Synthesis of linked carbohydrates and evaluation of their binding for 16S RNA by mass spectrometry

Article abstract of DOI:10.1016/j.bmcl.2003.09.005

A library of linked molecules were synthesized from the common sugar moieties existing in the natural amino glycosides. These linked molecules were screened against bacterial 16S RNA for their binding affinity using a mass spectrometry-based technology. Some of these compounds exhibited low micromolar affinity and could serve as leads for further development as antibacterial agents.

Full text of DOI:10.1016/j.bmcl.2003.09.005

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3915–3918
Synthesis of Linked Carbohydrates and Evaluation of Their
Binding for 16S RNA by Mass Spectrometry
Baogen Wu, Jun Yang, Dale Robinson, Steve Hofstadler, Rich Griffey, Eric E. Swayze
and Yun He*
Ibis Therapeutics, A Division of Isis Pharmaceuticals, Inc., 2292 Faraday Av., Carlsbad, CA 92008, USA
Received 19 July 2003; accepted 4 September 2003
Abstract—A library of linked molecules were synthesized from the common sugar moieties existing in the natural amino glycosides.
These linked molecules were screened against bacterial 16S RNA for their binding affinity using a mass spectrometry-based tech-
nology. Some of these compounds exhibited lowmicromolar affinity and could serve as leads for further development as anti-
bacterial agents.
# 2003 Published by Elsevier Ltd.
The potential of RNA as a newdrug target has recently
come to the core, with the recognition that RNA mole-
cules can adopt complex three-dimensional structures
that, as with proteins, enable the design of specific
ligands.1À7 Another reason for the present interest
comes from the fact that many pathogenic agents, such
as retroviruses, encode their genetic information in
RNA strands.8À10 Amino glycoside antibiotics (Fig. 1)
have long been used as very efficient drugs against
Gram-positive and Gram-negative bacteria, and against
mycobacterial infections.11 These molecules, however,
impair hearing and kidney functions at high doses and
resistant strains are appearing at an increasing rate.12À15
In the meanwhile, their complex chemical structures
have impeded studies to discover aminoglycoside ana-
logues with improved pharmaceutical properties. It is of
great interest to find compounds with improved prop-
erties and simplified chemical structures.16À23
target site for these antibiotics. The oligonucleotide
mimics the binding affinity and specificity of aminogly-
cosides binding to the ribosome.27,28 The structure of
the RNA oligonucleotide complex with the aminogly-
coside paromomycin has been determined by NMR and
X-ray crystallography.29 The 27 nucleotide construct
has been used by several research groups in the RNA
binding studies (Fig. 2).
In search for newand more specific compounds, we
have developed mass-spectrometry based high-through-
put screening technologies.1,30À32 These technologies
allowus to discover ligands that bind to the target RNA
and to carry out SAR studies around the lead
The aminoglycoside antibiotics are thought to function
by binding to the decoding region of bacterial 16S
ribosomal RNA, thus causing premature termination
and mistranslation of proteins and consequently, bac-
terial death.24À26 The interaction between aminoglyco-
sides and the decoding region was recently characterized
using a 27-nucleotide RNA molecule containing the
Figure 1. Structures of neomycin B, paromomycin and tobramycin.
*Corresponding author at current address: Department of Medicinal
Chemistry, GNF, 10675 John Jay Hopkins Drive, San Diego, CA
92121, USA. Tel.: +1-858-332-4706; fax: +1-858-332-4513; e-mail:
yhe@gnf.org
Figure 2. Sequence of the Escherichia coli 16S RNA A-site.
0960-894X/$ - see front matter # 2003 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2003.09.005

