Studies on the synthesis of neamine-dinucleosides and neamine-PNA conjugates and their interaction with RNA

Article abstract of DOI:10.1016/j.bmcl.2008.09.062

Two types of neamine derivatives, neamine-dinucleotide conjugates 8a-g and neamine-PNA conjugates 12a-c and 14a-d, were synthesized. Compound 8a-g were synthesized by the condensation of azido-neamine with dinucleotide-5′-carboxylic acids, followed by reduction and deprotection. Compound 12a-c and 14a-d were synthesized by the similar strategy. The binding affinities of conjugates 8a-g, 12a-c, and 14a-d towards 16S RNA, 18S RNA, and TAR RNA were evaluated by SPR. It indicates that conjugates 12a-c and 14a-d interact with 16S, 18S RNA at the same level as that of neamine, 14a and 14d show about twofold binding affinities to TAR RNA compared to that of neamine. However, the neamine-dinucleotide conjugates 8a-g exhibit very weak binding affinities to 16S, 18S, and TAR RNA, computer modelling results that negative-negative electrostatic repulsion of phosphate group in compound 8a-g and RNA leads to a sharp decrease of the binding affinities compared with that of neamine, neamine-nucleoside and neamine-PNA conjugates.

Full text of DOI:10.1016/j.bmcl.2008.09.062

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5355–5358
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Studies on the synthesis of neamine-dinucleosides and neamine-PNA
conjugates and their interaction with RNA
*
Hui Mei , Lei Xing , Li Cai, Hong-Wei Jin, Peng Zhao, Zhen-Jun Yang , Liang-Ren Zhang, Li-He Zhang
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xueyuan Road #38, Beijing 100083, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 June 2008
Revised 13 September 2008
Accepted 16 September 2008
Available online 19 September 2008
Two types of neamine derivatives, neamine-dinucleotide conjugates 8a–g and neamine-PNA conjugates
12a–c and 14a–d, were synthesized. Compound 8a–g were synthesized by the condensation of azido-
neamine with dinucleotide-50-carboxylic acids, followed by reduction and deprotection. Compound
12a–c and 14a–d were synthesized by the similar strategy. The binding affinities of conjugates 8a–g,
12a–c, and 14a–d towards 16S RNA, 18S RNA, and TAR RNA were evaluated by SPR. It indicates that con-
jugates 12a–c and 14a–d interact with 16S, 18S RNA at the same level as that of neamine, 14a and 14d
show about twofold binding affinities to TAR RNA compared to that of neamine. However, the neamine-
dinucleotide conjugates 8a–g exhibit very weak binding affinities to 16S, 18S, and TAR RNA, computer
modelling results that negative–negative electrostatic repulsion of phosphate group in compound 8a–g
and RNA leads to a sharp decrease of the binding affinities compared with that of neamine, neamine-
nucleoside and neamine-PNA conjugates.
Keywords:
Dinucleotide
PNA
Conjugate
SPR
RNA binding
Ó 2008 Elsevier Ltd. All rights reserved.
Aminoglycosides, such as neomycin or paramycin, are well-
known natural products as clinical used important antibiotics.
The mechanism of their anti-bacteria activities is the interaction
of these molecules and ribosome RNA of bacteria, accordingly
interfering with protein biosynthesis.1 Unfortunately, aminoglyco-
sides are far from ideal antibiotics due to their toxicities and ad-
verse affects which are caused by the lack of selectivity towards
different RNAs.2 Recently, many efforts have been made to design
specific RNA binders. With the determination of structure of RNA-
aminoglycoside complex by X-ray or NMR,3 there have been more
specific RNA binders since then.4
and synthesized two peptide nucleic (PNA) analogue based mole-
cules which connected with arginine through a linker. CD spectra
indicate that both designed molecules do interact with TAR and
they were active against HIV-1 at micro-molar level.8
Based on the rich of adenine base in the binding pocket of A-site
rRNA, we synthesized a series of neamine-nucleoside conjugates to
improve their binding affinity and specificity to 16S and 18S RNA.9
It was found that compound 1 (Fig. 1) could bind specifically to 16S
RNA and computer simulation indicated that the thymine moiety
of compound 1 could form hydrogen bonding with adenine residue
of 16S RNA.
Other study shows that PNAs (peptide nucleic acid), which are
DNA analogues with a 2-aminoethyl-glycine linkage replacing
the phosphodiester backbone, are capable of sequence specific rec-
ognition of DNA and RNA, obeying the Watson–Crick hydrogen
bonding or/and Hoogsteen schemes.5 Short PNA probes were
shown to be able to disturb and finally to bind folded RNA struc-
tures.6 Gregory Upert and coworkers synthesized a series of con-
strained cyclic PNA derivatives containing the octameric PNA
fragment 50-GTCCCAGA-30, and UV-monitored thermal denatur-
ation studies demonstrate that these cyclic PNAs are able to
strongly interact with their trans-activation response (TAR) RNA
target, very likely through the formation of a 6-bp stable complex.7
Based on the NMR structure of TAR RNA, Raphael Terreux designed
To understand more information on the interaction between
the designed molecules and RNA, we synthesized neamine-dinu-
cleosides (type I) as well as neamine-PNA heteroconjugates (type
II), aiming to increase their binding affinity and selectivity towards
RNA. Herein, the synthesis and their binding properties to 16S RNA,
18S RNA, and TAR RNA were reported.
Synthesis of dinucleosides-50-carboxylic acids 6a–d. Dinucleosides
were synthesized through the phosphoramidite strategy (Scheme
1).10 Phosphoramidite 4 was coupled with the free 50-hydroxyl
group of deoxynucleoside 3 followed by iodine oxidation and
selective removal of the DMTr group using trifluoroacetic acid to
afford dinucleosides 5 in 76–84% yield. The structures of 5a–d
were confirmed by 1H, 13C, 31P NMR, and HRMS. The 50-OH of dinu-
cleosides 5a–d were oxidized by 2,2,6,6-tetramethyl-1-piperidi-
nyloxyl (TEMPO) and [bis(acetoxy)-iodo]benzene (BAIB) in
acetonitrile/water (1:1, less than 1 mL) at ꢀ40 °C to give dinucleo-
sides-50-carboxylic acids 6a–d in good yield (78–85%).11
* Corresponding author. Tel./fax: +86 10 82802503.
E-mail address: yangzj@bjmu.edu.cn (Z.-J. Yang).
The authors contributed equally to this paper.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.062

