Neomycin B-cyclen conjugates and their Zn(II) complexes as RNA-binding agents

Article abstract of DOI:10.1016/j.jinorgbio.2015.11.029

Three new conjugates featuring the aminoglycoside antibiotic neomycin B linked to the 1,4,7,10-tetraazacyclododecane (cyclen) macrocycle via alkyl chains of varying lengths were synthesized from suitably protected derivatives of these precursors via conve

Full text of DOI:10.1016/j.jinorgbio.2015.11.029

Neomycin B-cyclen conjugates and their Zn(II) complexes as RNA binding
agents
Bopha Kong, Tanmaya Joshi, Matthew J. Belousoff, Yitzhak Tor, Bim
Graham, Leone Spiccia
PII:
DOI:
Reference:
S0162-0134(15)30130-6
doi: 10.1016/j.jinorgbio.2015.11.029
JIB 9860
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
1 October 2015
9 November 2015
30 November 2015
Please cite this article as: Bopha Kong, Tanmaya Joshi, Matthew J. Belousoff, Yitzhak
Tor, Bim Graham, Leone Spiccia, Neomycin B-cyclen conjugates and their Zn(II)
complexes as RNA binding agents, Journal of Inorganic Biochemistry (2015), doi:
1
0.1016/j.jinorgbio.2015.11.029
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ACCEPTED MANUSCRIPT
Neomycin B-Cyclen Conjugates and their Zn(II)
Complexes as RNA Binding Agents
a
a*
b
c
d*
Bopha Kong, Tanmaya Joshi, Matthew J. Belousoff, Yitzhak Tor, Bim Graham, and Leone
a*
Spiccia
a) School of Chemistry, Monash University, Victoria 3800, Australia.
b) Department of Microbiology, Monash University, Victoria 3800, Australia.
c) Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman
Drive, La Jolla, CA, 92093-0332, USA.
d) Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic 3052, Australia.
“
In memory of Professor Graeme Hanson and his contributions to Bioinorganic Chemistry and
Electron Paramagnetic Resonance”
*
Corresponding authors:
tanmaya.joshi@monash.edu; bim.graham@monash.edu; leone.spiccia@monash.edu.
Fax: +61 3 9905 4597 (T.J. and L.S.); +61 3 9903 9582 (B.G.)
Key words: aminoglycoside derivatives, antibiotics, 1,4,7,10-tetraazacyclodecane, RNA, zinc(II)
complexes
1

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Abstract
Three new conjugates featuring the aminoglycoside antibiotic neomycin B linked to the 1,4,7,10-
tetraazacyclododecane (cyclen) macrocycle via alkyl chains of varying lengths were synthesized from
suitably protected derivatives of these precursors via conventional peptide coupling protocols. The final
1
products were characterized by H NMR spectroscopy, mass spectrometry, and elemental analysis.
FRET-based measurements examining the ability of the compounds to displace coumarin-labelled
kanamycin A or neomycin B from Dy547-labelled prokaryotic ribosomal A-site RNA revealed that
they bind to the A-site with slightly higher affinities than the parent aminoglycoside (e.g., IC at pH 7
5
0
=
1.42–2.30 µM vs. 2.35 µM for neomycin B). This is attributed to the higher overall positive charge of
the conjugates, resulting from protonation of the macrocylic amines. Consistent with a predominantly
electrostatic mode of interaction, the binding affinities of the conjugates were found to increase with
decreasing pH, reflecting a greater degree of protonation at lower pH. The zinc(II) complexes of the
neomycin B-cyclen conjugates were found to bind to A-site RNA with even higher affinities (IC =
50
0
.85–1.32 µM), due to the Zn(II)-cyclen motif forming coordinative (and/or electrostatic) interactions
with the uracil bases and/or phosphate groups within the A-site. These results highlight the potential for
the nucleic acid-binding properties of aminoglycosides to be tuned via the covalent attachment of metal
complexes, which could ultimately prove useful to the development of new anti-bacterial and anti-viral
agents.
2

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Introduction
Aminoglycosides are naturally occurring compounds that consist of an aminocyclitol ring linked to
1
aminosugars. The discovery of the antibiotic activity of these compounds and the seminal findings on
their interactions with RNA have together opened up new avenues in the design of small molecule-
1
-11
based RNA-targeting therapeutics.
Because of their polycationic nature (arising from the presence
of multiple protonated amine groups), combined with the conformational “plasticity” of RNA, the
1
2
aminoglycosides are fairly promiscuous RNA binders. Their bactericidal activity, primarily limited to
aerobic Gram-negative bacteria, derives from their ability to bind to the so-called “A-site” of the
decoding region (i.e., the site of codon-anticodon recognition) of bacterial 16S ribosomal RNA within
the 30S ribosomal subunit (Figure 1), which leads to a loss of translational fidelity during protein
2
, 13-15
synthesis.
However, studies have shown that aminoglycosides can also bind to a number of RNA
targets with high affinity, including group I introns and the hammerhead, hairpin and hepatitis delta
2
virus ribozymes. Of perhaps greatest interest, several aminoglycosides bind to a number of RNA
16
motifs found within the HIV-1 genome, namely loops of the HIV-1 dimerisation initiation site (DIS),
1
7
the junction of HIV-1 rev response element (RRE) and the major groove of the HIV-1 transactivation
1
8-21
response element (TAR),
agents.
indicating that there is scope to develop aminoglycoside-based anti-viral
3

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Figure 1. Structure of the 16S prokaryotic ribosomal A-site (left, the binding site of the aminoglycoside
2
2
Neomycin B shown in red) and (right) Neomycin B binding to a 16S prokaryotic ribosomal construct
1
2
(
PDB entry: 2A04, graphic prepared using PyMOL).
The widespread use of aminoglycosides as antibiotic drugs has been compromised by their
4
-7, 9-10
toxicity and the emergence of bacterial resistance.
As a result, synthetic modifications to
aminoglycosides have been investigated in an attempt to produce derivatives that not only display
higher affinities and selectivity for specific therapeutically-significant RNA motifs, but which are also
4
-7, 9-10, 23
less prone to metabolic alterations by aminoglycoside-modifying enzymes.
By way of
example, conjugation of chloramphenicol and linezolid to the aminoglycoside, neomycin B, enhances
the binding affinity for HIV-1 TAR by approximately 10-fold relative to the parent aminoglycoside
2
4
(
Figure 2). A library of neomycin-dipeptide conjugates with enhanced HIV-1 RRE binding affinities
2
5
have also been developed. To date, one of the strongest competitive inhibitors of the interaction
between RRE and its cognate binding partner, the rev protein, is a neomycin-acridine conjugate (Neo
1
7
B-acridine, Figure 2) that exhibits an affinity for RRE similar to that of rev itself.
4

