Rapid solid-phase syntheses of a peptidic-aminoglycoside library

Article abstract of DOI:10.1016/j.tet.2018.07.012

A library of mono- and di-amino acid peptidic-aminoglycosides (PAs), with kanamycin and neomycin as the model aminoglycosides, was systematically and rapidly synthesized via solid phase peptide synthesis. Aminoglycosides were first converted into N-Boc pr

Full text of DOI:10.1016/j.tet.2018.07.012

Accepted Manuscript
Rapid solid-phase syntheses of a peptidic-aminoglycoside library
Casey Kukielski, Krishnagopal Maiti, Sayantan Bhaduri, Sandra Story, Dev P. Arya
PII:
S0040-4020(18)30809-3
10.1016/j.tet.2018.07.012
TET 29672
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 24 April 2018
Revised Date: 3 July 2018
Accepted Date: 5 July 2018
Please cite this article as: Kukielski C, Maiti K, Bhaduri S, Story S, Arya DP, Rapid solid-phase
syntheses of a peptidic-aminoglycoside library, Tetrahedron (2018), doi: 10.1016/j.tet.2018.07.012.
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Graphical Abstract
Rapid Solid-phase Syntheses of a Peptidic-
Aminoglycoside Library
Leave this area blank for abstract info.
Casey Kukielskia, Krishnagopal Maitia, Sayantan Bhadurib , Sandra Storyb and Dev P Aryaa,b,*
aLaboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, Clemson,
South Carolina 29634, United States
bNUBAD, LLC, Greenville, South Carolina 29605, United States.

1
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Tetrahedron
journal homepage: www.elsevier.com
Rapid Solid-phase Syntheses of Peptidic-Aminoglycoside Library
Casey Kukielskia, Krishnagopal Maitia, Sayantan Bhadurib Sandra Storyb, and Dev P Aryaa,b,*
aLaboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, Clemson, South Carolina 29634, United States
bNUBAD, LLC, Greenville, South Carolina 29605, United States
*Corresponding author. Tel.: (864) 656-1106; fax: +0-000-000-0000; e-mail: dparya@clemson.edu
ABSTRACT
ARTICLE INFO
Article history:
Received
Received in revised form
Accepted
A library of mono- and di-amino acid peptidic-aminoglycosides (PAs), with kanamycin and neomycin as the model
aminoglycosides, was systematically and rapidly synthesized via solid phase peptide synthesis. Aminoglycosides
were first converted into N-Boc protected carboxylic acids and fourteen L-amino acids were then used in the
diversification of the full library. The approach outlined describes a rapid synthetic procedure where >200 PA
compounds can be synthesized in a few months with 85-95% purity. UV thermal denaturation assessed the binding
stabilization by PAs to model human and bacterial A-site rRNA sequences. Significant differences were found in
thermal melting profiles among PAs that were attributed to specific amino acid sequences. Neomycin PAs lead to a
Available online
much larger variation in the stabilization of A-site rRNA sequences (ꢀ
Tm = 2.6-17.1 oC) as compared to kanamycin
o
PAs (
ꢀTm = 0.4-4.3 C). Kanamycin PAs had little activity against Gram-negative and Gram-positive bacteria as
compared with neomycin PAs that had significant antibacterial activity with MIC ranging from 2 – 16 µM.
Keywords:
Aminoglycoside
Deoxystreptamine
Kanamycin
Neomycin
Peptidic library
1. Introduction
Aminoglycosides have been at the forefront of
antimicrobial therapy for the past seven decades. In addition to
their use as antibiotics for the treatment of Gram-positive and
Gram-negative bacterial infections, aminoglycosides are useful as
scaffolds for the recognition of nucleic acids.1,2,3
Aminoglycosides bind to the A-site of the 16S bacterial
ribosomal RNA (rRNA) and perturb protein synthesis causing
mistranslation.4 Prevalent use of aminoglycosides provides the
selective pressure for the development of resistance via
aminoglycoside modifying enzymes (AMEs) and more recently,
target mutations such as rRNA methylation.5 Various chemical
modifications have been reported to address the issues of
aminoglycoside resistance and target recognition of the bacterial
16S rRNA. The conjugation of peptides and aminoglycoside has
been reported to show evasion of action of AMEs by
circumventing AME modification of the aminoglycoside core
unit in addition to building unique scaffolds for RNA and DNA
recognition. We report herein solid phase chemical synthesis to
gain rapid access to structurally well-defined aminoglycoside-
dipeptide conjugate library, with kanamycin and neomycin
serving as model aminoglycosides. Coincident binding of peptide
and aminoglycoside to rRNA may further the diversity of RNA
stabilization that can be achieved using different amino acid
combinations. In general, the centrally located scaffold present in
most of the clinically important aminoglycoside antibiotics is the
2-deoxystreptamine (DOS) moiety6 (Figure 1).
Figure 1. A. 4,5-deoxystreptamine subclass (neomycin,
paromomycin, ribostamycin); B. 4,6-deoxystreptamine subclass
(amikacin, kanamycin, tobramycin, gentamicin, sisomicin,
netilmicin).
Based on the substitution pattern of the 2-DOS ring,
aminoglycosides are broadly divided into two different
subclasses, the 4,5- and 4,6-disubstituted DOS. Neomycin and
paromomycin belong to 4,5-disubstituted subclass and

