Probing the specificity of aminoglycoside-ribosomal RNA interactions with designed synthetic analogs

Article abstract of DOI:10.1021/ja972599h

The binding of neomycin B and related aminoglycoside antibiotics to the prokaryotic ribosomal RNA decoding region has been investigated using a recently developed surface plasmon resonance assay. A number of naturally occurring aminoglycosides containing a neamine or neamine-like substructure bind specifically to a model of the site of the ribosomal decoding region RNA. This recognition event is the basis of the antibacterial activity of this class of compounds. A series of analogs was designed and synthesized to probe the role of neomycin ring IV (2,6-dideoxy-2,6-diamino-β-L- idopyranose). The binding results indicate that the positive charge presented on the idose ring is necessary for specific binding in vitro and cannot be replaced by amines attached via flexible linkers. However, the antibiotic activity (minimum inhibitory concentration) of the analog where ring IV is replaced with a diamine tail is the same as neomycin B in a liquid culture assay against Escherichia coli.

Full text of DOI:10.1021/ja972599h

J. Am. Chem. Soc. 1998, 120, 1965-1978
1965
Probing the Specificity of Aminoglycoside-Ribosomal RNA
Interactions with Designed Synthetic Analogs
Phil B. Alper, Martin Hendrix, Pamela Sears, and Chi-Huey Wong*
Contribution from the Department of Chemistry and The Skaggs Institute of Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
ReceiVed July 30, 1997
Abstract: The binding of neomycin B and related aminoglycoside antibiotics to the prokaryotic ribosomal
RNA decoding region has been investigated using a recently developed surface plasmon resonance assay. A
number of naturally occurring aminoglycosides containing a neamine or neamine-like substructure bind
specifically to a model of the site of the ribosomal decoding region RNA. This recognition event is the basis
of the antibacterial activity of this class of compounds. A series of analogs was designed and synthesized to
probe the role of neomycin ring IV (2,6-dideoxy-2,6-diamino-â-L-idopyranose). The binding results indicate
that the positive charge presented on the idose ring is necessary for specific binding in Vitro and cannot be
replaced by amines attached Via flexible linkers. However, the antibiotic activity (minimum inhibitory
concentration) of the analog where ring IV is replaced with a diamine tail is the same as neomycin B in a
liquid culture assay against Escherichia coli.
Introduction
Targeting RNA sequences using small molecule drugs is a
topic of significant interest. To this end, the interactions
necessary for an RNA recognition event to occur need to be
understood at the molecular level. Aminoglycoside antibiotics,
as a class, have long been known to bind RNA. They exert
their antibacterial effects at least in part by binding to specific
target sites in the bacterial ribosome.1 For the structurally
related antibiotics neamine (1), ribostamycin (2), neomycin B
(3), and paromomycin (4) (Figure 1), the binding site has been
localized to the A-site of the prokaryotic 16S ribosomal decoding
region RNA,2 which is shown in Figure 2. Binding of
aminoglycosides to this RNA target interferes with the fidelity
of mRNA translation and results in miscoding and truncation,
leading ultimately to bacterial cell death. The biological
properties of naturally occurring and synthetic aminoglycoside
antibiotics have been reviewed.3
There is also considerable biochemical data describing the
interaction of this class of compounds with a variety of other
biologically relevant RNA sequences. Apart from 16S riboso-
mal RNA2 these include two HIV-1 mRNA regulatory domains,
the Rev response element (RRE)4 and the transactivation
response element (TAR),5 as well as the group I intron6 and
the hammerhead ribozyme.7 In addition, numerous RNA
sequences which bind particular aminoglycosides have been
Figure 1. Structures of representative aminoglycoside antibiotics.
derived from in Vitro selection experiments.8 Among ami-
noglycoside-RNA interactions, the binding of neomycin type
antibiotics to 16S RNA stands out because it is linked to the
biological activity of this class of compounds. This interesting
mode of action of aminoglycosides has prompted investigation
of their RNA recognition capabilities in model systems,9
combinatorial synthesis of derivatives as possible inhibitors of
RNA recognition,10 and semisynthesis of known antibiotics to
(5) Mei, H.-Y.; Galan, A. A.; Halim, N. S.; Mack, D. P.; Moreland, D.
W.; Sanders, K. B.; Truong, H. N.; Czarnik, A. W. Bioorg. Med. Chem.
Lett. 1995, 5, 2755.
(6) (a) von Ahsen, U.; Davies, J.; Schroeder, R. Nature 1991, 353, 368.
(b) von Ahsen, U.; Schroeder, R. Nucleic Acids Res. 1991, 19 (9), 2261.
(c) von Ahsen, U.; Davies, J.; Schroeder, R. J. Mol. Biol. 1992, 226 (4),
935. (d) von Ahsen, U.; Noller, H. Science 1993, 260, 1500.
(7) (a) Stage, T. K.; Hertel, K. J.; Uhlenbeck, O. C. RNA 1995, 1, 95.
(b) Clouet-d’Orval, B.; Stage, T. K.; Uhlenbeck, O. C. Biochemistry 1995,
34, 11186.
(8) (a) Wang, Y.; Rando, R. R. Chem. Biol. 1995, 2, 281. (b) Lato, S.
M.; Boles, A. R.; Ellington, A. D. Chem. Biol. 1995, 2, 291. (c) Wallis, M.
G.; von Ahsen, U.; Schroeder, R.; Famulok, M. Chem. Biol. 1995, 2, 543.
(d) Famulok, M.; Huttenhofer, A. Biochemistry 1996, 35, 4265. (e) Wang,
Y.; Killian, J.; Hamasaki, K.; Rando, R. R. Biochemistry 1996, 35, 12338.
(9) Hendrix, M.; Alper, P. B.; Priestley, E. S.; Wong, C.-H. Angew.
Chem., Int. Ed. Engl. 1997, 36, 95.
(1) (a) Tanaka, N. Mechanism of Action of Aminoglycoside Antibiotics.
Handbook of Experimental Pharmacology; Springer-Verlag: New York,
1982; Vol. 62, p 221. (b) Cundliffe, E. Recognition Sites for Antibiotics
Within rRNA. In The Ribosome; Hill, W. E., et al., Eds.; American Society
of Microbiology: Washington, DC, 1990; p 479. (c) Noller, H. F. Annu.
ReV. Biochem. 1991, 60, 191.
(2) (a) Moazed, D.; Noller, H. F. Nature 1987, 327, 389. (b) Puhroit, P.;
Stern, S. Nature 1994, 370, 659. (c) Recht, M. I.; Fourmy, D.; Blanchard,
S. C.; Dahlquist, K. D.; Puglisi, J. D. J. Mol. Biol. 1996, 262, 421. (d)
Miyaguchi, H.; Narita, H.; Sakamoto, K.; Yokoyama, S. Nucleic Acids Res.
1996, 24, 3700.
(3) Price, K. E.; Godfrey, J. C.; Kawaguchi, H. AdV. Appl. Microbiol.
1974, 18, 191.
(4) (a) Zapp, M. L.; Stern, S.; Green, M. R. Cell 1993, 74, 969. (b)
Werstuck, G.; Zapp, M. L.; Green, M. R. Chem. Biol. 1996, 3, 129.
S0002-7863(97)02599-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/24/1998

1966 J. Am. Chem. Soc., Vol. 120, No. 9, 1998
Alper et al.
Figure 3. Schematic representation of the binding of paromomycin
to the target fragment of 16S ribosomal RNA based on the study
reported by Puglisi et al.l3
Figure 2. Sequences of the RNA molecules used in this study.
understand their function and improve their activity.11 Also,
other investigators have synthesized non-aminoglycoside mol-
ecules that bind RNA.12
Recently, it has been shown that a short RNA hairpin con-
taining the residues 1404-1412 and 1488-1497 of the Esch-
erichia coli ribosomal RNA (AS-wt, Figure 2) retains the
aminoglycoside binding properties of the site embedded in the
prokaryotic ribosome.2c,d A critical base for specific aminogly-
coside binding is U1495 which, in the wild type sequence, is
engaged in a noncanonical U:U base pair. Replacement of
U1495 with A leads to complete loss of specific aminoglycoside
binding.2c,d For this reason, AS-U1495A can serve as a negative
control for in Vitro binding studies.
Puglisi and co-workers have determined the solution structure
of the AS-wt complex with 4 by NMR.13 A representation of
the pertinent intermolecular contacts is shown in Figure 3.
Paromomycin (4) rings I and II fit into a pocket created by
the asymmetrical bulge. Ring II (2-amino-2-deoxy-D-glucose)
stacks against the G1491-C1409 base pair, and ring I (2-
deoxystreptamine) spans across the major groove, making
hydrogen bonds to N7 of G1494 and O6 of U1495. On the
other hand, the structural data indicated that ring IV (2,6-
dideoxy-2,6-diamino idose) had an ill-defined position in the
complex. The apparent dynamic nature of this residue raised
questions regarding its contribution to the binding event. The
L-idose moiety either could contribute to the specificity of the
interaction or might be merely a platform to present additional
charges increasing only the affinity.
Figure 4. Target molecules to study the role of ring IV of neomy-
cin B.
plasmon resonance based assay14 and compared RNA binding
affinities for AS-wt and AS-U1495A. To address the contribu-
tion of ring IV, we synthesized a series of neomycin B deriv-
atives modified in the idose ring. To examine whether the
amino groups of ring IV need to be displayed on a rigid plat-
form, the L-idose ring was replaced with an acyclic side chain
presenting either one (5) or two (6) amines (Figure 4). To
dissect the role of specific charges while maintaining the native
idose ring, either both (7) or one (8) of the amines was replaced
with a hydroxyl group.
We reasoned that these modifications would provide a
test for probing the ionic contribution, since hydrogen bond
donor abilities, electronegativity, and steric demand are similar
for both functional groups. In order to learn how well the model
system correlates with in ViVo activity, we checked the new
compounds for antibacterial activity against three bacterial
strains using the disk method (Kirby-Bauer technique).15a The
minimum inhibitory concentrations (MICs) of the compounds
were then determined using the broth dilution technique.15b
We have investigated the specificity of aminoglycoside
binding to the A-site RNA with a series of naturally occurring
aminoglycosides (1-4) using our recently developed surface
Results
(10) Park, W. K. C.; Auer, M.; Jaksche, H.; Wong, C.-H. J. Am. Chem.
Soc. 1996, 118, 10150.
(11) (a) Wang, H.; Tor, Y. Bioorg. Med. Chem. Lett. 1997, 7, 1951. (b)
Wang, H.; Tor, Y. J. Am. Chem. Soc. 1997, 119, 8734.
Our retrosynthetic analysis (Scheme 1) led us to fragments
10 and 11 as suitable precursors for the key pseudotrisaccharide
unit 9 from which all desired compounds can be constructed.16
(12) (a) Ratmeyer, L. S.; Vinayak, R.; Zon, G.; Wilson, W. D. J. Med.
Chem. 1992, 35 (5), 966. (b) Wilson, W. D.; Ratmeyer, L.; Zhao, M.;
Strekowski, L.; Boykin, D. Biochemistry 1993, 32, 4098. (c) Wilson, W.
D.; Ratmeyer, L.; Cegla, M. T.; Spychala, J.; Boykin, D.; Demounynck,
M.; Lhomme, J.; Krishnan, G. New J. Chem. 1994, 18, 419. (d) McCon-
naughie, A. W.; Spychala, J.; Zhao, M.; Boykin, D.; Wilson, W. D. J. Med.
Chem. 1994, 37, 1063. (e) Zhao, M.; Ratmeyer, L.; Peloquin, R. G.; Yao,
S.; Kumar, A.; Spychala, J.; Boykin, D. W.; Wilson, W. D. Bioorg. Med.
Chem. 1995, 3 (6), 785. (f) Fernandez-Saiz, M.; Schneider, H.-J.; Sartorius,
J.; Wilson, W. D. J. Am. Chem. Soc. 1996, 118, 4739.
(14) Hendrix, M.; Priestley, E. S.; Joyce, G. F.; Wong, C.-H. J. Am.
Chem. Soc. 1997, 119, 3641.
(15) For antibiotic testing protocols, see: (a) Disk testing protocols:
Phillips, I.; Williams, D. In Laboratory Methods in Antimicrobial Chemo-
therapy; Garrod, L., Ed.; Churchill Livingstone Press: Edinburgh, 1978;
pp 3-30. (b) MIC determination: Waterworth, P. M. In Laboratory Methods
in Antimicrobial Chemotherapy; Garrod, L., Ed.; Churchill Livingstone
Press: Edinburgh, 1978; pp 31-40, Barry, A. L. The Antimicrobic
Susceptibility Test: Principles and Practice; Lea and Febiger: Philadelphia,
1976. (c) Standards: Lorian, V. Antibiotics in Laboratory Medicine, 2nd
ed.; Williams and Wilkins: Baltimore, 1986.
(13) Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D. Science
1996, 274, 1367.

