The Synthesis of L-aminosugar and the studies of L-pyranoses on the ring III of pyranmycins.

Article abstract of DOI:10.1021/ol026588r

The synthesis of a novel class of aminoglycoside, pyranmycin, and a convenient method for the preparation of 6-amino-L-idopyranosides were reported. One of the members in the reported pyranmycin families, TC010, has prominent activity against Escherichia

Full text of DOI:10.1021/ol026588r

ORGANIC
LETTERS
2002
Vol. 4, No. 23
3997-4000
The Synthesis of L-Aminosugar and the
Studies of L-Pyranoses on the Ring III
of Pyranmycins
Jinhua Wang,† Jie Li,† David Tuttle,† Jon Y. Takemoto,‡ and
,†
Cheng-Wei Tom Chang*
Department of Chemistry and Biochemistry and Department of Biology,
Utah State UniVersity, 0300 Old Main Hill, Logan, Utah 84322-0300
chang@cc.usu.edu
Received July 23, 2002
ABSTRACT
The synthesis of a novel class of aminoglycoside, pyranmycin, and a convenient method for the preparation of 6-amino-L-idopyranosides were
reported. One of the members in the reported pyranmycin families, TC010, has prominent activity against Escherichia coli, Staphylococcus
aureus, and Bacillus megaterium. We also discovered that the 4C chair conformation on ring III of pyranmycin is essential for the antibacterial
1
activity.
Neomycin belongs to a group of aminoglycoside antibiotics
containing a 4,5-disubstituted 2-deoxystreptamine core and
has been used against both gram-positive and gram-negative
bacteria for more than fifty years.1,2 The neomycin class of
antibiotics (Figure 1) exert its antibacterial activity by binding
Although neomycin is still widely used for the treatment of
serious infections, its usefulness is significantly hampered
by the rapid emergence of drug resistance3,4 and its relatively
high cytotoxicity.1
Many neomycin analogues containing a ring III furanose,
aiming to increase the antibacterial activity, compensate
resistance from aminoglycoside-modifying enzymes, and
reduce the cytotoxicity, have been reported.5-11 However,
(1) Hooper, I. R. Aminoglycoside Antibiotics; Springer-Verlag: New
York, 1982.
(2) Haddad, J.; Kotra, L. P.; Mobashery, S. In Glycochemistry Principles,
Synthesis, and Applications; Wang, P. G., Bertozzi, C. R., Eds.; Marcel
Dekker: New York, 2001; pp 307.
(3) Cohen, M. L. Science 2002, 257, 1050-1055.
(4) Neu, H. C. Science 2002, 257, 1064-1072.
(5) Alper, P. B.; Hendrix, M.; Sears, P.; Wong, C.-H. J. Am. Chem. Soc.
1998, 120, 1965-1978.
(6) 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.
Figure 1. Structures of neomycin class antibiotics.
(7) Yoshikawa, M.; Ikeda, Y.; Takenaka, K. Chem. Lett. 1984, 2097-
2100.
selectively to the A-site 16S ribosomal RNA of bacteria, and
thereby inhibit the protein synthesis of these microorganisms.
(8) Girodeau, J.-M.; Pineau, R.; Masson, M.; Le Goffic, F. J. Antibiot.
1984, 37, 150-158.
(9) Woo, P. W. K.; Haskell, T. H. J. Antibiot. 1982, 35, 692-702.
(10) Kumar, V.; Jones, G. S., Jr.; Blacksberg, I.; Remers, W. A. J. Med.
Chem. 1980, 23, 42-49.
† Department of Chemistry and Biochemistry.
‡ Department of Biology.
10.1021/ol026588r CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/15/2002

