Conjugate of neamine and 2-deoxystreptamine mimic connected by an amide bond

Article abstract of DOI:10.1016/j.bmcl.2011.06.084

An amino-functionalized 2-deoxystreptamine (2-DOS) mimic was conjugated by an amide bond to a neamine moiety containing a carboxylic acid in ring II. A library of A-site RNA and its mutants was prepared to examine RNA binding characteristics of the additi

Full text of DOI:10.1016/j.bmcl.2011.06.084

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4713–4715
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Conjugate of neamine and 2-deoxystreptamine mimic connected
by an amide bond
⇑
Venkatareddy Udumula, Maruthi Chittapragada, Jorden B. Marble, Daniel L. Dayton, Young Wan Ham
Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
An amino-functionalized 2-deoxystreptamine (2-DOS) mimic was conjugated by an amide bond to a nea-
mine moiety containing a carboxylic acid in ring II. A library of A-site RNA and its mutants was prepared
to examine RNA binding characteristics of the additional 2-DOS moiety attached to neamine. The 2-DOS
mimic conjugated to the neamine increased binding affinity up to 200-folds compared to that of neamine.
The conjugate binds to native A-site RNA and its mutants with up to 6-fold difference in sequence
selectivity.
Received 27 May 2011
Revised 16 June 2011
Accepted 17 June 2011
Available online 29 June 2011
Keywords:
2-Deoxystreptamine mimic
Neamine
Ó 2011 Elsevier Ltd. All rights reserved.
Aminoglycosides
RNA recognition
RNA is increasingly recognized as a practical drug target due to
their important biochemical functions and unique, but well-de-
fined, three-dimensional structures such as loops, bulges, and
pseudo-knots.1–3 However, it remains a formidable challenge to
design small molecules to selectively recognize certain RNA se-
quences or sites.
Aminoglycosides, a large class of clinically important antibiot-
ics, received considerable interest due to their capability to bind
to various RNA targets and have been used as a model in under-
standing the governing principles of RNA recognition by small mol-
ecules.4–17 Unfortunately, they have shown a high degree of
binding promiscuity and often lack specificity,18,19 which is largely
attributed to conformational flexibility rendered by glycosidic
bonds and multiple amino groups present in aminoglycosides
resulting in electrostatically driven binding interaction with target
RNAs.
crystal structures between 2-DOS of various aminoglycosides and
the 16S ribosomal A-site RNA.20 Due to this hydrogen bonding
capability, it has been speculated that 2-DOS may be utilized to de-
sign novel RNA binding ligands capable of sequence selective
recognition.
Recently we have reported a 2-DOS mimic capable of amide
connectivity in place of glycosidic linkage.22 Based on this 2-DOS
mimic, we report herein preparation of conjugate 1, a neamine
and a 2-DOS mimic connected by an amide bond (Fig. 1). It was
hypothesized that the newly introduced amide bond would
provide improved binding affinity and sequence selectivity in
RNA recognition possibly through additional hydrogen bonding
Although aminoglycosides as a whole demonstrate poor bind-
ing selectivity, 2-deoxystreptamine (2-DOS), the highly conserved
central aminocyclitol scaffold of aminoglycosides, demonstrates
sequence-specific recognition capability toward 50-GU-30 sequence
within the context of various aminoglycosides.20 Similarly, 2-DOS
alone, without an aminosugar attached, was shown to recognize
50-GU-30 although its binding affinity was low (ꢀ1 mM).21 These
results indicate that 2-DOS binding is not exclusively driven by
electrostatic interactions known to promote non-specific interac-
tions, but rather by hydrogen bonding interactions to a certain de-
gree. In fact, two conserved hydrogen bonds were evident in
Figure 1. Recognition of A-site RNA by neamine and design of conjugate 1 between
neamine and a 2-DOS mimic connected by an amide bond. Sequences inside blue
rectangle (will be randomly mutated) are expected to interact with the additional
2-DOS unit (blue) connected by an amide bond. The 5-OH (⁄) in ring II of neamine is
omitted in the conjugate.
⇑
Corresponding author. Tel.: +1 801 422 1939; fax: +1 801 422 0153.
E-mail address: yham@chem.byu.edu (Y.W. Ham).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.084