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Figure 2. Structures of the reported RNA-binding neomycin B conjugates, Neo B-chloramphenicol
and Neo B-acridine, and the neomycin B-cyclen conjugates, C1–C3 and Zn(II)-C1–Zn(II)-C3,
developed in this study.
Herein, we describe the synthesis and A-site binding properties of a series of new neomycin B
conjugates featuring the polyazamacrocycle, 1,4,7,10-tetraazacyclododecane (cyclen) appended to their
D-ribose ring, as well as the corresponding Zn(II) complexes (C1–C3 and Zn(II)-C1–Zn(II)-C3, Figure
2
). Given that previous studies have shown that the high affinity of aminoglycosides for RNA is
associated predominantly with the formation of complementary electrostatic interactions between
protonated amine groups on the aminoglycosides and the polyanionic sugar-phosphate backbone of
3
, 12, 26-27
RNA,
it was hypothesised that the tethered cyclen macrocycle, with its additional ionisable
amino groups, might enhance affinity for the A-site RNA motif. Additionally, we assessed whether
5

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complexation of Zn(II) by cyclen might serve to enhance the affinity still further by allowing the
conjugates to form coordination bonds with the RNA target. Previous studies have shown that
Zn(II)-cyclen complexes form reasonably strong interactions with phosphate groups as well as the
2
8-31
deprotonated imide groups of the nucleobases, uracil and thymine.
Results and Discussion
Synthesis of neomycin B-cyclen conjugates. A convergent synthetic approach was used to access the
conjugates (Scheme 1). Firstly, Boc-protected cyclen derivatives incorporating a single carboxylic acid
pendant arm (5–7), as well as a Boc-protected neomycin B derivative featuring a solitary free amino
1
group at the 5′′ position of the central D-ribose ring (8), were prepared and characterized by H NMR
and IR spectroscopy, and mass spectrometry. These two components were then linked together under
standard HBTU/HOBt-mediated amide coupling conditions. Following purification by silica gel
column chromatography, the Boc-protected forms of the desired conjugates (9–11) were isolated in
1
good yield (70−85%) and high purity. The H NMR spectra showed numerous resonances in the
aliphatic region (1.0–6.0 ppm) characteristic of neomycin B, with additional signals in the upper field
region ascribed to alkyl CH protons. The appearance of additional broad singlet and multiplets
2
1
3
between  2.0–4.0 ppm were attributed to the CH protons of the cyclen ring. The C NMR spectra of
2
9
–11 each showed a signal between  170.5–170.9 ppm, corresponding to an amide C=O and
confirming successful amide coupling of 5–7 to 8. Formation of 9–11 was further corroborated by the
+
presence of the expected parent [M+H] signals at m/z = 1727.9, 1754.5 and 1784.0, respectively, in the
low-resolution electrospray ionization (ESI) mass spectrum.
Following trifluoroacetic acid (TFA)-mediated removal of the Boc groups and subsequent
preparative RP-HPLC purification, the final conjugates, C1–C3 (Figure 2), were obtained in high
purity, albeit in low yield (35–40%). The identity of the conjugates was confirmed by high-resolution
6

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+
ESI MS analysis, with parent [M+H] signals observed at m/z = 826.4998, 854.5283, 882.5649 for C1,
C2 and C3, respectively. The protonation state and number of TFA counterions was established by
elemental analysis.
Scheme 1. Synthesis of neomycin-cyclen conjugates, C1–C3. Reagents and conditions: (a) ethyl -
bromoalkanoate, K CO , CH CN, Δ, 48–72 h, 73–85%; (b) 1 M NaOH, EtOH, rt, 15 h, ca. 90%; (c)
2
3
3
cyclen carboxylic acids 5–7, HBTU/HOBt, DIPEA, dry CH Cl , rt, 20 h, 72–82%; (d) TFA/CH Cl
2
2
2
2
(1:1), rt, 5 h.
Displacement binding studies. Displacement binding of the RNA targets was probed using the
3
2-33
Dy547-labelled RNA/coumarin-labelled aminoglycoside system previously described by Tor et al.
Figure 3 illustrates the working concept for this FRET-based displacement binding assay. When the
aminoglycoside-coumarin is photoexcited, energy is transferred to the Dy547 dye. Energy transfer
7

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manifests itself as quenching of the coumarin fluorescence and a reduction of the excited state lifetime,
accompanied by an increase in fluorescence emission from the nearby Dy547 molecule. The binding
affinity of a test compound can be established by adding it in increasing concentration to the pre-
formed FRET complex and measuring the accompanying fluorescence changes. Gradual displacement
of the RNA-bound aminoglycoside-coumarin by the test compound results in the coumarin and Dy547
no longer being close enough for FRET to occur, leading to an increase in observed fluorescence
3
2-33
emission from the coumarin and a decrease in that from Dy547.
Figure 3. FRET-based displacement-style assay for determining A-site binding affinity. Conditions: λ
ex
3
2-33
=
400 nm, λ = 473 nm (coumarin) and 561 nm (Dy547).
em
In this work, either kanamycin A- or neomycin B-coumarin (donor) was used in combination
with the Dy547 A-site RNA (acceptor). As expected, titration of C1–C3 and their corresponding Zn(II)
complexes, Zn(II)-C1–Zn(II)-C3, into this FRET-active system resulted in an increase in the coumarin
emission (473 nm) and a concurrent reduction in Dy547 emission (561 nm) when excited at 400 nm
(see Figure S1 for a representative titration dataset). Plotting of the fractional fluorescence saturation at
8

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5
61 nm against the concentration of the test compound yielded displacement curves (see Figure S2 for
the corresponding dose response curve), from which the 50% inhibition concentration (IC ) values
5
0
were calculated (see experimental section for more details).
Figure 4 shows the dose response curves for the displacement of kanamycin A-coumarin from
Dy547 A-site by neomycin B and the conjugates C1–C3, with the corresponding IC values presented
5
0
in Table 1. Compared to neomycin B (IC = 0.68 ± 0.09 μM), the conjugates exhibit slightly higher
5
0
affinity for the A-site (IC = (0.51 ± 0.06) to (0.31 ± 0.10) μM).
5
0
Figure 4. Dose response curves (measured at 561 nm) for the displacement of kanamycin A-coumarin
from the Dy547 A-site by neomycin B and conjugates C1–C3. Conditions: [Dy547 A-site] = 1 μM,
[
[
kanamycin A-coumarin] = 0.5 μM, [cacodylate] = 20 mM, (pH = 7.0 @ 21 °C), [NaCl] = 100 mM,
EDTA] = 0.5 mM.
9

