A new class of branched aminoglycosides: Pseudo-pentasaccharide derivatives of neomycin B

Article abstract of DOI:10.1021/ol035213i

[Equation presented] The clinically important antibiotic neomycin B was modified at position C5″ by adding one extra sugar ring in the β-configuration, and the observed pseudo-pentasaccharides were tested against various bacterial strains, including patho

Full text of DOI:10.1021/ol035213i

ORGANIC
LETTERS
2003
Vol. 5, No. 20
3575-3578
A New Class of Branched
Aminoglycosides:
Pseudo-Pentasaccharide Derivatives of
Neomycin B
Micha Fridman,† Valery Belakhov,† Sima Yaron,‡ and Timor Baasov*,†
Department of Chemistry and Institute of Catalysis Science and Technology and
Department of Food Engineering and Biotechnology, TechnionsIsrael Institute of
Technology, Haifa 32000, Israel
chtimor@tx.technion.ac.il
Received June 30, 2003
ABSTRACT
The clinically important antibiotic neomycin B was modified at position C5′′ by adding one extra sugar ring in the
â-configuration, and the
observed pseudo-pentasaccharides were tested against various bacterial strains, including pathogenic and resistant strains. The designed
antibiotics show antibacterial activity superior to that of neomycin B against pathogenic and resistant bacterial strains.
Aminoglycoside antibiotics are clinically important drugs that
function through binding to specific sites in prokaryotic
ribosomal RNA (rRNA) and affecting the fidelity of protein
synthesis.1 However, rapid emergence of resistance and high
human toxicity has limited their use and resulted in the need
for novel structures devoid of these limitations.2 Hence, many
structural analogues of natural aminoglycosides have been
synthesized over the past decade.3
the construction of diverse analogues as potential new anti-
biotics.4 Some of the designed structures showed considerable
antibacterial activities. Unlike this strategy, we hypothesized
that since aminoglycoside antibiotics exert their antibacterial
activity by selectively recognizing and binding to rRNA, it
is likely that keeping the whole antibiotic constitution intact
while adding an additional recognition/binding elements will
result in superior binding to rRNA and probably better anti-
In the majority of these studies, a minimal structural motif,
which is common for a series of structurally related ami-
noglycosides, has been identified and used as a scaffold for
(4) For representative studies, see: (a) Kumar, V.; Jones, G. S., Jr.;
Blacksberg, I.; Remers, W. A. J. Med. Chem. 1980, 23, 42-49. (b)
Yoshikawa, M.; Ikeda, Y.; Takenaka, K. Chem. Lett. 1984, 13, 2097-2100.
(c) Girodeau, J.-M.; Pineau, R.; Masson, M.; Le Goffic, F. J. Antibiot. 1984,
37, 150-158. (d) Alper, P. B.; Hendrix, M.; Sears, P.; Wong, C.-H. J. Am.
Chem. Soc. 1998, 120, 1965-1978. (e) Greenberg, W. A.; Priestley, E. S.;
Sears, P. S.; Alper, P. B.; Rosenbohm, C.; Hendrix, M.; Hung, S.-C.; Wong,
C.-H. J. Am. Chem. Soc. 1999, 121, 6527-6541. (f) Wang, J.; Li, J.; Tuttle,
D.; Takemoto, J. Y.; Chang, C.-W. T. Org. Lett. 2002, 4, 3997-4000. (g)
Hanessian, S.; Tremblay, M.; Swayze, E. Tetrahedron 2003, 59, 983-993
and references therein.
† Department of Chemistry and Institute of Catalysis Science and
Technology.
‡ Department of Food Engineering and Biotechnology.
(1) Davis, B. D. Microbiol. ReV. 1987, 51, 341-350.
(2) Wright, G. D.; Berghuis, A. M.; Mobashery, S. AdV. Exp. Med. Biol.
1998, 456, 27-69.
(3) For a recent review, see: Ye, X.-S.; Zhang, L.-H. Curr. Med. Chem.
2002, 9, 929-939.
