Synthesis of neomycin analogs to investigate aminoglycoside-RNA interactions

Article abstract of DOI:10.1055/s-2003-40351

A series of novel aminoglycoside oligosaccharide analogs containing a 2,5-dideoxystreptamine core scaffold was prepared to study aminoglycoside binding to the small subunit of 16S rRNA. A set of monosaccharide building blocks carrying amino groups in diff

Full text of DOI:10.1055/s-2003-40351

LETTER
1323
Synthesis of Neomycin Analogs to Investigate Aminoglycoside-RNA
Interactions
Synthesis of
N
e
eomycin A
t
nalogs
e
to Investig
r
ate
A
minoglyco
H
side-RNA Interac
.
tions Seeberger,*a Michael Baumann,a,1a Guangtao Zhang,a,1b Takuya Kanemitsu,a Eric E. Swayze,b
Steven A. Hofstadler,b Richard H. Griffeyb
a
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge MA 02139, USA
Fax +1(617)2537929; E-mail: seeberg@mit.edu
b
Ibis Therapeutics, 2292 Faraday Avenue, Carlsbad, CA 92008
Received 28 May 2003
Dedicated to the Memory of Prof. Ray Lemieux.
to the small subunit of the 16S rRNA was studied using
a high throughput mass spectroscopy assay to determine
the effect of the 5-hydroxyl group on streptamine on the
affinity and selectivity of RNA binding.
Abstract: A series of novel aminoglycoside oligosaccharide ana-
logs containing a 2,5-dideoxystreptamine core scaffold was pre-
pared to study aminoglycoside binding to the small subunit of
16S rRNA. A set of monosaccharide building blocks carrying
amino groups in different positions and conformations around the
pyranose ring was utilized in the assembly of oligosaccharides.
These aminoglycoside analogs were analyzed for their RNA-bind-
ing capacity using a high throughput mass spectroscopy assay.
Key words: aminoglycosides, antibiotics, oligosaccharides, RNA-
targeting
RNA is a class of biopolymers with high structural and
functional diversity that constitutes an attractive therapeu-
tic target.2 Small molecules that serve as high affinity
RNA ligands have shown remarkable selectivity.3 Ami-
noglycosides, a clinically important class of antibiotics,
act by binding to bacterial RNA.4 High affinity binding of
different aminoglycosides to specific sites in larger RNA
molecules including ribosomal RNA, the hammerhead
ribozyme5 or smaller RNAs such as 16S rRNA and 23S
rRNA has been extensively studied.6,7 The interaction of
aminoglycoside analogs with RNA has provided a de-
tailed understanding of critical interactions responsible
for selective aminoglycoside-RNA binding.4,8,9 Since
Figure 1
electrostatic interactions play a key role in aminoglyco- 2,5-Dideoxystreptamine is structurally closely related to
side-RNA recognition, the introduction of additional 2-deoxystreptamine, that constitutes the core of many
amine groups significantly improved binding while de- aminoglycosides, including Neomycin B (Figure 1).
creasing selectivity. Screening of a series of aminoglyco- Synthetic access to the fully protected 2,5-dideoxy-
side analogs against the 16S rRNA, revealed a 1,3- streptamine core 2 from 111 was achieved in 89% yield by
hydroxyamine binding motif with micromolar affinity.10 treatment with NaN3/Tf2O (Scheme 1).12
To study some of the factors that govern binding affinity
and selectivity of the A and B rings of neomycin to RNA
a series of neomycin analogs had been prepared. The need
for new antibiotics due to increasing bacterial resistance
against currently used compounds mandates the search for
new drug materials.
Scheme 1
Here, we describe the synthesis of a series of aminoglyco-
side analogs containing a simplified 2,5-dideoxy-
Thioglycoside monosaccharide building blocks 7, 8, and
streptamine scaffold in place of the naturally occurring 2-
9
were prepared from glucose and glucosamine
deoxystreptamine core. Binding of these oligosaccharides
(Scheme 2).13 Other building blocks were prepared as pre-
viously described.14 The monosaccharides modules carry
amino groups in different positions and conformations
around the pyranose ring. Diazide 2 served as the core
Synlett 2003, No. 9, Print: 11 07 2003.
Art Id.1437-2096,E;2003,0,09,1323,1326,ftx,en;S05503ST.pdf.
© Georg Thieme Verlag Stuttgart · New York

