Glycodiversification for the optimization of the kanamycin class aminoglycosides

Article abstract of DOI:10.1021/jm050368c

In an effort to optimize the antibacterial activity of kanamycin class aminoglycoside antibiotics, we have accomplished the synthesis and antibacterial assay of new kanamycin B analogues. A rationale-based glycodiversification strategy was employed. The a

Full text of DOI:10.1021/jm050368c

J. Med. Chem. 2005, 48, 6271-6285
6271
Glycodiversification for the Optimization of the Kanamycin Class
Aminoglycosides
Jinhua Wang, Jie Li, Hsiao-Nung Chen, Huiwen Chang, Christabel Tomla Tanifum, Hsiu-Hsiang Liu,
Przemyslaw G. Czyryca, and Cheng-Wei Tom Chang*
Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, Utah 84322-0300
Received April 19, 2005
In an effort to optimize the antibacterial activity of kanamycin class aminoglycoside antibiotics,
we have accomplished the synthesis and antibacterial assay of new kanamycin B analogues.
A rationale-based glycodiversification strategy was employed. The activity of the lead is
comparable to that of commercially available kanamycin. These new members, however, were
found to be inactive against aminoglycoside resistant bacteria. Molecular modeling was used
to provide the explanation. Thus, a new strategy for structural modifications of kanamycin
class aminoglycosides is suggested.
Introduction
mycin B analogues by glycosylation of the O-6 OH of
neamine (rings I and II) (Figure 3).
Aminoglycoside antibiotics have been used as a treat-
ment against infectious diseases for over 60 years,1
although the prevalence of aminoglycoside resistant
bacteria has significantly reduced their effectiveness.2
Nevertheless, aminoglycoside antibiotics are still a
valuable resource against serious infections. With the
unraveled structural information involving the ami-
noglycoside-bound rRNA molecules3 and the details of
the resistance mechanisms, especially the information
obtained from the X-ray structural studies of aminogly-
coside-modifying enzymes,4 a growing interest has
resurfaced into the development of new aminoglycoside
antibiotics to counteract the problem caused by ami-
noglycoside resistant bacteria.5
The design of 17 is to examine the importance of 4′′-
OH group. The designs of 18 and 19 are to confirm the
advantage of 6′′-CH3. The designs of 16 and 20 can be
used to establish the effectiveness of an axial 4′′-OH.
Incorporation of D-fucose as in the design of 21 can be
used to further demonstrate the effect of axial 4′′-OH
group, while 23 containing L-fucose can be used as a
comparison. If the importance of 6′′-CH3 is established,
22 should be the most active compound compared to
kanamycin.
The syntheses of the corresponding glycosyl donors
for the preparation of 16-19, 21, and 23 are analogous
to the reported procedures.7 The synthesis of the gly-
cosyl donor for the preparation of 20 began from 24
(Scheme 1).6,8 Hydrolysis of the acetyl groups, followed
by protection of 4,6-diol with benzylidene, afforded 25.
After benzyl protection of 2-OH, compound 26 was
treated with Me3N-BH3 in THF generating 27.9 A two-
step epimerization of 4-OH using Tf2O and then t-Bu4-
NOAc yielded the designed glycosyl donor 28.
The synthesis of glycosyl donor 35 started from phenyl
2,3,4-tri-O-acetyl-1-thio-R-D-fucopyranose, 29 (Scheme
2).8 Hydrolysis of the acetyl groups, followed by the
isopropylidene protection of the 3,4-diol, gave 30. Pro-
tection of 2-OH followed by acid-mediated deprotection
generated 31, which was subjected to a two-step epimer-
ization to produce 32. Hydrolysis of the acetyl groups
of 32, followed by selective benzoylation of the 4-OH,
generated 34. Incorporation of azido group at the C-3
position completed the synthesis of designed glycosyl
donor.