Aminoglycoside-Ribosomal RNA Interactions
Scheme 1. Retrosynthetic Analysis
J. Am. Chem. Soc., Vol. 120, No. 9, 1998 1967
Scheme 2a
a Conditions: (a) (i) Bu2SnO, toluene, azeotropic H2O removal, (ii)
BnBr, TBAI, 110 °C; (b) Swern oxidation; (c) NaBH4, MeOH; (d)
NaH, allyl bromide, DMF; (e) DMF, 1 N HCl, ∆; (f) pNBzCl, pyridine.
Compound 10 can be prepared from 1 which in turn is available
by acidic hydrolysis of neomycin B (3).17
The choice of a suitable nitrogen protecting group was crucial
to our effort. Past synthetic work on the aminoglycosides has
relied on the use of alkyl carbamates18 (including benzyloxy-
carbonyl groups, cyclic carbamates,16,19 and tert-butyloxycar-
bonyl groups11a,20) and trifluoroacetamides21 for the protection
of the various primary amines. In our experience, the presence
of multiple Cbz groups makes NMR spectra of the intermediates
difficult to interpret, presumably due to the slow interconversion
of rotamers. The stability of ethyl and cyclic carbamates can
cause severe problems during deprotection, and the solubility
characteristics of polycarbamoylated aminoglycosides are not
always compatible with the requirements for glycosidation. The
lability associated with the trifluoroacetamide and Boc groups
made strategies utilizing these groups unattractive. Finally, none
of the acyl-type protecting groups address the issue of protecting
the acidic NH that is formed upon acylation of a primary amine.
These problems can be overcome by the use of azides as
nitrogen protecting groups. To this end, we have introduced a
metal-catalyzed version22 of the original diazo transfer protocol23
which allows the convenient conversion of amines to azides
with retention of stereochemistry. Using this protocol, neamine
(1) was converted into tetraazidoneamine, which was regiose-
lectively acetylated to afford 10.22
The next building block, ribose donor 11, was constructed
in a seven-step sequence starting with the 1,2-O-isopropyliden-
exylose (12) following a route analogous to that reported by
Umezawa (Scheme 2). Stannyl ester activation and subsequent
benzylation provided 13. A two-step oxidation/reduction
sequence24 served to invert the stereochemistry at the 3 position
and provide 15. Alkylation with allyl bromide served to install
the allyl group to afford 16. Finally, the acetonide was removed
to afford 17.
The p-nitrobenzoyl ester was installed in the anomeric
position to give 11 rather than the acetate which was employed
by Umezawa and co-workers.16 The reason for this was that
the attempted condensation of acceptor 10 with the anomeric
acetate led to poor conversions, presumably due to the reversible
nature of this glycosidation. However, the p-nitrobenzoyl group
solved this problem since p-nitrobenzoic acid precipitates from
the reaction mixture and thus shifts the equilibrium in favor of
the condensation (Scheme 3).
Reaction of 10 and 11 provided the desired â-linked pseudo-
trisaccharide 18 in 63% yield along with an additional 18% of
the R anomer which could be equilibrated to the desired product
by resubjecting it to the glycosidation conditions. The anomeric
configuration of 18 was assigned on the basis of the observed
coupling constants of the H1′′ proton.25 The protecting groups
were subsequently normalized to benzyl ethers to obtain the
key pseudotrisaccharide 9. The allyl group was then used in a
dual role. For access to the alkyl amino derivatives (Scheme
4), the allyl group of 9 could be converted to an aldehyde to
set up the introduction of nitrogen by reductive amination (Vide
infra). In order to construct glycosylated derivatives, a mild
two-step deallylation26 of 9 yielded a suitable acceptor for
subsequent glycosidation reactions (20).
(16) For the total synthesis of neomycin B, see: (a) Usui, T.; Umezawa,
S. J. Antibiot. 1987, 1464. (b) Usui, T.; Umezawa, S. Carbohydr. Res. 1987,
133.
(17) Ford, J. H.; Bergy, M. E.; Brooks, A. A.; Garrett, E. R; Alberti, J.;
Dryer, J. R.; Carter, H. E. J. Am. Chem. Soc. 1955, 77, 5311.
(18) (a) Suami, T.; Nishiama, S.; Ihikawa, Y.; Katsura, S. Carbohydr.
Res. 1976, 52, 187. (b) Suami, T.; Nishiyama, S.; Ishikawa, Y.; Katsura, S.
Carbohydr. Res. 1977, 53, 239. (c) Suami, T.; Nishiyama, S.; Ishikawa,
Y.; Katsura, S. Carbohydr. Res. 1977, 56, 415. (d) Canas-Rodriguez, A.;
Ruiz-Proveda, S. G. Carbohydr. Res. 1977, 58, 379. (e) Suami; T.;
Nishiyama, S; Ihikawa, Y.; Katsura, S. Carbohydr. Res. 1978, 65, 57. (f)
Pfeiffer; F. R.; Schmidt, S. J.; Kinzig; C. M.; Hoover, J. R. E.; Weisbach,
J. A. Carbohydr. Res. 1979, 72, 119.
(19) (a) Umezawa, S.; Koto, S.; Tatsuta, K.; Hineno, H.; Nishimura, Y.;
Tsumura, T. Bull. Chem. Soc. Jpn. 1969, 42, 537. (b) Umezawa, S.; Takagi,
Y.; Tsuchia, T. Bull. Chem. Soc. Jpn. 1971, 44, 1411. (d) Kumar, V.;
Remers, W. A. J. Org. Chem. 1978, 43, 3327. (d) Kumar, V.; Jones, G. S.,
Jr.; Blacksberg, I.; Remers, W. A.; Misiek, M.; Pursiano, T. A. J. Med.
Chem. 1980, 23, 42. (e) Sharma, M. N.; Kumar, V.; Remers, W. A. J.
Antibiot. 1982, 35, 905.
(20) (a) Grapsas, I.; Cho, Y. I.; Mobashery, S. J. Org. Chem. 1994, 59,
1918. (b) Roestamadji, J.; Grapsas, I.; Mobashery, S. J. Am. Chem. Soc.
1995, 117, 80.
(21) (a) Tsuchia, T.; Takagai, Y.; Umezawa, S. Tetrahedron Lett. 1979,
51, 4951. (b) Reid, R. J.; Mizsak, S. A.; Reineke, L. M.; Zurenko, G. E.;
Stern, K. F.; Magerlein, B. J. J. Med. Chem. 1981, 24, 1487.
(22) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett. 1996,
37, 6029.
For the preparation of 5 and 6, compound 9 was cleaved to
the key aldehyde 21 by ozonolysis. Compound 21, in turn, was
reductively aminated with the mono-Cbz adduct of 1,3-
diaminopropane to give 22. By contrast, use of unprotected
1,3-diaminopropane resulted in the formation of an aminal,
which was not reduced. The deprotection proceeded Via a two-
step sequence to yield the desired 6. First, the azides were
reduced Via Staudinger reaction, and the six remaining protecting
(24) (a) Yanagisawa, H.; Kinoshita, M.; Nadaka, S.; Umezawa, S. Bull.
Chem. Soc. Jpn. 1970, 43, 246. (b) Wanatabe, I.; Tsuchia, T.; Takase, T.;
Umezawa, S.; Umezawa, H. Bull. Chem. Soc. Jpn. 1977, 50, 2369.
(25) The desired â anomer featured no coupling to H2′′ whereas the R
anomer had a coupling constant of 4 Hz.
(23) Vasella, A.; Witzig, C.; Chiara, J.-L.; Martin-Lomas, M. HelV. Chim.
Acta 1991, 74, 2073.
(26) Lamberth, C.; Bednarski, M. D. Tetrahedron Lett. 1991, 32, 7369.

1968 J. Am. Chem. Soc., Vol. 120, No. 9, 1998
Alper et al.
Scheme 3a
was constructed by allylation of 30 at C6 to afford 31.30
Attempts to introduce nitrogen at C6 of 30 by activating the 6
position as the mesylate followed by displacement with azide
proved to be fruitless due to intramolecular participation of the
anomeric methyl sulfide which was followed by attack at the
anomeric center to yield a product with sulfur at the 6 position.
However, introduction of an amino substituent at C6 was even-
tually achieved through a chemoselective Swern oxidation fol-
lowed by reductive amination with allylamine to afford 32.
Subsequent deallylation31 and Cbz protection led to the desired
donor 33.
The tetrasaccharide core was assembled by glycosidation of
20 with the glycosyl donors 31 and 33 to yield the protected
pseudotetrasaccharides 34 and 36, respectively (Scheme 6). Both
reactions proceeded with complete selectivity for the desired â
anomer, presumably due to the triaxial conformation of the
donor in solution (evident by the small coupling constants of
the ring protons). This conformation would lead to a severe
(1,3) diaxial interaction if the product was formed in the R
configuration. Compound 34 was deallylated26 to afford 35 and
then subjected to our standard two-step deprotection protocol
to afford 2′′′,6′′′-didesamino-2′′′,6′′′-dihydroxyneomycin B (7).
Analogous deprotection of 36 yielded 2′′′-desamino-2′′′-hy-
droxyneomycin B (8).
The RNA binding properties of 1-8 were analyzed using a
surface plasmon resonance based assay that was recently
developed in these laboratories.14 This assay allows the
determination of both affinity and specificity of small molecule-
RNA interactions. For the analysis, the compounds were
injected over a matrix containing RNA that was immobilized
through a biotin tag, and the equilibrium binding values were
recorded at various concentrations. A sample of binding curves
is shown in Figure 5.
Nonlinear curve fitting was then used to determine the values
of the dissociation constants (KD). The specificity of binding,
i.e., the ability to discriminate between different RNA sequences,
was evaluated by comparing the binding to the target sequence
(AS-wt) and the negative control (AS-U1495A). The ratio of
KD(AS-U1495A) to KD(AS-wt) was taken to be a representation
of the specificity of the binding event. The data are summarized
in Table 1.
The compounds were assayed for biological activity against
three bacterial reference strains, E. coli (ATCC 25922),
Pseudomonas aeruginosa (ATCC 27853), and Staphylococcus
aureus (ATCC 25923), using the Kirby-Bauer disk method,15a
in which paper disks containing known amounts of antibiotic
are placed on plates inoculated with bacterial cultures, and the
diameters of the zones of inhibition (DZI), apparent as clear
regions around the disks, are measured after overnight growth.
Figure 6 shows a representative disk assay.
a Conditions: (a) 11, BF3OEt2, CH2Cl2; (b) LiOH, H2O, MeOH; (c)
BnBr, NaH, DMF; (d) (i) bis(methyldiphenylphosphine)(COD)IrIPF6
activated by hydrogen, THF, (ii) OsO4, Me4NO‚2H2O, CH2Cl2.
groups were reductively cleaved using sodium in ammonia. The
direct treatment of azido-protected aminoglycosides with Na/
NH3 led to mixtures of products containing deaminated deriva-
tives which presumably arose from the homolysis of the C-N
bond following the one-electron reduction of an azide.
Synthesis of 5 proved to be somewhat more challenging.
Attempts to use benzylamine as the nitrogen source in order to
avoid extra steps led to an unexpected deprotection problem.
During attempted dissolving metal reduction, the N-benzyl group
was reduced to the corresponding inert Birch product. To
circumvent this problem, compound 21 was converted to 23
by reductive amination with p-methoxybenzylamine followed
by carbamoylation with ZOSu. The PMB group was then
oxidatively cleaved using CAN to afford 24, and the standard
two-step deprotection protocol was used to obtain the desired
analog 5.
Attention was then focused on the idose synthesis (Scheme
5). A number of approaches to L-ido and L-gulo configured
systems27 have been described in the literature. However, none
of the known methods were concise, with the possible exception
of Paulsen’s elegant rearrangement of peracetylated D-glucose
to D-idose,27a but to prepare the L-derivative would require the
expensive L-glucose pentaacetate.
A low-temperature hydroboration of 2528 with excess borane
followed by oxidation of the carbon boron bond yielded a
mixture of 26 and 27 which could be separated by chromatog-
raphy, but separation was achieved much more readily after
closure to the anhydrosugar 28.29 Compound 28 was remark-
ably stable, but proved to be labile to a sulfur nucleophile under
TMSOTf promotion. The equilibrium was shifted toward the
otherwise disfavored open form due to the strength of the re-
sulting O-Si bond. Removal of the TMS group led to a 1:18
mixture of the anomers 29 and 30. The C6 oxygenated donor
Table 2a gives the zone diameters measured for the three
bacterial strains. The zone diameters measured for the strains
with known antibiotics are well within the accepted limits.15a
The minimum inhibitory concentrations were determined via
(29) It should be noted that attempts to introduce nitrogen at the 6 position
in 27 yielded discouraging results. Introduction of azide Via activation as
the mesylate and displacement was sluggish and low yielding. Subsequent
attempts to generate the thioglycoside with TMSOTf and MeSTMS led to
ring opening, and attempted hydrolysis by treatment with aqueous H2SO4
led to the decomposition of the azide prior to liberation of the anomeric
center.
(30) Use of benzyl bromide instead of allyl bromide as an electrophile
led to decomposition under conditions necessary for reaction while Lewis
acid promoted benzylation with benzyl 2,2,2-trichloroacetimidate led to
reclosure to 28.
(27) (a) Paulsen, H. Methods Cabohydr. Chem. 1972, 6, 142-148. (b)
Horton, D.; Tsai, J.-H. Methods Carbohydr. Chem. 1980, 8, 177. (c)
Andrews, G. C.; Crawford, T. C.; Bacon, B. E. J. Org. Chem. 1981, 46,
2976. (d) Medakovic, D. Carbohydr. Res. 1994, 253, 299.
(28) Semeria, D.; Philippe, M.; Delaumeny, J.-M.; Sepulchre, A.-M.;
Gero, S. D. Synthesis 1983, 710.
(31) Laguzza, B.; Ganem, B. Tetrahedron Lett. 1981, 22, 1483.

Aminoglycoside-Ribosomal RNA Interactions
J. Am. Chem. Soc., Vol. 120, No. 9, 1998 1969
Scheme 4a
a Conditions: (a) (i) O3, CH2Cl2, (ii) DMS; (b) mono-Cbz-1,3-diaminopropane, AcOH, pH 6, MeOH, NaBH3CN; (c) (i) PMe3, THF, H2O, 1 N
NaOH, (ii) Na, NH3, THF, (iii). Amberlite CG-50 cation exchange chromatography; (d) (i) PMB-NH2, AcOH, pH 6, MeOH, NaBH3CN, (ii)
ZOSu, CH2Cl2; (e) acetonitrile-H2O (9:1), CAN.
Scheme 5a
Discussion
The available NMR structure of the paromomycin-AS-wt
complex13 suggested that the pseudodisaccharide portion of the
molecule was mainly responsible for recognition. However,
the Biacore data on neamine (1), ribostamycin (2), and neomycin
B (3) suggested that the idose ring was responsible for a large
portion of both affinity and specificity (Table 1). This observa-
tion made a good case for studying the role of this ring in the
binding event.
To probe this role, the neomycin B analogs have the idose
ring replaced with a flexible monoamine tail (5) or a diamine
tail (6) or with idose analogs containing only hydroxy groups
(7) or one amine group (8). In contrast to previous observations,
all of the aminoglycosides tested here using surface plasmon
resonance14 show some degree of specificity in the recognition
of AS-wt. As expected, the overall affinity correlates with the
net charge of the molecule, neomycin B being the tightest binder.
The binding data for compounds 1-8 can be compared by
starting with neamine (1) and observing the changes in binding
that occur as functionality is added to the molecule. Neamine
(1) binds the AS-wt sequence with a dissociation constant of
7.8 µM and approximately 4-fold specificity relative to the AS-
U1495A sequence. Ribostamycin (2) adds a ribose ring to this
core at the 5 position of 2-deoxystreptamine and has 3-fold lower
affinity with specificity similar to that of neamine while retaining
the same overall charge. Addition of an uncharged idose
platform to ribostamycin (2) to generate 7 results in a molecule
which has virtually the same RNA binding profile. This result
implies that the idose ring by itself (without amines) does not
contribute to the affinity or specificity of binding.
a Conditions: (a) (i) BH3‚THF, THF, 0 °C, (ii) HOOH, NaOH; (b)
AcOH, concentrated HCl, 70 °C; (c) (i) MeSTMS, TMSOTf, CH2Cl2,
(ii) TBAF; (d) DMF, NaH, allyl bromide; (e) allylamine, AcOH, pH
6, NaBH3CN; (f) (i) Wilkinson’s catalyst, acetonitrile-H2O (84:16),
distillation, (ii) Z-OSu, CH2Cl2.
the broth dilution technique,15b and the results are shown in
Table 2b. The results for neomycin are within accepted ranges
for sensitive E. coli strains.15c It is worth noting that some
testing protocols recommend overnight growth of the cultures,
rather than the minimum time required to obtain good growth
of the control. MIC values were tested for several of the control
antibiotics using overnight growth, and the MIC values observed
were 2-4× greater than those observed for 4-6 h growth.
Addition of an amine at a defined position on the idose
platform to give compound 8 improves affinity by 40-fold in
relation to 7 and makes the interaction relatively more specific.
Addition of a second amine to this platform to generate the
parent agent neomycin B (3) results in further improvement of
the overall affinity, without affecting specificity. When a single
positive charge on a flexible ethyl tether rather than a saccharide