2
kanamycin, tobramycin, amikacin, belong to 4,6- disubstituted
subclass (Figure 1).
included to identify the effect of these modifications on the
ACCEPTED MANUSCRIPT
electronics of the parent ring structure. We also report the
antibacterial activity of select kanamycin and neomycin PAs and
their effects upon binding human and E. coli A-site rRNA as
evaluated by UV thermal denaturation.
To circumvent issues of aminoglycoside resistance,
chemical modification of aminoglycosides has been used as a
viable strategy, with a new drug plazomicin7 recently receiving
FDA approval. Major challenges associated with chemical
syntheses of aminoglycoside-based novel antibiotics mirror the
inherent difficulties related to synthesis and purification of pure
and structurally well-defined carbohydrates. Challenges include:
(i) the presence of multiple hydroxy and amino groups with
comparable reactivity (ii) regio- and stereoselective glycosidic
bonds (iii) multi-step building block synthesis (iv) the need for
manipulation of numerous protecting groups and (v) tedious
column purification for multiple intermediates. Therefore, due to
the structural diversity of natural aminoglycosides, synthesis of
modified aminoglycoside libraries remains an arduous challenge.
A common strategy employed over the past decade has been to
maintain the core aminoglycoside scaffold and incorporate
additional binding/recognition moieties to synthesize structural
analogs of natural aminoglycoside antibiotics.
2. Results and discussion
2.1. Synthetic strategy
Retrosynthetic analysis of covalently linked peptidic-
kanamycin library is given in Figure 2. The scheme is designed
as a combination of both solid and solution phase synthesis.
Various synthetic protocols for the production of 5′′-
modified neomycin class of aminoglycosides have been reported
for modification of antibacterial activity and nucleic acid binding.
8-24
Optimization of nucleic acid binding has been achieved by
conjugating (via C5′′-OH modification) B-DNA minor groove
binding ligand Hoechst 3325825-27 and intercalators such as
fluorescein, 28-30 pyrene, 31-34 naphthalene diimide, anthraquinone,
methidium carboxylic acid37 for selective recognition of A-
35, 36
and B-form DNA and binding to different nucleic acid structures.
38
Novel perylene-neomycin conjugates have been utilized for
selective targeting of nucleic acid structures such as human
telomeric G-quadruplex DNA through base stacking and groove
39
recognition.
Various triazole linked neomycin dimers and
benzimidazole-neomycin conjugates through C5′′-OH group
modification with varying linker length have been used for
selective recognition of quadruplex, 40 duplex RNA, 41, 42 miRNA,
43
44-47
HIV TAR-RNA.
Moreover, various neomycin B dimers
tethered via triazole, urea and thiourea linkages were found to be
poor substrates for AMEs and selectively bind bacterial A site
rRNA over human A site rRNA. 48, 49 Nucleic acid binding chains
Figure 2. Retrosynthesis of peptidic-kanamycin conjugate
library.
such as PNA and DNA can be included in these target binding
In solution phase, kanamycin acid monomer 6, as the precursor
for solid phase synthesis, could be synthesized from kanamycin
amine 5, which in turn can be formed from commercially
available kanamycin A, 1 in 4 steps. Simultaneously, different
mono- and di- amino acid conjugates could rapidly be
50-52
approaches.
These reports suggest that improvements in
rRNA recognition can be attained by chemical modifications of
naturally-occurring aminoglycosides.
Most of the synthetic approaches described above are
performed using solution-phase synthesis. Compared to solid-
phase synthesis, solution-phase synthesis is a slow process as it
involves multiple purification steps during the course of
synthesized
by
modifying
the
standard
9-
fluorenylmethoxycarbonyl (Fmoc)-based solid phase peptide
synthetic strategy. 54 Amino acid conjugates can then be coupled
with kanamycin acid monomer 6, followed by global
deprotection to achieve a large PA library. The solid phase
synthesis was carried out using Fmoc-PAL-PEG-PS resin by
modifying Fmoc-based solid phase peptide synthetic strategy as
described in the results and discussion section. This modified
solid phase strategy could potentially be employed for the rapid
synthesis of large libraries consisting of various Fmoc-protected
amino acids, and various aminoglycosides by reducing the time
and manual labor drastically. PAs were synthesized on the resin
beads using HCTU (2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-
tetramethylaminium hexafluorophosphate) as coupling reagent
and DIPEA (N,N-Diisopropylethylamine) as base. Successful
global deprotection was achieved by using TFA (trifluoroacetic
acid)/m-cresol (8/2 v/v) mixture. All these conjugates were
synthesized (as shown in the schemes below) and characterized
synthesis. Thus,
a methodology for rapid production of
compound libraries followed by antibacterial screening and
ribosomal binding analysis is highly desirable. We recently
published a comprehensive approach for studying rRNA binding
affinity and antibacterial activity of peptidic-neomycin library to
identify novel rRNA binding antimicrobials. 53 Several PAs were
quickly identified that bind with high affinity and greater
selectivity to the E. coli A-site rRNA than the parent
aminoglycoside as compared to the mammalian A-site. These
promising results led us to expand the approach to include PAs
using kanamycin, which belongs to 4,6-deoxystreptamine
subclass. Herein, we successfully report the solid-phase assisted
rapid synthesis of a 210 member mono- and diamino-cid
peptidic-kanamycin library by using fourteen L-amino acids. A
comparison of NMR spectra of neomycin and kanamycin
conjugates with their precursor acid and PA derivative is

3
by NMR spectroscopy and mass spectrometry (MALDI-TOF) as
reported in the supporting information.
Scheme 2. Synthesis of kanamycin acid derivative 6, the
precursor for solid phase synthesis of kanamycin conjugates.
ACCEPTED MANUSCRIPT
In comparison, neomycin amine 8 (Scheme 3) was
obtained after hydrogenation of neomycin azide 7 for 12 h,
followed by filtration and column purification to afford amine 8
in 90% yield. Neomycin amine 8 was further reacted with
succinic anhydride to furnish the neomycin acid derivative 9, in
good yield.
2.2. Synthesis of kanamycin/neomycin acid derivative
In order to synthesize peptidic-kanamycin conjugates, our first
goal was to prepare kanamycin acid derivative 6, which was
initiated by selective protection of amino groups in kanamycin A,
1 with tert-butoxylcarbonyl (Boc) groups to obtain 2 as shown in
Scheme 1.
Scheme 1. Solution-phase synthesis of Kanamycin-azide 4.
Scheme 3. Synthesis of neomycin acid monomer 9: Precursor for
solid phase synthesis of neomycin conjugates.
The primary hydroxy group (6”-OH of ring III) of compound
2
was
then
selectively
protected
with
2,4,6-
triisopropylbenzenesulfonyl group, which was further substituted
by azide group via SN2 displacement reaction in order to achieve
the kanamycin azide derivative 4. RW.ERROR - Unable to find reference:13049
Hydrogenation of azide derivative furnished 6”-deoxy-6”-amino
kanamycin 5 which was reacted with succinic anhydride to yield
the amide bond linked kanamycin acid derivative 6 as shown in
Scheme 2. Using solvent conditions of methanol : ethyl acetate
(1:1, v/v) which completely solubilized kanamycin azide 4,
reduction was performed, and the reaction was monitored hourly
after the addition of Pd/C mixture. TLC showed ~80%
conversion after 1 h and complete conversion of 4 to 5 after 2 h.
A quick filtration to avoid the amine exposure to atmosphere was
performed to remove excess Pd/C and then dried under vacuum
for 1 h. The product 5 was then immediately reacted with
succinic anhydride in order to afford the final product, i.e.,
kanamycin acid derivative 6. Measured amount (1.05 equiv.) of
succinic anhydride was added in the reaction mixture to get
quantitative conversion of the amine 5 to the corresponding acid
6 and to avoid formation of any undesired kanamycin dimer
adducts (Scheme 2). Compound 6 was purified by extraction
with water and ethyl acetate and no column chromatography was
performed. The presence of a free carboxyl functional group in 6
allows us to use this key intermediate for further modifications
using solid phase peptide chemistry.
2.3. Synthesis of kanamycin/neomycin peptidic conjugates
With the kanamycin/neomycin acid monomers 6 and 9 in
hand, the solid phase synthesis of the peptidic-kanamycin or
neomycin library was carried out using Fmoc-PAL-PEG-PS resin
by modifying Fmoc-based solid phase peptide synthetic strategy
as shown in Scheme 4. The general protocol for solid-phase
synthesis of peptidic-kanamycin library is detailed in the
supporting information. This modified solid phase strategy was
successfully employed for the rapid syntheses of a 210-member
library consisting of mono- and diamino acid-kanamycin
conjugates (DPA 1900-2110) and neomycin conjugates (DPA
1167, 1261, 1267, 1276 and 1281) (shown in the supporting
information) by reducing the manual labor needed in solution
phase synthesis. Fifteen L-amino acids (β-alanine, arginine,
asparagine, aspartic acid, cysteine, histidine, leucine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine,
lysine and valine) were studied in the full library. Growing
peptide chain was attached to Fmoc-PAL-PEG-PS resin via
amide linkage. Kanamycin/neomycin acid derivative 6 and 9
were coupled with different mono- and diamino acid conjugates
on the resin beads using HCTU (2-(6-Chloro-1H-benzotriazole-
1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate)
as
coupling reagent and DIPEA (N,N-Diisopropylethylamine) as
base.
With the initial success, our next goal was removal of all
molecules from the solid support and global deprotection to
achieve peptidic-kanamycin/neomycin conjugate libraries.
Successful global deprotection and cleavage from the solid
support was accomplished by using
a mixture of TFA
(trifluoroacetic acid)/Phenol/water/TIPS (triisopropylsilane)
(88/5/5/2 v/v). The detailed experimental procedures and
characterization data (NMR and MALDI-TOF) of all the
compounds are shown in the supporting information. Complete
characterization including 1H, 13C NMR and MALDI were