The kanamycin B analogues from the corresponding
glycosyl donor were prepared according to the reported
procedure (Schemes 3 and 4).7 The designed kanamycin
B analogues were tested against Escherichia coli (ATCC
25922) and Staphylococcus aureus (ATCC 25923) using
kanamycin B as the control (Table 1).10
Design and Synthesis of New Kanamycin B
Analogues
Our group has prepared libraries of aminosugars
(azidosugars), which enable a modular approach for the
construction of libraries of novel aminosugar-containing
glycoconjugates with the original carbohydrate compo-
nent replaced by a synthetic one. This strategy is termed
glycodiversification.6 Following this concept, our group
has synthesized a library of kanamycin B analogues
with structural variation at ring III (Figure 1).7 We have
established the preliminary structure activity relation-
ship (SAR) (Figure 2): (i) An equatorial amino group is
preferred over an equatorial hydroxyl group at C-3′′. (ii)
At the C-4′′ position, the presence of an axial NH2
decreases the activity. (iii) Deoxygenation at C-6′′ (6′′-
CH3) provides better activity than CH2NH2 and CH2-
OH groups. However, there are some structural features
whose effectiveness is still required to be established,
for example, the effect of having an equatorial OH
versus an axial one at the C-4′′ position, the importance
of having 6′′-CH3 group, and the effect of 4′′-deoxygen-
ation. To address these questions and to confirm the
observed activity, we decided to synthesize more kana-
The SAR of the kanamycin B analogues in Table 1
demonstrates that an axial OH group is superior to an
equatorial OH at the C-4′′ position (entries 7 and 4, 21
* To whom correspondence should be addressed. Phone: 435-797-
3545. Fax: 435-797-3390. E-mail: chang@cc.usu.edu.
10.1021/jm050368c CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/02/2005

6272 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Wang et al.
Figure 1. Structures of Kanamycin class aminoglycosides.
5). The presence of the 6′′-CH3 group appears to be
important. However, replacing the 6′′-CH3 group with
H does not abolish the antibacterial activity. Rather,
such modification seems to enhance the activity (entries
4 and 5, 18 vs 19). The observation, however, requires
further investigation. Finally, as predicted, 22 is the
most active compound, which could be employed as the
lead for further modification.
Combined SAR information allows us to identify 4′′-
OH as the optimal site for further modification on ring
III. At the beginning, four designs including 45-48,
where the R group represents the point of diversifica-
Figure 2. Summary of the SAR of ring iii of kanamycin B
analogues.
vs 18). A hydroxyl group (or amino group) at the C-4′′
position is essential for activity (entries 3 and 10, 17 vs
Figure 3. Structures of additional kanamycin class aminoglycosides.
Scheme 1. Synthesis of Glycosyl Donora
a (a) (1) NaOMe, MeOH, (2) PhCH(OMe)2, TsOH, DMF; (b) BnBr, NaH, TBAI, THF; (c) BH3-Me3N, AlCl3, 4 Å molecular sieves, THF;
(d) (1) Tf2O, CH2Cl2, pyridine, (2) Bu4NOAc, CH2Cl2.

Optimization of Aminoglycosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6273
Scheme 2. Synthesis of Glycosyl Donora
a (a) (1) NaOMe, MeOH, (2) Me2C(OMe)2, TsOH‚H2O, acetone; (b) (1) BnBr, NaH, TBAI, THF, (2) HOAc, TFA, H2O; (c) (1) Tf2O, py,
CHCl2, (2) n-Bu4N+-AcO-; (d) NaOMe, MeOH; (e) BzCl, DIPEA, DMAP, CH2Cl2; (f) (1) Tf2O, py, CH2Cl2, (2) NaN3, DMF.
Scheme 3a
a The ratios, including those in the following tables, are measured on the basis of the integral ratio of the ring I anomeric proton (H-1′).
(a) Glycosyl donor, NIS, TfOH, Et2O:CH2Cl2 (3:1); (b) NaOMe, MeOH:THF (5:1).
Scheme 4a
a (a) (1) PMe3, NaOH, THF, (2) H2, Pd(OH)2/C, HOAc, H2O (3) Dowex 1X8-200 (Cl- form).
tion, were envisioned (Figure 4). Nevertheless, there are
several problems associated with the first three designs.
For example, it is very difficult to introduce an equato-
rial 4′′-NH2 group. Attempts to use reductive amination
and nucleophilic substitution for the synthesis of 45 (R
) (CH2)4N3) were unsuccessful. The presence of an acid-

6274 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Wang et al.