1970 J. Am. Chem. Soc., Vol. 120, No. 9, 1998
Alper et al.
Scheme 6a
a Conditions: (a) 31, NIS, AgOTf (cat), 3 Å MS, CH2Cl2; (b) (i) bis(methyldiphenylphosphine)(COD)Ir(I)PF6 activated by hydrogen, THF, (ii)
OsO4, Me4NO‚2H2O, CH2Cl2; (c) (i) PMe3, THF, H2O, 1 N NaOH, (ii) Na, NH3, THF, EtOH, (iii) Amberlite CG-50 anion exchange chromatography;
(d) 33, NIS, AgOTf (cat), 3 Å MS, CH2Cl2.
Table 1. In Vitro Binding Data for the Natural Antibiotics and
Synthetic Analogs Used in This Study
KD(AS-wt)a
(µM)
KD(AS-U1495A)a
(µM)
specificity
factorb
compd
1
2
3
4
5
6
7
8
7.8
25
0.019
0.20
1.7
0.26
28
0.70
95
31
90
0.38
2.7
10
1.6
123
14
4
4
20
14
6
6
4
19
1
streptomycin
74
a All KD values were determined in duplicate except for KD(AS-wt)
of 4 and 8 which were determined in triplicate. The deviation from the
mean was <100% in all cases. The standard deviation for KD(AS-wt)
of 4 and 8 was 29% and 49%, respectively. Solution conditions: 150
mM NaCl, 10 mM HEPES (pH 7.4), 3.4 mM EDTA. b KD(AS-
U1495A)/Ki(AS-wt).
sequence exhibit a much reduced binding affinity and no
specificity for the native sequence over the AS-U1495A mutant.
The MIC data for these compounds in E. coli (Table 2b)
indicate that the in ViVo activity does not always correlate well
with the in Vitro binding data. Compounds 5 and 6 both have
significantly lower binding affinities toward the AS-wt RNA
than neomycin and show considerably higher nonspecific
binding to the AS-U1495A species (Table 1), yet have very
nearly the same antimicrobial activity as neomycin B (3) itself.
Neamine, which appears to bind better to AS-wt than ribosta-
mycin, shows inferior antimicrobial activity. These discrepan-
cies may reflect different uptake dynamics of the different
compounds, or perhaps a slightly different conformation of the
ribosome in ViVo. Figure 7 shows the published13 representative
structure of paromomycin interacting with AS-wt on the left
and a possible structure of 6 interacting with the same model
sequence.
Figure 5. Titration curves for compounds 1-8 binding to AS-wt.
platform is attached to ribostamycin (2) to give 5, the binding
affinity goes up by an order of magnitude but specificity is
unaffected. The addition of another positive charge on an even
more flexible linker (6) gives diminishing returns on the affinity
without affecting the specificity. These results indicate that,
within the scope of this model, the rigid scaffold of the idose
ring is necessary in order to preserve the specificity exhibited
by neomycin B.
Paromomycin (4) which differs from neomycin B (3) only
in position 6′ can best be compared to 8, which has the same
number of charges. Compared to 8, paromomycin (4) shows
somewhat higher affinity but lower specificity. The control
antibiotic streptomycin was used to demonstrate that unrelated
aminoglycoside antibiotics which are not known to bind to this
The presumption that the binding mode is similar for the two
molecules can be justified by the near identity of rings I, II,

Aminoglycoside-Ribosomal RNA Interactions
J. Am. Chem. Soc., Vol. 120, No. 9, 1998 1971
Table 2. Antibacterial Activities of Aminoglycosides
A. Diameters of Zones of Inhibition (DZI), mma
antibiotic amount (nmol) E. coli S. aureus Ps. aeruginosa
1
2
3
4
5
6
7
8
200
33
33
33
33
33
33
33
18.5
16.5
20.5
18
18.5
19
18.5
14.5
21.5
19.5
18.5
21
NI
NI
9.5
NI
NI
NI
NI
NI
16.5
19
11.5
19.5
B. Minimum Inhibitory Concentrations (MICs) against
E. coli ATCC 25922b
antibiotic
MIC (µM)
MIC (µg/mL)
1
2
3
4
5
6
7
8
50
12.5
1.6
6.25
3.1
1.6
26
8
1.5
5.5
2.3
1.4
10
2.6
12.5
3.1
a The zones of inhibition as determined by the Kirby-Bauer disk
method are given. In the case of neomycin, 30 µg (33 nmol) of
neomycin sulfate was used per disk. For all other compounds, the molar
amount was kept constant at 33 nmol, except for the neamine standard,
which was increased 6-fold due to its low activity. b The minimum
inhibitory concentrations are given in both µM and µg/mL. (Weight/
mL was calculated on the basis of the predicted molecular weights of
the compounds’ sulfate salts.) NI ) no inhibition.
experiments were performed as described previously.14 Solution
conditions: 150 mM NaCl, 10 mM HEPES (pH 7.4), 3.4 mM EDTA.
KD Determination from the Binding Curves. KD values were
determined by fitting to the equation
Figure 6. Representative Kirby-Bauer disk assay. (A, top) Clockwise,
from top left: neomycin (33 nmol); negative control; compound 6 (33
nmol); neamine (200 nmol). (B, bottom) Clockwise from top left:
compound 8 (33 nmol); compound 7 (33 nmol); compound 6 (33 nmol);
compound 5 (33 nmol).
c
c
c
equiv ) a
+
+
+ ... + b
(
)
KD(1) + c KD(2) + c KD(3) + c
and III and the comparison between neomycin B and the
synthetic analogs regarding their 13C-NMR shifts and coupling
constants (Table 3). The 5′-phosphate of A1493 makes a fairly
long range contact with the 6′-OH of paromomycin, but since
neomycin-like structures feature a 1,3-hydroxyamine motif9
between the 4 and 6 positions, this interaction may well be closer
for this class of molecules. The structure shows that the diamine
tail is in an area rich in potential phosphate contacts, with the
5′-phosphates of U1406, C1407, A1408, G1488, and G1489 all being
candidates. This may mean that the interaction of a simple
doubly positively charged appendage with a highly electrone-
gative major groove of RNA is enough to orient rings I, II, and
III into their binding pocket.
wherein c ) concentration, a ) adjustment factor to adjust the value
of response units considered to be 1 equivalent, b ) correction to adjust
the baseline to 0, and KD(1), KD(2), ... ) stepwise dissociation
constants.
The fitting routine of the program Kaleidagraph was used for all
calculations. The starting values for a and b were set to 1 and 0,
respectively. The number of KD values used in the fitting was adjusted
depending on the observed range of equivalents bound but generally
varied from 3 to 4.
Antimicrobial Testing. Kirby-Bauer Disk Test. These tests were
performed exactly as described.15a Reference strains E. coli ATCC
25922, S. aureus ATCC 25923, and Ps. aeruginosa ATCC 27853 were
obtained as packs of lyophilized pellets (Difco), which were freshly
reconstituted every few days. To make the antibiotic disks, paper disks
(6 mm diameter, BBL Microbiology Systems) were wetted through
with 20 µL of solution containing an appropriate amount (usually 33
nmol) of antibiotic. The wet disks were placed in a desiccator
overnight, and used the next day.
Minimal Inhibitory Concentration (MIC) Testing. E. coli ATCC
25922 was grown in Mueller-Hinton broth (cation-adjusted, BBL
Microbiology Systems) to an optical density of approximately 0.5
(absorbance read at 600 nm), and then diluted to an OD600 of 0.1.
Samples of antibiotic were prepared in Mueller-Hinton broth, typically
a series of 2-fold dilutions from 0.1 mM to <1 µM. A 50 mL sample
of the diluted culture was added to 1 mL of each of the antibiotic
samples, and the cultures were allowed to grow at 37 °C for 4-6 h, at
which point the negative control sample (no antibiotic) typically had
an absorbance of 1.2-1.5. The absorbance of each sample was read
(λ ) 600 nm), and MIC was considered to be the lowest antibiotic
concentration at which the absorbance was less than 1% of the
control.
The antimicrobial activity of compounds 5 and 6 holds
promise for the design of novel antibiotics. It is apparent that
gross changes are tolerated in the structure of aminoglycoside
antibiotics without significant effect on biological activity. This
observation should allow design of structurally simpler mol-
ecules which could possibly address issues of drug resistance.
Experimental Section
Unless otherwise stated, all reactions were performed under an Ar
atmosphere with the assistance of magnetic stirring. THF and CH2Cl2
were distilled under Ar prior to use from benzophenone ketyl and CaH2,
respectively. All other solvents and reagents were purchased anhydrous
and used as received. NMR spectra were recorded using either a Bruker
AMX-500 or a Bruker DRX-600 instrument. Synthesis of the
biotinylated RNA and surface plasmon resonance detected binding

1972 J. Am. Chem. Soc., Vol. 120, No. 9, 1998
Alper et al.
Figure 7. A model of paromomycin (4) bound to AS-wt created on the basis of the coordinates from Puglisi13 (left) and possible representation
of 6 bound to AS-wt (right). The trans-1,3-hydroxyamine motif of ring II points toward the phosphate group of A1493. The amine tail also interacts
with phosphate groups.
5-O-Benzyl-1,2-O-isopropylidene-r-D-xylofuranose (13). 1,2-O-
Isopropylidene-R-D-xylofuranose (12) (4.2 g, 22.08 mmol) was dis-
solved in toluene (120 mL) and treated with Bu2SnO (5.76 g, 23.19
mmol). The reaction was then refluxed overnight with azeotropic
removal of water. The Dean-Stark trap was then removed and replaced
with a standard reflux condenser. The reaction was treated with BnBr
(5.66 g, 33.12 mmol) and kept at 110 °C for 7 h. Upon addition of
EtOAc and water, a solid formed which was filtered. The organic phase
was washed with saturated sodium bicarbonate solution and brine and
dried over Na2SO4. Chromatography of the resulting oil using a
gradient of 25% to 30% to 35% EtOAc in hexane afforded 4.01 g (65%)
of the title compound as an oil which solidified after standing under
vacuum: 1H NMR (CDCl3, 500 MHz) δ 1.31 (s, 3H, acetonide methyl),
1.48 (s, 3H, acetonide methyl), 3.68 (s, 1H, OH), 3.90 (dd, 2H, J1 )
11 Hz, J2 ) 4 Hz, H5a), 3.93 (dd, 2H, J1 ) 11 Hz, J2 ) 4 Hz, H5b),
4.25 (dd, 1H, J1 ) 7 Hz, J2 ) 4 Hz, H4), 4.27 (m, 1H, H3), 4.50 (d,
1H, J ) 4 Hz, H2), 4.60 (ABq, 2H, J ) 12 Hz, ∆ν ) 29.7 Hz,
PhCH2O), 5.97, (d, 1H, J ) 4 Hz, H1), 7.25-7.4 (m, 5H, C6H5); 13C
NMR (CDCl3, 125 MHz) δ 26.1, 26.7, 68.1, 74.0, 76.3, 78.0, 85.2,
104.8, 111.5, 127.8, 128.0, 128.5, 137.0; HRMS for C15H20O5 (M +
Na) calcd 303.1208, found 303.1201.
5-O-Benzyl-3-oxo-1,2-O-isopropylidene-r-D-xylofuranose (14).
Methylene chloride (100 mL) was cooled to -78 °C, and DMSO (2.79
g, 35.76 mmol) was added, followed by oxalyl chloride (2.18 g, 17.16
mmol). The reaction was allowed to stir for 20 min at this temperature
and then treated with a solution of 13 (4.01 g, 14.3 mmol) in 30 mL
of CH2Cl2. The reaction was allowed to slowly warm to -35 °C and
was kept at that temperature for 15 min before the addition of
triethylamine (7.24 g, 71.5 mmol). The reaction was allowed to warm
to room temperature, extracted with saturated sodium bicarbonate
solution and saturated NaCl solution, and dried over Na2SO4. Flash
chromatography on 200 mL of silica gel using a gradient of 0% to
0.5% to 1% to 1.5% MeOH in CHCl3 afforded 3.2 g (80.4%) of the
title compound: 1H NMR (CDCl3, 500 MHz) δ 1.43 (s, 3H, acetonide
methyl), 1.46 (s, 3H, acetonide methyl), 3.72-3.75 (m, 2H, H5a and
H5b), 4.35 (dd, J1 ) 4 Hz, J2 ) 1 Hz, 1H, H2), 4.45 (m, 1H, H4), 4.51
(ABq, J ) 12 Hz, ∆ν ) 15.75 Hz, PhCH2O), 6.13 (d, J ) 4 Hz, H1),
7.2-7.4 (m, 5H, C6H5); 13C NMR (125 MHz) δ 27.2, 27.6, 70.0, 73.6,
76.7, 79.8, 103.5, 114.1, 127.4, 127.8, 128.4, 128.5, 137.3; HRMS for
C15H18O5 (M + Na) calcd 301.1052, found 303.1043.
5-O-Benzyl-1,2-O-isopropylidene-r-D-ribofuranose (15). Com-
pound 14 (3.2 g, 11.5 mmol) was dissolved in 50 mL of anhydrous
methanol and treated with NaBH4 (218 mg, 5.75 mmol). The reaction
was allowed to stir for 1 h and then quenched with water. The solvent
was removed, and the reaction was partitioned between EtOAc and
saturated sodium bicarbonate solution. The organic phase was dried
with brine and Na2SO4. Flash chromatography on 120 mL of silica
gel using a gradient of 25% to 30% to 35% to 40% EtOAc in hexane
afforded 2.53 g (79%) of the title compound: 1H NMR (CDCl3, 500
MHz) δ 1.37 (s, 3H, acetonide methyl), 1.56 (s, 3H, acetonide methyl),
2.42 (d, 1H, J ) 10 Hz, OH), 3.64 (dd, 1H, J1 ) 11 Hz, J2 ) 4.5 Hz,
H5a), 3.79 (dd, 1H, J1 ) 11 Hz, J2 ) 2.5 Hz, H5b), 3.92 (m, 1H, H4),
3.3.97 (m, 1H, H3), 4.56 (dd, 1H, J1 ) 4.5 Hz, J2 ) 3.5 Hz, 1H, H2),
4.60 (s, 2H, PhCH2O), 5.84 (d, 1H, J ) 3.5 Hz, H1), 7.27-7.37 (m,
4H, C6H5); 13C NMR (CDCl3, 125 MHz) δ 26.4, 26.5, 68.5, 71.7, 73.5,
78.3, 79.7, 104.1, 112.6, 127.6, 127.7, 128.4, 137.8; HRMS for
C15H20O5 (M + Na) calcd 303.1208, found 303.1200.
3-O-Allyl-5-O-benzyl-1,2-O-isopropylidine-r-D-ribofuranose (16).
Compound 15 (500 mg, 1.784 mmol) was dissolved in 10 mL of DMF
and cooled to ice bath temperature. The reaction was treated with
sodium hydride (47 mg, 1.963 mmol) followed by allyl bromide (647
mg, 5.352 mmol). After 20 min, another 20 mg of NaH was added.