4
performed on ten compounds, DPA 1167, 1261, 1267, 1276,
1281, 1966, 2060, 2066, 2075 and 2081 (Figure 3).
2.4. Synthesis of deprotected kanamycin/neomycin acid
derivative
ACCEPTED MANUSCRIPT
In order to perform comparative NMR studies of the
synthesized kanamycin/neomycin conjugates with their precursor
acid derivatives, deprotection (removal of tert-butoxycarbonyl
groups) of kanamycin and neomycin acid derivatives (6 and 9)
was carried out in the presence of trifluoroacetic acid (TFA) to
obtain the corresponding deprotected kanamycin and neomycin
acid derivatives 10 and 11 (Scheme 5). The formation of
compounds 10 and 11 was confirmed by NMR spectroscopy and
mass spectrometry (MALDI-TOF). The characterization data are
shown in the supporting information.
Scheme 5. Synthesis of deprotected kanamycin and neomycin
acid derivative.
Scheme
4.
Solid-phase
syntheses
of
peptidic-
kanamycin/neomycin conjugates.
2.5. NMR studies of kanamycin/neomycin conjugates with their
precursor acid derivative
A comparative study of the synthesized kanamycin and
neomycin conjugates with their corresponding acid precursor
were then undertaken by NMR spectroscopy. The protons of
aminoglycoside kanamycin A and neomycin B were assigned and
55, 56
confirmed as known in the literature.
We first assigned
anomeric protons and carbons for the acid derivative 10 and 11
by using 2D NMR spectroscopy. The HMQC (1H-13C 2D NMR)
spectra for the compound 10 and 11 are shown in Figure 4 and 5,
respectively. For clarity, we designated the cyclic rings for both
the systems (kanamycin and neomycin) as I-IV as shown in the
Figure 6. In 1H NMR spectrum of kanamycin acid derivative 10,
anomeric protons for the ring III and II appeared at 5.09 (J = 3.3
Hz) and 5.68 ppm (J = 3.9 Hz), as a doublet, respectively. The
anomeric carbons for the ring III and II in compound 10,
appeared at 100.7 and 96.8 ppm, respectively, in 13C NMR
spectrum of 10. On the other hand, the anomeric protons for the
pyranosyl ring II and IV in neomycin acid derivative 11,
resonated at 5.89 and 5.26 ppm as an apparent singlet,
respectively, whereas the anomeric proton of furanoside ring III
1
appeared at 5.34 ppm as a doublet (J = 3.6 Hz), in the H NMR
spectrum of 11. In 13C NMR spectrum of 11, anomeric carbons
for the pyranosyl ring II and IV appeared at 94.9 and 95.7 ppm,
respectively, whereas the anomeric carbon for the furanoside ring
III resonated at 109.13 ppm in 13C NMR spectrum of 11.
Figure
3.
Solid-phase
syntheses
of
peptidic-
Based on the assignment of anomeric protons and carbons of
kanamycin and neomycin acid derivative, we similarly assigned
the chemical shifts of their amino acid components. The 2D
NMR specta (HMQC) of the conjugates are shown in the
supporting information. Comparative NMR studies were carried
out with the PAs and their precursor acid derivative and
kanamycin/neomycin conjugates: Some of the representatives are
shown such as DPA 1167, 1261, 1267, 1276, 1281, 1966, 2060,
2066, 2075 and 2081.

5
1
kanamycin/neomycin sulphate and are given as stack plots of H
NMR spectra (Figures 7 and 8).
ACCEPTED MANUSCRIPT
Figure 7. Stack plot for 1H NMR spectra (in D2O) of kanamycin
sulphate, kanamycin acid derivative 10 and its various peptidic
conjugates.
Figure 4. HMQC spectrum kanamycin acid derivative 10.
1
Figure 8. Stack plot for H NMR (in D2O) spectra of neomycin
sulphate, neomycin acid derivative 11 and its various peptidic
Figure 5. HMQC spectrum neomycin acid derivative 11.
conjugates.
The resonance values for the anomeric protons and
carbons for both kanamycin and neomycin derivatives are shown
in Table 1 and 2, respectively. A small upfield shift of anomeric
H1 protons of the ring II in all the kanamycin conjugates was
observed as compared to its acid derivative 10. On the other
hand, a small downfield shift of anomeric H1 protons of the ring
II in all the neomycin conjugates was observed as compared to its
acid derivative 11. These results suggest that for both the 4,6-
and 4,5-DOS aminoglycosides, substitutions by different amino
acids at the primary hydroxy group do not substantially or
differentially perturb the electronics of the base rings. The amino
acid chains can then be expected to interact with the enzyme or
ribosomal RNA without electronically perturbing the functional
groups present in the natural aminoglycosides, that are required
for binding to the eubacterial rRNA A-site.
Figure 6. Structures of (a) kanamycin (a) and (b) neomycin
conjugates, designated with different ring systems, where R
represents hydroxy group for acid derivative; and amino acids
moiety for the conjugates.
The stack plots of 1H spectra of kanamycin and neomycin PAs
with their corresponding acid precursor is shown in Figure 7 and
8, respectively.