Table 1. Minimum Inhibitory Concentration (MIC)
MIC (µg/mL)
Kanamycin exerts its antibacterial activity by binding
to rRNA and is a highly negatively charged molecule
due to its phosphodiester backbone. It is our expectation
that by introducing a more positively charged side chain
at the O-4′′ position, an increase in the antibacterial
activity can be obtained. Therefore, we propose the syn-
thesis of several new kanamycin B analogues with modi-
fication at O-4′′ position following the design of 48 (Fig-
ure 5). Since 6-deoxy-3-aminoglucopyranose is harder to
prepare, we used 6-deoxyglucopyranose for the model
studies (49-53). After the identification of the optimal
structural component at O-4′′, the desired 6-deoxy-3-
aminoglucopyranose was prepared (55). The design of
54 contains an enlarged side chain that hopefully may
render it a poor substrate for the aminoglycoside-modify-
ing enzymes. Therefore, if the high potency of such an
analogue can be maintained, this new aminoglycoside
antibiotic may generate activity against resistant strains
of bacteria (Figure 6).
entry
compd
E. coli
S. aureus
1
2
kanamycin B
2
2
16
32
32
3
17
inactive
inactive
4
18
inactive
inactive
5
19
32
64
6
20
4
1
7
21
32
16
8
22
2
2
9
23
inactive
12
inactive
2
10
5 (ref 7)
labile tertiary 4′′-OH in the design of 46 may hinder its
synthesis. On the basis of our and others’ 5e experience,
kanamycin analogues with a galacto-configuration, as
in the design of 47, will degrade under acidic conditions
more easily than those with the gluco-configuration.
Therefore, we decided to employ 48 as the template for
introducing modifications at O-4′′.
Figure 4. Possible designs of kanamycin B analogues with O-4′′ modifications.
Figure 5. Structures of kanamycin class aminoglycosides bearing O-4′′ modification.
Figure 6. Concept for the design of kanamycin analogues against resistant bacteria.

Optimization of Aminoglycosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6275
Scheme 5. Synthesis of the Donors for the Preparation of Kanamycin B with O-4′′ Modificationsa
(a) AllBr, NaH, THF, TBAI; (b) (1) BH3, THF, (2) NaOH, H2O2; (c) TSCl, CH2Cl2, Et3N, DMAP; (d) NaN3, DMF; (e) NaN3, CeCl3‚7H2O,
CH3CN/H2O (9:1); (f) Ac2O, CH2Cl2, Et3N, DMAP; (g) (1) Tf2O, py, CH2Cl2, (2) NaN3, DMF.
Scheme 6
The syntheses of glycosyl donors for the model studies
of the designed kanamycin analogues started from 56
(Scheme 5).6 The 4-OH can be alkylated with allyl, (R)-
glycidyl, and (S)-glycidyl groups, yielding 57, 61, and
64, respectively. The designed glycosyl donor, 59, can
be synthesized via hydroboration followed by azido
substitution from 57, while the glycosyl donors 62, 63,
65, and 66 can be prepared from 61 and 64 via azide-
induced ring opening and acetylation or azido substitu-
tion. The designed donor 69 can be synthesized by
repeating the glycidylation and the azide-induced ring-
opening processes. The kanamycin B analogues from the
corresponding glycosyl donors were prepared as before
(Schemes 6 and 7).
Results and Conclusion
The additional kanamycin B analogues were assayed
as described previously (Scheme 7).10 From the results
of antibacterial assay, we noticed that there is no
significant difference in the activity of analogues with
various side chains at O-4′′, although such side chains
can revive the activity compared to the corresponding
inactive parent compound 18. However, to our surprise,
when one of the side chains was attached to the lead,
no increase in antibacterial activity (entry 8, 55 vs 22)
was observed. No activity was obtained when these
analogues were tested against aminoglycoside resistant
bacteria.11 The results were disappointing; thus, we
used molecular modeling studies of the kanamycin
analogues for providing a rationale. The kanamycin
analogues were docked to the RNA binding site, and
selected structures were docked to the kanamycin
kinase type III (APH(3′)-IIIa) as well.12 The scoring
function was based on the electrostatic interactions, the
molecular mechanics using the Amber 96 force field as

6276 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Wang et al.
Scheme 7a
a The lower are the values, the better is the binding affinity to rRNA.
implemented in HyperChem 7.0, and the solvent-
accessible surface methodology to account for the hydra-
tion effects. The function was developed as a part of the
de novo drug design package and not specifically for the
calculation of absolute binding affinity. Therefore, it
evaluates the relative binding affinities rather than the
absolute binding scores.