Aminoglycoside-Ribosomal RNA Interactions
J. Am. Chem. Soc., Vol. 120, No. 9, 1998 1973
Table 3. 13C NMR Shifts and Coupling Constants of Neomycin B and the Synthetic Analogs
C1
C2
C3
C4
C5
C6
C1′
C2′
C3′
C4′
C5′
C6′
neo B
51.4
51.3
51.3
51.3
51.3
29.9
29.5
29.5
29.5
29.5
49.9
49.9
49.9
49.9
49.9
77.3
76.8
76.9
76.9
76.7
86.3
86.2
86.2
86.3
86.3
74.0
74.0
74.0
73.9
73.9
97.0
97.1
97.1
97.0
97.0
55.0
55.0
55.0
54.9
54.9
69.6
69.5
69.5
69.5
69.5
72.1
72.0
72.0
72.0
72.0
70.8
70.9
70.9
70.9
70.9
41.6
41.5
41.5
41.6
41.6
5
6
7
8
J (H2ax, H1) (Hz)
J (H2eq, H1) (Hz)
J (H2ax, H3) (Hz)
J (H2eq, H3) (Hz)
neo B
12.6
12.6
12.6
12.6
12.6
4.1
4.1
4.1
4.1
4.1
12.6
12.6
12.6
12.6
12.6
4.1
4.1
4.1
4.1
4.1
5
6
7
8
J (H2ax, H2eq) (Hz)
J (H3, H4) (Hz)
J(H4, H5) (Hz)
J (H5, H6) (Hz)
neo B
12.6
12.6
12.6
12.6
12.6
broad
10.5
10.4
10.3
10.2
broad
10.1
9.9
10.3
10.2
9.4
5
6
7
8
9.2
9.3
J (H1, H6) (Hz)
J (H1′, H2′) (Hz)
J (H2′, H3′) (Hz)
J (H3′, H4′) (Hz)
neo B
10.4
10.7
10.6
10.6
10.4
4.0
4.0
3.9
4.0
4.0
10.8
10.8
10.9
9.2
9.3
9.5
9.4
9.3
5
6
7
8
10.9
J (H4′, H5′) (Hz)
J (H5′, H6′a) (Hz)
J (H6′a, H6′b) (Hz)
neo B
6.7
6.4
6.4
6.3
6.4
13.6
13.6
13.2
13.7
13.7
5
6
7
8
9.3
9.5
9.4
9.3
After all starting material was consumed, the reaction was quenched
with AcOH and the solvent was removed. The residue was taken up
in EtOAc, washed with water, saturated sodium bicarbonate solution,
and brine, and dried over Na2SO4. Flash chromatography on 70 mL
of silica gel using a gradient of 12% to 15% to 18% to 20% EtOAc in
hexane afforded 555 mg (97%) of the title compound: 1H NMR
(CDCl3, 500 MHz) δ 1.36 (s, 3H, acetonide methyl), 1.58 (s, 3H,
acetonide methyl), 3.61 (dd, 1H, J1 ) 11 Hz, J2 ) 4 Hz, H5a), 3.79
(dd, 1H, J1 ) 11 Hz, J2 ) 2 Hz, H5b), 3.85 (dd, 1H, J1 ) 9 Hz, J2 )
4.5 Hz, H3), 4.07 (dddd, 1H, J1 ) 12.5 Hz, J2 ) 6 Hz, J3 ) J4 ) 1.5
Hz, 1H, H4), 4.12-4.17 (m, 2H, CH2CHCH2O), 4.60 (ABq, 2H, J )
12 Hz, ∆ν ) 45 Hz, PhCH2O), 4.60 (dd, 1H, J1 ) J2 ) 4 Hz, H2),
5.21 (ddd, 1H, J1 ) 11.5 Hz, J2 ) J3 ) 1.5 Hz, CH2CHCH2O), 5.28
(ddd, 1H, J1 ) 17.5 Hz, J2 ) J3 ) 1.5 Hz, CH2CHCH2O), 5.78 (d, 1H,
J ) 4 Hz, H1), 5.36-5.46 (m, 1H, CH2CHCH2O), 5.27-7.36 (m, 5H,
C6H5); 13C NMR (CDCl3, 125 MHz) δ 26.4, 26.7, 67.8, 71.6, 73.5,
77.3, 77.4, 77.8, 103.9, 112.8, 118.0, 127.6, 127.7, 128.3, 134.4, 138.0;
HRMS for C18H24O5 (M + Na) calcd 343.1521, found 343.1513.
1,2-O-(4-Nitrobenzoyl)-3-O-allyl-5-O-benzyl-r/â-D-ribofura-
nose (11). Compound 16 (757 mg, 2.36 mmol) was dissolved in 15
mL of dioxane and treated with 5 mL of 1 N HCl solution. The reaction
was then warmed to 80 °C for 1.5 h and cooled back to room
temperature (RT). The acid was quenched by addition of solid sodium
bicarbonate, and the solvent was removed. The residue was partitioned
between water and EtOAc. The water layer was further extracted twice
with EtOAc, and the combined organic extracts were dried over MgSO4.
The solvent was removed, and the residue was treated with pyridine
(15 mL), 4-nitrobenzoyl chloride (1.04 g, 5.60), and a few crystals of
DMAP. The reaction was stirred overnight, and the solvent was
removed. The residue was taken up in EtOAc and washed with water,
saturated CuSO4 solution followed by saturated ammonium chloride
solution, and brine. The combined organic phases were dried over
MgSO4, and the solvent was removed. The residue was chromato-
graphed over 50 mL of silica gel using 10% to 12% to 15% EtOAc in
hexane to afford 910 mg (68%) (over two steps) of the product as a
chromatographically separable mixture (approximately 4:1) of anomers.
Data for the â anomer: 1H NMR (CDCl3, 500 MHz) δ 3.73 (dd, 1H,
J1 ) 11 Hz, J2 ) 3 Hz, H5a), 3.86 (dd, 1H, J1 ) 11 Hz, J2 ) 2.5 Hz,
H5b), 4.05-4.18 (m, 2H, CH2CHCH2O), 4.40 (ddd, 1H, J1 ) 8 Hz, J2
) J3 ) 3 Hz, H4), 4.53 (s, 2H, PhCH2O), 4.63 (dd, 1H, J1 ) 8 Hz, J2
) 4.5 Hz, H3), 5.15-5.28 (m, 2H, CH2CHCH2O), 5.70 (d, 1H, J )
4.5 Hz, H2), 5.75-5.86 (m, 1H, CH2CHCH2O), 6.56 (s, 1H, H1), 7.20-
7.30 (m, 5H, C6H5), 8.00-8.35 (m, 8H, C6H4NO2); 13C NMR (CDCl3,
125 MHz) δ 68.5, 72.3, 73.5, 75.0, 75.8, 82.1, 99.4, 118.1, 123.5, 123.7,
127.6, 127.8, 128.4, 130.9, 131.0, 133.6, 134.5, 137.7, 150.6, 150.8,
163.0 163.5; HRMS for C29H26N2O11 (M + Na) calcd 601.1434; found
601.1447. Data for the R anomer: 1H NMR (CDCl3, 500 MHz) δ
3.70 (dd, 2H, J1 ) 3.5 Hz, J2 ) 3 Hz, 2H, H5a,b), 4.05-4.10 (m, 2H,
CH2CHCH2O), 3.70 (dd, J1 ) 6.5 Hz, J2 ) 3 Hz, 1H, H3), 4.55-4.60
(m, 3H, H4 and PhCH2O), 5.22-5.37 (m, 2H, CH2CHCH2O), 5.47
(dd, J1 ) 6 Hz, J2 ) 4 Hz, 1H, H2), 5.77-5.86 (m, 1H, CH2CHCH2O),
6.81 (d, J ) 4 Hz), 7.35-7.42 (m, 5H, C6H5), 8.08-8.30 (m, 8H, C6H4-
NO2); 13C NMR (CDCl3, 125 MHz) δ 69.3, 72.1, 73.3, 73.7, 75.6, 84.9,
95.9, 117.3, 123.6, 127.7, 127.9, 128.5, 130.7, 131.0, 134.0, 134.4,
135.1, 137.5, 150.7, 163.5, 163.6; MS for C29H26N2O11 (M + Na) calcd
601, found 601; for C29H26N2O11 (M + Cl-) calcd 613, found 613.
6,3′,4′-Tri-O-acetyl-3′′-O-allyl-5′′-O-benzyl-1,3,2′,6′-tetranzidori-
bostamycin (18). Compound 11 (3.5 g, 6.18 mmol) and compound
10 (1.34 g, 2.43 mmol) were dissolved in 15 mL of CH2Cl2 and cooled
in an ice bath. Then, BF3‚OEt2 (922 mg, 6.5 mmol) was added Via
syringe, and the reaction was allowed to stir for 4.5 h. By this time a
large amount of precipitate had formed. The reaction was quenched
by addition of triethylamine until the solution became homogeneous.
Chloroform was added and the reaction was extracted with saturated
NaHCO3 solution and brine and dried over Na2SO4. Chromatography
over 200 mL of silica gel using a gradient of 5% to 10% to 15% to
20% to 25% to 30% EtOAc in hexane yielded 2.02 g of the donor,
1.47 g of the â anomer (63%) and 0.43 g of the R anomer (18%). Data
for the â anomer: 1H NMR (CDCl3, 500 MHz) δ 1.61 (ddd, 1H, J1 )
J2 ) J3 ) 13 Hz, H2 eq), 2.05 (s, 3H, COCH3), 2.08 (s, 3H, COCH3),
2.14 (s, 3H, COCH3), 2.37 (ddd, 1H, J1 ) 13 Hz, J2 ) J3 ) 4.5 Hz,
H2 ax), 3.10 (dd, 1H, J1 ) 11 Hz, J2 ) 3.5 Hz, H2′), 3.22 (dd, 1H, J1
) 13.5 Hz, J2 ) 5.5 Hz, H6′a), 3.32 (dd, 1H, J1 ) 13.5 Hz, J2 ) 3 Hz,
H6′b), 3.42 (ddd, 1H, J1 ) 13 Hz, J2 ) 10 Hz, J3 ) 4.5 Hz, H1), 3.49