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H. Mei et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5355–5358
NH2
N3
O
O
HO
HO
H2N
HO
HO
H2N
NH2
T
N3
NH2
O
O
N3
N3
O
HO
HO
OH
H2N
NH
OH
NH
HN
NH2
H2N
O
O
HO
N
n
OH
2
O
H
Neamine
2
a
b
c
1
. n = 2; . n = 3; . n = 6
OH
NH2
O
NH2
O
H2N
HO
HO
NH2
H2N
NH2
O
HO
HO
NH2
OH
OH
O
NH2
NH
B1
n
HN
B2 or/and B1
H2N
O
O
HN
O
O
HO
N
n
N
O
P
O
B2
H
n = 1, 2;
n = 2,3,6;
O
O
B1 or B2 = uracil,
thymine, adenine
Type II
B1, B2 = uracil,
thymine
Type I
OH
Figure 1. Structures of aminoglycosides, neamine, neamine-dinucleotide conjugates (type I) and neamine-PNA conjugates (type II), and aminated azido-neamines.
B1
B
B
O
O
B1
HO
O
HO
HO
O
O
OAc
CN
B2
CN
B2
i
ii
3
O
+
O P O
O
O P O
O
76-84%
78-85%
DMTrO
NC
O
O
O
a. B1 = B2 = T;
b. B1 = B2 = U;
c. B1 = U, B2 = T;
O
OAc
OAc
5
P N
6
O
B = U or T
4
1
2
d
. B = T, B = U;
Scheme 1. Synthesis of 50-carboxyl-di-nucleotide 6a–6d. Reagents and conditions: (i) a—1H-Tetrazole/CH2Cl2; b—I2/THF/H2O/Py; c—3% CF3COOH/CH2Cl2; (ii) TEMPO/BAIB,
H2O/CH3CN, 35–40 °C.
Synthesis of neamine-dinucleotide 8a–g and neamine-PNA conju-
gates 12a–c and 14a–d. Condensation of azidodisaccharide 2 and
compound 6 was run with DCC/HOBt strategy. After removal of
cyanoethyl and acetyl group with concd aq ammonia, compound
7 was afforded in 36–53% yield. The azido group of intermediate
7 was reduced with H2S, followed by HPLC purification to obtain
neamine-dinucleosides conjugates 8a–g in 76–85% yield (Scheme
2).
zoyl of compound 10 with TFA followed with saturated ammonia
in methanol, the intermediate 11 was reduced by H2S to yield com-
pound 12a–c (Scheme 3). Neamine-di-PNA conjugates 13a–d were
obtained by condensation of 11a–b with 9a–b, respectively, in the
presence of DCC/HOBT. After deprotection and reduction as de-
scribed above, 14a–d were afforded in good yield (Scheme 4).
Study on the interaction of target compounds with 16S, 18S, and
TAR RNA by SPR. The interaction between aminoglycoside and
RNA has been successfully observed by Surface Plasmon Resonance
Neamine-PNA conjugates were synthesized by the following
procedure. Compound 2a was condensed with 9a–c, respectively,
to give 10a–c in good yield.9b,12 After deprotection of Boc and ben-
(SPR).13 The dissociation constants (Kd values in
lM) were calcu-
lated from the slope of the Scatchard plot.14 The dissociation con-
N3
NH2
O
O
N3
H2N
HO
HO
HO
HO
NH2
N3
N
O
3 O
H2N
OH
NH
OH
NH
n
NH
B1
n
O
B1
O
a. B1 = B2 = T, n = 2;
i
N
ii
O
O
2 + 6
H
b. B1 = B2 = U, n = 2
36-53%
76-85%
c. B1 = U, B2 = T, n = 2;
.Et3N
B2
O
.Et3N
B2
O
P OH
O
1
. B = T, B2 = U, n = 2
d
e
O P OH
O
1
2
. B = B = T, n = 3;
O
O
O
f. B1 = B2 = T, n = 6
g. B1 = B2 = U, n = 6
8
OH
7
OH
Scheme 2. Synthesisof amino disaccharide-dinucleotide conjugates. Reagents and conditions: (i) a—DCC, HOBt, DMF, 0 °C to rt; b—concd NH4OH, rt, then HPLC; (ii) a—H2S,
Pyridine/Et3N/H2O, rt, then HPLC; b—1 N HCl.