From the binding scores, we noticed that there is no
significant difference among compounds with the same
total number of amino groups although the extra amino
group on the side chain seems to lower the activity,
while the binding score suggests otherwise. When fitting
54 into the binding site of the kinase, we found that
the compound can still bind to the active site, following
a conformational change in the side chain attached at
O-4′′. The conformations of rings I and II remain largely
unchanged. The result suggests that the sites of modi-
fications, primarily on rings I and II, are still susceptible
to enzyme-catalyzed reactions such as acetylation, phos-
class antibiotics. Results from molecular modeling and
antibacterial assay both suggest that there is no sig-
nificant difference in the antibacterial activity due to
the variation of functional groups (OH or NH2) and
stereocenter. However, we did notice a discrepancy: an
extra amino group on the side chain seems to lower the
activity while increasing the binding score. The impor-
tance of employing real molecules in a whole cell based
assay has also been highlighted because binding affinity
studies using aminoglycoside and fragment of RNA
molecules will likely generate results as predicted by
molecular modeling and, thus, overemphasize the im-
portance of amino group(s). Finally, the lack of activity
of 54 against aminoglycoside resistant bacteria suggests
that the attachment of a large but flexible side chain
at the O-4′′ position is an ineffective design. Perhaps a
rigid functionality where we are currently devoting our
effort will be a better design.
phorylation, and adenylation. This can explain the
ineffectiveness of 54 that is equipped with an enlarged
but flexible side chain (Figure 7).
Experimental Section
Proton magnetic resonance spectra were recorded using a
JEOL 270 or Bruker 400 spectrometer. Chemical shifts (δ)
were reported as parts per million (ppm) downfield from
tetramethylsilane, and coupling constants were given in cycles
per second (Hz). Splitting patterns were designated as s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet. 13C
spectra were obtained using the JEOL 270 spectrometer at
In conclusion, we have synthesized complex analogues
of kanamycin B. Through a chemical glycodiversification
strategy, a library of structurally diverse glycosyl donors
can be readily incorporated onto a given aglycon (neam-
ine derivative) whereas an enzymatic method of syn-
thesis may not be viable because of the constrained
substrate acceptance by glycosyltransferase. Although
the expected activity against aminoglycoside resistant
bacteria was not observed, we have outlined a rationale-
based model for the development of novel kanamycin
68 MHz or Bruker 400 spectrometer at 100 MHz. Routine 13
C
NMR spectra were fully decoupled by broad-band waltz
decoupling. All NMR spectra were recorded at ambient tem-
perature unless otherwise noted. Results from low-resolution
fast atom bombardment (LRFAB) and high-resolution fast
atom bombardment (HRFAB) or high-resolution matrix-as-

Optimization of Aminoglycosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6277
Figure 7. Binding of 54 in APH(3′)-IIIa.
sisted laser desorption ionization (MALDI) were provided by
the Mass Spectrometry Facilities, University of California,
Riverside.
Chemical reagents and starting materials were purchased
from Aldrich Chemical Co. or Acros Chemical Co. and were
used without purification unless otherwise noted. Dichlo-
romethane was distilled over CaH2. Other solvents were used
without purification. Column chromatography was carried out
by using silica gel (60 Å, 230 mm × 450 mm mesh, Sorbent
Tech.) unless otherwise noted.
was washed with a solution of hexanes/ether (95/5) and
collected as a light-yellowish solid (0.92 g, 1.94 mmol, 73%).
1H NMR (270 MHz, CDCl3) δ 7.3-7.6 (m, 15H), 5.57 (s, 1H),
4.93 (d, J ) 10.0 Hz, 1H, PhCH2O), 4.81 (d, J ) 10.0 Hz, 1H,
PhCH2O), 4.75 (d, J ) 9.6 Hz, 1H, H-1), 4.38 (dd, J ) 10.9 Hz,
J ) 4.3 Hz, 1H), 3.7-3.8 (m, 2H), 3.4-3.5 (m, 2H), 3.36 (dd, J
) 8.9 Hz, J ) 9.6 Hz, 1H); 13C NMR (68 MHz, CDCl3) δ 137.3
(s), 136.8 (s), 132.9 (s), 132.4 (s), 129.2 (s), 128.66 (s), 128.58
(s), 128.4 (s), 128.28 (s), 128.18 (s), 126.1 (s), 101.5 (s), 88.7
(s), 79.7 (s), 79.1 (s), 75.8 (s), 71.1 (s), 68.7 (s), 67.0 (s); LRFAB
m/e 476 ([M + H]+); HRFAB calcd for C26H26N3O4S ([M + H]+)
m/e 476.1644, measured m/e 476.1665.