1974 J. Am. Chem. Soc., Vol. 120, No. 9, 1998
Alper et al.
(ddd, 1H, J1 ) 13 Hz, J2 ) 10 Hz, J3 ) 4.5 Hz, H3), 3.58 (dd, 1H, J1
) 10.5 Hz, J2 ) 4.5 Hz, H5′′a), 3.68 (dd, 1H, J1 ) J2 ) 10 Hz, H4),
3.82 (dd, 1H, J1 ) 10.5 Hz, J2 ) 2.5 Hz, H5′′b), 3.85 (dd, 1H, J1 ) J2
) 10 Hz, H5), 3.90 (dd, 1H, J1 ) 12.5 Hz, J2 ) 6 Hz, CH2CHCH2O),
4.00 (dd, 1H, J1 ) 12.5 Hz, J2 ) 5.5 Hz, 1H, CH2CHCH2O), 4.16-
4.22 (m, 2H, H3′′ and H4′′), 4.38-4.42 (m, 1H, H5′), 4.58 (ABq, 2H,
J ) 11.5 Hz, ∆ν ) 51.2 Hz, PhCH2O), 4.86 (dd, 1H, J1 ) J2 ) 10 Hz,
H4′), 4.96 (dd, 1H, J1 ) J2 ) 10 Hz, H6), 5.09 (dd, 1H, J1 ) 10 Hz,
J2 ) 1.5 Hz, CH2CHCH2O), 5.16 (dd, J1 ) 17 Hz, J2 ) 1.5 Hz, 1H,
CH2CHCH2O), 5.29 (d, 1H, J ) 3 Hz, H2′′), 5.38 (dd, 1H, J1 ) 11
Hz, J2 ) 9.5 Hz, H3′), 5.40 (s, 1H, H1′′), 5.63-5.74 (m, 1H,
CH2CHCH2O), 6.07 (d, 1H, J ) 3.5 Hz, H1′), 7.2-7.35 (m, 5H, C6H5),
8.15-8.35 (m, C6H4NO2); 13C NMR (CDCl3, 125 MHz) δ 20.7, 20.9,
31.3, 50.9, 58.1, 58.9, 61.0, 69.0, 69.2, 69.4, 70.2, 73.5, 75.1, 75.9,
76.2, 76.6, 80.4, 82.6, 96.1, 107.8, 118.1, 123.7, 127.8, 127.9, 128.6,
130.9, 133.5, 134.7, 137.7, 150.8, 163.5, 169.7, 170.0; HRMS for
C40H45N13O16 (M + Na) calcd 986.3005. found 986.3035. Data for
the R anomer: 1H NMR (CDCl3, 500 MHz) δ 1.58 (ddd, 1H, J1 ) J2
) J3 ) 13 Hz, H2 eq), 2.04 (s, 3H, COCH3), 2.10 (s, 3H, COCH3),
2.14 (s, 3H, COCH3), 2.38 (ddd, 1H, J1 ) 13 Hz, J2 ) J3 ) 4.5 Hz,
H2 ax), 3.18 (dd, 1H, J1 ) 13.5 Hz, J2 ) 4.5 Hz, H6′a), 3.30-3.37
(m, 2H, H6′b, H2′), 3.43 (ddd, 1H, J1 ) 12 Hz, J2 ) 10 Hz, J3 ) 4.5
Hz, H3), 3.50 (ddd, 1H, J1 ) 12.5 Hz, J2 ) 10 Hz, J3 ) 4.5 Hz, H1),
3.57 (dd, 1H, J1 ) J2 ) 9.5 Hz, H4), 3.58 (dd, 1H, J1 ) 11 Hz, J2 )
4 Hz, H5′a), 3.71 (dd, 1H, J1 ) 11 Hz, J2 ) 2.5 Hz, H5′b), 3.80 (dd,
1H, J1 ) J2 ) 9.5 Hz, H5), 3.88-4.02 (m, 2H, CH2CHCH2O), 4.08
(dd, J1 ) 7.5 Hz, J2 ) 5 Hz, 1 H, H3′′), 4.22-4.26 (m, 1 H, H4′′),
4.42-4.47 (m, 1H, H5′), 4.58 (ABq, 2H, J ) 12 Hz, ∆ν ) 43.5 Hz,
PhCH2O), 4.92-4.99 (m, 2H, H6, H4′), 5.08-5.19 (m, 2H, CH2-
CHCH2O), 5.47-5.44 (m, 2H, H1′, H3′), 5.58 (d, 1H, J ) 4 Hz, H1′′),
5.67 (dd, 1H, J1 ) J2 ) 5 Hz, H2′′), 5.67-5.76 (m, 1H, CH2CHCH2O),
7.28-7.42 (m, 5H, C6H5), 8.23-8.35 (m, 2H, C6H4NO2); 13C NMR
(CDCl3, 125 MHz) δ 20.57, 20.63, 21.1, 31.5, 50.5, 58.1, 58.6, 61.0,
68.7, 69.0, 69.4, 70.3, 71.5, 72.0, 73.5, 73.6, 75.8, 79.4, 80.0, 82.5,
97.4, 103.0, 118.1, 123.7, 127.8, 128.4, 130.4, 131.1, 133.6, 134.7,
137.6, 150.8, 164.2, 169.6, 169.9, 170.0; HRMS for C40H45N13O16 (M
+ Cs) calcd 1096.2162, found 1096.2119.
3′′-O-Allyl-5′′-O-benzyl-1,3,2′,6′-tetraazidoribostamycin (19). Com-
pound 18 (1.47 g, 1.525 mmol) was dissolved in a mixture of MeOH
and dioxane, 1:1 (30 mL). The reaction was then treated with a solution
of LiOH (384 mg, 9.151 mmol) in 10 mL of H2O. The mixture was
allowed to stir overnight at room temperature, and the solvent was
removed. The reaction was partitioned between EtOAc and saturated
NaHCO3 and extracted three times with EtOAc. The combined organic
phases were dried over MgSO4 and purified on 100 mL of silica gel
using 50% to 55% to 60% EtOAc in hexane to afford 947 mg (93%)
of product as a white foam: 1H NMR (CD3OD, Bruker AMX-500) δ
1.35 (ddd, 1H, J1 ) J2 ) J3 ) 12.5 Hz, H2 eq), 2.19 (ddd, J1 ) 12.5
Hz, 1H, J2 ) J3 ) 4.5 Hz, H2 ax), 3.02 (dd, 1H, J1 ) 10.5 Hz, J2 )
4 Hz, H2′), 3.27 (dd, 1H, J1 ) 10 Hz, J2 ) 9 Hz, H4′), 3.34-3.45 (m,
3H, H1, H3, H6′a), 3.46-3.54 (m, 1H, H5), 3.50 (dd, 1H, J1 ) 13 Hz,
J2 ) 2.5 Hz, H6′b), 3.58 (dd, 1H, J1 ) 11 Hz, J2 ) 5.5 Hz, H5′′a),
3.61-3.65 (m, 2H, H4, H6), 3.72 (dd, 1H, J1 ) 11 Hz, J2 ) 3 Hz,
H5′′b), 3.84 (dd, 1H, J1 ) 10.5 Hz, J2 ) 9 Hz, H3′), 3.98 (dddd, 1H,
J1 ) 12.5 Hz, J2 ) 6Hz, J3 ) J3 ) 1.5 Hz, CH2CHCH2O), 4.01 (dd,
1H, J1 ) 7 Hz, J2 ) 4.5 Hz, H3′′), 4.06-4.15 (m, 3H, H5′, H4′′, CH2-
CHCH2O), 4.31 (dd, 1H, J1 ) 4.5 Hz, J2 ) 1 Hz, H2′′), 4.57 (ABq,
2H, J ) 12 Hz, ∆ν ) 25.3 Hz, PhCH2O), 5.15 (ddd, J1 ) 10.5 Hz, J2
) J3 ) 1.5 Hz, 1H, CH2CHCH2O), 5.27 (ddd, 1H, J1 ) 17 Hz, J2 )
J3 ) 1.5 Hz, CH2CHCH2O), 5.33 (d, 1H, J ) 1 Hz, H1′′), 5.86-5.94
(m, 1H, CH2CHCH2O), 5.91 (d, 1H, J ) 4 Hz, H1′), 7.25-7.40 (m,
5H, C6H5); 13C NMR (CD3OD, 125 MHz) δ 33.1, 52.6, 61.3, 61.8,
64.8, 71.6, 72.3, 72.4, 72.6, 73.1, 74.3, 74.5, 77.2, 77.4, 79.1, 81.4,
85.4, 97.9, 110.6, 117.8, 128.7, 129.0, 129.4, 135.9, 139.4; HRMS for
C27H36N12O10 (M + Cs) calcd 821.1732, found 821.1726.
and the solvent was removed. The reaction was picked up in EtOAc
and washed with water twice. The organic phases were combined and
dried over MgSO4 and purified on 100 mL of silica gel using 10% to
12.5% to 15% EA/H to afford 1.24 g, 84% of product: 1H NMR
(CDCl3, 500 MHz) δ 1.43 (ddd, 1H, J1 ) J2 ) J3 ) 12.5 Hz, H2 eq),
2.26 (ddd, 1H, J1 ) 12.5 Hz, J2 ) J3 ) 4.5 Hz, H2 ax), 3.20-3.27 (m,
2H, H5, H2′), 3.30 (dd, 1H, J1 ) 13.5 Hz, J2 ) 5 Hz, H6′a), 3.35-
3.45 (m, 3H, H1, H3, H4′), 3.30 (dd, 1H, J1 ) 13.5 Hz, J2 ) 2.5 Hz,
H6′b), 3.58 (dd, 1H, J1 ) 10.5 Hz, J2 ) 4.5 Hz, H5′′a), 3.60-3.72 (m,
3H, H4, H6, H5′′b), 3.72-3.82 (m, 2H, CH2CHCH2O), 3.84 (dd, 1H,
J1 ) J2 ) 5.5 Hz, H3′′), 3.92 (dd, 1H, J1 ) 5 Hz, J2 ) 3.5 Hz, H2′′),
3.98 (dd, 1H, J1 ) 10 Hz, J2 ) 9 Hz, H3′), 4.15-4.22 (m, 2H, H4′′,
H5′), 4.42-4.90 (m, 10 H, PhCH2O), 5.12 (ddd, J1 ) 10.5 Hz, J2 ) J3
) 1.5 Hz, 1H, CH2CHCH2O), 5.12 (ddd, J1 ) 17 Hz, J2 ) J3 ) 1.5
Hz, 1H, CH2CHCH2O), 5.12 (d, 1H, J ) 3Hz, H1′′), 5.75-5.84 (m,
1H, CH2CHCH2O), 5.96 (d, 1H, J ) 3.5Hz, H1′), 7.2-7.4 (m, 25 H,
C6H5); 13C NMR (CDCl3, 125 MHz) δ 32.2, 51.1, 59.6, 60.4, 63.5,
70.2, 70.9, 71.0, 72.3, 73.3, 74.9, 75.1, 75.5, 76.1, 78.5, 80.1, 80.5,
80.8, 81.2, 83.3, 96.0, 107.3, 116.8, 127.5, 127.8, 127.9, 128.1, 128.3,
128.4, 134.5, 137.4, 137.8, 138.0, 138.2; HRMS for C55H60N12O10 (M
+ Cs) calcd 1181.3610, found 1181.3641.
6,3′,4′,3′′,5′′-Penta-O-benzyl-1,3,2′,6′-tetraazidoribostamycin (20).
Bis(methyldiphenylphoshine)(1,5-cyclooctadiene)iridium(I) hexafluo-
rophosphate (40 mg, 0.05 mmol) was suspended in THF (5 mL), and
H2 was bubbled through this suspension for 20 min. The resulting
clear solution was transferred Via syringe into a solution of compound
9 (1.24 g, 1.18 mmol) in THF (15 mL). After 1 h, a quantitative
conversion to a slightly less polar material was observed by TLC (25%
EtOAc in hexane). The solvent was removed, and the residue was
corotary evaporated with CH2Cl2 several times. The reaction was then
taken up in CH2Cl2 (30 mL) and treated with trimethylamine N-oxide
dihydrate (197 mg, 1.77 mmol), and a solution of OsO4 in tBuOH
(enough solution to deliver 3 mg of OsO4, 0.012 mmol). After the
reaction was complete (overnight) the solvent was removed and the
residue was purified over 100 mL of silica gel using 20% to 25% to
30% EtOAc in hexane to obtain 1.11 g (93.3%) of the title compound
as a colorless oil: 1H NMR (CDCl3, 500 MHz) δ 1.45 (ddd, 1H, J1 )
J2 ) J3 ) 12.5 Hz, H2 eq), 2.28 (ddd, J1 ) 12.5 Hz, 1H, J2 ) J3 ) 4.5
Hz, H2 ax), 2.35 (d, 1H, J ) 4 Hz, OH), 3.21 (dd, 1H, J1 ) 10.5 Hz,
J2 ) 4 Hz, H2′), 3.25 (dd, 1H, J1 ) J2 ) 9 Hz, H5), 3.21 (dd, 1H, J1
) 13 Hz, J2 ) 5 Hz, H6′a), 3.35-3.44 (m, 3H, H1, H3, H4′), 3.47
(dd, 1H, J1 ) 13 Hz, J2 ) 2.5 Hz, H6′b), 3.57 (dd, 1H, J1 ) 10.5 Hz,
J2 ) 4 Hz, H5′′a), 3.61 (dd, 1H, J1 ) J2 ) 9 Hz, H4 or H6), 3.65 (dd,
1H, J1 ) J2 ) 9 Hz, H4 or H6), 3.72 (dd, 1H, J1 ) 10.5 Hz, J2 ) 3
Hz, H5′b), 3.92 (dd, 1H, J1 ) 4 Hz, J2 ) 3 Hz, H2′′), 3.97 (dd, 1H, J1
) 10.5 Hz, J2 ) 4 Hz, H3′), 4.00-4.06 (m, 2H, H3′′, H4′′), 4.15-
4.20 (m, 1H, H5′), 4.39 (ABq 2H, J ) 11.5, ∆ν ) 23.6 Hz, PhCH2O),
4.52 (d, 1H, J ) 12.5 Hz, PhCH2O), 4.60 (dd, 2H, J1 ) J2 ) 11 Hz,
PhCH2O), 4.76 (d, 1H, J ) 11 Hz, PhCH2O), 4.80-4.90 (m, 4H
PhCH2O), 5.45 (d, 1H, J ) 3 Hz, H1′′), 5.98 (d, 1H, J ) 4 Hz, H1′),
7.13-7.40 (m, 25H, C6H5); 13C NMR (CDCl3, 125 MHz) δ 32.3, 51.1,
59.6, 60.6, 63.5, 70.5, 70.6, 70.9, 72.9, 73.3, 74.9, 75.37, 75.41, 76.0,
78.5, 80.1, 81.6, 82.2, 83.0, 83.5, 127.5, 127.6, 127.8, 128.0, 128.1,
128.4, 128.5, 137.1, 137.4, 137.76, 137.78, 138.1; HRMS for
C52H56N12O10 (M + Cs) calcd 1141.3297, found 1141.3267.
3′′-O-(2-Oxoethyl)-6,3′,4′,3′′,5′′-penta-O-benzyl-1,3,2′,6′-tetraazi-
doribostamycin (21). Compound 9 (112 mg, 107 µmol) was dissolved
in CH2Cl2 (5 mL) and cooled to -78 °C. Ozone was passed through
the solution until the blue color persisted. Then DMS (66 µL, 1.07
mmol) was added to the reaction, and the mixture was stirred at ambient
temperature for 2 days. The solvent was removed, and the residue
was chromatographed over 50 mL of silica gel using a 25% to 30% to
35% to 40% gradient of EtOAc in hexane to afford 83 mg (74%) of
the title compound as an oil: 1H NMR (CDCl3, 500 MHz) δ 1.44 (ddd,
1H, J1 ) J2 ) J3 ) 12.5 Hz, H2 eq), 2.17 (ddd, 1H, J1 ) 12.5 Hz, J2
) J3 ) 4.5 Hz, H2 ax), 3.19-3.25 (m, 2H, H5, H2′), 3.30 (dd, 1H, J1
) 11 Hz, J2 ) 5 Hz, H6′a), 3.36-3.45 (m, 3H, H4′, H1, H3), 3.48
(dd, 1H, J1 ) 11 Hz, J2 ) 2 Hz, H6′b), 3.59 (dd, 1H, J1 ) 10 Hz, J2
) 4 Hz, H5′′a), 3.62-3.78 (m, 5H, H4, H6, H5′′b, OCH2CHO), 3.80
(dd, 1H, J1 ) J2 ) 4.5 Hz, H3′′), 3.92 (dd, 1H, J1 ) 4.5 Hz, J2 ) 3.5
Hz, H2′′), 3.99 (dd, 1H, J1 ) 9.5 Hz, J2 ) 9 Hz, H3′), 4.15-4.22 (m,
3′′-O-Allyl-6,3′,4′,3′′,5′′-penta-O-benzyl-1,3,2′,6′-tetraazidoribos-
tamycin (9). Compound 19 (974 mg, 1.414 mmol) was dissolved in
20 mL of DMF and treated with 8 mL of BnBr. The solution was
cooled using an ice bath and treated with sodium hydride (204 mg,
8.484 mmol) in one portion. The cooling bath was then removed and
the reaction was stirred for 1 h. AcOH was added to quench the NaH,