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Table 1. IC50 values for the displacement of kanamycin A-coumarin from the Dy547 A-site by
neomycin B and the conjugates C1–C3.
Conjugate
IC (μM)
50
C1
C2
C3
0.51 ± 0.10
0.45 ± 0.08
0.31 ± 0.05
0.68 ± 0.09
Neomycin B (control)
Conditions: [Dy547 A-site] = 1 μM, [kanamycin A-coumarin] = 0.5 μM, [cacodylate] = 20 mM, (pH =
.0 @ 21 °C), [NaCl] = 100 mM, [EDTA] = 0.5 mM. Values represent the mean ± SD of 3
experiments.
7
The degree of protonation (and hence average overall charge) of the kanamycin A and
neomycin B derivatives is expected to vary significantly with pH. Thus, the strength of binding
between the kanamycin A-coumarin/neomycin B derivatives and the target A-site will be dependent on
pH. The affinity of neomycin B itself for the bacterial A-site has previously been found to be pH
3
4
dependent. For the neomycin B derivatives, protonation of amino groups would see the neomycin B
moiety existing predominantly in the 5+ protonation state, and cyclen in the 2+ protonation state, at
2
7, 35-36
physiological pH.
Introduction of cyclen is thus expected to increase the overall positive charge
of the conjugates, and enhance their electrostatic interactions (charge-assisted hydrogen bonding
interactions) with the negatively charged A-site. It should be noted that any given pH, both the
kanamycin A-coumarin and neomycin B derivatives will exist as a mixture of species with different
protonation states, with the measured displacement constant (IC50 value) representing the overall
average contribution of all species present.
With the above in mind, we further examined how the additional amino groups present in
cyclen might influence the binding event. Previously, it was noted that conjugate C1, which has an
ethyl spacer between the neomycin B and cyclen, has the lowest binding affinity for the A-site (see
Table 1). Thus, C1 along and neomycin B were further tested to see if binding affinity could be
improved under different pH conditions. Figures S3 and S4 (Supporting Information) illustrate the
1
0

ACCEPTED MANUSCRIPT
displacement plots for neomycin B and C1 at pH values between 6.0−8.5, with the corresponding IC50
values presented in Table 2.
Table 2. IC values for the displacement of kanamycin A-coumarin from Dy547 A-site by C1 and
5
0
neomycin B at pH 6.0−8.5.
C1
Neomycin B
pH
IC (μM)
IC (μM)
5
0
50
6
6
7
7
8
8
.0
.5
.0
.5
.0
.5
0.35 ± 0.09
0.42 ± 0.01
0.51 ± 0.09
0.61 ± 0.08
0.72 ± 0.01
0.88 ± 0.08
0.63 ± 0.09
0.65 ± 0.07
0.68 ± 0.10
0.73 ± 0.07
0.85 ± 0.05
1.20 ± 0.07
Conditions: [Dy547 A-site] = 1 μM, [kanamycin A-coumarin] = 0.5 μM, [cacodylate] = 20 mM, (pH =
.0–8.5 @ 21 °C), [NaCl] = 100 mM, [EDTA] = 0.5 mM. Values represent the mean ± SD of 3
experiments.
6
The strength of the interaction between C1 and the Dy547 A-site was found to steadily increase
with decreasing pH (see also Figure 5); the IC for C1 decreased by up to 0.2 μM per unit decrease in
5
0
pH (0.35–0.88 μM for pH 6.0–8.5). Binding of neomycin B was also strongest at lower pH, but below
pH 7.5 the IC values decreased only slightly with further reductions in pH. Overall, the fact that, at
5
0
each tested pH, C1 exhibited lower IC values than neomycin B indicates that C1 is a better A-site
5
0
binder compared to unmodified neomycin B. We postulate that the additional cationic charge
introduced into C1–C3 by the amino groups present on the cyclen macrocycle helps to promote the
displacement of kanamycin A-coumarin from the A-site, thereby resulting in a marginal improvement
in binding ability. The length of the carbon spacer between neomycin B and cyclen in the conjugate did
not have much of an effect on the strength of binding.
1
1

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Figure 5. Plot of the variation in IC50 values (μM) with pH for C1 and neomycin B. Conditions:
[
Dy547 A-site] = 1 μM, [kanamycin A-coumarin] = 0.5 μM, [cacodylate] = 20 mM, (pH = 6.0–8.5 @
2
1 °C), [NaCl] = 100 mM, [EDTA] = 0.5 mM.
Further studies were conducted to investigate if the conjugates can also displace the stronger
3
2-33
aminoglycoside A-site binder, neomycin B-coumarin.
Figure 6 shows the displacement plots for
C1–C3 and Zn(II)-C1–Zn(II)-C3, with the corresponding IC values presented in Table 3.
5
0
Table 3. IC values for the displacement of neomycin B-coumarin from Dy547 A-site by C1–C3 and
5
0
the corresponding Zn(II) metal complexes.
Conjugate
IC (μM)
Complex
IC (μM)
5
0
50
C1
C2
C3
1.77 ± 0.09
1.42 ± 0.26
2.30 ± 0.22
2.35 ± 0.31
Zn(II)-C1
Zn(II)-C2
Zn(II)-C3
1.13 ± 0.09
1.32 ± 0.19
0.85 ± 0.04
Neomycin B
Conditions: [Dy547 A-site] = 1 μM, [neomycin B-coumarin] = 0.5 μM, [cacodylate] = 20 mM, (pH =
.0 @ 21 °C), [NaCl] = 100 mM, [EDTA] = 0.5 mM (no EDTA for metal complex titration). Values
represent the mean ± SD of 3 experiments.
7
1
2