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B. Wu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3915–3918
compounds. We have been using the 27-nucleotide con-
struct for the discovery of RNA binding molecules with
potential application in developing novel antibacterial
agents.22,23
three steps from 15 by allylation, epoxidation and ami-
nation. Again, the corresponding dimer 18 was obtained
in excellent yield and purity by reacting 17 with one
equivalent of 16 followed by the deprotection of the Boc
and acetonide protecting groups.
Several major classes of natural aminoglycosides have
been discovered. These include the neomycin class,
kanamycin class and gentamicin class. Many of these
aminoglycosides have a common 2-deoxystreptamine
(2-DOS, 4) moiety, which suggest the important role of
this moiety. Previous studies have established the direct
involvement of the neomycin A ring and paromomycin
A ring in causing the misreading in the bacterial trans-
lation. Both A rings were shown to contribute sig-
nificantly to the binding affinity and specificity for the
target 16S RNA.25 We have found that 2-DOS and
neomycin A ring binds to the target 16S RNA with
micromolar affinity. Since these aminosugar moieties
bind to the target with two or more binding sites, it was
perceived that linking these aminosugar moieties with a
proper linker could enhance the binding affinity and
specificity. Recently, Wong and coworkers have synthe-
sized a series of neamine dimers that showed significant
binding to the target RNA and exhibited lowmicro-
molar MIC activity against bacteria.33 Encouraged by
their results, we synthesized a library of linked mole-
cules with different linker length based on these motifs
to study their binding properties for the 16S RNA.
The synthesis of the paromomycin A-ring dimers 26–28
started from readily available N-acetyl glucosamine
(Scheme 2). Selective allylation of N-acetyl glucosamine
.
with allyl alcohol and BF3 Et2O gave the a-anomer in
71%. The acetate was removed with calcium carbonate
and the C4 and C6 hydroxy groups were protected as an
acetonide. Protection of the amino group in 21 with Boc
followed by epoxidation led to 22 in 78% overall yield,
which was converted to the corresponding monomers
23–25 with different lengths of linkers. The correspond-
ing dimers 26–28 were then obtained by reacting with
the corresponding linker amines and removal of the
protecting groups.
The synthesis of the 4-O substituted 2-DOS dimers 11–
13 and the related intermediates is shown in Scheme 1.
2-DOS (4) was first treated with TfN3 followed by the
protection of the adjacent hydroxy groups as an acet-
onide. The free hydroxy group was then allowed to
react with allyl bromide. Expoxidation of the resulted
allyl ether led to the formation of the corresponding
epoxide as a mixture of two diastereomers in a 1:1 ratio.
For the purpose of diversity synthesis and quick
screening, all the linked compounds were synthesized as
a mixture of diastereomers and used directly for the
RNA binding assays in this study. Reaction of epoxide
7 with three different diamines gave the corresponding
intermediates 8–10 with proper linkers that are ready
for coupling with another sugar moiety. Although reac-
tion of two equivalents of epoxide 7 with one equivalent
of linker diamine gave the corresponding dimers 11–13,
it was more efficient to react 8–10 with 7, which gave the
desired products in excellent yields and purity after
deprotection. These compounds were used directly for
our MS-based screening assay.
The synthesis of the 5-O substituted 2-DOS dimer 18
started from 4 again (Scheme 1). The selective intro-
duction of susbtituents at 5-hydroxy group was not a
trivial task. No efficient process has been reported in the
literature. We have developed a highly efficient and
practical route towards this problem. The two amino
groups in 2-DOS were first protected with Boc. Treat-
ment of 14 with BOMCl in the presence of DIEA and
catalytic amount of TBAI led to the formation of the
desired intermediate 15 in 68% yield together with its
regioisomer (15%). In a similar fashion to the synthesis
of 8–10, the desired intermediate 17 was prepared in
Scheme 1. Synthesis of 2-deoxystreptamine (2-DOS) dimers. Reagents
and conditions: (a) TfN3, CuSO4, MeOH, Et3N, 85%; (b)
Me2C(OMe)2, PPTS, DMF, 100 ꢀC, 98%; (c) NaH, allyl bromide,
DMF, 0 ꢀC, 93%; (d) Me3P, NaOH, MeOH, 88%; (e) Boc2O, NaOH,
dioxane, 90–95%; (f) mCPBA, CH2Cl2, NaHCO3, 82%; (g) 10 equiv
MeNH(CH2)4NHMe, EtOH, reflux, 95%; (h) 1.0 equiv epoxide (7 or
16), EtOH, reflux, 90–95%; (i) 4.0 M HCl, dioxane, 90–95%; (j)
BOMCl, EtNiPr2, TBAI, 80 ꢀC, 68% plus 15% regioisomer; (k) NaH,
DMF, glycidol triflate, 85%.