10.1021/ol035213i CCC: $25.00
© 2003 American Chemical Society
Published on Web 09/06/2003

that all the functional groups of aminoglycosides utilized for
binding are identical in both, with the exception of two,
which are not employed for binding in the antibiotic-
resistance enzyme.11 One of these groups is the C5′′-OH of
neomycin B, which is phased toward the second substrate,
ATP, and may have a crucial role for the formation of the
reactive ternary complex prior the phosphorylation step.
Therefore, we anticipated that incorporation of gross changes
such as addition of an extra rigid sugar ring in this region
might have a dramatic effect on the formation of the precise
ternary complex required for enzymatic catalysis. Taken
together with the relative ease of derivatizing a primary
alcohol, we selected position C5′′ in neomycin B and
prepared the new generation of pseude-pentasaccharides 2-5
with the expectation that they will function better than
neomycin B against both the resistant and non-resistant
organisms.
Our strategy for the construction of 2-5 (Schemes 1 and
2) was to convert commercial neomycin B to a common
Figure 1.
Scheme 1a
bacterial performance. Enhancement of RNA binding by
using dimerized aminoglycosides,5 bifunctional aminoglyco-
sides,6 and amino-aminoglycosides7 supports this hypothesis.
To this end, we have modified neomycin B (1, Figure 1)
by adding an additional sugar as a ring V at C5′′-OH and
prepared a series of pseudo-pentasaccharides 2-5. These
structures keep the whole antibiotic constitution intact as a
recognition element to the rRNA, while the extended sugar
ring (V) of each structure was designed in a manner that
incorporates either cis-1,2-dimine (2), flexible 1,3-diamine
(3), cis-1,3-hydroxyamine (4), or ribofuranose ring (5) as
potential functionalities directed for the recognition of the
phosphodiester bond of RNA.8-10
In selecting the modification site in neomycin B and the
degree of modification, we have taken into consideration the
following recently found structural information. Superposi-
tion of neomycin B bound to the aminoglycoside kinase
APH(3′) ternary complex with ADP,11 as well as paromo-
mycin I (containing C6′-OH instead of C6′-NH2 as in
neomycin B) bound to A-site bacterial ribosome,12 reveals
a Reagents and conditions: (a) (i) TBDPSCl, pyridine, DMAP,
60 °C; (ii) 2,2-dimethoxypropane, acetone, CSA; (iii) BzCl,
pyridine, DMAP; (iv) AcOH/H2O 9:1, THF, 60 °C, 55% for four
steps. (b) (i) Tf2O, pyridine; (ii) NaN3, DMF, HMPA, 63% for two
steps; (iii) HF/pyridine; (iv) ClAcCl, pyridine, 91% for two steps.
(c) (i) Anisaldehyde-dimethylacetal, CSA, THF; (ii) BzCl, pyri-
dine; (iii) AcOH/H2O 9:1, THF, 60 °C, 58% for three steps. (d) (i)
Tf2O, pyridine; (ii) NaN3, DMF, HMPA, 86% for two steps.
(5) Michael, K.; Wang, H.; Tor, Y. Bioorg. Med. Chem. 1999, 7, 1361-
1371.
acceptor (14) to which the donors 6-913 can be connected.
The protecting groups used in this study served admirably
in terms of the ease of attachment and removal and sur-
vivability under the reaction conditions, whereas the thiogly-
coside-NIS glycosidation method14 proved to be both rapid
and efficient. The N-phth and ester protections at C-2 of the
monosaccharide donors (6-9) were designed to allow,
through neighboring group participation, selective â-glyco-
side bond formation between rings V and III.
(6) Sucheck, S. J.; Wong, A. L.; Koeller, K. M.; Boehr, D. D.; Draker,
K.-a.; Sears, P.; Wright, G. D.; Wong, C.-H. J. Am. Chem. Soc. 2000, 122,
523-524.
(7) Wang, H.; Tor, Y. Angew. Chem., Int. Ed. Engl. 1993, 32, 109-
111.