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1
3

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Figure 6. Displacement of neomycin B-coumarin from Dy547 A-site by neomycin B, C1–C3, and
Zn(II)-C1–Zn(II)-C3. Conditions: [Dy547 A-site] = 1 μM, [neomycin B-coumarin] = 0.5 μM,
[
cacodylate] = 20 mM, (pH = 7.0 @ 21 °C), [NaCl] = 100 mM, [EDTA] = 0.5 mM (no EDTA for
metal complex titration).
As expected, larger IC values were obtained, since neomycin B-coumarin is harder to displace
5
0
3
2-33
from the A-site.
The addition of Zn(II) ion to C1–C3 enhanced their affinity for the A-site, but to
varying degrees; Zn(II)-C2 showed only marginally improved affinity, while Zn(II)-C3 bound the A
site two-fold more strongly than its non-metallated counterpart. This is consistent with the fact that
2
9-31
28-30
anionic phosphates,
and uracil and thymine bases in RNA and DNA, respectively,
bind strongly
to Zn(II)-cyclen motifs via coordinative and/or electrostatic interactions. The presence of uracil bases
near the aminoglycoside binding site (Figure 1) may explain the enhanced binding affinities of the
Zn(II)-loaded neomycin B conjugates. The high affinity of Zn(II)-C3 for the A-site may stem from the
fact that this conjugate includes the longest carbon spacer, which might allow the Zn-cyclen moiety to
better position itself to bind to a uracil base.
Of note, in our case, a lower IC value was obtained for the displacement of neomycin B-
5
0
2
3
coumarin by neomycin B (2.35 ± 0.31 μM) compared to the reported value of 4.4 ± 0.3 μM.
1
4

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Conclusion
Three new neomycin B-cyclen conjugates have been developed that exhibit higher affinities for the
bacterial ribosomal A-site than neomycin B itself, with IC values lying in the sub-micromolar-to-
5
0
micromolar range. We attribute the stronger binding to the increased overall positive charge of the
conjugates arising from the presence of the protonated cyclen ring. The A-site affinities of each of the
compounds was found to be dependent on pH and, therefore, by implication, their protonation state –
the lower the pH (higher the charge), the higher the affinity for the A-site. The Zn(II) complexes of the
conjugates bound to the A-site even more strongly than the conjugates, which may be due to the
cationic Zn(II)-cyclen moiety forming coordinative (and/or electrostatic) interactions with the
phosphates or deprotonated uracil bases present within the A-site. We are currently investigating the
ability of the conjugates and their Zn(II) complexes to hydrolytically-cleave selected bonds within the
A-site, as well as their affinity towards other (potentially) therapeutically-relevant RNA targets, in
particular the HIV-1 TAR, RRE and DIS motifs.
Experimental Section
Materials. All chemicals were of reagent grade quality or better, obtained from commercial suppliers
and used without further purification. Solvents were used as received or distilled using standard
procedures. Deionised water was used for all reactions in aqueous solution. Column chromatography
was performed using Silica gel 60 (0.040–0.063 mm mesh, Merck). The Dy547-labelled A-site RNA
oligonucleotide (deprotected and purified) was purchased from Dharmacon. Standard ZnCl solution
2
was purchased from Merck. Chemicals for preparing buffer solutions were purchased from Sigma-
Aldrich (enzyme free-grade). Autoclaved water was used for all fluorescence titrations.
1
13
Instrumentation and methods. H and C NMR spectra were recorded in deuterated solvents at room
temperature on either Bruker spectrometers (AC 200, AM 300, Avance DR 400) or Varian
1
5

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Spectrometers (Mercury 300 and 500). The chemical shifts, δ, are reported in ppm (parts per million).
The signals from the residual protons of deuterated solvent were used as an internal reference. The
abbreviations for the peak multiplicities are as follows: s (singlet), d (doublet), dd (doublet of doublets),
t (triplet), q (quartet), m (multiplet), and br (broad). ESI mass spectrometry was performed on a
Micromass Platform II Quadrupole Mass Spectrometer fitted with an electrospray source. The capillary
voltage was 3.5 eV and the cone voltage was 3.5 eV. High-resolution mass spectra were recorded with
a Bruker BioApex II 47e FT-ICR MS fitted with an Analytica Electrospray Source. Samples were
–
1
introduced via a syringe pump at a rate of 1 μL min and the capillary voltage was set at 200 V.
–
1
Infrared spectra were recorded on neat samples at a resolution of 4.0 cm using an Agilent
Technologies Cary 630 FTIR (ATR) or a Perkin-Elmer FTIR 1600 series spectrometer. Peak intensities
are given as broad (br), very strong (vs), strong (s), medium (m) and weak (w). Reverse phase-HPLC
analyses were performed using a Jupiter C18 Column (5 μm particle size, 300 Å pore size, 4.6 × 250
mm). Solution A consisted of 0.1% TFA in water and solution B of 0.1% TFA in MeCN.
Chromatographic separations were performed used a gradient from 0–100% B over 40 min, with a flow
–
1
rate of 1 mL min and UV detection at λ = 210 nm. Microanalyses were carried out by the Campbell
Microanalytical Laboratory, University of Otago, New Zealand.
3
7
Syntheses. The compounds 1,4,7-tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclododecane (1),
3
32-33
5
’-Amino-N-Boc-protected neomycin B (8), kanamycin A-coumarin (12)
and neomycin B-
3
2-33
coumarin (13)
were synthesised according to literature procedures, with all characterisation data in
agreement with that reported previously.
-(2-(Ethoxycarbonyl)ethyl)-4,7,10-tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclododecane
2). To a stirred mixture of compound 1 (1.03 g, 2.2 mmol) and anhydrous K CO (1.78 g, 13 mmol) in
1
(
2
3
CH CN (15 mL) was added ethyl bromoacetate (0.35 mL, 3.1 mmol) in CH CN (15 mL) in a dropwise
3
3
fashion. The mixture was then heated to reflux. After 48 h, the reaction mixture was cooled to room
temperature, filtered and the solvent removed under reduced pressure to afford 2 as a yellow oil, which
1
6