H. Mei et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5355–5358
5357
N3
O
NH2
O
N3
HO
HO
H2N
O
HO
HO
N3
B
NH2
N
3 O
H2N
B1
O
O
i
iii
B1
2a
+
OH
OH
N
HN
O
HN
69~82%
63~71%
BocHN
COOH
O
O
N
9
N
N
RHN
N
H
H2N
H
a. B = T; b. B = U; c. B = ABz
12
10. R = Boc; 11. R = H
a. B1 = T; b. B1 = U; c. B1 = A
ii. 69-70%
Scheme 3. General synthesis of neamine-PNA conjugates 12a–c. Reagents and conditions: (i) DCC, HOBt, DMF, rt; (ii) TFA, DCM, rt; (iii) H2S, Pyridine/Et3N/H2O, rt.
NH2
N3
O
O
N3
HO
HO
HO
HO
H2N
11a
11b
,or
N3
NH2
N3 O
B1
NH2 O
B1
O
ii
i
B2
B2
+
OH
OH
76~97%
45~55%
H2N
O
HN
O
O
HN
O
O
O
O
9a, or 9b
N
N
N
N
BocHN
N
N
N
N
H
H
H
H
14
13
1
2
1
2
1
2
1
2
a
b
c
d
. B = T, B = T; . B = U, B = U; . B = U, B = T; . B = T, B = U
Scheme 4. General synthesis of neamine-PNA conjugates 14a–d. Reagents and conditions: (i) DCC, HOBt, HBTU, DMF, rt; (ii) a—TFA, DCM, rt; b—H2S, Pyridine/Et3N/H2O, rt.
stants (Kd) of compounds 8a–g, 12a–c, and 14a–d to 16S, 18S, and
TAR RNA were listed in Table 1, respectively. Neamine-PNA conju-
gates 12a and 14a indicate the stronger binding activities to 16S
and 18S RNA compared with the activity of neamine, the other
conjugates interact with 16S or 18S RNA at the same level as that
of neamine. However, 14a and d show about twofold binding affin-
ities to TAR RNA compared to that of neamine. It seems that the
structure of PNA unit in compounds 12a–c and 14a–14d gives
some contributions to the interaction with 16S, 18S and TAR
RNA. Neamine-dinucleosides 8a–g conjugates show as much as
10–30 higher dissociation constants than that of neamine, with
less relationship with the kind of nucleobase or the length of linker.
Compared with compound 1, 8a–g displayed a sharp decrease of
binding affinity to 16S RNA. This implied that some modified func-
tion-groups in 8a–g decrease significantly the binding affinity of
aminoglycoside to the A-site of 16S RNA. It was reported that
16S RNA is highly discriminatory of the structure and conforma-
tion of aminoglycoside and their derivatives.15 Obviously, the addi-
tional amino groups in compounds 12a–c and 14a–d may increase
the electrostatic interactions with RNA, on the contrary, phosphate
groups in compounds 8a–g make the repulsion between such
interactions.
Study on computer modelling. For elucidating the binding mode
of neamine-dinucleosides and neamine-PNA conjugates to 16S
and 18S RNA, we carried out molecular docking study for com-
pounds 12a, 14a, and 8e to RNA using the program AutoDock
3.0. The target structure of 16S RNA was extracted from the protein
databank (PDB code 1PBR). Because there is no crystal structure for
human 18S ribosomal RNA, the target structure of it was produced
from molecular dynamics simulation. Binding ability of compound
to 16S and 18S RNA has been evaluated by the free energy. And the
conformation with the lowest free energy was used as the final
docked conformation for analysis. In this model, compounds 12a,
14a, and 8e can fit into the major groove of 16S or 18S ribosomal
RNA and the dinucleosides and PNA parts bind towards the bulge