(8) For the interaction of unstructured diamines with RNA, see: (a)
Yoshinari, K.; Yamazaki, K.; Komiyama, M. J. Am. Chem. Soc. 1991, 113,
5899-5901. (b) Komiyama, M.; Yoshinari, K. J. Org. Chem. 1997, 62,
2155-2160. (c) See, also: Kirk, S. R.; Tor, Y. Chem. Commun. 1998, 147-
148.
(9) For the favorable interaction of the â-hydroxyamine moiety in sugars
with the phosphodiester group and the Hoogsteen face of guanine residues
in RNA, see: Hendrix, M.; Alper, P. B.; Priestley, E. S.; Wong, C.-H.
Angew. Chem., Int. Ed. Engl. 1997, 36, 95-98.
(10) For the interaction of ribofuranoses, see: (a) Han, M. J.; Yoo, K.
S.; Cho, T. J.; Chang, J. Y.; Cha, Y. J.; Nam, S. H. Chem. Commun. 1997,
163-164. (b) Han, M. J.; Yoo, K. S.; Kim, K. H.; Lee, G. H.; Chang, J. Y.
Macromolecules 1997, 30, 5408-5415. (c) Han, M. J.; Yoo, K. S.; Kim,
Y. H.; Chang, J. Y. Tetrahedron Lett. 2002, 43, 5597-5600.
(11) Fong, D. H.; Berghuis, A. M. EMBO J. 2002, 21, 2323-2331.
(12) Carter, A. P.; Clemons, W. M.; Brodersen, D. E.; Morgan-Warren,
R. J.; Wimberly, B. T.; Ramakrishnan, V. Nature 2000, 407, 340-348.
(13) Monosaccharides 8 and 9 were prepared by standard methods. All
new compounds exhibited satisfactory spectral and analytical data. Yields
refer to spectroscopically and chromatographically homogeneous materials.
(14) Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J. H. Tetrahedron
Lett. 1990, 31, 1331-1334.
3576
Org. Lett., Vol. 5, No. 20, 2003

protected compounds were subjected to a two-step depro-
tection, removal of all the ester and phthalimido groups by
treatment with methylamine (33% solution in EtOH) and
reduction of all the azido groups by Staudinger reaction, to
furnish the final products 2-5 with excellent purity and
isolated yields.18
Scheme 2a
The new analogues were tested for antibacterial activities
against both Gram-negative and Gram-positive bacteria,
including pathogenic and resistant strains,19 and the minimal
inhibitory concentrations (MIC)20 were determined using a
microdilution assay with neomycin B and kanamycin as
controls (Table 1).
From the MIC values, it turns out that among the four
analogues, only compound 5 having a ribose substituent at
ring V is as potent as neomycin B against E. coli strains.
The activity of this analogue against E. coli XL1(pET9d)
having kanamycin resistance is even more impressive,
exhibiting better activity than neomycin B. Analogue 5 is
also effective against Gram-positive bacteria, Staphylococcus
epidermidis and Bacilus subtilis. Furthermore, 5 demonstrates
better activity than other analogues against pathogenic
bacterium Salmonella Virchow that is resistant to kanamycin
and neomycin B. In this case 5 is about 5 times more
effective than kanamycin and 2 times more effective than
neomycin B. Finally, we also examined the susceptibility of
enterobacterium Pseudomonas aeuriginosa, which is often
very difficult to treat, sometimes requiring use of a combina-
tion of aminoglycosides with other antibiotics.2,21 Interest-
ingly, in this particular case, while 5 demonstrates activity
close to that of neomycin B, the 2-glucosamino derivative 4
is even more effective than 5 and the diamino derivative 3
is superior to both.
a Reagents and conditions: (a) (i) TfN3, Et3N, CuSO4, in CH2Cl2/
MeOH/H2O 3:10:3; (ii) TBDMSCl, pyridine, DMAP; (iii) Ac2O,
pyridine, DMAP; (iv) HF/pyridine, 57% for the four steps. (b) NIS,
TfOH; 6 f 15a (81%), 7 f 15b (89%), 8 f 15c (58%), 9 f 15d
(71%). (c) For 2-4: (i) MeNH2 (33% in EtOH); (ii) PMe3, NaOH
0.1 M, THF/H2O 3:1. For 5: (i) NaOMe/MeOH; (ii) PMe3, NaOH
0.1 M, THF/H2O 3:1, afforded 2 (81%), 3 (96%), 4 (84%), and 5
(70%).