6
Table 1. Resonance values of anomeric protons and carbons of
kanamycin derivatives.
ACCEPTED MANUSCRIPT
Compound
Anomeric H1 for
ring II (ppm)
Anomeric H1′ for
ring III (ppm)
Anomeric C1 for
ring II (ppm)
Anomeric C1′ for
ring III (ppm)
Kanamycin sulphate
5.48
5.68
5.02
5.09
97.47
96.77
100.08
100.70
Kanamycin acid
derivative 10
Kan CV (DPA 2081)
5.62
5.56
5.57
5.56
5.65
5.07
5.03
5.05
5.04
5.07
97.07
97.06
97.25
97.27
97.05
100.77
100.78
100.81
100.81
100.72
Kan FV (DPA 2075)
Kan VY (DPA 2066)
Kan LY (DPA 2060)
Kan TD (DPA 1966)
Table 2. Resonance values of anomeric protons and carbons of neomycin derivatives.
Compound
Anomeric
Anomeric H1′ Anomeric H1′′ Anomeric C1 Anomeric
Anomeric C1′′
H1 for ring for ring III for ring IV for ring II C1′ for ring for ring IV
II (ppm)
5.98
(ppm)
5.40
(ppm)
5.25
(ppm)
95.15
III (ppm)
110.18
(ppm)
95.19
Neomycin sulphate
Neomycin acid
5.89
5.34
5.26
94.92
109.13
95.69
derivative 11
Neo CV (DPA 1281)
5.96
5.95
5.96
5.96
5.38
5.37
5.39
5.39
5.27
5.26
5.28
5.28
94.89
94.88
94.89
94.87
109.39
109.39
109.45
109.44
95.77
95.76
95.74
95.75
Neo FV (DPA 1276)
Neo VY (DPA 1267)
Neo LY (DPA 1261)
Neo TD (DPA 1167)
5.97
5.40
5.28
94.88
109.40
95.73
2.6. Thermal denaturation analysis of kanamycin/neomycin
conjugates binding human and E. coli A site rRNA
peptides when bound with the human A-site (Figure 12),
whereas upon binding with the E. coli A site, the melting
temperature (Tm) was found to vary from 71.3 to 79.5 oC (Figure
13). In both cases for the human and E. coli rRNA, neomycin
conjugate Neo LY exhibited the maximum increment of
The effects of select kanamycin and neomycin PAs with
a 27-base model of E. coli and human A-site rRNA (Figure 9)
was evaluated by UV thermal denaturation. The melting
temperature (Tm) for the human and E. coli A-site was found to
be 51.5 and 62.4 oC, respectively (Table 3). Kanamycin sulphate
o
denaturation temperature by 10.8 and 17.1 C, respectively. The
UV thermal denaturation studies reveal that conjugation of amino
acids to neomycin impart greater stabilization of both human and
E. coli A-site rRNA as compared to the corresponding
kanamycin PAs. This was most dramatic for the PAs Kan LY and
Neo LY that had the greatest ꢀTm of 4.3 oC and 17.1 oC
respectively of the target rRNA structures. To the best of our
knowledge, such large stabilizations of rRNA by tunable
aminoglycoside variants, such as PAs shown here, have never
been reported.
o
showed little change in Tm (-1 C) upon binding to the human A
o
site, whereas a small change in Tm (2.1 C) when bound with the
E. coli A site was observed (Figure 10 and 11). Neomycin
o
sulphate imparted a larger change in Tm of 3.7 C upon binding
with the E. coli A site. Kanamycin acid derivative (10) and
kanamycin-peptides exhibited a change in Tm that ranged from
o
o
0.4 to 2.6 C upon binding with human A-site (Tm of 51.5 C),
where kanamycin conjugate Kan LY showed the highest Tm of
o
54.1 C. A similar trend was observed with kanamycin-peptides
bound to the E. coli A site with increases in Tm of 1.1 to 4.3 C
o
o
with Kan LY yielding the highest Tm of 66.7 C as compared to
E. coli A site alone (Tm of 62.4 oC). The change in Tm varied from
2.6 to 10.8 oC with neomycin acid derivative (11) and neomycin-

7
ACCEPTED MANUSCRIPT
Figure 9. Secondary structures of the E. coli and human A-site
rRNA models used in this study.
Table 3. UV melting temperature (Tm, oC) of human A-site
rRNA and E. coli A-site rRNA bound to PAs
.
Tm
Human A-site rRNA
E. coli A-site rRNA
51.5
62.4
Tm
ꢀTm
Tm
ꢀTm
Figure 11. Thermal melting profiles of E. coli A-site rRNA upon
binding with kanamycin PAs.
Compounds
human
A-site
50.5
E. coli
A-site
64.5
Kanamycin sulphate
Kanamycin acid
derivative (10)
-1.0
+0.4
+2.1
+1.1
51.9
63.5
Kan TD (DPA 1966)
Kan LY (DPA 2060)
KanVY (DPA 2066)
Kan FV (DPA 2075)
Kan CV (DPA2081)
53.9
54.1
52.7
52.3
52.2
+2.4
+2.6
+1.2
+0.8
+0.7
64.4
66.7
64.8
65.0
65.2
+2.0
+4.3
+2.4
+2.6
+2.8
Neomycin sulphate
Neomycin acid
50.5
57.0
-1.0
+5.5
66.1
72.8
+3.7
+10.4
derivative (11)
Neo TD (DPA 1167)
61.8
62.3
+10.3
+10.8
71.3
79.5
+8.9
Neo LY (DPA 1261)
+17.1
Neo VY (DPA1267)
Neo FV (DPA 1276)
Neo CV (DPA1281)
59.0
54.1
58.4
+7.5
+2.6
+6.9
76.0
72.4
75.2
+13.6
+10.0
+12.8
Figure 12. Thermal melting profiles of human A-site rRNA upon
binding with neomycin PAs.
Figure 10. Thermal melting profiles of human A-site rRNA upon
binding with kanamycin PAs.
Figure 13. Thermal melting profiles of E. coli A-site RNA upon
binding with neomycin PAs.