ACCEPTED MANUSCRIPT
was purified via column chromatography (eluent: n-hexane/EtOAc, 2:1 v/v) (R = 0.47). Yield: 0.75 g
f
1
t
(
85%). H NMR (500 MHz, CDCl ) δ (ppm): 1.12 (t, 3H, J = 7.0 Hz, ethyl CH ), 1.30 (s, 27H, Bu
3
3
CH ), 2.76−2.79 (m, 4H, cyclen ring CH ), 3.19−3.27 (m, 8H, cyclen ring CH ), 3.35−3.42 (m, 8H,
3
2
2
1
3
cyclen ring CH and acetate CH ), 4.06 (q, 2H, J = 7.0 Hz, ethyl CH ). C NMR (125 MHz, CDCl ) δ
2
2
2
3
t
(
ppm): 14.3 (ethyl CH ), 28.5 ( Bu CH ), 47.1, 47.4, 47.7, 50.0, 51.2, 53.5 (cyclen CH ), 54.9 (acetate
3
3
2
t
CH ), 60.3 (ethyl CH ), 79.5 ( Bu C), 155.3, 155.7, 156.1 (Boc group C=O), 170.6 (ester C=O). IR
2
2
–
1
(
cm ): 3363br w, 2979s, 2933s, 1736s, 1686s, 1461s, 1417s, 1367s, 1250s, 1162s, 1029m, 980m,
+
+
9
11m, 887w, 859m, 756s, 666m. ESI-MS (m/z): 559.2 [M+H] , 581.2 [M+Na] .
-(4-(Ethoxycarbonyl)butyl)-4,7,10-tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclododecane
3). Compound 3 was synthesised from 1 (0.51 g, 1.1 mmol) and ethyl 4-bromobutyrate (0.43 g, 2.2
1
(
mmol) following the method described for compound 2. The reaction mixture was refluxed for 72 h,
and following work-up, 3 obtained as a yellow oil, which was purified via column chromatography
1
(
eluent: n-hexane/EtOAc, 2:1 v/v) (R = 0.51). Yield: 0.59 g (73%). H NMR (500 MHz, CDCl ) δ
f
3
t
(
ppm): 1.22 (t, 3H, J = 7.5 Hz, CH ), 1.43 (s, 27H, Bu CH ), 1.81−1.86 (m, 2H, butyl CH ), 2.25 (t,
3 3 2
2
3
H, J = 4.0 Hz, butyl CH ), 2.39 (t, 2H, J = 4.0 Hz, butyl CH ), 2.52−2.55 (m, 4H, cyclen ring CH ),
2 2 2
.2−3.45 (m, 8H, cyclen ring CH ), 3.4−3.5 (m, 4H, cyclen ring CH ), 4.10 (q, 2H, J = 7.8 Hz, ethyl
2
2
13
t
CH ). C NMR (125 MHz, CDCl ) δ (ppm): 14.5 (ethyl CH ), 24.1(butyl CH ), 28.5 ( Bu CH ), 31.8
2
3
3
2
3
(butyl CH ), 49.6, 50.6, 51.9, 52.3, 54.8, 55.5 (cyclen CH ), 59.6 (butyl CH ), 61.8 (ethyl CH ), 79.9
2 2 2 2
t
t
+
+
(
Bu C), 155.7 ( Bu C=O), 173.3 (ester C=O). ESI-MS (m/z): 587.4 [M+H] 609.3 [M+Na] .
-(6-(Ethoxycarbonyl)hexyl)-4,7,10-tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclododecane
4). Compound 4 was synthesised from 1 (0.50 g, 1.1 mmol) and ethyl 6-bromohexanoate (0.47 g, 2.2
1
(
mmol) following the method described for compound 2. The reaction mixture was refluxed for 72 h,
and, following work-up, 4 was isolated as a yellow oil, which was purified via column chromatography
1
(
eluent: n-hexane/EtOAc, 2:1 v/v) (R = 0.45). Yield: 0.51 g (75%). H NMR (200 MHz, CDCl ) δ
f
3
t
(
ppm): 1.21−1.30 (m, 5H, ethyl CH and hexyl CH ) 1.44 (s, 27H, Bu CH ), 1.56−1.70 (m, 4H, hexyl
3
,
2
3
1
7

ACCEPTED MANUSCRIPT
CH ), 2.29 (t, 2H, J = 7.4 Hz, hexyl CH ), 2.43−2.69 (m, 6H, hexyl CH and cyclen ring CH ),
2
2
2
2
1
3
3
.17−3.59 (m, 12H, cyclen ring CH ), 4.12 (q, 2H, J = 7.1 Hz, ethyl CH ). C NMR (50 MHz, CDCl )
2 2 3
t
δ (ppm): 14.5 (ethyl CH ), 21.3 (hexyl CH ), 23.7 (hexyl CH ), 24.9 (hexyl CH ), 28.7 ( Bu CH ), 34.3
3
2
2
2
3
(
hexyl CH ), 47.83, 48.2, 48.4, 50.3, 52.6, 53.8 (cyclen ring CH ), 55.2 (hexyl CH ), 60.5 (ethyl CH ),
2 2 3 2
t
-1
7
2
9.6 ( Bu C), 155.6, 155.9, 156.3 (Boc group C=O), 173.8 (ester C=O). IR (cm ): 3373br w, 2977s,
935s, 1736s, 1690s, 1463s, 1416s, 1392s, 1366s, 1251s, 1167s, 1032m, 978w, 860w, 757m. ESI-MS
+
(
m/z): 615.4 [M+H] .
1
-(2-Carboxyethyl)-4,7,10-tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclododecane
(5).
Compound 2 (0.75 g, 1.3 mmol) was added to a solution of 1 M NaOH/ EtOH (20 mL, 1:1 v/v) and the
reaction mixture stirred at room temperature for 4–6 h. The solvent was then removed under reduced
pressure, the residue redissolved in 10% citric acid solution and the pH adjusted to 5. The product was
extracted into EtOAc (2  30 mL), the combined organic layers dried over Na SO and filtered, and the
2
4
solvent then removed under reduced pressure. Compound 5 was obtained as a yellow oil. Yield: 0.62 g
1
t
(
88%). H NMR (500 MHz, CDCl ) δ (ppm): 1.44 (s, 27H, Bu CH ), 2.38−2.42 (m, 4H, cyclen ring
3
3
CH ), 2.87−2.96 (m, 8H, cyclen ring CH ), 3.24−3.33 (m, 4H, cyclen CH ), 3.51 (s, 2H, acetate CH ).
2
2
2
2
1
3
t
C NMR (125 MHz, CDCl ) δ (ppm): 28.5 ( Bu CH ), 47.4, 49.9, 51.5, 51.9 (cyclen CH ), 54.0
3
3
2
t
–1
(
acetate CH ), 79.9 ( Bu C), 155.5, 156.1 (Boc group C=O), 173.4 (COOH). IR (cm ): 3491br w,
2
2
974s, 2931s, 1672s, 1460m, 1414s, 1364s, 1247s, 1152s, 1031m, 975m, 886m, 812m, 673w. ESI-MS
+
+
(
m/z): 531.3 [M+H] , 553.3 [M+Na] .
-(4-Carboxybutyl)-4,7,10-tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclododecane
1
(6).
Compound 6 was synthesised from 3 (0.52 g, 0.84 mmol) following the method described for
1
compound 5, and was obtained as yellow oil. Yield: 0.46 g (93%). H NMR (500 MHz, CDCl ) δ
3
t
(
ppm): 1.33 (s, 27H, Bu CH ), 1.75−1.85 (m, 2H, butyl CH ), 2.42 (t, 2H, J = 7.5 Hz, butyl CH ), 2.34
3
2
2
(
t, 2H, J = 7.5 Hz, butyl CH ), 2.51−2.58 (m, 4H, cyclen ring CH ), 3.21−3.31 (m, 8H, cyclen ring
2
2
1
3
CH ), 3.40−3.48 (m, 4H, cyclen ring CH ). C NMR (125 MHz, CDCl ) δ (ppm): 19.1(butyl CH ),
2
2
3
2
1
8