The diazido monosaccharides 6 and 7, having D-allo and
D-gluco configurations, respectively, were constructed from
the common D-galactose derivatives (Scheme 1) by selec-
tively inverting the configurations at C3 and C4 (in 6) and
at C4 (in 7). The diol 12 was prepared from the known
thioglycoside 1015 in four steps (selective silylation of the
primary hydroxyl, acetonide formation at C3-OH and
C4-OH, benzoylation, and removal of the acetonide) without
isolation of intermediate products in an overall yield of 55%.
Simultaneous triflation of both hydroxyls in 12 was followed
by nucleophilic displacement with azide (without isolation
of the intermediate ditriflate) to afford the corresponding
diazide (63% isolated yield for two steps). Desilylation was
then followed with a chloracetylation step to produce the
allo-donor 6. Alternatively, selective protection of C6 and
C4 hydroxyls in the galactoside 11 by p-methoxybenzylidene,
followed by benzoylation and hydrolysis of the benzylidene,
gave the diol 13 in an overall 58% yield for three steps. This
diol was then subjected to a similar triflation and azidation
steps as for 12 to afford the 4,6-diazido donor 7 in an isolated
yield of 86% for two steps.
Although we still did not examine the similar analogues
with the plain pyranose ring and with the furanose ring
bearing amino group(s),22 the observed preliminary data of
2-5 indicate that merely increasing the number of amino
(16) These steps included perazidation of the commercial neomycin B
(Sigma) with TfN3 according to the procedure of Wong,4a,b selective
silylation of the primary hydroxyl at C5′′, acetylation of all the remaining
hydroxyls, and desilylation as depicted in Scheme 2.
(17) Purity and exclusive â-stereochemistry of new glycosidic bonds in
1
15a,b were confirmed by H NMR spectroscopy (15a: H-1, δ 4.86 ppm,
J1,2 ) 8.0 Hz. 15b: H-1, δ 4.81 ppm, J1,2 ) 7.5 Hz. 15c: H-1, δ 5.62
ppm, J1,2 ) 8.5 Hz. 15d: H-1, δ 5.95 ppm, J1,2 ) 4.5 Hz).
(18) Complete NMR assignments for the monosaccharides 6-9, along
with the protected pseudo-pentasaccharides 15a-d, and selected data for
the unprotected 2-5 are given in Supporting Information.
(19) Resistant strains included E. coli XL1(pET9d), Pseudomonas
aeuriginosa (ATCC 27853), and Salmonella Virchow (SV49). E. coli XL1-
(pET9d) is an antibiotic-sensitive laboratory strain of E. coli that harbors
plasmid pET9d with the cloned orf2 gene, which codes for aminoglycoside
kinase APH(3′). P. aeuriginosa is a Gram-negative pathogen. The aph(3′)-
IIb gene, which codes for APH(3′), is a chromosomal gene that was found
in many clinical isolates of P. aeruginosa, including the ATCC 27853 strain,
and likely accounts at least partly for the resistance of Pseudomonas to
aminoglycosides (Hachler, H.; Santanam, P.; Kayser, F. H. Antimicrob.
Agen. Chemother. 1996, 40, 1254-1256). S. Virchow (SV49) is a clinical
multidrug-resistant strain obtained from poultry and found to be resistant
to streptomycin, tetracycline, ampicillin, sulfa, kanamycin, and neomycin.
The mechanism(s) of resistance of this strain is still not known.
(20) Phillips, I.; Williams, D. In Laboratory Methods in Antimicrobial
Chemotherapy; Gerrod, L., Ed.; Churchill Livingstone Press: Edinburg,
1978; pp 3-30.