Aminoglycoside-Ribosomal RNA Interactions
J. Am. Chem. Soc., Vol. 120, No. 9, 1998 1975
2H, H4′′, H5′), 4.46-4.90 (m, 10H, PhCH2O), 5.58 (d, 1H, J1 ) 3.5
Hz, H1′′), 5.93 (d, 1H, J1 ) 3.5 Hz, H1′), 7.23-7.37 (m, 25H, C6H5);
13C NMR (CDCl3, 125 MHz) δ 32.3, 51.1, 59.6, 60.4, 63.5, 69.9, 71.0,
72.7, 73.4, 74.9, 75.0, 75.2, 75.5, 76.0, 78.5, 78.9, 80.0, 80.7, 80.8,
81.0, 83.4, 96.0, 106.7, 127.4, 127.6, 127.7, 127.8, 127.9, 128.1, 128.4,
137.5, 137.6, 137.7, 138.0, 200.4; MS for C54H58N12O11 (M + Cs) calcd
1183, found 1183 (the peak was too weak for an exact match).
N-CH2CH2-O, H5′′b, H5′, H5), 4.04 (dd, 1H, J1 ) 10.9 Hz, J2 ) 9.5
Hz, H3′), 4.11 (dd, 1H, J1 ) 7.2 Hz, J2 ) 4.6 Hz, H3′′), 4.18 (dd, 1H,
J1 ) 10.4 Hz, J2 ) 9.9 Hz, H4), 4.18-4.21 (m, 1H, H4′′), 4.48 (dd,
1H, J1 ) 4.6 Hz, J2 ) 1.7 Hz, H2′′), 5.45 (d, 1H, J ) 1.6 Hz, H1′′),
6.06 (d, 1H, J ) 3.9 Hz, H1′); 13C NMR (CDCl3, 500 MHz) δ 25.1
(NH2CH2CH2CH2NH-), 29.5 (C2), 38.0 (NH2CH2CH2CH2NH-), 41.5
(C6′), 46.0 (NH2CH2CH2CH2NH-), 48.8 (N-CH2CH2-O), 49.9 (C3),
51.3 (C1), 55.0 (C2′), 62.3 (C5′′), 66.5 (N-CH2CH2-O), 69.5 (C3′),
70.9 (C5′), 72.0 (C4′), 74.0 (C6), 76.9 (C4), 78.4 (C3′′), 82.6 (C4′′),
86.2 (C5), 97.1 (C1′), 112.0 (C1′′); MS: for C22H46N6O10 (M + H)
calcd 555, found 555; for C23H45N5O14 (M - H) calcd 553, found 553.
3′′-O-(2-N-(3-N-Cbz-propylamino)-ethylamino-6,3′,4′,3′′,5′′ -penta-
O-benzyl-1,3,2′,6′-tetraazidoribostamycin (22). Compound 21 (50
mg, 48 µmol) was suspended in MeOH (2 mL). A solution of mono-
CBZ-propylenediamine (81 mg, 389 µmol) was made up in MeOH (2
mL) and acidified with glacial acetic acid until pH 6 (pH paper). This
solution was then added to the aldehyde mixture, and to this was added
THF until homogeneity was achieved. The reaction was treated with
an excess of solid NaCNBH3, and the amination was complete in
minutes. The reaction was diluted with ethyl acetate and extracted
with 1 N NaOH twice. The organic phases were dried over MgSO4,
and the solvent was removed. The residue was purified on 50 mL of
silica gel using a gradient of 2% to 3% to 4% to 5% MeOH in CHCL3
to afford 32 mg (54%) of the title compound: 1H NMR (CDCl3, 500
MHz) δ 1.42 (ddd, 1H, J1 ) J2 ) J3 ) 12.5 Hz, H2 eq), 1.45-1.53
(m, 2H, NHZCH2CH2CH2NH-), 2.24 (ddd, 1H, J1 ) 12.5 Hz, J2 ) J3
) 4.5 Hz, H2 ax), 2.50-2.58 (m, 2H, NHZCH2CH2CH2NH-), 2.55-
2.66 (m, 2H, N-CH2CH2-O), 3.09-3.22 (m, 2H, NHZCH2CH2CH2-
NH-), 3.18-3.31 (m, 4H, H5, H2′, N-CH2CH2-O), 3.30 (dd, 1H, J1
) 13.5 Hz, J2 ) 5.5 Hz, H6′a), 3.34-3.42 (m, 2H, H1, H3), 3.41 (dd,
1H, J1 ) J2 ) 9.5 Hz, H4′), 3.48 (dd, 1H, J1 ) 13.5 Hz, J2 ) 2.5 Hz,
H6′b), 3.56 (dd, 1H, J1 ) 10.5 Hz, J2 ) 4 Hz, H5′′a), 3.59-3.69 (m,
3H, H4, H6, H5′′b), 3.78 (dd, 1H, J1 ) J2 ) 5 Hz, H3′′), 3.93 (dd, 1H,
J1 ) 5 Hz, J2 ) 3.5 Hz, H2′′), 3.98 (dd, 1H, J1 ) J2 ) 9.5 Hz, H3′),
4.12-4.21 (m, 2H, H5′, H4′′), 4.42-4.55 (m, 3H, PhCH2O), 4.56-
4.63 (m, 2H, PhCH2O), 4.73-4.90 (m, 5H, PhCH2O), 5.05-5.10 (m,
2H, PhCH2O), 5.45-5.50 (m, 1H, NHZCH2CH2CH2NH-), 5.55 (d,
1H, J ) 3.5 Hz, H1′′), 5.95 (d, 1H, J ) 3.5 Hz, H1′), 7.23-7.37 (m,
30 H, C6H5); 13C NMR (CDCl3, 125 MHz) δ 29.1, 29.7, 32.2, 39.9,
47.5, 49.1, 51.1, 59.6, 60.4, 63.5, 6.4, 69.1, 70.3, 70.9, 72.3, 73.3, 74.9,
75.0, 75.4, 76.2, 78.1, 78.5, 80.1, 80.4, 80.8, 81.1, 83.3, 96.0, 107.1,
127.5, 127.6, 127.7, 127.9, 128.1, 128.3, 128.4, 128.5, 136.8, 137.6,
137.60, 137.83, 138.1, 156.4; HRMS for C65H74N14O12 (M + Cs) calcd
1375.4665, found 1375.4709.
3′′-O-(2-N-(p-Methoxybenzyl)(Cbz)ethylamino)-6,3′,4′,3′′,5′′-penta-
O-benzyl-1,3,2′,6′-tetraazidoribostamycin (23). Compound 21 (76
mg, 72 µmol) was suspended in MeOH (2 mL). A solution of
p-methoxybenzylamine (99 mg, 720 µmol) was made up in MeOH (2
mL) and acidified with glacial acetic acid until pH 6 (pH paper). This
solution was then added to the aldehyde mixture, and to this was added
THF until homogeneity was achieved. The reaction was treated with
an excess of solid NaCNBH3, and the amination was over in a matter
of minutes. The reaction was diluted with ethyl acetate and extracted
with 1 N NaOH twice. The organic phases were dried over MgSO4,
and the solvent was removed. The residue was purified on 50 mL of
silica gel using a gradient of 2% to 3% to 4% to 5% MeOH in CHCL3.
The resulting amine was then dissolved in CH2Cl2 and treated with
ZOSu (22 mg, 86 µmol). The reaction mixture was then directly
chromatographed on 50 mL of silica gel using a gradient of 5% to
10% to 15% ethyl acetate in hexane to afford 65 mg (69%) of the title
compound: 1H NMR (CDCl3, 500 MHz) δ 1.43 (ddd, 1H, J1 ) J2 )
J3 ) 12.5 Hz, H2 eq), 2.25 (ddd, 1H, J1 ) 12.5 Hz, J2 ) J3 ) 4.5 Hz,
H2 ax), 3.22 (dd, 1H, J1 ) 10.5 Hz, J2 ) 4 Hz, H2′), 3.16-3.35 (m,
4H, N-CH2CH2-O), 3.30 (dd, 1H, J1 ) 13.5 Hz, J2 ) 5.5 Hz, H6′a),
3.30-3.44 (m, 3 H, H1, H3, H4′), 3.47 (dd, 1H, J1 ) 13.5 Hz, J2 )
2.5 Hz, H6′b), 3.45-3.55 (m, 1H, H5′′a), 3.58-3.61 (m, 3H, H4, H6,
H5′′b), 3.62-3.71 (m, 4H, OMe, H3′′), 3.83-3.93 (m, 1H, H2′′), 3.97
(dd, 1H, J1 ) 10.5 Hz, J2 ) 9 Hz, H3′), 4.03-4.15 (m, 1H, H4′′),
4.15-4.21 (m, 1H, H5′), 4.32-4.52 (m, 5H, PhCH2O), 4.59 (d, J )
12 Hz, 2H, PhCH2O), 4.71-4.89 (m, 5H, PhCH2O), 5.14 (s, 2H,
PhCH2O), 5.48-5.52 (m, 1H, H1′′), 5.92-5.98 (m, 1H, H1′), 6.76 (dd,
J1 ) 17.5 Hz, J2 ) 8 Hz, C6H4OMe), 7.04 (dd, J1 ) 61 Hz, J2 ) 8 Hz,
2H, C6H4OMe), 7.14-7.37 (m, 30 H, C6H5); 13C NMR (CDCl3, Bruker
125 MHz) 32.2, 45.6, 46.5, 50.78, 50.82, 51.1, 55.2, 59.6, 60.5, 63.5,
67.2, 68.8, 70.2, 70.3, 70.9, 72.36, 72.39, 73.3, 74.9, 75.06, 75.10, 75.4,
76.11, 76.15, 78.3, 78.5, 80.1, 80.6, 80.78, 80.84, 81.2, 81.4, 83.3, 96.0,
107.4, 107.5, 113.8, 127.5, 127.6, 127.7, 127.8, 127.9, 128.0, 128.3,
128.4, 128.7, 129.4, 129.8, 137.5, 137.8, 138.1; HRMS for C70H75N13O13
(M + Cs) calcd 1438.4662, found 1438.4597.
3′′-O-(2-N-Cbz-ethylamino-6,3′,4′,3′′,5′′-penta-O-benzyl-1,3,2′,6′-
tetraazidoribostamycin (24). Compound 23 (65 mg, 50 µmol) was
dissolved in a mixture of acetonitrile and water (9:1, 4 mL) and treated
with CAN (136 mg, 249 µmol). After 4.5 h, the reaction was quenched
by addition of a 1 N solution of Na2S2O4. The aqueous layer was
extracted twice with ethyl acetate, and the pooled organic phases were
dried over MgSO4. Chromatography of the residue over 40 mL of
silica gel using a gradient of 15% to 20% to 25% to 30% ethyl acetate
in hexane afforded 49 mg (83%) of product: 1H NMR (CDCl3, 500
MHz) δ 1.42 (ddd, 1H, J1 ) J2 ) J3 ) 12.5 Hz, H2 eq), 2.26 (ddd,
1H, J1 ) 12.5 Hz, J2 ) J3 ) 4.5 Hz, H2 ax), 3.05-3.27 (m, 6H, H5,
H2′, NHZCH2CH2-O), 3.31 (dd, 1H, J1 ) 13.5 Hz, J2 ) 5 Hz, H6′a),
3.34-3.43 (m, 2H, H1, H3), 3.42 (dd, 1H, J1 ) J2 ) 9.5 Hz, H4′),
3.48 (dd, 1H, J1 ) 13.5 Hz, J2 ) 2.5 Hz, H6′b), 3.54 (dd, 1H, J1 )
10.5 Hz, J2 ) 4 Hz, H5′′a), 3.56-3.66 (m, 3H, H4, H6, H5′′b), 3.71
(dd, 1H, J1 ) J2 ) 5 Hz, H3′′), 3.89 (dd, 1H, J1 ) 5 Hz, J2 ) 3 Hz,
H2′′), 3.97 (dd, J1 ) 10 Hz, J2 ) 9.5 Hz, 1H, H3′), 4.07-4.12 (m, 1H,
H4′′), 4.17-4.22 (m, 1H, H5′), 4.42-4.53 (m, 3H, PhCH2O), 4.59 (d,
J ) 12 Hz, 2H, PhCH2O), 4.73-4.89 (m, 5H, PhCH2O), 5.06 (s, 2H,
PhCH2O), 5.13-5.18 (m, 1H, NHZCH2CH2-O), 5.52 (d, 1H, J ) 3
Hz, H1′′), 5.91 (d, 1H, J ) 3.5 Hz, H1′), 7.10-7.7.45 (m, 30H, C6H5);
13C NMR (CDCl3, 125 MHz) δ 29.7, 32.3, 40.9, 51.1, 59.5, 60.4, 63.5,
66.6, 68.9, 70.2, 70.9, 72.4, 73.3, 74.9, 75.0, 75.5, 76.2, 78.3, 78.5,
80.1, 80.2, 80.7, 81.0, 83.3, 96.0, 107.1, 127.3, 127.6, 127.7, 127.8,
3′′-O-(2-N-(3-Propylamino)ethylaminoribostamycin (6). Com-
pound 22 (45 mg, 36 µmol) was dissolved in THF (5 mL) and treated
with H2O (500 µL) and 1 N NaOH (50 µL). A solution of PMe3 in
THF (159 µL of a 1 N solution) was added, and the reaction was
allowed to stir for 10 h. The reaction mixture was then loaded onto a
50 mL column of silica gel and eluted with a gradient of 0% to 2.5%
to 5% to 10% concentrated NH3 in MeOH. The product fractions were
pooled and coevaporated with THF (three times). THF (7 mL) was
added via syringe to a dry three neck flask equipped with a Dewar
condenser. Then ammonia (∼20 mL) was condensed into the reaction
vessel. A chunk of Na (93 mg, 4 mmol) was then allowed to dissolve
in the ammonia for 15 min. Then a solution of the polyamine in a
mixture of EtOH and THF (500 µL each) was added in one portion
and washed down with THF. The reaction was stirred until the blue
color was discharged. Then an aqueous solution of ammonium formate
(235 mg, 3.7 mmol) was added, and the ammonia was allowed to
evaporate overnight. The remaining solvent was removed in Vacuo,
and the residue was loaded onto a column of Amberlite CG-50 cation
+
exchange resin (0.5 cm × 7 cm) in its NH4 form and eluted with a
linear gradient of 0% to 7.5% NH3 in H2O (100 mL of each in a gradient
maker). After lyophilization, neutralization, and relyophilization, 21.5
mg (75%) of 6‚6HCl salt was obtained: 1H NMR (D2O, pD 2 with
Cl- as counterinon, 500 MHz) δ 1.95 (ddd, 1H, J1 ) J2 ) J3 ) 12.6
Hz, H2 eq), 2.09-2.17 (m, 2H, NH2CH2CH2CH2NH-), 2.53 (ddd, 1H,
J1 ) 12.6 Hz, J2 ) J3 ) 4.1 Hz, H2 ax), 3.13 (dd, 2H, J1 ) J2 ) 7.9
Hz, NH2CH2CH2CH2NH-), 3.23 (dd, 2H, J1 ) J2 ) 8.0 Hz, NH2CH2-
CH2CH2NH-), 3.33 (dd, 1H, J1 ) 13.2 Hz, J2 ) 6.4 Hz, H6′a), 3.32-
3.39 (m, 2H, N-CH2CH2-O), 3.40 (ddd, 1H, J1 ) 12.6 Hz, J2 ) 10.6
Hz, J3 ) 4.1 Hz, H1), 3.44-3.52 (m, 2H, H2′, H6′b), 3.52 (dd, 1H, J1
) J2 ) 9.5 Hz, H4′), 3.60 (ddd, 1H, J1 ) 12.6 Hz, J2 ) 10.4 Hz, J3 )
4.1 Hz, H3), 3.72-3.78 (m, 2H, H6, H5′′a), 3.88-4.01 (m, 5H,