4714
V. Udumula et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4713–4715
interactions. The neamine moiety was used as a building block for
the conjugate preparation so that it may act as an anchor to bring
the additional 2-DOS moiety in close proximity to the sequence in-
side the blue rectangle (Fig. 1).
Compound
1 was prepared starting from asymmetrically
protected cyclohexanol 222 following the procedures shown in
Scheme 1. 2,5-dideoxystreptamine (2,5-DDOS) was used as the 2-
⁄
DOS mimic and the 5-OH in ring II of neamine (marked by in
Fig. 1) was omitted in the synthesis of the compound 1 as it pro-
jects out of RNA helix and therefore does not interact with any
nucleotide according to crystal structure. After protection of the
hydroxyl group of 2 to methoxy group and transformation of two
azides to NHBoc, the MOM protected hydroxyl group of the cyclo-
hexanol 2 was transformed to NH2 through two consecutive Mits-
unobu reactions. The amino group of 5 was further coupled with 6
to yield 7. In the coupling reaction, moderate heat was applied to
ensure completion of the amide coupling between 5 and 6 using
HATU/HOAt as coupling agents. After transformation of the two
NHBoc groups to azides, a glycosylation reaction was performed
Figure 2. Sequences of RNA targets. RNA1 is the native A-site sequence and RNA2–
8 are mutants. A1492 of the native A-site sequence is substituted by 2-AP by 2-
aminopurine (2-AP) for each RNA.
serve as a good tool to study preferred sequence by the 2-DOS mi-
mic attached to neamine in the context of fully base-paired dsRNA.
For each target RNA, 2AP, a fluorescent adenine analogue, was
introduced in place of A1492 to monitor binding of compound 1.
In order to confirm that the 2-AP modified A-site RNA could be
used for the binding assay, we treated RNA1 (the A-site RNA with
2-AP replacement at A1492) with neomycin, which is known to
form a 1:1 complex with A-site RNA. The titration exhibited a typ-
ical sigmoidal curve (Fig. 3) and its Kd value was estimated to be
using the procedure developed by Fugedi.23 The desired
9 was obtained as the major product with the approx. ratio of 2:1
for :b. Conjugate 1 was obtained after deprotection of the acetyl
a-anomer
a
groups and reduction of the azides to amines.
Compound 1 was tested for its RNA binding toward the native
A-site RNA sequence (RNA1) and its mutants (RNA2–8) (Fig. 2).
The mutants were constituted by replacing the 50-CG-30/50-GC-30
pair (red in Fig. 1) with random sequences. Neamine interacts with
several nucleotides of A-site RNA through four hydrogen bonds and
two electrostatic interactions (Fig. 1).20 All six interactions are con-
served when other bulkier aminoglycosides (gentamycin B, kana-
mycin A, ribostamycin, lividomycin A, and neomycin B, all of
which contain neamine as a part of their structures) bind to the
same A-site RNA and neamine recognizes the same RNA sequence
in the context of these aminglycosides.20 Based on this observation,
the neamine that was used as a base to which to attach the addi-
tional 2-DOS mimic was employed to serve as an anchor to bring
its attached 2-DOS mimic to a close proximity of the mutated
sequences. Therefore, the library of A-site RNA and its mutants will
1.5
l
M by Scatchard analysis. This value is in good agreement with
M for the complex of neomycin
the literature-reported Kd of 0.5
l
and A-site RNA.24 Then, several select RNAs (RNAs 3, 5, and 7) were
then treated with neomycin to ensure that mutation of the stem
sequences above the 2-DOS binding region did not cause detrimen-
tal effect in neomycin binding. We anticipated that the mutation
would have a negligible effect in binding affinity as the mutation
was made on the sequences that are not recognized by neomycin.
The mutations minimally affected the binding affinity of the RNAs
as demonstrated by the measured Kd values found to be between
1.1 and 2.4
curves.
lM. All three RNAs demonstrated sigmoidal binding
Then, compound 1 was titrated with each target RNA to assess
its binding affinity and sequence selectivity toward different target
(1) MeOTf
(1) DIAD, PPh3, PNBA,
NHBoc
N3
N3
BocHN
HO
THF, 70 oC
OCH3
(2) HCl/MeOH
MOMO
OH (3) Pd/C, H2,
NEt3, Boc2O
3
2
(2) K2CO3, MeOH
91% for 2 steps
85% for 3 steps
(1) PPh3, DIAD, DPPA
BocHN
H2N
NHBoc
OCH3
BocHN
HO
NHBoc
OCH3
THF, 70 o
C
N3
N3
O
HATU, HOAt,
DIPEA, DMF,
50 oC, 72%
5
4
(2) Pd/C, H2
83 %
OH
HO
N3
6
Tf2O/DMS, 4Å MS
Et2O:CH2Cl2 (5:2)
N3
N3
N3
OCH3
N3
O
AcO
AcO
N
3O
N3
N
H
R
R
N3
N3
O
9
O
O
AcO
SPh
AcO
HO
OCH3
N
H
8
N3
(1) K2CO3, MeOH
7
(2) P(n-Bu)3, MeOH, H2O
63%
a: R= NHBoc
b: R= N3
NH2
(1) TFA, CH2Cl2
(2) TfN3, K2CO3,
MeOH, CH2Cl2
65%
H2N
O
NH
2H2N
NH2
O
HO
HO
88%
H2N
OCH3
N
H
1
O
Scheme 1. Synthesis of the compound 1.