The neomycin acceptor 14 was readily accessible in four
chemical steps from the commercial neomycin B (Scheme
2) in an overall yield of 57%.16 NIS-promoted coupling of
14 with thioglycosides 6-9 furnished the designed protected
pseudo-pentasaccharides 15a-d in 58-89% yields.17 These
(21) Haddad, J.; Kotra, L. P.; Liano-Sotel, B.; Kim, C.; Azucena, E. F.,
Jr.; Liu, M.; Vakulenko, S. B.; Chow, C. S.; Mobashery, S. J. Am. Chem.
Soc. 2002, 124, 3229-3237.
(15) Pozsgay, V.; Jennings, H. J. Tetrahedron Lett. 1987, 28, 1375-
1376.
Org. Lett., Vol. 5, No. 20, 2003
3577

Table 1. MICs of 1-5 against Various Bacterial Strains
MIC (µg/mL)
bacterial strain
E. coli (R47-100)
E. coli (ATCC 25922)
E. coli XL1 blue (pET9d)
Staphylococcus epidermidis (ATCC 12228)
Bacilus subtilis (ATCC 6633)
Salmonella virchow (SV49)
KANa
NDb
ND
260-270
ND
ND
500-570
450-500
1
2
3
4
5
4-5.5
8-10
85
95
40-50
40-50
>200
1.5-1.8
3.5-4
>1250
30-35
35-40
25-30
>200
1.4-1.8
1.4-1.8
>1250
40-50
4.5-6
10-11
35-45
0.2-0.4
0.6-0.8
75-125
60-65
50-60
0.3-0.4
0.8-0.9
200-250
55-60
>200
5.5-7
8.5-10
>1250
110-130
Pseudomonas aeuriginosa (ATCC 27853)
a KAN ) kanamycin. b ND ) not determined.
groups on the natural drug may not lead to an increase in
antibacterial activity, even though the binding affinity of
these analogues to RNA is likely to increase in vitro.5-7
However, the excellent activities observed for the amino
derivatives 3 and 4 against Pseudomonas but significantly
weak activities against other bacterial strains imply that the
structural and functional requirements for this family of drugs
are not similar in order to reach analogous high antibacterial
performance against different organisms. While this is in
agreement with earlier reported data on other aminoglycoside
analogues,2,21 clearly further structure-activity studies within
more diverse structures at ring V, along with more structural
data on the interaction between aminoglycosides and rRNA,
are required to better understand this issue in detail.22
of additional rigidity into the natural drug while the parent
structure remains intact leads to improved antibacterial
performance. This research thus provides a new direction
for the development of novel antibiotics that target at once
both the bacterial RNA and resistance-causing enzymes.
Acknowledgment. We thank Dr. Smadar Shulami for
antibacterial tests and insightful discussions, Dr. Yael Balazs
for technical support in monitoring oligosaccharides by
NMR, and Shay Yogev for help in preparation of donor 7.
This research was supported by the Israel Science Foundation
founded by the Israel Academy of Sciences and Humanities
(Grant 214/00) and in part by the Foundation for Promotion
of Research at the Technion. V.B. acknowledges the financial
Support by the Center of Absorption in Science, the Ministry
of Immigration Absorption and the Ministry of Science and
Arts, Israel (Kamea Program).
In summary, the neomycin B derivatives prepared in this
study represent a new class of branched aminoglycoside
antibiotics that show antibacterial activity superior to that
of neomycin B against pathogenic and resistant bacterial
strains. Here we demonstrate that the precise incorporation
Supporting Information Available: Selected procedures
and complete analytical data for compounds 6-9, 15a-d,
and selected data for 2-5. This material is available free of
charge via the Internet at http://pubs.acs.org.
(22) Studies on the complete structure-activity relationship of ring V
of designer pseudo-pentasaccharides are currently under investigation. We
also intend to examine these newly designed structures as substrates/
inhibitors of aminoglycoside-modifying enzymes to validate the postulated
reduced interaction.
OL035213I
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Org. Lett., Vol. 5, No. 20, 2003