1324
P. H. Seeberger et al.
LETTER
for the construction of aminoglycoside analogs by glyco-
sylation of one or both hydroxyl groups using thioethyl
glycosides activated by treatment with NIS/AgOTf.15
Even in the presence of excess glycosylating agent, main-
ly disaccharides 11–14 and only small amounts of trisac-
charides 15 and 16 were obtained (Scheme 3, Table 1).16
Glycosylation of disaccharide 11 with 9 afforded trisac-
charide 17 in 80% yield (Scheme 4).
Scheme 4
Table 1 Fully Protected Di- and Trisaccharides 11–16
R1
N3
H
R2
H
R3
R4
Yield (%)
11
12
13
14
15
16
N3
OBn
OBn
N3
59
87
34
60
6
N3
H
N3
N3
OBn
N3
H
OBn
OBn
N3
H
N3
H
OBn
OBn
N3
N3
8
Following assembly of the aminoglycoside analogs 11–17
in fully protected form, Pd(OH)2 catalyzed hydrogenation
relieved the oligosaccharides from the benzyl ether pro-
tective groups, while reducing all azides to amines.17 Ac-
cess to aminoglycosides 18–24 (Scheme 2) required
careful removal of any traces of ethane thiol, the leaving
group of the glycosylation reaction. The addition of acid
to the reaction mixture prevented catalyst inhibition by the
amine groups present in the products. The fully deprotect-
ed aminoglycosides 18–24 were obtained in 5–10 hours
(Table 2).
Scheme 2 i) a) PhCH(OMe)2, p-TsOH; b) BnBr, TBAI, NaH; ii)
a) AcOH, b) MsCl (79%); c) NaN3 (67%); iii) a) TFA (68%), b)
N2H4·Ac (94%); TfN3 (92%); iv) a) Tf2O, b) NaN3 (80%); v) a(Tf2O,
b) NaNO2, c) Tf2O, d) NaN3 (27%, 4 steps).
Table 2 Deprotected Di- and Trisaccharides 18–24
R1
R2
H
R3
R4
R5
–
R6
–
Yield (%)
18
19
20
21
22
23
24
NH2
H
NH2
NH2
OH
OH
OH
NH2
NH2
OH
OH
OH
49
57
38
8
NH2
H
–
–
NH2
OH
NH2
H
–
–
H
OH
–
–
H
NH2
NH2
NH2
NH2
H
H
H
59
NH2
H
NH2 quant.
NH2 78
NH2
Scheme 3
Synlett 2003, No. 9, 1323–1326 ISSN 1234-567-89 © Thieme Stuttgart · New York