8
2.7. Antibacterial activity of kanamycin- and neomycin- PAs
ACCEPTED MANUSCRIPT
Antibacterial activity for kanamycin and neomycin PAs were
assessed using two Gram-positive and two Gram-negative
reference strains. Similar to the effects observed in rRNA A-site
stabilization, kanamycin PAs were much weaker antibacterial
compounds with only DPA 2081 exhibiting significant activity
against the S. aureus and MRSA strain (Table 4). In contrast, E.
coli and S. aureus were susceptible to several of the neomycin
PAs with MIC values ranging from as low as 4 µM to 64 µM.
DPA 1281 was the most potent PA with MIC of 8 and 4 for E.
coli and S. aureus, respectively. Notably, the MRSA strain was
inhibited 30 - 37% with four of the neomycin PAs and up to 52%
with DPA 1281 at 64 µM.
Table 4. Minimum inhibitory concentration (MIC µM) of
kanamycin and neomycin PAs against bacterial strains and
(percent growth inhibition) for each bacterial strain.
DPA#
E. coli
S.
P.
MRSA
aureus aeruginosa
ATCC
25922
ATCC ATCC
ATCC
1960649
25923
27853
MIC of Kanamycin and Kanamycin Pas
Kanamycin sulphate
4
4
>64
>64
(11±1)
(25±3)
Kanamycin acid
derivative (10)
>64
(23±2)
>64
>64
>64
(32±3)
>64
33±2
>64 (0)
>64 (0)
>64 (0)
2.8. Docking studies of kanamycin/neomycin conjugates with
model E. coli A site RNA
(85±10) (13±1)
Kan TD (DPA 1966) >64 (0) >64 (0) >64 (0)
Kan LY (DPA 2060) >64 (0) >64 (0) >64 (0)
KanVY (DPA 2066) >64 (0) >64 (0) >64 (0)
Kan FV (DPA 2075) >64 (0) >64 (0) >64 (0)
Molecular docking studies were performed with few of the
conjugates binding to bacterial A-site model RNA. The model
(PDB 1pbr) used was the A-site of E. coli 16S ribosomal RNA. 57
The docking studies were performed with two kanamycin
conjugates (Kan LY and Kan CV) and two neomycin conjugates
(Neo LY and Neo CV) which showed significant change in
melting temperature (Tm) in UV denaturation experiments Figure
14. Analysis of the docked structures reveals that number of
interactions (hydrogen bonding, van der Waals) between amino
acid side chains of neomycin conjugates with E. coli A-site RNA
are much more pronounced compared to that of kanamycin
conjugates. Both the neomycin conjugates (Neo LY and Neo CV)
fit well inside the grooves of A-site RNA, and the hydroxy group
of tyrosine residue in Neo LY makes a close H-bond contact with
phosphate backbone between U5 and C6. In contrast, the
structure of Kan LY leaves tyrosine residue orienting away from
the groove, leaving the hydroxyl group far from the backbone.
Kan CV (DPA 2081) >64
(19±1)
>64
(40±4)
>64 (0)
>64
(30±6)
DPA#
E. coli
S.
P.
MRSA
aureus aeruginosa
ATCC ATCC
25923
ATCC
25922
ATCC
1960649
27853
MIC of Neomycin and Neomycin PAs
Neomycin sulphate
2
4
2
>64
(52±3)
>64
64
Neomycin acid
derivative (11)
2
>64 (0)
>64 (0)
>64 (0)
>64 (0)
>64 (0)
(a)
(b)
Neo TD (DPA 1167) 64
Neo LY (DPA 1261) 16
Neo VY (DPA 1267) 16
Neo FV (DPA 1276) 32
32
4
>64
(34±2)
>64
(30±4)
>64
(36±0)
>64
4
8
(37±2)
Neo CV (DPA 1281)
8
4
>64
>64
(14±1)
(52±4)
(d)
(c)
Figure 14. Docked structures of A-site bacterial RNA binding
with (a) Kan LY (DPA 2060); (b) Kan CV (DPA 2081); (c) Neo
LY (DPA 1261) and (d) Neo CV (DPA 1281)

9
Assisted Laser Desorption/Ionization (MALDI). 1H and 13C
3. Conclusion
ACCEPTED MANUSCRIPT
NMR spectral analyses were performed on a spectrometer
operating at 300 MHz, 500 MHz, and 75 MHz, 125 MHz,
respectively in D2O solutions unless otherwise stated. Chemical
shifts are reported with respect to solvent residual water peak at
A
210-member library of peptidic-kanamycin/neomycin
conjugates was successfully synthesized via a rapid and efficient
solid-phase protocol for developing DNA/RNA-targeted
therapeutics.
Various
mono-
and
di-amino
acid
1
4.79 ppm for H NMR. Coupling constants (J) are reported in
kanamycin/neomycin conjugates were rapidly synthesized. As
compared to the synthesis of neomycin acid monomer 9,
synthesis of kanamycin acid monomer 6 resulted in the formation
of various side products upon prolonged exposure to the
atmosphere. However, subtle change in the reaction solvent
conditions allowed us to isolate pure kanamycin amine within 2h
which was then immediately coupled with succinic anhydride to
afford kanamycin acid monomer 6 as the key precursor for the
solid phase synthesis. The data suggest that for the examples
studied here, the amino acid variations do not lead to a significant
perturbation in the electronic behavior of the core
aminoglycoside ring, allowing it to retain its specificity in
binding and function.
Hz. Standard abbreviations s, d, t, dd, br s, app s, m refer to
singlet, doublet, triplet, doublet of doublet, broad singlet,
apparent singlet, multiplet, respectively.
Antibacterial Screening and Minimal Inhibitory Concentration
(MIC) Determination.
Initial screening of the library of kanamycin-peptide conjugates
and neomycin-peptide conjugates against exponential phase
microbial cultures was performed at single point concentration of
12.5 µM and 6.25 µM respectively, in duplicate wells using 96-
well polystyrene microplates along with the reference compounds
kanamycin or neomycin sulfate and kanamycin or neomycin
acids. The same volume (10 µL) of sterile water with 1% DMSO
was used for antibiotic addition was added to the broth
(background control) and microbial culture (growth control).
Plates were incubated in a humidified incubator at 37⁰C for 15-
20 hours. The percent growth inhibition was calculated using the
formula:
Thermal denaturation analysis of PAs bound to human and E.
coli A-site rRNA identified several kanamycin and neomycin
peptidic conjugates that improved rRNA stability as compared to
kanamycin or neomycin alone demonstrating the utility of our
approach in modulating rRNA binding by simple amino acid
variations. Molecular docking studies of select peptidic
conjugates bound to the model bacterial A-site rRNA accurately
predicted differential binding stabilities of neomycin PAs as
compared to kanamycin PAs. These observations corroborated
with the significant antibacterial activity of the neomycin PAs
against aminoglycoside sensitive S. aureus and E. coli. Broader
antibacterial screens and synergy profiles of these peptidic
kanamycin- and neomycin conjugates on several Gram-positive
and Gram-negative bacterial strains and their binding affinities
for the bacterial 16S ribosomal A-site RNA using high-
throughput screening are currently underway in our laboratory
and will be reported in due course.
MIC values for select kanamycin-peptide conjugates and
neomycin-peptide conjugates were determined for exponential
phase bacterial cultures by the microdilution method in triplicate
according to the Clinical Laboratory Standards protocol. Stock
test compounds were prepared at ten times the final concentration
in 10% dimethylsulfoxide (DMSO) followed by a serial 1:2
dilution. For each dilution, a ten-microliter aliquot of stock test
compound was combined with 90 µl diluted bacterial suspension
(~3 x 105 cells per mL) in cation-adjusted Mueller-Hinton broth.
Final test compound concentrations from serial 2-fold dilutions
ranged from 64 µM to 1 µM. Plates were incubated in a
humidified incubator at 37oC for 15-20 h. After incubation with
test compound, growth was measured by absorbance at 595 nm
using a TecanM100Pro plate reader. The percent growth
inhibition at each concentration was averaged from triplicate
assays calculated from the formula above.
Previous approaches to synthesize modified aminoglycosides
in our hands and from other groups have been performed in
solution phase. A typical output for such solution phase reactions
is ~1 compound per week per person, and would be significantly
more time consuming, if multiple amino acids or fragments were
attached to the aminoglycoside. Using a non-automated solid
phase synthesis setup, we are able to synthesize up to 10
compounds per week per person, yielding
a
ten-fold
improvement in output, in addition to the savings of solvent and
silica needed for column purification. As more complex
conjugates are synthesized and automation is considered, the
increase in output could be further expanded to orders of
magnitude. The rapid approach described in this work for solid-
phase synthesis of various modified aminoglycoside conjugates
and high-throughput screening could pave the way for identifying
the optimal peptide chain residues and linker length required for
developing novel antibiotics against bacterial, viral, and fungal
pathogens containing potential nucleic acid binding sites.
4.1. Kanamycin acid derivative (10).
To a solution of compound 6 (0.1g, 0.102 mmol) in
dichloromethane (1 mL) trifluoroacetic acid (0.5 mL) was added
and stirred at room temperature for 3 h. The solvent was removed
under vacuum and the residue was dissolved in deionized water
(2 mL) and washed with ethyl acetate (3
× 10 mL).
Lyophilization of the aqueous solution produced deprotected
kanamycin acid derivative 10, as a white solid powder (0.055 g,
92%); 1H NMR (D2O, 300 MHz) δ 5.68 (d, J = 3.9 Hz, 1 H), 5.09
(d, J = 3.3 Hz, 1 H), 3.96-3.75 (m, 7 H), 3.67-3.32 (m, 9 H), 3.20-
3.13 (m, 1 H), 2.63 (d, J = 6.6 Hz, 2 H), 2.55 (d, J = 5.7 Hz, 2 H),
2.06-1.97 (m, 1 H), 1.32-1.23 (m, 1 H); 13C NMR (D2O, 75 MHz)
δ 175.55, 163.22, 162.75, 118.26, 114.40, 100.70, 96.77, 83.62,
77.62, 73.33, 71.89, 71.04, 70.97, 70.80, 68.71, 68.13, 66.66,
54.66, 49.95, 48.14, 40.45, 39.23, 30.42, 29.64, 27.52; MS
(MALDI-TOF) m/z calcd for C22H42N5O13 [M + H]+ 584.3, found
584.4.
4. Experimental section
General information. Solvents were dried and distilled according
to literature procedures. Chemicals were purchased from
commercial sources and were used without further purifications.
Silica gel (100-200 mesh and 230-400 mesh) was used for
column chromatography and TLC analysis was performed on
commercial plates coated with silica gel 60 F254. Visualization of
the spots on TLC plates was achieved by UV radiation or
spraying ethanolic solution of ninhydrin and acetic acid. Mass
spectra were obtained from Q-TOF instrument by Matrix
4.2. Kan CV (DPA 2081).