ACCEPTED MANUSCRIPT
t
2
8.8 ( Bu CH ), 32.2 (butyl CH ), 47.9, 48.5, 50.0, 50.4 51.5, 52.1 (cyclen CH ), 55.7 (butyl CH ), 80.0
3 2 2 2
t
–1
(
Bu C), 15.7, 156.2 (Boc group C=O), 177.0 (COOH). IR (cm ): 3312br w, 2975s, 2932s, 1678s,
+
1
5
463s, 1416s, 1367s, 1251s, 1163s, 974w, 910w, 859w, 757s, 632s. ESI-MS (m/z): 559.4 [M+H] ,
+
81.4 [M+Na] .
1
-(6-Carboxyhexyl)-4,7,10-tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclododecane
(7).
Compound 7 was synthesised from 4 (0.60 g, 0.97 mmol) following the method described for
1
compound 5, and obtained as a yellow oil. Yield: 0.41 g (89%). H NMR (500 MHz, CDCl ) δ (ppm):
3
t
1
.32 (m, 2H, hexyl CH ), 1.44 (s, 27H, Bu CH ), 1.63 (m, 2H, hexyl CH ), 2.29 (t, 2H, J = 7.0 Hz,
2
3
2
hexyl CH ), 2.50−271 (m, 4H, cyclen ring CH ), 2.77−2.89 (m, 2H, hexyl CH ), 3.2–3.43 (m, 8H,
2
2
2
1
3
cyclen ring CH ), 3.50−3.67 (m, 4H, cyclen ring CH ). C NMR (125 MHz, CDCl ) δ (ppm): 24.1
2
2
3
t
(
hexyl CH ), 25.8 (hexyl CH ), 27.9 (hexyl CH ), 29.0 ( Bu CH ), 35.2 (hexyl CH ), 47.5, 48.4, 51.2,
2 2 2 3 2
t
5
2.8, 53.9, 55.6 (cyclen ring CH ), 58.6 (hexyl CH ), 79.6 ( Bu C), 156.5 (Boc group C=O), 178.2
2 2
–
1
(
COOH). IR (cm ): 3460br w, 2974s, 2932s, 1680s, 1459m, 1412s, 1364s, 1247s, 1152s, 976w, 858w,
+
+
7
72m, 639w. ESI-MS (m/z): 587.4 [M+H] 609.3 [M+Na] .
-{2-[5’-(N-Boc-protected neomycin B)carboxamido]ethyl}-4,7,10-tris(tert-butoxycarbonyl)-
,4,7,10-triazacyclododecane (9). Compound 5 (0.090 g, 0.16 mmol), HBTU (0.172 g, 0.33 mmol),
1
1
HOBt (0.045 g, 0.33mmol) and DIPEA (0.043 g, 0.33 mmol) were added to dry CH Cl (10 mL). The
2
2
solution was stirred for 30 min before the addition of the 8 (0.200 g, 0.16 mmol). The solution was
stirred at room temperature for 20 h, after which the solvent was removed under reduced pressure. The
crude product was purified by column chromatography (eluent: EtOAc) (R = 0.6), to give 9 as a white
f
1
t
solid. Yield: 0.24 g (82%). H NMR (500 MHz, MeOD) δ (ppm):1.45 (m, 81H, Bu CH ), 2.01 (m,
3
2
H), 2,41 (s, 1H), 2.80−3.03 (m, 8H, cyclen CH ), 3.17−3.61 (m, 24H), 3.74 (br s, 2H), 3.74 (s, 2H),
2
1
3
3
.89 (m, 2H), 4.10 (m, 2H), 4.57 (s, 1H), 5.07 (s, 1H), 5.40 (s, 1H). C NMR (125 MHz, MeOD) δ
(ppm): 28.1, 35.9, 41.2, 42.2, 42.9, 50.5, 50.7, 53.5, 53.9, 54.4, 55.7, 59.4, 62.3, 70.9, 71.5, 73.5, 75.1,
7
6.8, 79.2, 79.5, 79.9, 80.6, 82.9, 85.9, 86.7, 109.8, 113.9, 156.1, 156.9, 157.2, 157.4, 157.6, 170.5. IR
1
9

ACCEPTED MANUSCRIPT
–
1
(
cm ): 3422 br s, 2979m, 2531m, 2476w, 2373w, 2345w, 2105w, 1686s, 1522s, 1474m, 1458m,
1
1
420m, 1394m, 1368s, 1275w, 1252m, 1169s, 1045m, 946w, 916w, 850m, 777w. ESI-MS (m/z):
+
727.9 [M+H] .
1
-{4-[5’-(N-Boc-protected neomycin B)carboxamido]butyl}-4,7,10-tris(tert-butoxycarbonyl)-
1
,4,7,10-triazacyclododecane (10). Compound 10 was synthesised from 6 (0.187 g, 0.34 mmol) and 8
(0.406 g, 0.34 mmol) following the method described for 9. The crude product was purified by column
1
chromatography (eluent: EtOAc) (R = 0.59). Yield: 0.44 g (75%). H NMR (500 MHz, MeOD) δ
f
t
(
ppm): 1.26 (m, 2H, butyl CH ), 1.42 (m, 81H, Bu CH ), 1.81 (t, J=7 Hz, 2H, butyl CH ), 1.96 (s,
2
3
2
1
1
5
4
8
H), 2.01 (s, 1H), 2.28−2.35 (m, 8H, cyclen CH ), 2.67−2.73 (m, 10H, cyclen CH ), 2.67−2.73 (m,
2 2
0H), 3.19 (t, J = 9.0 Hz, 2H), 3.29−3.61 (m, 16H), 3.75 (br s, 1H), 3.90−4.11 (m, 2H), 4.28 (s, 1H),
1
3
.06 (s, 1H), 5.39 (s, 1H). C NMR (125 MHz, MeOD) δ (ppm): 24.9, 28.3, 34.8, 35.9, 41.4, 42.4,
2.9, 50.7, 50.9, 53.7, 53.9, 54.6, 55.9, 59.7, 59.9, 62.6, 71.3, 71.8, 73.9, 75.5, 77.1, 79.7, 79.9, 80.1,
0.9, 83.1, 86.0, 86.9, 110.1, 114.2, 156.3, 156.9, 157.4, 157.6, 157.9, 170.8. ESI-MS (m/z): 1754.5
+
[
M+H]
1
-{6-[5’-(N-Boc-protected neomycin B)carboxamido]hexyl}-4,7,10-tris(tert-butoxycarbonyl)-
1
,4,7,10-triazacyclododecane (11). Compound 11 was synthesised from 7 (0.300 g, 0.50 mmol) and 8
(0.608 g, 0.50 mmol) following the method described for 9. The crude product was purified by column
1
chromatography (eluent: EtOAc) (R = 0.57). Yield: 0.66 mg (72%). H NMR (500 MHz, MeOD) δ
f
t
(
ppm): 1.28 (t, J = 7.0 Hz, 2H, hexyl CH ), 1.31−1.54 (m, 87H, Bu CH and hexyl CH ), 1.69 (m, 2H,
2
3
2
hexyl CH ), 2.33−2.39 (m, 4H, hexyl CH ), 2.69 (br s, 6H, cyclen CH ), 3.18−3.59 (m, 18H), 3.76 (m,
2
2
2
13
6
H), 4.07−4.12 (m, 3H), 5.01 (s, 1H), 5.48 (s, 2H), 6.23 (d, J = 10 Hz, 2H). C-NMR (125MHz,
MeOD) δ (ppm): 25.5, 26.5, 27.8, 28.4, 35.8, 36.8, 41.3, 42.1, 42.8, 50.3, 50.9, 53.6, 53.8, 54.3, 55.7,
5
1
9.5,59.7, 59.9, 62.5, 71.0, 71.7, 73.8, 75.4, 76.9, 79.6, 79.8, 80.0, 80.6, 82.9, 86.0, 86.5, 110.0, 114.0,
–
1
56.1, 156.7, 157.2, 157.5, 157.8, 170.6. IR (cm ): 3422br s, 2979s, 2934s, 2508w, 1692s, 1522s,
2
0