only two examples use D-glucopyranose as the ring III
component via R and â linkages.12,13 These glucopyranose
incorporated adducts are less active than neamine, and no
following work has been reported since. Because the
glycosidic bond of a furanose is more acid sensitive than
that of a pyranose,14,15 replacing the furanose ring (III) with
an aminopyranose, which leads to the design of pyranmycin,
will have two conceivable advantages (Figure 2). First, it
Wong and co-worker have reported the studies of the roles
of the L-idose (ring IV) of the neomycin.5 From the molecular
modeling of the scaffold of pyranmycin, we discovered that
a â-glycosidic bond between rings II and III is crucial in
orienting the necessary conformation of rings I and II.
Therefore, an analogue of the ring IV idose of neomycin,
6-amino-6-deoxy-L-idose, was selected as the ring III of
pyranmycin with the expectation that the presence of the
2-acetyl group instead of the amino (azido) group will favor
the formation of the â-glycosidic bond.
We began our synthesis of L-aminoidose from commer-
cially available diacetone-D-glucose (Scheme 1). Benzylation
Scheme 1. Synthesis of the 6-Amino-6-deoxy-l-idopyranose
Donora
Figure 2. Structural relationship between neomycin class amino-
glycoside antibiotics and pyranmycin.
may increase the acid-stability and, therefore, reduce the
required orally administered dose of drug and the associated
cytotoxicity. Second, from the NMR and X-ray crystal-
lography studies,16-19 there is a relatively large cavity within
the RNA target to accommodate the conformational change,
especially on the ring IV of neomycin. This could allow the
introduction of more complex structural components, such
as pyranose derivatives, to enhance the antibacterial activity.
The resulting artificial aminoglycosides could also become
poor substrates for the resistance enzymes, and thereby re-
gain the antibacterial activity against drug resistant bacteria
as indicated by Mobashery and colleagues.20
Encouraged by the advantages of our pyranmycin design
and the newly developed method for the preparation of
L-aminosugar generated from our laboratory, we quickly
assembled several pyranmycins with various ring III L-
glycopyranoses. Herein, we wish to report our findings in
the synthesis of L-aminoidopyranose, and the preparation and
antibacterial studies of pyranmycin with L-pyranose at the
ring III.
a Conditions: (a) (i) BnBr, NaH, nBu4NI, THF, (ii) TsOH,
MeOH, 65%; (b) (i) TsCl, (ii) NaN3, 75%; (c) (i) (COCl)2, DMSO,
DIPEA, (ii) NaBH4, 37%; (d) (i) 60% HOAc, (ii) Ac2O, 43%; (e)
(i) H2NNH2-HOAc, (ii) CCl3CN, DBU, 10%.
of the C-3 hydroxyl group, followed by the regioselective
deprotection of the O-5,6-isopropylidene group, provided the
diol, 1. A regioselective tosylation of the C-6 hydroxyl group
and an azide substitution using NaN3 incorporated the azido
at C-6. In the attempts to invert the chirality of the C-5
hydroxyl group, we were pleased to find out that the standard
procedure employed in our laboratory, Swern oxidation then
NaBH4 reduction, can achieve our objective conveniently,
providing compound 3. The finding offers a practical method
of making L-sugars from D-sugar in addition to the modifica-
tion, mainly azide incorporation, on the C-6 position. The
observed stereoselectivity of the NaBH4 reduction can be
(11) Kumar, V.; Remers, W. A. J. Org. Chem. 1978, 43, 3327-3331.
(12) Endo, T.; Perlman, D. J. Antibiot. 1972, 25, 681-682.
(13) Suami, T.; Nishiyama, S.; Ishikawa, Y.; Katsura, S. Carbohydr. Res.
1977, 56, 414-418.
Scheme 2. Syntheses of Glycosyl Donorsa
(14) Bochkov, A. F.; Zaikov, G. E. Chemistry of the O-Glycosidic
Bond: Formation and CleaVage; Pergamon Press: New York, 1979.
(15) Shallenberger, R. S.; Birch, G. G. Sugar Chemistry; The Avi
Publishing Co., Inc.: New York, 1975.
(16) Fourmy, D.; Recht, M. I.; Puglisi, J. D. J. Mol. Biol. 1998, 277,
347-362.
(17) Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D. Science
1996, 274, 1367.
(18) Fourmy, D.; Yoshizawa, S.; Puglisi, J. D. J. Mol. Biol. 1998, 277,
333-345.
(19) Ma, C.; Baker, N. A.; Joseph, S.; McCammon, J. A. J. Am. Chem.
Soc. 2002, 124, 1438-1442.
(20) 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.
a Conditions: (a) H2NNH2-HOAc, DMF; (b) CCl3CN, DBU,
CH2Cl2.
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Org. Lett., Vol. 4, No. 23, 2002