V. Udumula et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4713–4715
4715
NH2
I
O
HO
HO
II
H2N
H2N
O
NH2
HO
O
O
OH
III
H2N
HO
O
OH
NH2
HO
IV
O
Neomycin
Figure 3. Chemical structure of neomycin and titration curve between RNA1 and neomycin. The four rings of neomycin are indicated as I–IV. Neomycin is known to form a
1:1 complex with A-site RNA and demonstrated a sigmoidal binding curve.
which is yet a formidable challenge to achieve for medicinal chem-
ists. It binds to the native RNA sequence and all its mutants with
somewhat similar binding affinity. The biggest difference in se-
quence selectivity was observed to be 6-fold between RNAs 5
and 7.
Supplementary data
Supplementary data (detailed experiment procedures for syn-
thesis and spectral data of all compounds, and fluorescence bind-
ing assay condition and procedure) associated with this article
can be found, in the online version, at doi:10.1016/
j.bmcl.2011.06.084.
References and notes
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RNA sequences. All RNAs demonstrated sigmoidal regression
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show fluorescence decreases at the given concentrations in the
same assay conditions (up to overall 35
lM) (not shown). Based
on the dissociation constant (approx. 100
lM) reported for the
complex between neamine and the native A-site RNA from a foot-
print assay, attachment of an additional 2-DOS mimic to neamine
increased binding affinity by 35- to 200-fold to sub to low micro-
molar range, which is predictable as the additional 2-DOS mimic
has two amino groups that can interact with the negatively
charged phosphate backbone of RNA target. It was found that
RNA5 is the preferred target of compound 1 and demonstrated
six times stronger binding affinity when compared to RNA7.
In conclusion, we have prepared conjugate 1 by connecting an
amino-functionalized 2-DOS analogue with neamine moiety mod-
ified to have a carboxylic acid functional group by an amide bond.
A library of A-site RNA and its mutants was prepared to examine
RNA binding characteristic of the additional 2-DOS moiety at-
tached to neamine. The additional 2-DOS analogue attached to
neamine was observed to predictably increase binding affinity up
to 200-fold when compared with neamine. The conjugate did not
demonstrate exclusive recognition of a single target RNA sequence,
23. Fugedi, P.; Tatai, J. Org. Lett. 2007, 9, 4647.
24. Fourmy, D.; Recht, M. I.; Puglisi, J. D. J. Mol. Biol. 1998, 277, 347.