10
1H NMR (D2O, 500 MHz) δ 5.62 (d, J = 4 Hz, 1 H), 5.07 (app
s, 1 H), 4.54-4.52 (m, 1 H), 4.12-4.10 (m, 1 H), 3.98-3.91 (m, 6
H), 3.78-3.68 (m, 5 H), 3.59-3.45 (m, 6 H), 3.20-3.14 (m, 3 H),
2.62-2.59 (m, 4 H), 2.00-1.93 (m, 1 H), 1.26-1.24 (m, 1 H), 0.95
(d, J = 6.5 Hz, 6 H); 13C NMR (D2O, 75 MHz) δ 175.92, 175.01,
173.88, 172.21, 163.23, 162.76, 118.26, 114.39, 100.78, 97.07,
83.69, 78.41, 75.71, 75.17, 72.02, 70.79, 68.73, 67.99, 66.66,
65.12, 59.41, 55.57, 54.70, 54.34, 49.98, 47.97, 44.52, 42.52,
40.31, 29.73, 27.54, 25.22, 17.68, 16.21; MS (MALDI-TOF) m/z
calcd for C30H57N8O14S [M + H]+ 785.4, found 785.6.
(m, 1 H), 1.23-1.22 (m, 1 H), 1.19 (d, J = 6 Hz, 3 H); 13C
ACCEPTED MANUSCRIPT
NMR (D2O, 75 MHz) δ 176.47, 175.67, 174.78, 174.27, 172.13,
163.21, 162.74, 118.25, 114.39, 100.72, 97.05, 83.70, 78.14,
71.96, 70.84, 68.73, 68.19, 66.75, 66.66, 59.34, 54.34, 52.33,
49.86, 44.52, 42.52, 40.70, 35.29, 30.47, 29.86, 29.00, 18.72; MS
(MALDI-TOF) m/z calcd for C30H55N8O17 [M + H]+ 799.4, found
799.6.
4.7. Neomycin acid derivative (11)
To a solution of compound 9 (0.1g, 0.076 mmol) in
dichloromethane (1 mL) trifluoroacetic acid (0.5 mL) was added
and stirred at room temperature for 3 h. The solvent was removed
under vacuum and the residue was dissolved in deionized water
4.3. Kan FV (DPA 2075)
1H NMR (D2O, 300 MHz) δ 7.37-7.23 (m, 5 H), 5.56 (d, J =
3.6 Hz, 1 H), 5.03 (d, J = 3.3 Hz, 1 H), 4.58 (t, J = 7.5 Hz, 1 H),
4.00-3.85 (m, 6 H), 3.76-3.66 (m, 3 H), 3.58-3.37 (m, 8 H), 3.23-
3.16 (m, 2 H), 3.04 (d, J = 7.5 Hz, 1 H), 2.84 (s, 1 H), 2.50 (app
s, 4 H), 2.00-1.93 (m, 1 H), 1.25-1.22 (m, 1 H), 0.88 (d, J = 6.6
Hz, 6 H); 13C NMR (D2O, 75 MHz) δ 175.37, 175.24, 174.57,
173.24, 163.21, 162.74, 162.27, 136.13, 129.14, 128.79, 127.24,
118.26, 114.39, 110.53, 100.78, 97.06, 83.63, 78.80, 72.92,
72.02, 70.92, 70.77, 70.59, 68.74, 68.11, 66.57, 59.18, 55.19,
54.69, 54.34, 49.94, 47.87, 40.21, 39.23, 37.07, 30.57, 30.37,
29.84, 27.57, 18.26, 17.52; MS (MALDI-TOF) m/z calcd for
C36H61N8O14 [M + H]+ 829.4, found 829.6.
(2 mL) and washed with ethyl acetate (3
× 10 mL).
Lyophilization of the aqueous solution produced deprotected
neomycin acid derivative 11, as a white solid powder (0.048 g,
88%); 1H NMR (D2O, 300 MHz) δ 5.89 (app s, 1 H), 5.34 (d, J =
3.6 Hz, 1 H), 5.26 (s, 1 H), 4.35-4.19 (m, 5 H), 4.12-4.05 (m, 2
H), 4.02-3.85 (m, 2 H), 3.80 (s, 1 H), 3.75-3.33 (m, 12 H), 2.65-
2.45 (m, 5 H), 1.95-1.82 (m, 1 H); 13C NMR (D2O, 75 MHz) δ
178.80, 175.92, 163.56, 163.09, 162.62, 162.15, 122.11, 118.24,
114.38, 110.51, 109.13, 95.69, 94.92, 84.82, 80.82, 77.04, 75.23,
73.38, 72.16, 70.20, 70.08, 69.95, 68.04, 67.58, 67.39, 53.11,
50.77, 49.53, 48.47, 40.71, 40.44, 39.85, 31.01, 30.70, 27.88; MS
(MALDI-TOF) m/z calcd for C27H52N7O15 [M + H]+ 714.4, found
714.6.
4.4. Kan VY (DPA 2066)
1H NMR (D2O, 500 MHz) δ 7.16 (d, J = 8.5 Hz, 2 H), 6.82 (d,
J = 8 Hz, 2 H), 5.57 (s, 1 H), 5.05 (s, 1 H), 4.58-4.54 (m, 1 H),
3.98-3.88 (m, 6 H), 3.77-3.70 (m, 2 H), 3.60-3.33 (m, 9 H), 3.22-
3.14 (m, 3 H), 2.93-2.88 (m, 1 H), 2.60-2.52 (m, 4 H), 1.96-1.91
(m, 1 H), 1.28-1.21 (m, 1 H), 0.75 (d, J = 5 Hz, 6 H); 13C NMR
