal filtration and washed twice with auto-
claved H2O. The desalted solutions were lyophilized, and the re-
moval of TFA counterions was confirmed by 13C NMR spectroscopy.
A-Site Binding assay: Aminoglycoside titration procedures, bind-
ing curves, and the curve fitting equation can be found in the Sup-
porting Information.
Minimum inhibitory concentration (MIC) determinations: MIC
values for aminoglycosides were determined using broth micro-
dilution in accordance with Clinical Laboratory Standards Institute
guidelines.[20]
Parent aminoglycoside crystal structures: PyMOL representations
of all aminoglycoside/A-site crystal structures with modification
sites and relevant bases highlighted can be found in the Support-
ing Information.
Acknowledgements
The authors thank the US National Institutes of Health for gener-
ous financial support (grant no.: GM069773 to Y.T., HD071600 to
V.N.) and Dr. Yun Xie (AAAS Science & Technology Policy Fellow,
US National Institutes of Health) for advice regarding the A-site
binding assay.
Keywords: amikacin · aminoglycosides · antibiotics · A-site
RNA · methicillin-resistant Staphylococcus aureus (MRSA)
[1] a) M. P. Mingeot-Leclercq, Y. Glupczynski, P. M. Tulkens, Antimicrob.
Agents Chemother. 1999, 43, 727–737; b) K. Poole, J. Pharm. Pharmacol.
2001, 53, 283–294; c) K. Lewis, Antimicrob. Agents Chemother. 2001, 45,
999–1007; d) S. B. Vakulenko, S. Mobashery, Clin. Microbiol. Rev. 2003,
16, 430–450; e) G. F. Liou, S. Yoshizawa, P. Courvalin, M. Galimand, J.
Mol. Biol. 2006, 359, 358–364.
[2] a) E. Durante-Mangoni, R. Zarrilli, Future Microbiol. 2011, 6, 407–422;
b) A. Sꢁnchez, S. Gattarello, J. Rello, Semin. Respir. Crit. Care Med. 2011,
32, 151–158.
[3] J. Li, C. R. Rayner, R. L. Nation, R. J. Owen, D. Spelman, K. E. Tan, L. Lio-
lios, Antimicrob. Agents Chemother. 2006, 50, 2946–2950.
[4] a) T. Strateva, D. Yordanov, J. Med. Microbiol. 2009, 58, 1133–1148;
b) N. L. Werner, M. T. Hecker, A. K. Sethi, C. J. Donskey, BMC Infect. Dis.
2011, 11, 187–193.
[5] a) H. Liu, S. G. Mulholland, Am. J. Med. 2005, 118 (Suppl. 7A), 14S–20S;
b) K. L. Leach, S. J. Brickner, M. C. Noe, P. F. Miller, Ann. N. Y. Acad. Sci.
2011, 1222, 49–54.
[6] a) D. J. Newman, G. M. Cragg, K. M. Snader, J. Nat. Prod. 2003, 66, 1022–
1037; b) D. J. Newman, G. M. Cragg, J. Nat. Prod. 2007, 70, 461–477.
[7] a) D. J. Payne, M. N. Gwynn, D. J. Holmes, D. L. Pompliano, Nat. Rev.
Drug Discovery 2007, 6, 29–40; b) W. R. J. D. Galloway, A. Bender, M.
Welch, D. R. Spring, Chem. Commun. 2009, 2446–2462; c) J. R. Miller,
G. L. Waldrop, Expert Opin. Drug Discovery 2010, 5, 145–154.
[8] a) J. G. Silva, I. Carvalho, Curr. Med. Chem. 2007, 14, 1101–1119; b) M. E.
Falagas, A. P. Grammatikos, A. Michalopoulos, Expert Rev. Anti-Infect.
Ther. 2008, 6, 593–600.
[13] K. F. Blount, F. Zhao, T. Hermann, Y. Tor, J. Am. Chem. Soc. 2005, 127,
9818–9829.
[14] A. V. Dix, L. Fischer, S. Sarrazin, C. P. H. Redgate, J. D. Esko, Y. Tor, Chem-
BioChem 2010, 11, 2302–2310.
[15] a) Y. Xie, A. V. Dix, Y. Tor, J. Am. Chem. Soc. 2009, 131, 17605–17614;
b) Y. Xie, A. V. Dix, Y. Tor, Chem. Commun. 2010, 46, 5542–5544.
[16] M. Kaul, C. M. Barbieri, D. S. Pilch, J. Am. Chem. Soc. 2006, 128, 1261–
1271.
[17] J. D. Dutcher, M. N. Donin, J. Am. Chem. Soc. 1952, 74, 3420–3422.
[18] T. J. Baker, M. Tomioka, M. Goodman, Org. Synth. 2002, 78, 91–94; Org.
Synth. 2004, 10, 266–269.
[19] W. M. Huston, M. P. Jennings, A. G. McEwan, Mol. Microbiol. 2002, 45,
1741–1750.
[20] Performance Standards for Antimicrobial Susceptibility Testing, 19th Infor-
mational Supplement M100-S19, Clinical and Laboratory Standards
Institute, Wayne, PA, USA, 2008.
Received: March 19, 2012
[9] R. S. Edson, C. L. Terrell, Mayo Clin. Proc. 1999, 74, 519–528.
Published online on May 25, 2012
1244
www.chemmedchem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 1237 – 1244