1976 J. Am. Chem. Soc., Vol. 120, No. 9, 1998
Alper et al.
127.9, 128.0, 128.1, 128.3, 128.4, 136.5, 137.5, 137.6, 137.7, 138.0,
156.3; HRMS for C62H67N13O12 (M + Cs) calcd 1318.4086, found
1318.4032.
79.3, 81.8, 82.4, 99.6, 127.6, 127.7, 127.9, 128.0, 128.3, 128.4, 128.5,
137.9, 138.0, 138.6; HRMS for C27H28O5 (M + Cs) calcd 565.0991,
found 565.1015.
3′′-O-Ethyl-2-aminoribostamycin (5). The deprotection was carried
out starting with compound 24 in the exact manner as the preparation
of compound 6 to afford the title substance in 33% yield. It should be
noted that this is a result from a single experiment where there was a
problem with the reduction of the azides and a better yield can probably
be obtained: 1H NMR (D2O, pD 2 adjusted with DCl, 500 MHz) δ
1.41 (ddd, 1H, J1 ) J2 ) J3 ) 12.6 Hz, H2 eq), 2.24 (ddd, 1H, J1 )
12.6 Hz, J2 ) J3 ) 4.1 Hz, H2 ax), 3.26 (dd, 2H, J1 ) J2 ) 4.9 Hz,
NH2CH2CH2-O), 3.32 (dd, 1H, J1 ) 13.6 Hz, J2 ) 6.4 Hz, H6′a), 3.41
(ddd, 1H, J1 ) 12.6 Hz, J2 ) 10.7 Hz, J3 ) 4.1 Hz, H1), 3.44-3.52
(m, 2H, H2′, H6′b), 3.51 (dd, 1H, J1 ) J2 ) 9.3 Hz, H4′), 3.60 (ddd,
1H, J1 ) 12.6 Hz, J2 ) 10.5 Hz, J3 ) 4.1 Hz, H3), 3.71-3.78 (m, 2H,
H5′′a, H6), 3.83-3.92 (m, 2H, NH2CH2CH2-O), 3.93 (dd, 1H, J1 )
12.6 Hz, J2 ) 2.8 Hz, H5′′b), 3.93-4.00 (m, 1H, H5′), 3.98 (dd, 1H,
J1 ) J2 ) 10.1 Hz, H5), 4.03 (dd, 1H, J1 ) 10.8 Hz, J2 ) 9.3 Hz,
H3′), 4.10 (dd, 1H, J1 ) 7.2 Hz, J2 ) 4.5 Hz, H3′′), 4.13-4.20 (m,
2H, H4, H4′′), 4.46 (dd, 1H, J1 ) 4.5 Hz, J2 ) 1.4 Hz Hz, H2′′), 5.44
(dd, 1H, J1 ) 1.4 Hz, H1′′), 6.05 (dd, 1H, J1 ) 4 Hz, H1′); 13C NMR
(CDCl3, 125 MHz) δ 29.5 (C2), 40.8 (NH2CH2CH2-O), 41.5 (C6′),
49.9 (C3), 51.3 (C1), 55.0 (C2′), 62.2 (C5′′), 67.5 (NH2CH2CH2-O),
69.5 (C3′), 70.9 (C5′), 72.0 (C4′), 74.0 (C6), 74.9 (C2′′), 76.8 (C4),
78.3 (C3′′), 82.7 (C4′′), 86.2 (C5), 97.1 (C1′), 112.0 (C1′′); MS for
C19H39N5O10 (M + H) calcd 498, found 498; for C19H39N5O10 (M -
H) calcd 496, found 496.
1,6-Anhydro-2,3,4-Tri-O-benzylidopyranoside (28). R,O-Methyl-
2,3,4-O-benzyl-5,6-dianhydroglucopyranoside (25) (5.62 g, 12.098
mmol) was dissolved in THF (20 mL) and cooled in an ice/water bath.
The reaction was then treated with a 1 M solution of BH3‚THF in THF
(50.9 mL, 50.9 mmol). The hydroboration was complete after an hour,
and the reaction mixture was then slowly dripped into a cooled flask
containing concentrated HOOH (18.1 mL) in 1 N NaOH (181 mL).
The aqueous layer was extracted three times with EtOAc, and the
organic phases were back-extracted with water. The EtOAc solution
was dried over MgSO4, and the solvent was removed. The residue
was dissolved in 50 mL of AcOH and treated with 10 drops of 12 N
HCl. The reaction was warmed to 70 °C and allowed to proceed for
1 h after which time the solvent was removed and the residue was
purified by column chromatography over 200 mL of silica gel using
10% to 12.5% to 15% EtOAc in hexane to obtain 2.74 g (51% or 80%
per step) of the product as an oil which solidifies upon standing under
vacuum.
Methyl 2,3,4-Tri-O-benzyl-1-deuxy-â-1-thioidopyranoside (30).
Compound 28 (1.31 g, 2.820 mmol) was dissolved in 15 mL of CH2-
Cl2 and treated with (methylthio)trimethylsilane (1.07 g, 8.460 mmol)
and trimethylsilyl trifluoromethanesulfonate (1.25 g, 5.640 mmol) and
stirred for 40 h. The reaction was then quenched by addition of an
excess of triethylamine and was subsequently treated with a 1 M
solution of TBAF in THF (15 mL). After the desilylation was complete,
the reaction was diluted wtih EtOAc and extracted three times with 1
N NaOH and once with water. The EtOAc solution was dried over
MgSO4, and the solvent was removed. The residue was purified by
column chromatography over 100 mL of silica gel using 30% to 35%
to 40% to 45% EtOAc in hexane to obtain the R anomer first (70 mg,
5%) and then the â anomer (1.20 g, 88.5%).
Methyl 2,3,4-Tri-O-benzyl-1-deoxy-r-1-thioidopyranioside (29):
1H NMR (CDCl3, 500 MHz) δ 2.17 (s, 3H, SCH3), 3.51 (dd, 1H, J1 )
J2 ) 4.5 Hz, H2), 3.55 (dd, 1H, J1 ) J2 ) 4.5 Hz, H3), 3.71 (dd, 1H,
J1 ) 12 Hz, J2 ) 4.5 Hz, H6a), 3.76 (dd, 1H, J1 ) J2 ) 4.5 Hz, H3),
3.94 (dd, 1H, J1 ) 12 Hz, J2 ) 7 Hz, H6b), 4.30-4.35 (m, 1H, H5),
4.40-4.78 (m, 6H, PhCH2O), 5.13 (d, 1H, J ) 4 Hz, H1), 7.20-7.40
(m, 15H, C6H5); 13C NMR (CDCl3, 125 MHz) δ 14.3, 62.0, 69.6, 72.6,
73.2, 75.4, 75.7, 77.1, 83.6, 127.8, 127.9, 128.1, 128.2, 128.4, 128.5,
137.6, 137.7, 137.8.
Methyl 2,3,4-Tri-O-benzyl-1-deoxy-â-1-thioidopyranioside (30):
1H NMR (CDCl3, 500 MHz) δ 1.89 (dd, 1H, J1 ) 9.5 Hz, J2 ) 3.5 Hz,
OH), 2.24 (s, 3H, SCH3), 3.25-3.27 (m, 1H, H4), 3.51-3.57 (m, 2H,
H2, H6a), 3.66 (dd, 1H, J1 ) J2 ) 3 Hz, H3), 3.83 (ddd, 1H, J1 ) 8
Hz, J2 ) 4 Hz, J3 ) 2 Hz, H6b), 4.00 (ddd, 1H, J1 ) 11.5 Hz, J2 ) 8
Hz, J3 ) 3.5 Hz, H5), 4.22-4.39 (m, 3H, PhCH2O), 4.55-4.64 (m,
3H, PhCH2O), 4.79 (d, 1H, J ) 1.5 Hz, H1), 7.14-7.38 (m, 15H, C6H5);
13C NMR (CDCl3, 125 MHz) δ 14.7, 62.7, 70.7, 71.7, 71.8, 72.1, 73.2,
75.3, 77.2, 85.1, 127.8, 127.9, 128.0, 128.1, 128.3, 128.4, 128.5, 137.4,
137.6, 137.7; HRMS for C28H32O5S (M + Cs) calcd 613.1025, found
613.1051.
Methyl 2,3,4-Tri-O-benzyl-1-deoxy-â-1-thio-6-deoxy-6-(allyloxy)-
idopyranoside (31). Compound 30 (245 mg, 510 µmol) was dissolved
in 3 mL of DMF and treated with NaH (24 mg, 1.02 mmol) followed
by allyl bromide (185 mg, 1.53 mmol). After being stirred overnight,
the reaction was quenched by additon of MeOH and the solvent was
removed in Vacuo. The resulting residue was partitioned between
EtOAc and H2O. The organic phases were then dried over MgSO4,
and the solvent was removed. Chromatography over 50 mL of silica
gel using a gradient of 15% to 20% to 25% EtOAc in hexane afforded
160 mg (60%) of the title compound: 1H NMR (CDCl3, 500 MHz) δ
2.22 (s, 3H, SCH3), 3.34-3.47 (m, 1H, H4), 3.45-3.48 (m, 1H, H2),
3.60-3.65 (m, 2H, H3, H6a), 3.71 (dd, 1H, J1 ) 10 Hz, J2 ) 6 Hz,
H6b), 3.92-3.97 (m, 2H, H3, CH2CHCH2O), 3.99-4.05 (m, 1H, CH2-
CHCH2O), 4.28 (s, 2H, PhCH2O), 4.31 (ABq, 2H, J ) 12 Hz, ∆ν )
49.7 Hz, PhCH2O), 4.51-4.58 (m, 2H, PhCH2O), 4.77 (d, 1H, J ) 1.5
Hz, H1), 5.12-5.28 (m, 2H, CH2CHCH2O), 5.82-5.92 (m, 1H,
CH2CHCH2O), 7.05-7.38 (m, 15H, C6H5); 13C NMR (CDCl3, Bruker
AMX-500) δ 14.5, 69.5, 71.0, 71.8, 71.9, 72.1, 72.3, 73.0, 75.1, 76.2,
84.9, 1 16.8, 127.7, 127.8, 127.9, 128.2, 128.4, 128.5, 134.8, 137.8,
138.0, 138.1; HRMS for C31H36O5S (M + Cs) calcd 653.1338, found
653.1366.
Methyl 2,3,4-Tri-O-benzyl-r-glucopyranoside (26): 1H NMR
(CDCl3, 500 MHz) δ 1.63 (dd, 1H, J1 ) 7.5 Hz, J2 ) 5.5 Hz, OH),
3.36 (s, 3H, OCH3), 3.50 (dd, 1H, J1 ) 9.5 Hz, J2 ) 3.5 Hz, H2), 3.52
(dd, 1H, J1 ) J2 ) 9.5 Hz, H4), 3.62-3.67 (m, 1H, H6a), 3.67-3.72
(m, 1H, H5), 3.74-3.79, (m, 1H, H6b), 4.01 (dd, 1H, J1 ) J2 ) 9.5
Hz, H3), 4.56 (d, 1H, J ) 3.5 Hz, H1), 4.65 (dd, 1H, J1 ) J2 ) 12 Hz,
PhCH2O), 4.85 (dd, 1H, J1 ) J2 ) 11.5 Hz, PhCH2O), 4.92 (ABq, 2H,
J ) 11 Hz, ∆ν ) 49.3 Hz, PhCH2O), 7.25-7.40 (m, 15H, C6H5); 13
C
NMR (CDCl3, 125 MHz) δ 55.2, 61.8,70.6, 73.4, 75.0, 75.8, 79.9, 81.9,
98.1, 127.6, 127.9, 128.0, 128.1, 128.4, 128.5, 138.1, 138.7; HRMS
for C28H32O6 (M + Cs) calcd 597.1253, found 597.1265.
Methyl 2,3,4-Tri-O-benzyl-â-idopyranioside (27): 1H NMR (CDCl3,
500 MHz) δ 2.74 (dd, 1H, J1 ) 9 Hz, J2 ) 5 Hz, OH), 3.48 (dd, 1H,
J1 ) 8 Hz, J2 ) 3 Hz, H2), 3.48 (s, 3H, OCH3), 3.64 (dd, 1H, J1 ) 8
Hz, J2 ) 5.5 Hz, H4), 3.80-3.87 (m, 1H, H6a), 3.88-3.94, (m, 1H,
H6b), 3.97 (ddd, 1H, J1 ) J2 ) J3 ) 5.5 Hz, H5), 4.05 (dd, 1H, J1 )
J2 ) 8 Hz, H3), 4.53 (d, 1H, J ) 3 Hz, H1), 4.54-4.83 (m, 6H,
PhCH2O), 7.25-7.40 (m, 15H, C6H5); 13C NMR (CDCl3, 125 MHz) δ
56.9, 63.1, 73.7, 73.8, 74.9, 75.0, 76.9, 77.8, 78.2, 99.9, 127.8, 127.9,
128.0, 128.1, 128.4, 128.5, 137.7, 138.2, 138.3; HRMS for C28H32O6
(M + Na) calcd 465.2277, found 487.2108.
Methyl 2,3,4-Tri-O-benzyl-1-deoxy-â-thio-6-deoxy-6-(allylamino)-
idopyranoside (32). DMSO (1.3 g, 3.39 mmol) was dissolved in CH2-
Cl2 (20 mL), and the solution was cooled to -78 °C. The reaction
was treated with 2 M oxalyl chloride in CH2Cl2 (2.21 mL, 4.42 mmol),
and the reaction was allowed to stir for 15 min. Then, a solution of
compound 30 (1.63 g, 3.39 mmol) in CH2Cl2 (10 mL) was added
dropwise Via syringe. The reaction was allowed to proceed at -78 °C
for 45 min, then triethylamine (1.72 g, 16.96 mmol) was added, and
the reaction was allowed to warm to room temperature. The reaction
was diluted with EtOAc and extracted twice with water. The organic
phases were dried over MgSO4, and the solvent was removed. The
residue was dissolved in methanol (15 mL). A solution of allylamine
(1.94 g, 33.9 mmol) was neutralized to pH 6 (pH paper) using glacial
acetic acid, and this solution was added to the solution of the aldehyde.
1,6-Anhydro-2,3,4-Tri-O-benzylidopyranoside (28): 1H NMR
(CDCl3, 500 MHz) δ 3.48 (dd, 1H, J1 ) 8 Hz, J2 ) 1.5 Hz, H2), 3.66-
3.75, (m, 2H, H4, H6a), 3.78 (dd, 1H, J1 ) J2 ) 8 Hz, H3), 4.13 (d,
1H, J ) 8 Hz, H6b), 4.39 (dd, 1H, J1 ) J2 ) 4.5 Hz, H5), 4.60-4.88
(m, 6H, PhCH2O), 5.30 (d, 1H, J ) 1.5 Hz, H1), 7.25-7.40 (m, 15H,
C6H5); 13C NMR (CDCl3, 125 MHz) δ 65.5, 73.0, 73.1, 73.2, 75.5,