(D2O, 125 MHz) δ 175.71, 175.56, 175.26, 173.54, 163.41,
163.12, 162.84, 154.33, 130.48, 128.46, 117.47, 115.45, 115.15,
112.83, 100.81, 97.25, 83.66, 78.64, 73.12, 71.95, 70.96, 70.74,
70.58, 68.70, 68.10, 66.60, 59.95, 54.72, 54.58, 54.33, 49.92,
47.92, 40.21, 39.20, 35.91, 30.51, 30.21, 29.60, 27.58, 18.06,
17.17; MS (MALDI-TOF) m/z calcd for C36H61N8O15 [M + H]+
845.4, found 845.6.
4.8. Neo CV (DPA 1281)
1H NMR (D2O, 300 MHz) δ 5.96 (s, 1 H), 5.38 (s, 1 H), 5.27
(s, 1 H), 4.53 (t, J = 6.3 Hz, 1 H), 4.34-4.22 (m, 5 H), 4.10-3.92
(m, 5 H), 3.82 (s, 1 H), 3.76-3.69 (m, 2 H), 3.58-3.35 (m, 11 H),
2.90 (d, J = 5.7 Hz, 2 H), 2.63-2.48 (m, 5 H), 1.96-1.83 (m, 1 H),
0.94 (d, J = 6.9 Hz, 6 H); 13C NMR (D2O, 75 MHz) δ 175.84,
175.30, 174.76, 172.16, 163.17, 162.70, 118.26, 114.40, 110.53,
109.39, 95.77, 94.89, 84.85, 80.61, 77.32, 75.21, 73.37, 72.26,
70.29, 70.15, 69.89, 68.10, 67.56, 67.35, 59.43, 55.46, 53.17,
50.81, 49.54, 48.49, 41.35, 40.48, 39.96, 30.62, 30.26, 29.78,
27.86, 25.40, 18.33, 17.61; MS (MALDI-TOF) m/z calcd for
C35H67N10O16S [M + H]+ 915.4, found 915.5.
4.5. Kan LY (DPA 2060)
1H NMR (D2O, 300 MHz) δ 7.14 (d, J = 8.7 Hz, 2 H), 6.82 (d,
J = 8.4 Hz, 2 H), 5.56 (d, J = 3.6 Hz, 1 H), 5.04 (d, J = 2.4 Hz, 1
H), 4.55 (dd, J = 5.4 Hz, J = 10.2 Hz, 1 H), 4.10 (dd, J = 5.7 Hz,
J = 9.3 Hz, 1 H), 3.97-3.84 (m, 5 H), 3.77-3.67 (m, 2 H), 3.60-
3.31 (m, 8 H), 3.22-3.14 (m, 3 H), 2.92-2.83 (m, 1 H), 2.54-2.50
(m, 4 H), 1.95-1.82 (m, 1 H), 1.46-1.39 (m, 3 H), 1.27-1.22 (m, 1
H), 0.82 (d, J = 6.3 Hz, 3 H), 0.77 (d, J = 6.3 Hz, 3 H); 13C NMR
(D2O, 75 MHz) δ 175.73, 175.42, 175.21, 174.73, 163.21,
162.74, 154.33, 130.51, 128.47, 118.26, 115.40, 114.39, 100.81,
97.27, 83.65, 78.70, 73.11, 71.96, 70.97, 70.76, 70.58, 68.71,
68.11, 66.60, 54.73, 54.34, 52.96, 49.94, 47.94, 40.22, 39.50,
39.21, 35.82, 30.48, 30.24, 27.59, 24.12, 21.87, 20.72; MS
(MALDI-TOF) m/z calcd for C37H63N8O15 [M + H]+ 859.4, found
859.6.
4.9. Neo FV (DPA 1276)
1H NMR (D2O, 300 MHz) δ 7.35-7.22 (m, 5 H), 5.95 (s, 1 H),
5.37 (s, 1 H), 5.26 (s, 1 H), 4.58 (t, J = 7.5 Hz, 1 H), 4.31-4.21
(m, 5 H), 4.10-3.97 (m, 5 H), 3.81 (s, 1 H), 3.74-3.67 (m, 2 H),
3.58-3.38 (m, 11 H), 3.03 (d, J = 7.2 Hz, 2 H), 2.53-2.50 (m, 5
H), 1.96-1.92 (m, 1 H), 0.88 (d, J = 6.6 Hz, 6 H); 13C NMR (D2O,
75 MHz) δ 175.29, 175.24, 174.36, 173.24, 163.17, 162.70,
162.23, 136.09, 129.15, 128.80, 127.27, 118.26, 114.39, 109.39,
95.76, 94.88, 84.83, 80.58, 77.35, 75.21, 73.37, 72.25, 70.28,
70.14, 69.88, 68.08, 67.55, 67.35, 59.19, 55.20, 54.34, 53.16,
50.80, 49.54, 48.48, 40.46, 39.94, 37.18, 30.63, 30.23, 29.89,
27.87, 18.23, 17.55; MS (MALDI-TOF) m/z calcd for
C41H71N10O16 [M + H]+ 959.5, found 959.7.
4.6. Kan TD (DPA 1966)
1H NMR (D2O, 300 MHz) δ 5.65 (d, J = 3.6 Hz, 1 H), 5.07 (d,
J = 2.7 Hz, 1 H), 4.72 (t, J = 6.3 Hz, 1 H), 4.30-4.23 (m, 5 H),
3.94-3.87 (m, 4 H), 3.84-3.70 (m, 2 H), 3.57-3.37 (m, 6 H), 3.19-
3.13 (m, 2 H), 2.94-2.86 (m, 2 H), 2.68-2.65 (m, 4 H), 2.03-1.95
4.10. Neo VY (DPA 1267)
1H NMR (D2O, 500 MHz) δ 7.15 (d, J = 8 Hz, 2 H), 6.83 (d, J
= 8.5 Hz, 2 H), 5.96 (s, 1 H), 5.39 (s, 1 H), 5.28 (s, 1 H), 4.57-
4.54 (m, 1 H), 4.35-4.22 (m, 5 H), 4.13-3.93 (m, 5 H), 3.82 (s, 1