explained with the Felkin-Ahn model. Deprotection of the
O-1,2-isopropylidene group using HOAc/TFA/H2O solution,
followed by peracetylation afforded the acetyl 6-azido-6-
deoxy-L-idopyranose, 4.
The acetyl 6-azido-6-deoxy-L-idopyranose, 4, was con-
verted into a glycosyl trichloroacetimidate after hydrolysis
of the anomeric acetyl group using hydrazine acetate fol-
lowed by reaction with trichloroacetonitrile. A similar pro-
cedure was used for the preparation of designed ribostamycin
analogues with L-rhamnose and L-glucose as the ring III
sugars (Scheme 2). Acid-catalyzed glycosylation of these
three glycosyl donors with neamine acceptor5 afforded the
designed protected pyranmycins (Scheme 3). These three
TC010, and TC015, with excellent purity and with minimum
effort devoted to column chromatography purification. The
three analogues were assayed against E. coli, Staphylococcus
aureus, and Bacillus megaterium, and the minimum inhibi-
tory concentrations (MIC)22 were determined using neo-
mycin, ribostamycin, and neamine as the control (Table 1).
Table 1. MIC of Pyranmycins
MIC (µM)
compd
E. coli
S. aureus
B. megaterium
neomycin B
ribostamycin
neamine
TC009
2
5
2
12
ND
inactive
23
NDa
ND
ND
ND
2
36
inactive
9
Scheme 3. Synthesis of the Designed Pyranmycinsa
TC010
TC015
inactive
inactive
ND
a ND: not determined.
Among the three members in pyranmycin families with
L-sugar on ring III, TC009 contains a L-rhamnopyranose,
TC010 contains a 6-amino-6-deoxy-L-idopyranose, and
TC015 has a L-glucopyranose. TC010 is almost as active
as ribostamycin while TC009 and TC015 are not active at
all. From the coupling constant of the 1H NMR, we learned
that all of the trichloroacetimidate glycosyl donors from
L-rhamnose, L-glucose, and 6-amino-6-deoxy-L-idopyranose
have a 1C4 chair conformation. Interestingly, after the
glycosylation and final syntheses, the 6-amino-6-deoxy-L-
idopyranose on TC010 adopts a 4C1 chair conformation (J1′′,2′′
of the L-idopyranose changes from 0.0 to 7.24 Hz) while
1
the L-rhamnopyranose on TC009 remains in its C4 chair
conformation (J1′′,2′′ of the L-rhamnopyranose varies from 2.0
to 2.97 Hz).
In an effort to provide an explanation for the lack of
activity of TC009, we carried out molecular modeling for
both TC009 and TC010 (Figure 3). The results show that a
a Conditions: (a) BF3-OEt2, CH2Cl2, 4 Å MS; (b) (1) K2CO3,
MeOH, (2) PMe3, THF/H2O, (3) H2, Pd(OH)2/C.
Figure 3. Optimized conformations of ribostamycin, TC009, and
TC010.
protected pyranmycins were subjected to the final synthesis,
hydrolysis of acetyl groups, reduction of azido with Staudinger
reaction, and deprotection of benzyl groups with hydrogena-
tion. We modified the procedure for the final synthesis from
the literature21 and obtained the final products, TC009,
4C1 chair conformation will preserve the desired intra-
molecular hydrogen bond between 2′′-OH and 2′-NH2 in
(21) Sucheck, S. J.; Greenberg, W. A.; Tolbert, T. J.; Wong, C.-H. Angew.
Chem., Int. Ed. 2000, 39, 1080-1084.
Org. Lett., Vol. 4, No. 23, 2002
3999