Aminoglycoside-Ribosomal RNA Interactions
J. Am. Chem. Soc., Vol. 120, No. 9, 1998 1977
The reaction was then treated with NaCNBH3 (213 mg, 3.4 mmol).
The transformation was complete within 15 min. The solvent was
removed, and the reaction was taken up in EtOAc. The organic phases
were dried over MgSO4, and the solvent was removed. The residue
was purified by column chromatography over 100 mL of silica gel
using 5% to 6% to 7% MeOH in CHCL3 to obtain 1.20 g (68%) of the
title compound as an oil: 1H NMR (CDCl3, 500 MHz) δ 2.23 (s, 3H,
SCH3), 2.53 (dd, 1H, J1 ) 12.5 Hz, J2 ) 3.5 Hz, H6a), 3.16 (dd, 1H,
J1 ) 12.5 Hz, J2 ) 9 Hz, H6b), 3.18-3.26 (m, 3H, H4 and
CH2CHCH2O), 3.49-3.51 (m, 1H, H2), 3.66 (dd, 1H, J1 ) J2 ) 3 Hz,
H3), 3.87-3.92 (m, 1H, H5), 4.26-4.61 (m, 6H, PhCH2O), 4.78 (d,
1H, J ) 1.5 Hz, H1), 5.03-5.17 (m, 2H, CH2CHCH2O), 5.78-5.88
(m, 1H, CH2CHCH2O), 7.14-7.38, (m, 15H, C6H5); 13C NMR (CDCl3,
125 MHz) δ 14.7, 49.6, 52.2, 70.8, 71.8, 72.0, 72.6, 73.2, 75.3, 75.9,
85.2, 116.2, 127.7, 127.8, 128.0, 128.3, 128.4, 128.5, 136.5, 137.5,
137.9; HRMS for C31H37NO4S (M + Na) calcd 542.2341, found
542.2353.
Methyl 2,3,4-Tri-O-benzyl-1-deoxy-â-1-thio-6-deoxy-6-(carboben-
zyloxyamino)idopyranoside (33). Compound 32 (894 mg, 1.72 mmol)
was dissolved in a mixture of acetonitrile and water (84/16) and brought
to reflux. A system was set up such that the solvent in the pot was
continuously being distilled off while fresh acetonitrile/water mixture
was added to replace the distillate. A suspension of Wilkinson’s catalyst
(300 mg, 1.720 mmol) in the acetonitrile/water mixture was added,
and the reaction was allowed to reflux vigorously. The reaction was
complete in 2 h, and the solvent was removed. The residue was
dissolved in CH2Cl2 and cooled with an ice bath. The reaction was
then treated with a solution of N-(benzyloxycarbonyloxy)succinimide
(536 mg, 2.15 mmol) in CH2Cl2 (5 mL). The reaction was complete
within 15 min. The solvent was removed, and the residue was
chromatographed over 100 mL of silica gel using 17.5% to 20% to
22.5% to 25% EtOAc in hexane to afford 706 mg (67%) of the title
compound as a colorless oil: 1H NMR (CDCl3, 500 MHz) δ 2.20 (s,
3H, SCH3), 3.20-3.23 (m, 1H, H4), 3.34-3.41 (m, 1H, H6a), 3.44-
3.52 (m, 2H, H6b, H2), 3.64 (dd, 1H, J1 ) J2 ) 2.5 Hz, H3), 3.79-
3.84 (m, 1H, H5), 4.20-4.36 (m, 3H, benzillic protons), 4.52-4.61
(m, 3H, PhCH2O), 4.74 (s, 1H, H1), 4.86-4.91 (m, 1H, CH2-NHZ),
5.02-5.1 (m, 2H, PhCH2O), 7.13-7.20 (m, 4H, C6H5), 7.26-7.37,
(m, 16H, C6H5); 13C NMR (CDCl3, 125 MHz) δ 14.6, 41.8, 66.6, 70.4,
71.7, 71.8, 72.0, 73.2, 75.1, 75.3, 85.0, 127.9, 128.0, 128.3, 128.4, 128.5,
136.6, 137.3, 137.5, 137.8, 156.4; HRMS for C36H39NO6S (M + Cs)
calcd 746.1552, found 746.1568.
Compound 34. Compound 20 (69.4 mg, 69 µmol) and 31 (97 mg,
186 µmol) were mixed and dried overnight over P2O5. Then CH2Cl2
(5 mL) was added Via syringe. The reaction was cooled to -10 °C
using an ice/salt bath, and NIS (46 mg, 20 µmol) was added. The
reaction was allowed to stir for 15 min, and then a catalytic amount of
AgOTf (∼2 mg) was added. The reaction assumed a purple color and
was allowed to proceed for 45 min before being quenched with
triethylamine. The reaction was then filtered through a pad of Celite,
and the solvent was removed. Chromatography of the residue over 50
mL of silica gel using a gradient of 10% to 15% to 20% to 25% ethyl
acetate in hexane afforded 50 mg (49%) of the desired product: 1H
NMR (CDCl3, 500 MHz) δ 1.42 (ddd, 1H, J1 ) J2 ) J3 ) 12.5 Hz,
H2 eq), 2.24 (ddd, 1H, J1 ) 12.5 Hz, J2 ) J3 ) 4.5 Hz, H2 ax), 3.10
(dd, 1H, J1 ) 10.5 Hz, J2 ) 4 Hz), 3.22-3.32 (m, 4H), 3.36-3.48 (m,
4H), 3.10 (dd, 1H, J1 ) 10 Hz, J2 ) 5.5 Hz), 3.59 (dd, 1H, J1 ) J2 )
3.5 Hz), 3.61-3.67 (m, 2H), 3.83 (dd, 1H, J1 ) 10.5 Hz, J2 ) 2 Hz),
3.84-3.97 (m, 5H), 4.00 (dd, 1H, J1 ) J2 ) 9.5 Hz), 4.10-4.25 (m,
4 H), 4.34-4.61 (m, 10H), 4.67-4.75 (m, 3H), 4.76-4.89 (m, 3H),
4.93 (d, 1H, J ) 11 Hz), 5.11-5.16 (m, 1H), 5.19-5.27 (m, 1H), 5.55
(d, J ) 4.5 Hz, 1H, H1′′), 5.79-5.89 (m, 1H), 6.14 (d, 1H, J ) 4 Hz,
H1′), 7.02-7.37 (m, 40H); 13C NMR (CDCl3, 125 MHz) δ 32.4, 51.2,
59.8, 60.4, 63.2, 69.4, 70.2, 70.8, 71.9, 72.1, 72.2, 72.4, 72.6, 73.2,
73.9, 74.0, 74.1, 74.8, 75.2, 75.3, 75.4, 76.5, 78.5, 80.1, 81.8, 82.0,
82.3, 83.9, 95.6, 100.5, 107.0, 116.9, 127.3, 127.4, 127.5, 127.6, 127.7,
127.8, 128.0, 128.1, 128.2, 128.4, 128.5, 134.8, 137.6, 137.7, 137.82,
137.84, 138.0, 138.4, 138.8; HRMS for C82H89N12O15 (M + Cs) calcd
1614.5625, found 1614.5539.
(5 mL), and H2 was bubbled through this suspension for 20 min. The
resulting clear solution was transferred via syringe into a solution of
compound 34 (50 mg, 34 µmol) in THF (15 mL). After 1 h, a
quantitative conversion to a slightly less polar material was observed.
The solvent was removed, and the residue was coevaporated with CH2-
Cl2 several times. The reaction was then taken up in CH2Cl2 (30 mL)
and treated with trimethylamine N-oxide dihydrate (19 mg, 0.17 mmol),
and a solution of OsO4 in tBuOH (20 µL of the 2.5 wt % commercial
preparation). After the reaction was over (overnight), the solvent was
removed and the residue was purified over 50 mL of silica gel using
15% to 20% to 25% to 30% EtOAc in hexane to obtain 41 mg (84%)
of the title compound: 1H NMR (CDCl3, 500 MHz) δ 1.41 (ddd, 1H,
J1 ) J2 ) J3 ) 16 Hz, H2 eq), 2.24 (ddd, 1H, J1 ) 16 Hz, J2 ) J3 )
5.5 Hz, H2 ax), 2.70-2.82 (m, 1H, OH), 3.15-3.23 m, 2H), 3.25-
3.43 (m, 6H), 3.47 (dd, 1H, J1 ) 16.5 Hz, J2 ) 2.5 Hz), 3.47-3.57
(m, 1H), 3.59 (dd, J1 ) J2 ) 11.5 Hz, 1H), 3.63-3.77 (m, 4H), 3.78-
3.84 (m, 1H), 3.89-4.03 (m, 3H), 4.15-4.21 (m, 1H), 4.23-4.49 (m,
8H), 4.52-4.72 (m, 7H), 4.78-4.90 (m, 4H), 5.52 (d, 1H, J ) 4.5 Hz,
H1′′), 5.98 (d, 1H, J ) 4.5 Hz), 7.06-7.37 (m, 40H); 13C NMR (CDCl3,
125 MHz) δ 32.3, 51.1, 59.6, 60.4, 62.6, 63.3, 69.3, 70.9, 72.1, 72.3,
73.0, 73.6, 74.0, 74.7, 74.9, 75.1, 75.4, 75.8, 76.0, 78.4, 80.0, 81.3,
81.5, 81.9, 83.4, 95.9, 99.9, 107.4, 127.3, 127.5, 127.6, 127.7, 127.8,
127.9, 128.0, 128.1, 128.2, 128.3, 128.4, 137.65, 137.69, 137.8, 137.9,
138.0, 138.6; HRMS for C79H85N12O15 (M + Cs) calcd 1574.5312,
found 1574.5397.
2′′′,6′′′-Didesamino-2′′′,6′′′-dihydroxyneomycin B (7). The depro-
tection of 35 (31 mg, 2.15 µmol) was carried out in the exact manner
as the preparation of compound 6 to afford 12.4 mg (76%) of 7‚4HCl:
1H NMR (D2O, adjusted with DCl, 600 MHz) δ 1.85 (ddd, 1H, J1 )
J2 ) J3 ) 12.6 Hz, H2 eq), 2.24 (ddd, 1H, J1 ) 12.6 Hz, J2 ) J3 ) 4.1
Hz, H2 ax), 3.24 (dd, 1H, J1 ) 13.7 Hz, J2 ) 6.3 Hz, H6′a), 3.32 (ddd,
1H, J1 ) 12.6 Hz, J2 ) 10.6 Hz, J3 ) 4.1 Hz, H1), 3.37-3.43 (m, 2H,
H2′, H6′b), 3.43 (dd, 1H, J1 ) J2 ) 9.4 Hz, H4′), 3.51 (ddd, 1H, J1 )
12.6 Hz, J2 ) 10.3 Hz, J3 ) 4.1 Hz, H3), 3.57-3.60 (m, 1H, H4′′′),
3.65 (dd, 1H, J1 ) 10.6 Hz, J2 ) 9.2 Hz, H6), 3.72-3.82 (m, 4H,
H6′′′a, H6′′′b, H5′′a, H2′′′), 3.85-3.97 (m, 5H, H5′′b, H5′, H5, H3′,
H5′′′), 3.99 (dd, 1H, J1 ) J2 ) 3.7 Hz, H3′′′), 4.06 (dd, 1H, J1 ) 10.3
Hz, J2 ) 9.2 Hz, H4), 4.14-4.18 (m, 1H, H4′′), 4.35 (dd, 1H, J1 ) 4.7
Hz, J2 ) 1.7 Hz, H2′′), 4.42 (dd, 1H, J1 ) 7.2 Hz, J2 ) 4.7 Hz, H3′′),
64.89 (d, 1H, J ) 1.3 Hz, H1′′′), 5.35 (d, 1H, J ) 1.7 Hz, H1′′), 5.98
(d, 1H, J ) 4 Hz, H1′); 13C NMR (CDCl3, 125 MHz) δ 29.5 (C2),
41.5 (C6′), 49.9 (C3), 51.3 (C1), 55.0 (C2′), 2.8 (C5′′ and (C2′′′ or
C2′′′)), 9.46 (C4′′′), 9.52 (C3′), 70.7 (C2′′′ or C6′′′), 70.9 (C5′), 71.1
(C3′′′), 72.0 (C4′), 74.0 (C6), 75.3 (C2′′), 76.8, 76.9 (C3′′, C4, C5′′′),
3.0 (C4′′), 6.1 (C5), 97.1 (C1′), 100.3 (C1′′′), 111.6 (C1′′); MS for
C23H44N4O15 (M + H) calcd 617, found 617; for C23H45N5O14 (M -
H) calcd 615, found 615.
Compound 36. Compound 20 (321 mg, 0.32 mmol) and compound
33 (312 mg, 0.510 mmol) were dried together with 3 Å MS (250 mg)
overnight. Then CH2Cl2 (5 mL) was added, and the reaction was cooled
to -10 °C using an ice/salt bath. After 30 min of stirring, NIS (125
mg, 0.56 mmol) was added, and the reaction was allowed to stir for 15
min. Then, a catalytic amount of AgOTf was added, and the reaction
was allowed to stir for 30 min prior to quenching with triethylamine.
The reaction was then filtered through a pad of Celite, and the solvent
was removed. Chromatography of the residue over 50 mL of silica
gel using a gradient of 10% to 15% to 20% to 25% ethyl acetate in
hexane afforded 175 mg (35%) of the desired product: 1H NMR
(CDCl3, 500 MHz) δ 1.35 (ddd, 1H, J1 ) J2 ) J3 ) 12.5 Hz, H2 eq),
2.17 (ddd, 1H, J1 ) 12.5 Hz, J2 ) J3 ) 4.5 Hz, H2 ax), 3.12 (dd, J1
) 10 Hz, J2 ) 3.5 Hz, 1H, H2′), 3.15 (dd, J1 ) J2 ) 9 Hz, 1H, H3′′′),
3.21-3.33 (m, 3H, H1, H3, H4′′′), 3.29 (dd, 1H, J1 ) 13.5 Hz, J2 )
4.5 Hz, H6′a), 3.34-3.49 (m, 4H, H5, H4′, H6′′′a, H6′′′b), 3.47 (dd,
1H, J1 ) 13.5 Hz, J2 ) 2.5 Hz, H6′b), 3.55-3.72 (m, 5H, H4, H6,
H5′′a, H5′′b, H2′′′), 3.77-3.83 (m, 1H, H5′′′), 3.95 (dd, 1H, J1 ) 4.5
Hz, J2 ) 4 Hz, H2′′), 4.00 (dd, 1H, J1 ) 10 Hz, J2 ) 9.5 Hz, H3′),
4.13-4.19 (m, 1H, H5′), 4.19-4.24 (m, 2H, H3′′, PhCH2O), 4.29-
4.34 (m, 2H, H4′′, PhCH2O), 4.38-5.12 (m, 17H, PhCH2O and H1′′′),
5.47-5.53 (m, 1H, CbzNH), 5.54 (d, 1H, J1 ) 4 Hz, H1′′), 5.99 (d,
1H, J1 ) 3.5 Hz, H1′), 7.02-7.37 (m, 40 H, C6H5); 13C NMR (CDCl3,
125 MHz) δ 32.2, 41.6, 51.2, 59.6, 60.3, 63.2, 66.5, 69.4, 70.9, 71.7,
Compound 35. Bis(methyldiphenylphoshine)(cyclooctadienyl)-
iridium(I) hexafluorophosphate (5 mg, 6 µmol) was suspended in THF

1978 J. Am. Chem. Soc., Vol. 120, No. 9, 1998
Alper et al.
72.4, 72.6, 73.3, 73.89, 73.93, 74.2, 74.3, 74.9, 75.1, 75.3, 75.8, 76.6,
78.5, 79.9, 81.3, 81.5, 82.1, 83.4, 95.9, 100.1, 107.2, 127.2, 127.5, 127.6,
127.7, 127.8, 127.9, 128.0, 128.2, 128.3, 128.4, 128.5, 136.6, 137.56,
137.60, 137.80, 137.81, 138.1, 138.6, 156.5; HRMS for C87H91N13O16
(M + Cs) calcd 1706.5761, found 1706.5849.
H3′), 4.11 (dd, 1H, J1 ) J2 ) 3.5 Hz, H3′′′), 4.18 (dd, 1H, J1 ) J2 )
10.2 Hz, H4), 4.23-4.28 (m, 2H, H4′′, H5′′′), 4.44 (dd, 1H, J1 ) 4.8
Hz, J2 ) 2.4 Hz, H2′′), 4.52 (dd, 1H, J1 ) 6.5 Hz, J2 ) 4.8 Hz, H3′′),
5.03 (d, 1H, J ) 1.2 Hz, H1′′′), 5.46 (d, 1H, J ) 2.4 Hz, H1′′), 6.09
(d, 1H, J ) 4 Hz, H1′); 13C NMR (CDCl3, 125 MHz) δ 29.5 (C2),
41.6 (C6′), 42.0 (C6′′′), 49.9 (C3), 51.3 (C1), 54.9 (C2′), 61.8 (C5′′),
69.5 (C3′), 70.0 (C4′′′), 70.2 (C2′′′), 70.9 (C5′), 71.0 (C3′′′), 72.01
(C4′), 72.04 (C5′′′), 73.9 (C6), 75.2 (C2′′), 76.7 (C4), 76.9 (C3′′), 83.1
(C4′′), 86.3 (C5), 97.0 (C1′), 100.1 (C1′′′), 111.7 (C1′′); MS for
C23H45N5O14 (M + H) calcd 616, found 616; for C23H45N5O14 (M -
H) calcd 614, found 614.
2′′′-Desamino-2′′′-hydroxyneomycin B (8). The deprotection of
compound 36 (60.7 mg, 39 µmol) was carried out in the exact manner
as the preparation of compound 6 to afford 21.6 mg (70%) of 8‚5HCl:
1H NMR (D2O, pD 2 adjusted with DCl, Bruker AMX-500) δ 1.95
(ddd, 1H, J1 ) J2 ) J3 ) 12.6 Hz, H2 eq), 2.24 (ddd, 1H, J1 ) 12.6
Hz, J2 ) J3 ) 4.1 Hz, H2 ax), 3.33 (dd, 1H, J1 ) 13.7 Hz, J2 ) 6.4
Hz, H6′a), 3.35-3.44 (m, 3H, H6′′′a, H1, H6′′′b), 3.46-3.51 (m, 2H,
H2′, H6′b), 3.52 (dd, 1H, J1 ) J2 ) 9.3 Hz, H4′), 3.60 (ddd, 1H, J1 )
12.8 Hz, J2 ) 10.2 Hz, J3 ) 4.1 Hz, H3), 3.71-3.74 (m, H4′′′), 3.77
(dd, 1H, J1 ) 10.4 Hz, J2 ) 9.3 Hz, H6), 3.80 (dd, 1H, J1 ) 12.4 Hz,
J2 ) 5.1 Hz, H5′′a), 3.85-3.88 (m, 1H, H2′′′), 3.95 (dd, 1H, J1 ) 12.4
Hz, J2 ) 3.0 Hz, H5′′b), 3.94-3.99 (m, 1H, H5′), 3.99 (dd, 1H, J1 )
10.2 Hz, J2 ) 9.3 Hz, H5), 4.04 (dd, 1H, J1 ) 10.9 Hz, J2 ) 9.3 Hz,
Acknowledgment. We thank Professor Puglisi for providing
the coordinates to his NMR structure and Ian Ollman for
assistance with the molecular modeling.
JA972599H