LETTER
Synthesis of Neomycin Analogs to Investigate Aminoglycoside-RNA Interactions
1325
(6) Sears, P.; Wong, C.-H. Angew. Chem. Int. Ed. 1999, 38,
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Chem. Soc. 1998, 120, 1965.
Binding of aminoglycoside analogs 18–24 to the 16S
rRNA A-site (Figure 2) was screened using a FT-ICR
mass spectrometric assay.18 The formation of non-cova-
lent complexes between and the synthetic oligosaccha-
rides and the RNA substrate at different concentrations
were determined and the binding affinities were calculat-
ed. Tight binding neamine served as a positive control
whereas 18–24 did exhibit only weak binding affinity to
the RNA substrate. All aminoglycosides were also tested
in a bacterial transcription/translation assay where only
neamine showed inhibitory activity while analogs 18–24
failed to inhibit translation.
(11) Kavadias, G.; Velkof, S.; Belleau, B. Can. J. Chem. 1978,
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(13) 7: Rf = 0.81 (hexanes–EtOAc, 1:1); 1H NMR (300 MHz,
CDCl3): d = 7.40–7.28 (m, 10 H), 5.25 (d, J = 3.0 Hz, 1 H),
4.83 (dd, J = 10.1, 10.1 Hz, 2 H), 4.79 (dd, J = 10.2, 10.2 Hz,
2 H), 4.51 (d, J = 9.4 Hz, 1 H), 4.44 (dd, J = 6.3, 10.2 Hz, 1
H), 4.27 (dd, J = 6.8, 10.4 Hz, 1 H), 3.91 (dd, J = 6.6, 6.6
Hz, 1 H), 3.69 (dd, J = 3.0, 9.3 Hz, 1 H), 3.58 (dd, J = 9.4,
9.4 Hz, 1 H), 3.07 (s, 3 H), 3.03 (s, 3 H), 2.75 (m, 2 H), 1.32
(t, J = 7.4 Hz, 3 H); 13C NMR (75 MHz, CD3Cl): d = 137.7,
137.0, 128.8, 128.6, 128.5, 128.4, 128.2, 85.9, 80.5, 76.1,
76.0, 74.1, 73.6, 67.1, 39.4, 37.8, 25.5, 15.5; HRMS (FAB+)
Calcd for m/z C24H32O9S3Na [(M + Na)+] 583.1106, found
583.1112. 9: Rf = 0.38 (hexanes–EtOAc, 1:1); 1H NMR (300
MHz, CDCl3): d = 7.41 –7.30 (m, 10 H), 4.90 (dd, J = 11.1,
11.1 Hz, 2 H), 4.57 (dd, J = 11.1,11.1 Hz, 2 H), 4.32 (d,
J = 9.5 Hz, 1 H), 3.74 (dd, J = 2.3, 4.5 Hz, 1 H), 3.78–3.65
(m, 2 H), 3.44 (dd, J = 4.7, 9.6 Hz, 1 H), 3.38 (dd, J = 6.7,
6.7 Hz, 1 H), 3.36 (m, 1 H), 2.82 (br s, 1 H), 2.75 (m, 2 H),
1.32 (t, J = 7.4 Hz, 3 H); 13C NMR (75 MHz, CD3Cl): d =
138.2, 137.8, 128.8, 128.7, 128.4, 128.3, 128.1, 128.0, 84.7,
84.5, 78.0, 75.5, 73.9, 72.6, 70.7, 65.6, 24.9, 15.3; HRMS
(FAB+) Calcd for m/z C22H27N3O4SNa [(M + Na)+]
452.1619, found 552.1624. 10: Rf = 0.53 (hexanes–Et2O,
1:2); 1H NMR (300 MHz, CDCl3): d = 7.40–7.32 (m, 10 H),
4.72 (dd, J = 11.6, 11.6 Hz, 2 H), 4.57 (s, 2 H), 4.24 (d,
J = 10.3 Hz, 1 H), 4.08 (d, J = 3.2 Hz, 1 H), 3.77–3.67 (m, 2
H), 3.54 (dd, J1 = 7.1, 7.1 Hz, 1 H), 3.40 (dd, J = 3.1, 9.4 Hz,
1 H), 2.75 (m, 2 H), 1.31 (t, J = 7.2 Hz, 3 H); 13C NMR (75
MHz, CD3Cl): d =138.0, 137.2, 128.8, 128.7, 128.5, 128.3,
128.1, 128.0, 84.2, 81.3, 77.2, 73.9, 72.1, 69.4, 65.8, 62.1,
24.6, 15.2; HRMS (FAB+) Calcd for m/z C22H27N3O4SNa
[(M + Na)+] 452.1619, found 552.1634.
Figure 2 Sequence and 2° structure of 16S rRNA A-site model.
In conclusion, a series of di- and trisaccharide aminogly-
coside analogs, containing a 2,5-dideoxystreptamine core
in place of the naturally occurring 2-deoxystreptamine
moiety were synthesized. These aminoglycoside analogs,
differing by just one hydroxyl group from neamine, had
markedly lower affinity for the 16S A-site target (> 10-
fold) and did not inhibit bacterial translation.
Acknowledgement
This research was supported by ISIS Pharmaceuticals and the NIH
(R41 AI50406). P.H.S. thanks GlaxoSmithKline (Scholar Award),
the Alfred P. Sloan Foundation (Fellowship) and Merck for finan-
cial support. We thank Dr. K. A. Sannes-Lowery for performing
the binding assays and Ms. L. M. Risen for transcription/translation
and antibacterial assays.
(14) Greenberg, W. A.; Priestley, E. S.; Sears, P. S.; Alper, P. B.;
Rodenbohm, C.; Hendrix, M.; Hung, S.-C.; Wong, C. J. Am.
Chem. Soc. 1999, 121, 6527.
(15) Garegg, P. J. Adv. Carbohydr. Chem. Biochem. 1997, 52,
179.
(16) Glycosylation. General procedure. Thioethyl glycoside (0.1
mmol) and acceptor 2 (0.1 mmol) were coevaporated with
toluene and dried under vacuum. Et2O (1.5 mL), CH2Cl2 (0.5
mL), toluene (0.2 mL), and 4 Å MS (40 mg) were added and
the mixture was cooled to –40 °C. Addition of NIS (0.071
mmol) and AgOTf (0.020 mmol) followed. The mixture was
stirred for 5 h at –40 °C, EtOAc (20 mL) was added and the
filtrate was washed with Na2S2O3 (5% aqueous, 5 mL) and
dried over MgSO4. After removal of the solvents, the residue
was purified by silica gel column chromatography to afford
the disaccharides as two diastereomers.
References
(1) (a) Present address: Aventis Pharma Deutschland GmbH,
65926 Frankfurt, Germany (b) Present address: Provid
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95.
Synlett 2003, No. 9, 1323–1326 ISSN 1234-567-89 © Thieme Stuttgart · New York

1326
P. H. Seeberger et al.
LETTER
an additional 2 h. The catalyst was filtered off and the
solvent was removed under reduced pressure. The residue
was dissolved in water (5 mL) and freeze dried to afford the
deprotected aminoglycoside..
(17) Deprotection. General procedure. Protected di- or
trisaccharide (0.052 mmol) was dried 48 h at high vacuum,
dissolved in MeOH (5 mL) and stirred at room temperature
with Pd(OH2)/C (60 mg) while bubbling hydrogen through
the mixture. After 5 h AcOH (2 mL, 50% aqueous) was
added to the solution and the mixture was hydrogenated for
(18) Hofstadler, S. A.; Griffey, R. H. Chem. Rev. 2001, 101, 377.
Synlett 2003, No. 9, 1323–1326 ISSN 1234-567-89 © Thieme Stuttgart · New York