ACCEPTED MANUSCRIPT
1
1
480w, 1458w, 1419m, 1393w, 1368s, 1278m, 1252s, 1168s, 1044m, 852m, 776w. ESI-MS (m/z):
+
784.0 [M+H] .
1
-{2-[5’-(Neomycin B)carboxamido]ethyl}-1,4,7,10-triazacyclododecane (C1). Compound 9
(
0.355 g, 0.21 mmol) was dissolved in TFA/CH Cl (10 mL, 1:1 v/v) and the solution was stirred at
2
2
room temperature for 5 h. The solvent was then removed under reduced pressure, and the crude residue
purified by preparatory HPLC (Jupiter C18 Prep Column (5 μm, 300 Å, 4.6 × 250 mm), monitored at
2
10 nm) using a linear gradient from 100% H O (0.1% TFA) to 85% H O: 15% CH CN over 40 min.
2 2 3
The product eluted between 19–23 min and was isolated as a white solid (TFA salt) following removal
1
of the solvent. Yield: 0.150 g (39%). H NMR (500 MHz, D O) δ (ppm): 1.18 (d, J = 7.0 Hz, 1H), 1.75
2
(d, J = 12.5 Hz, 1H), 2.35 (d, J = 8.5 Hz, 1H), 2.84 (br s, 6H), 3.02 (m, 8H), 3.15−3.52 (m, 12H), 3.59
(t, J = 10 Hz, 2H), 3.70 (br s, 1H), 3.77−3.89 (m, 4H), 3.97 (t, J = 9.5 Hz, 1H), 4.03−4.09 (m, 2H),
13
4
.18−4.22 (m, 3H), 5.15 (s, 1H), 5.25 (s, 1H), 5.84 (d, J = 4.0 Hz, 1H). C NMR (125 MHz, D O) δ
2
(ppm): 27.9, 40.1, 40.5, 41.9, 42.5, 44.5, 48.6, 49.3, 49.7, 50.9, 53.1, 55.2, 67.5, 67.6, 68.2, 70.2, 72.2,
–
1
7
3
1
3.0, 75.5, 77.0, 80.0, 84.6, 94.9, 95.5, 109.6, 112.0, 114.9, 117.8, 120.7, 173.5. IR (cm ): 3313m,
088m, 2874m, 2358w, 2338w, 2148w, 2039w, 2002w, 1669s, 1636w, 1559m, 1540m, 1522m,
457w, 1429m, 1321w, 1183s, 1125s, 1074w, 1049w, 1014m, 837m, 798s, 722s. High-resolution ESI-
+
MS (C H N O ): Calcd. 826.9403 [M+H] , Found 826.4998. Microanalysis: Found (%): C, 30.93;
3
3
67 11 13
H, 4.69; N, 7.99; F, 24.18. Calcd. for C H N O 8TFA8H O (%): C, 32.27; H, 4.87; N, 8.19; F,
3
3
67 11 13
2
2
4.22.
1
-{4-[5’-(Neomycin B)carboxamido]ethyl}-1,4,7,10-triazacyclododecane (C2). Compound C2
was synthesised from compound 10 (0.411 g, 0.23 mmol) following the method described for
1
preparation of C1. Yield: 0.156 g (35%). H NMR (500 MHz, D O) δ (ppm): 1.65 (br s, 2H), 1.76 (s,
2
1
H), 2.17 (t, J = 7.5 Hz, 4H), 2.34 (br s, 2H), 2.55 (s, 2H), 2.79 (br s, 6H), 2.98 (br s, 6H), 3.15−3.37
(m, 8H), 3.41−3.45 (m, 3H), 3.58 (s, 1H), 3.68 (s, 1H), 3.69 (s, 1H), 3.77−3.88 (m, 3H), 3.97 (t, J = 9.0
2
1