Phenyl 3-Azido-2,6-di-O-benzyl-3-deoxy-1-thio-â-D-glu-
copyranoside (27). A solution of 26 (0.2 g, 0.42 mmol) in THF
(15 mL) was stirred for 10 min over 4 Å molecular sieves.
Borane-trimethylamine complex (0.18 g, 2.52 mmol) was
added in one portion, followed by aluminum chloride (0.34 g,
2.52 mmol). After 4.5 h, additional borane-trimethylamine
complex (0.12 g, 1.68 mmol) and aluminum chloride (0.17 g,
1.26 mmol) were added, and the mixture was stirred overnight
at ambient temperature. The reaction mixture was filtered
through Celite, neutralized with 1 M H2SO4, diluted with
EtOAc, and washed with water, saturated NaHCO3(aq), and
brine, and then dried over Na2SO4(s). Removal of the solvent
followed by gradient column chromatography (hexanes/EtOAc
) 100:0 to 55:45) afforded the product (0.18 g, 0.38 mmol, 90%).
1H NMR (270 MHz, CDCl3) δ 7.2-7.5 (m, 15H), 4.91 (d, J )
10.2 Hz, 1H, PhCH2O), 4.74 (d, J ) 10.2 Hz, 1H, PhCH2O),
4.66 (d, J ) 9.6 Hz, 1H, H-1), 4.59 (d, J ) 11.9 Hz, 1H,
PhCH2O), 4.53 (d, J ) 11.9 Hz, 1H, PhCH2O), 3.7-3.8 (m, 2H),
3.4-3.6 (m, 3H), 3.32 (dd, J ) 9.2 Hz, J ) 8.9 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ 137.7 (s), 137.5 (s), 133.6 (s), 132.3
(s), 129.2 (s), 128.8 (s), 128.74 (s), 128.67 (s), 128.3 (s), 128.2
(s), 128.0 (s), 88.2 (s), 79.3 (s), 78.1 (s), 75.5 (s), 74.0 (s), 71.2
(s), 70.7 (s), 70.6 (s); LRFAB m/e 500 ([M + Na]+); HRFAB
calcd for C26H27N3O4SNa ([M + Na]+) m/e 500.1620, measured
m/e 500.1640.
Phenyl 3-Azido-4,6-O-benzylidene-3-deoxy-1-thio-â-D-
glucopyranoside (25). To a solution of 24 (1.2 g, 2.86 mmol)
in anhydrous MeOH (10 mL), 1 mL of NaOMe (2 M in MeOH)
was added. After completion of the reaction (1 h), the reaction
was quenched by addition of Amberlite IR-120 (H+) and
filtered. After removal of solvent, the crude triol was dissolved
in DMF. To the mixture were then added PhCH(OM2)2 (2.0
mL, 13.3 mmol) and a catalytic amount of TsOH‚H2O. The
reaction mixture was stirred at 60 °C for 0.5 h, and then the
solvent was removed with a rotovap. The product was pre-
cipitated by addition of saturated NaHCO3(aq) and collected
with a Hirsch funnel as a light-yellowish solid (1.1 g, 2.86
1
mmol, 99%). H NMR (270 MHz, CDCl3) δ 7.3-7.5 (m, 10H),
5.55 (s, 1H), 4.62 (d, J ) 9.6 Hz, 1H, H-1), 4.39 (dd, J ) 10.2
Hz, J ) 4.3 Hz, 1H), 3.76 (dd, J ) 10.2 Hz, J ) 9.9 Hz, 1H),
3.71 (dd, J ) 9.2 Hz, J ) 9.2 Hz, 1H), 3.5-3.6 (m, 2H), 3.38
(dd, J ) 9.2 Hz, J ) 9.2 Hz, 1H); 13C NMR (68 MHz, CDCl3)
δ 136.7 (s), 133.4 (s), 130.8 (s), 129.3 (s), 128.8 (s), 128.4 (s),
126.1 (s), 101.6 (s), 89.2 (s), 79.2 (s), 71.7 (s), 71.5 (s), 68.6 (s),
65.9 (s); LRFAB m/e 386 ([M + Na]+); HRFAB calcd for
C19H20N3O4S ([M + H]+) m/e 386.1175, measured m/e 386.1184.