11
H), 3.73-3.70 (m, 2 H), 3.62-3.34 (m, 11 H), 3.20-3.09 (m, 2 H),
2.60-2.49 (m, 5 H), 1.95-1.92 (m, 1 H), 0.80-0.77 (m, 6 H); 13C
NMR (D2O, 125 MHz) δ 175.57, 175.33, 174.95, 173.48, 163.37,
163.08, 162.80, 162.52, 154.38, 130.51, 128.25, 119.79, 117.47,
115.43, 115.15, 112.84, 109.45, 95.74, 94.89, 84.84, 80.49,
77.36, 75.24, 73.36, 72.25, 70.30, 70.13, 69.86, 68.08, 67.53,
67.33, 59.72, 54.57, 54.33, 53.15, 50.79, 49.54, 48.47, 40.45,
39.97, 36.12, 30.62, 30.15, 29.87, 27.85, 18.14, 17.34; MS
(MALDI-TOF) m/z calcd for C41H71N10O17 [M + H]+ 975.5,
found 975.7.
the corresponding pdbqt files. The pdb file (1pbr)60 of the
ACCEPTED MANUSCRIPT
receptor bacterial A-site RNA was taken from Protein Data Bank
and prepared for docking by deleting the bound ligand. Final
figures of the docked structures were made by using PyMol.
Acknowledgments
We thank the National Institutes of Health for financial
support (grants R42GM097917, AI114114) to DPA.
References and notes
4.11. Neo LY (DPA 1261)
1H NMR (D2O, 500 MHz) δ 7.14 (d, J = 8.5 Hz, 2 H), 6.83 (d,
J = 8.5 Hz, 2 H), 5.96 (s, 1 H), 5.39 (s, 1 H), 5.28 (s, 1 H), 4.58-
4.55 (m, 1 H), 4.35-4.22 (m, 5 H), 4.17-4.12 (m, 2 H), 4.02-3.92
(m, 3 H), 3.82 (s, 1 H), 3.72-3.69 (m, 2 H), 3.59-3.34 (m, 10 H),
3.20-3.13 (m, 2 H), 2.57-2.55 (m, 5 H), 1.94-1.87 (m, 1 H), 1.44-
1.38 (m, 3 H), 0.84 (d, J = 6 Hz, 3 H), 0.79 (d, J = 6 Hz, 3 H);
13C NMR (D2O, 125 MHz) δ 175.61, 175.31, 174.90, 174.69,
163.37, 163.09, 162.81, 154.36, 130.53, 128.27, 119.79, 117.48,
115.39, 115.16, 109.44, 95.75, 94.87, 84.82, 80.48, 77.38, 75.25,
73.35, 72.24, 70.29, 70.13, 69.86, 68.09, 67.53, 67.33, 54.33,
53.15, 52.79, 50.79, 49.54, 48.47, 42.51, 41.47, 40.45, 39.95,
39.65, 36.00, 30.57, 30.09, 27.84, 24.15, 21.85, 20.76; MS
(MALDI-TOF) m/z calcd for C42H73N10O17 [M + H]+ 989.6,
found 989.8.
1. Moazed, D.; Noller, H. F. Nature 1987, 6121, 389-
394.
2. Arya, D. P. In - Aminoglycoside Antibiotics: From
Chemical Biology to Drug Discovery. Wang, B., Ed.; -
John Wiley & Sons, Inc.: 2007; pp - 319 pp.
4.12. Neo TD (DPA 1167)
1H NMR (D2O, 500 MHz) δ 5.97 (s, 1 H), 5.40 (s, 1 H), 5.28
(s, 1 H), 4.73-4.70 (m, 1 H), 4.36-4.24 (m, 7 H), 4.13-4.10 (m, 1
H), 4.03-3.94 (m, 3 H), 3.83 (s, 1 H), 3.73-3.68 (m, 2 H), 3.55-
3.37 (m, 10 H), 2.96-2.92 (m, 1 H), 2.88-2.86 (m, 1 H), 2.69-2.68
(m, 4 H), 2.60-2.59 (m, 1 H), 1.94-1.87 (m, 1 H), 1.20 (d, J = 6
Hz, 3 H); 13C NMR (D2O, 125 MHz) δ 176.96, 175.38, 174.82,
174.48, 172.08, 163.37, 163.08, 162.80, 162.51, 119.80, 117.47,
115.15, 112.83, 109.40, 95.73, 94.88, 84.81, 80.51, 77.29, 75.21,
73.34, 72.23, 70.25, 70.14, 69.85, 68.10, 67.55, 67.34, 66.87,
66.74, 59.15, 54.33, 53.16, 50.78, 49.97, 48.47, 40.46, 39.93,
35.63, 30.56, 29.87, 29.00, 27.85, 18.72; MS (MALDI-TOF) m/z
calcd for C35H65N10O19 [M + H]+ 929.4, found 929.6.
3. Willis, B.; Arya, D. P. Adv. Carbohydr. Chem.
Biochem. 2006, 251-302.
4. Chambers, H. F.; DeLeo, F. R. Nat. Rev. Microbiol.
2009, 9, 629-641.
5. Husain, N.; Tkaczuk, K. L.; Tulsidas, S. R.;
Kaminska, K. H.; Cubrilo, S.; Maravic-Vlahovicek, G.;
Bujnicki, J. M.; Sivaraman, J. Nucleic Acids Res. 2010,
12, 4120-4132.
UV thermal denaturation experiments. UV thermal denaturation
experiments were carried out using a Cary 100E UV-Vis
spectrophotometer with a thermoelectrically controlled 12-cell
holder. The stability of spectrophotometer and wavelength
alignment were checked prior to initiation of each UV
experiment. The samples were prepared by dilution of a stock
o
RNA sample that was prepared by heating the RNA to 95 C for
10 minutes, followed by slow cooling to room temperature. The
samples were incubated at 4 oC for 12 h, followed by the addition
of ligands in 1:1 molar ratio and kept at 4 oC for another 1 h. All
the samples were analyzed in quartz cells of 1 cm path length. All
UV experiments were monitored at 260 nm, conducted at a
heating rate of 0.2 oC/min, data were recorded every 1 oC interval
and melting temperature was determined by sigmoidal fitting
analysis in origin software. The buffer used in the preparation of
samples was HEPES-NaCl buffer (10 mM HEPES, 50 mM NaCl,
0.09 mM EDTA in DEPC-treated H2O, pH 7.0).
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58
were performed using AutoDock Vina. Prepared pdb files of
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