B. Wu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3915–3918
3917
The synthesis of the neomycin A-ring dimers 35–37
started from 20. Selective tosylation and displacement
of the resulted tosylate converted the primary alcohol
into the corresponding azide. The azide was then
reduced with PMe3, and the amino groups were pro-
tected with Boc. Again, the terminal olefin was con-
verted into epoxide with mCPBA, which reacted with
three linker amines to give the corresponding inter-
mediates 32–34 and dimers 35–37 by following the same
procedures as described above.
The linked molecules were screened against 16S RNA
for their binding affinity using a mass spectrometry-
based assay and their estimated Kd are shown in Table
1.32 Data suggest that all the linked molecules tested
have improved binding affinity for 16S RNA compared
to the corresponding monomers. While the 2-DOS
dimmers (11–13, 18, 49) showed moderate improvement
over 2-DOS (4), the glucosamine dimmers acquired sig-
nificant improvement in potency over the corresponding
monomers (26–28 vs 51; 35–37 and 50 vs 52). In parti-
cular, 27 exhibited more than 35-fold improvement in
potency over 51. The cross-linked molecules (38–48)
between 2-DOS and glucosoamine also exhibited
improved binding affinity over the corresponding
monomers (4, 51 and 52). The data also suggest that the
glucosamine dimmers have higher potential for binding
affinity improvement as compared to the 2-DOS dim-
mers. In general, compounds with relatively longer lin-
kers (n=2 and 4) exhibited higher potency (11 vs 12 and
13, 26 vs 27 and 28, 41 vs 42 and 43, 44 vs 45 and 46).
Among these compounds, 12, 36, 37 and 45 showed
highest potency with Kd in the low mM range. Studies to
further improve the potency of these compounds for
The crossly linked molecules 38–48 were easily prepared
by following similar procedures as for the dimer synth-
esis described in Schemes 1 and 2 (Scheme 3). All these
linked compounds were obtained in excellent yields and
purity. To explore alternative linkers for the library
synthesis, compounds 49 and 50 were also synthesized,
which have a bisester linker and a piperazine linker
respectively.
For comparison, the aminosugar intermediates in their
free form were prepared by easily removing the acid labile
protecting groups with hydrochloric acid (Scheme 4).
Scheme 3. Synthesis of cross-linked molecules from 2-DOS, glucos-
amine and neomycin A ring, and dimers with piperazine and ester
linkers. Reagents and conditions: (a) EtOH, reflux, >95%; (b) HCl,
MeOH, >95%; (c) 0.5 equiv piperazine, ethanol, reflux, >95%; (d)
HO2C(CH2)3CO2H, PS-carbodiimide, EtNiPr2, 75%; (e) PMe3,
NaOH, MeOH, 90%.
Scheme 2. Synthesis of neomycin A ring and glucosamine dimers.
.
Reagents and conditions: (a) allyl alcohol, BF3 Et2O, 71%; (b)
Me2C(OMe)2, PPTS, 95%; (c) Ca(OH)2, 89%; (d) Boc2O, NaOH,
dioxane, 90–90%; (e) mCPBA, NaHCO3, 75–80%; (f) 10 equiv
MeNH(CH2)nNHMe, EtOH, reflux, 90–95%; (g) EtOH, epoxide (22 or
31) reflux, 90–95%; (h) HCl, MeOH, 90–95%; (i) TsCl, EtNiPr2, 82%;
(j) NaN3, 91%; (k) Ca(OH)2, 87%; (l) PMe3, NaOH, MeOH, 91%.
Scheme 4. (a) HCl, MeOH, 95%; (b) Me3P, NaOH, MeOH, 91%.

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B. Wu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3915–3918
Table 1. Binding affinity of the linked molecules for 16S RNA (mM)
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