region (except for 12a binding to 18S). As expected, some amino
groups of aminosugar interact strongly with the negative charges
of phosphate in RNA leading to strong electrostatic interaction.
Moreover, hydrogen bonding was also observed when 12a, 14a,
and 8e binding to 16S or 18S RNA. One of carbonyl oxygen is
strongly hydrogen bonded with the N4 atom of C1407 on the major
groove in 14a. Both of electrostatic interaction and hydrogen bond-
ing made all compounds binding to the pocket of RNA. However,
the phosphate group in 8e interacted with the phosphate negative
charges of residue G 1491 (16S RNA) or G 1405 (18S RNA). The neg-
ative–negative electrostatic repulsive interaction kept the dinucle-
osides part of 8e a tendency to far from the bulge region of RNA,
which was responsible for the lower binding affinity of the nea-
mine-dinucleosides conjugates to 16S or 18S RNA than the nea-
mine-PNA ones (Fig. 2).
Table 1
Interaction between neamine-dinucleotide, neamine-PNA conjugates and 16S, 18S, or
TAR RNA
Structure
Type Ia
Entry
Kd (16S)
Kd (18S)
S.F.*
Kd (TAR)
8a
8b
8c
8d
8e
8f
304
180
352
227
316
208
N/A#
11
548
404
1214
479
555
266
N/A#
26
1.80
2.24
3.45
2.11
1.76
1.28
N/A#
2.36
634
417
1000
476
533
115
N/A#
11
8g
Neamineb
Type IIa
12a
12b
12c
14a
14b
14c
14d
8
14
16
9
17
12
11
11
11
17
12
10
35
25
20
26
1.38
1.21
0.75
1.11
2.06
2.08
1.81
2.36
10
24
12
5
11
8
6
11
Neamineb
The free energy of the complexes reflected the binding affinity of
the compounds to 16S and 18S RNA. The negative free energy sug-
gested that the compounds 12a, 14a, and 8e could all bind to 16S
RNA and 18S RNA. Compared to compound 12a and 14a, the lower
*
Specific factor (S.F. = Kd 18S/Kd 16S). Kd in
Not available.
See Figure 2.
lM.
#
a
b
See Figure 1.

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H. Mei et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5355–5358
Figure 2. The binding mode of compound 14a (left), or 8e (right) with 16S RNA. Hydrogen bond between 14a and 16S RNA is shown as dotted line.
free energy for compound 8e shows its very weak binding interac-
tions with 16S and 18S RNA. Compound 12a and 14a showed the
similar binding affinity according to the free energy values. The cal-
culated results were in good agreement with SPR analysis.
References and notes
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In conclusion, two types of aminodisaccharide-dinucleosides
and aminodisaccharide-peptide nucleic acids conjugates were syn-
thesized by the condensation of azidodisaccharide and dinucleo-
sides/PNA monomer using aliphatic diamine as linker. Results
from the SPR evaluation suggested that aminodisaccharide-PNA
conjugate 12a–c and 14a–d were favourable for 16S, 18S, and
TAR RNA binding, while neamine-dinucleotides conjugate 8a–g
displayed a sharp decrease of binding affinity to 16S, 18S, and
TAR RNA. Molecular modelling indicated that the PNA unit in con-
jugates 12a–c and 14a–d gives some contributions for the interac-
tion between the designed compounds and RNA, but nucleotide
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.062.