Phenyl 3-Azido-2-O-benzyl-4,6-O-benzylidene-3-deoxy-
1-thio-â-D-glucopyranoside (26). To a solution of compound
25 (1.03 g, 2.67 mmol) in anhydrous THF (10 mL), BnBr (0.64
mL, 5.35 mmol), NaH (0.53 g, 13.4 mmol), and a catalytic
amount of TBAI were added. The reaction mixture was stirred
overnight. The excess BnBr was quenched by addition of
MeOH (0.5 mL). Then the reaction mixture was poured into a
solution of ice and EtOAc. The aqueous layer was extracted
with EtOAc. The combined organic layers were washed with
1 N HCl(aq), water, saturated NaHCO3(aq), and brine and then
dried over Na2SO4(s). After removal of solvents, the product
was crystallized and collected with a Hirsch funnel. The crystal
Phenyl 4-O-Acetyl-3-azido-2,6-di-O-benzyl-3-deoxy-1-
thio-â-D-galactopyranoside (28). To a solution of 27 (0.81
g, 1.69 mmol) and pyridine (0.41 mL, 5.08 mmol) in anhydrous
CH2Cl2 at 0 °C, Tf2O (0.57 mL, 3.38 mmol) was added slowly.
After being stirred for 30 min, the reaction mixture was diluted
with CH2Cl2, washed with water, saturated NaHCO3(aq), and

6278 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Wang et al.
brine, and then dried over Na2SO4(s). The solution was filtered
through glass wool and transferred into a solution of tetrabu-
tylammonium acetate (1.02 g, 3.38 mmol) in CH2Cl2. The
reaction mixture was stirred overnight while the solvent was
slowly evaporated with an aspirator. After completion of the
reaction, the reaction mixture was diluted with EtOAc, washed
with 1 N HCl, saturated NaHCO3(aq), and brine, and then dried
over Na2SO4(s). Removal of the solvent followed by purification
with gradient column chromatography (hexanes/EtOAc )
100:0 to 50:50) afforded the product (0.77 g, 1.48 mmol, 88%).
1H NMR (270 MHz, CDCl3) δ 7.3-7.6 (m, 15H), 5.48 (dd, J )
2.6 Hz, J ) 0.7 Hz, 1H, H-4), 4.97 (d, J ) 9.9 Hz, 1H, PhCH2O),
4.74 (d, J ) 9.9 Hz, 1H, PhCH2O), 4.73 (d, J ) 9.0 Hz, 1H,
H-1), 4.54 (d, J ) 11.6 Hz, 1H, PhCH2O), 4.47 (d, J ) 11.6 Hz,
1H, PhCH2O), 3.79 (ddd, J ) 6.2 Hz, J ) 6.2 Hz, J ) 0.7 Hz,
1H, H-5), 3.6-3.7 (m, 2H), 3.60 (dd, J ) 9.8 Hz, J ) 6.2 Hz,
1H, H-6), 3.52 (dd, J ) 9.8 Hz, J ) 6.2 Hz, 1H, H-6), 2.12 (s,
3H, CH3CO2); 13C NMR (68 MHz, CDCl3) δ 170.1 (s, CH3CO2),
137.8 (s), 137.8 (s), 133.8 (s), 132.1 (s), 129.2 (s), 128.8 (s),
128.68 (s), 128.64 (s), 128.4 (s), 128.2 (s), 128.1 (s), 127.9 (s),
88.6 (s), 76.9 (s), 75.7 (s), 73.9 (s), 68.7 (s), 68.5 (s), 65.6 (s),
20.9 (s, CH3CO2); LRFAB m/e 542 ([M + Na]+); HRFAB calcd
for C28H29N3O5SNa ([M + Na]+) m/e 542.1726, measured m/e
542.1732.