ACCEPTED MANUSCRIPT
Hz, 2H), 4.08 (s, 2H), 4.15 (m, 2H), 4.22 (t, J = 4.5 Hz, 1H), 5.14 (s, 1H), 5.25 (s, 1H), 5.82 (s, 1H).
1
3
C NMR (125 MHz, D O) δ (ppm): 19.3, 27.9, 33.0, 40.1, 40.5, 41.6, 41.9, 44.1, 47.7, 48.5, 49.6, 50.9,
2
5
1.6, 53.2, 67.4, 67.6, 68.1, 69.8, 70.2, 70.5, 72.3, 73.3, 75.2, 77.4, 80.4, 84.9, 94.9, 95.7, 109.5, 176.3.
–
1
IR (neat), (cm ): 3549w, 3348w, 3083m, 2879m, 2731m, 2543m, 1670s, 1541w, 1528w, 1429w,
1
281w, 1187s, 1127s, 1074w, 1050w, 1017w, 837m, 798m, 766w, 722s. High-resolution ESI-MS
+
(
C H N O ): Calcd. 854.0055 [M+H] , Found 854.5289. Microanalysis: Found (%): C, 32.21; H,
3
5
71 11 13
4
.58; N, 8.05; F, 28.97. Calcd. for C H N O 10TFA (%): C, 32.54; H, 4.22; N, 7.59; F, 28.07.
3
5
71 11 13
1
-{6-[5’-(Neomycin B)carboxamido]hexyl}-1,4,7,10-triazacyclododecane (C3). Compound C3
was synthesised from 11 (0.650 g, 0.36 mmol) following the method described for compound C1.
1
Yield: 0.28 g (40%). H NMR (500 MHz, D O) δ (ppm): 1.15 (s, 2H), 1.23 (s, 1H), 1.42−1.46 (m, 3H),
2
1
.56 (s, 1H), 1.74−1.78 (m, 2H), 2.14 (t, J = 7 Hz, 2H), 2.33−2.37 (m, 1H), 2.56 (s, 1H), 2.67 (br s,
H), 2.91 (d, J = 10.5 Hz, 12H), 3.18−3.45 (m, 12H), 3.584 (t, J = 10 Hz, 2H), 3.69 (s, 1H), 3.77−3.89
2
(m, 3H), 3.97 (t, J = 9 Hz, 1H), 4.08-4.16 (m, 4H), 4.23 (m, 1H), 5.14 (s, 1H), 5.25 (s, 1H), 5.82 (1H).
1
3
C NMR (125 MHz, D O) δ (ppm): 22.8, 25.3, 26.1, 28.4, 35.8, 40.1, 40.5, 41.4, 41.8, 42.4, 43.6, 48.6,
2
4
9
1
8.8, 49.7, 50.9, 52.5, 53.3, 67.4, 67.6, 68.3, 69.7, 70.2, 70.5, 72.4, 73.4, 75.8, 77.5, 80.5, 85.0, 95.0,
–
1
5.8, 109.4, 177.4 IR (neat), (cm ): 3372m, 3280m, 2925m, 2876m, 2362w, 2150w, 1670s, 1637w,
533w, 1459w, 1457w, 1429w, 1183s, 1125s, 1074w, 1050w, 1015w, 837m, 798m, 766w, 722s. High-
+
resolution ESI-MS (C H N O ): Calcd. 882.059 [M+H] , Found 882.5649. Microanalysis: Found
3
7
75 11 13
(
%): C, 33.49; H, 4.76; N, 8.11; F, 24.69. Calcd. for C H N O 8TFA5H O (%): C, 33.78; H, 4.97;
3
7
75 11 13
2
N, 8.18; F, 24.2.
Aminoglycoside binding studies. Binding studies were conducted using 1 μM solutions of Dy547 A-
site RNA in 20 mM calcodylate buffer (pH 7.0, 100 mM, NaCl, 0.5 mM EDTA). The Dy547 A-site
was annealed by heating solutions to 75 °C for 5 min, cooling to room temperature over 2 h, and then
to 0 °C for 30 min. They were then allowed to warm back to room temperature. For experiments
2
2

ACCEPTED MANUSCRIPT
involving the Zn(II) complexes of the conjugates, EDTA was removed from the buffer. These
complexes were prepared by combining C1–C3 with Zn(II) in a 1:1 molar ratio using a stock Zn(II)
solution made by dissolving a commercial standard solution of ZnCl in water.
2
The FRET-based displacement-style binding experiments were carried out at 21 °C on a Horiba
Fluoromax-4 luminescence spectrometer, using excitation and emission slit widths of 9 nm and with
background correction (spectrum of buffer was subtracted from each sample). In a typical run, 125 μL
of Dy547 A site solution (1 μM) was placed in a 0.5 mL quartz fluorescence cell with a path length of
1
.0 cm (Hellma GmbH & Co KG, Müllheim, Germany). To this, 0.66 μL of a 94.4 μM stock solution
of Kanamycin A-coumarin (12) or neomycin B-coumarin (13) was then added to reach a final
concentration of 0.5 μM for 12 or 13, in solution. The coumarin-Dy547 FRET system was excited at
4
00 nm and changes in emission at 561 nm were monitored after the addition of increasing amounts
(1 μL aliquots of 10 mM stock solution) of a conjugate or its Zn(II) complex, to assess the degree of
displacement of kanamycin- and neomycin-coumarin from Dy547 A-site. The OriginPro 8 software
was used to calculate IC50 values by fitting a dose response curve to the fractional fluorescence
saturation (F ) plotted against the log of aminoglycoside (AG) concentration (eq 1).
s
ꢋ
ꢋ
ꢋ
ꢂꢁ ꢃ ꢄꢅ ꢁꢇꢈꢉꢊ ꢌꢍꢅꢇꢎꢏ ꢊ ꢄꢇꢈꢉꢊ ꢌ
(1)
ꢀ
ꢁ
ꢆ
ꢐꢃ
F and F are the fluorescence intensity in the absence of aminoglycoside or at saturation, respectively,
o
∞
and n is the Hill coefficient or degree of cooperativity associated with binding. Error estimates were
generated from three sets of measurements.
Acknowledgment
This work was supported by funding from the Australian Research Council through a Future
Fellowship to BG (FT130100838), a Discovery Grant to LS, BG and YT (DP1094100) as well as a
Discovery Outstanding Researcher Award and Discovery Grant to LS (DP130100816).
2
3

ACCEPTED MANUSCRIPT
Supporting Information Available
Representative example of a FRET titration graph (Figure S1); typical dose response curve (Figure S2);
pH-dependence of the affinities of neomycin B and conjugate C1 for the A-site (Figures S3–S4).
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Highlights

Three new neomycin B derivatives were prepared which feature an alkyl chain-linked 1,4,7,10-
tetraazacyclododecane (cyclen) macrocycle.
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ACCEPTED MANUSCRIPT



Binding affinity of neomycin B-cyclen conjugates and their zinc(II) complexes for A-site RNA
was examined by FRET-based measurements.
Conjugates bind to the A-site with higher affinities than neomycin B due to their higher overall
positive charge, arising from protonated cyclen ring.
The Zn(II) complexes bind more strongly due to coordinative (and/or electrostatic) interactions
between cationic Zn(II)-cyclen moiety and phosphates or deprotonated uracil bases.
Figure for TOC:
Synopsis:
Three new derivatives of the aminoglycoside antibiotic neomycin B, incorporating the 1,4,7,10-
tetraazacyclododecane macrocycle, are reported together with an examination of the ability of these
compounds and their zinc(II) complexes to bind to the prokaryotic ribosomal A-site RNA via FRET-
based measurements.
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