TC010, which has been suggested to be crucial in orienting
the neamine core for binding toward the A-site of the 16S
RNA.17 To maintain an intramolecular hydrogen bond
mycin appears to be essential for the antibacterial activity.
This finding provides us a guideline for our future approach.
Interestingly, the 2,6-diamino-2,6-dideoxy-L-idose (ring IV)
1
1
between the ring III with a C4 chair conformation and the
of neomycin adopts an C4 chair conformation. Although
neamine core, a similar interaction between 2′′-OH and 2′-
NH2 was produced. However, a rather dramatic conforma-
tional difference, a 180° flipping on the ring III of TC009,
is observed, which may account for the lack of antibacterial
activity for TC009.
TC010 shows degradation in acidic media, the slower rate
of degradation provides us the support of using pyranose as
the substitution of furanose at the ring III of neomycin class
aminoglycoside antibiotics. We believe this new addition of
aminoglycoside can pave the way to the development of more
potent antibiotics against drug-resistant bacteria. We have
currently constructed a library of pyranmycins with both D-
and L-aminosugars as the ring III component. We expect to
generate more interesting structural activity relationships of
the new class of aminoglycosides soon, and provide valuable
information for developing a library of new antibacterial
agents.
We also carried out acid-degradation experiments for the
constructed analogue using neomycin as the control. Neo-
mycin and TC010 were dissolved in D2O purged with
anhydrous HCl (pH ca. 1) then sealed in the NMR tubes
and incubated at 37 °C. The controls were prepared from
neomycin and TC010 in neutral D2O. All four samples were
1
monitored with H NMR. The controlled samples showed
no sign of degradation during the 14-day incubation period
as indicated by the signals of the anomeric H-1′ and H-2
protons. However, the neomycin and TC010 in acidified D2O
underwent a time-dependent acid degradation (20%, 40%,
60%, and 80% degradation after 2, 6, 10, and 14 days for
neomycin, and 5%, 20%, 35%, and 50% for TC010,
respectively). After the incubation, the samples in acidic D2O
were pump-dried and assayed against E. coli. We observed
a significant decrease in the antibacterial activity of neomycin
and TC010. The MIC of acid-treated neomycin increases
from 2 µM to 50 µM, while the MIC of TC010 increases
from 9 µM to 40 µM.
Acknowledgment. We acknowledge the financial support
from the Utah State University (CURI grant) and the support
from the Department of Chemistry and Biochemistry, Utah
State University. We thank Mr. Mekki Bensaci from the
Department of Biology, Utah State University for technical
assistance. We also thank professor Hung-wen (Ben) Liu
from Department of Chemistry and Biochemistry, University
of Texas for providing valuable discussion and suggesting
the name, pyranmycin, for us. Mass spectrometry was
provided by the Washington University Mass Spectrometry
Resource, and the Mass Spectrometry Facilities, University
of California, Riverside.
In conclusion, we have reported a concise and convenient
method for the preparation of L-aminoidopyranose and the
synthesis of a novel class of aminoglycoside, pyranmycins.
The constructed aminoglycoside has promising antibacterial
Supporting Information Available: Experimental pro-
cedures for the preparation of compounds 2-7, 9-11,
4
1
activity. The C1 chair conformation on ring III of pyran-
TC009, TC010, and TC015 and the corresponding H and
13C NMR spectra; mass spectra for compounds 3, 4, 9-11,
TC009, TC010, and TC015. This material is available free
of charge via the Internet at http://pubs.acs.org.
(22) Methods for Dilution Antimicrobial Susceptibility Testing for
Bacteria that Grow Aerobically. Approved standard M7-A5, and Perfor-
mance Standards for Antimicrobial Disk Susceptibility Tests. Approved
standard M2-A7, National Committee for Clinical Laboratory Standards:
Wayne, PA.
OL026588R
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Org. Lett., Vol. 4, No. 23, 2002