Phenyl 3,4-Di-O-acetyl-2-O-benzyl-6-deoxy-1-thio-â-D-
allopyranoside (32). Refer to the procedure for the prepara-
tion of 28. 1H NMR (400 MHz, CDCl3) δ 7.3-7.6 (m, 10H),
5.79 (dd, J ) 3.0 Hz, J ) 2.7 Hz, 1H, H-3), 5.00 (d, J ) 9.7 Hz,
1H, H-1), 4.62 (d, J ) 11.1 Hz, 1H, PhCH2O), 4.56 (dd, J )
10.2 Hz, J ) 2.7 Hz, 1H, H-4), 4.41 (d, J ) 11.1 Hz, 1H,
PhCH2O), 3.98 (dq, J ) 10.2 Hz, J ) 6.2 Hz, 1H, H-5), 3.44
(dd, J ) 9.7 Hz, J ) 3.0 Hz, 1H, H-2), 2.12 (s, 3H, CH3CO2),
2.03 (s, 3H, CH3CO2), 1.24 (d, J ) 6.2 Hz, 3H, H-6); 13C NMR
(100 MHz, CDCl3) δ 170.4 (s, CH3CO2), 169.9 (s, CH3CO2),
137.2 (s), 133.0 (s), 132.8 (s), 129.0 (s), 128.6 (s), 128.4 (s), 128.2
(s), 128.0 (s), 83.8 (s), 75.0 (s), 72.15 (s), 72.06 (s), 70.9 (s), 67.4
(s), 21.0 (s, CH3CO2), 20.9 (s, CH3CO2), 17.8 (s); LRFAB m/e
453 ([M + Na]+); HRFAB calcd for C23H26O6SNa ([M + Na]+)
m/e 453.1348, measured m/e 453.1358.
Phenyl 2-O-Benzyl-6-deoxy-1-thio-â-D-allopyranoside
(33). A solution of 32 (0.76 g, 1.77 mmol) and NaOMe (1 M,
1.0 mmol) in MeOH (5 mL) was stirred at room temperature
till the complete consumption of starting material (∼2 h). Then
Amberlite 120H+ was added to quench the reaction. The
reaction mixture was filtered through Celite and washed with
EtOAc and MeOH. Removal of the solvent followed by puri-
fication with gradient column chromatography (hexanes/
EtOAc ) 90:10 to 40:60) afforded the product (0.55 g, 1.59
1
mmol, 90%). H NMR (400 MHz, CDCl3) δ 7.3-7.6 (m, 10H),
Phenyl 3,4-O-Isopropylidene-1-thio-â-D-fucopyrano-
side (30). To a solution of compound 298 (7.04 g, 18.4 mmol)
in anhydrous MeOH (30 mL), 2 mL of NaOMe (2 M in MeOH)
was added. After completion of the reaction (1 h), the reaction
was quenched by addition of Amberlite IR-120 (H+) and the
mixture was filtered. After removal of solvent, the crude triol
was dissolved in a solution of acetone (20 mL) and Me2C(OMe)2
(20 mL) containing a catalytic amount of TsOH‚H2O. The
reaction mixture was stirred overnight, and the reaction was
quenched by addition of Et3N (5 mL). After removal of solvents,
the oily crude produced was redissolved in EtOAc. The organic
solution was washed with 1 N HCl(aq), water, saturated
NaHCO3(aq), and brine, and then dried over Na2SO4(s). Removal
of the solvent followed by purification with gradient column
chromatography (hexanes/EtOAc ) 100:0 to 50:50) afforded
4.95 (d, J ) 9.8 Hz, 1H, H-1), 4.78 (d, J ) 11.4 Hz, 1H,
PhCH2O), 4.62 (d, J ) 11.4 Hz, 1H, PhCH2O), 4.18 (dd, J )
3.0 Hz, J ) 3.0 Hz, 1H, H-3), 3.70 (dq, J ) 9.4 Hz, J ) 6.2 Hz,
1H, H-5), 3.40 (dd, J ) 9.8 Hz, J ) 3.0 Hz, 1H, H-2), 3.21 (dd,
J ) 9.4 Hz, J ) 3.0 Hz, 1H, H-4), 1.33 (d, J ) 6.2 Hz, 3H,
H-6); 13C NMR (100 MHz, CDCl3) δ 137.5 (s), 132.0 (s), 129.0
(s), 128.8 (s), 128.5 (s), 128.4 (s), 127.6 (s), 83.6 (s), 73.1 (s),
72.91 (s), 72.89 (s), 69.3 (s), 18.1 (s); LRFAB m/e 369 ([M +
Na]+); HRFAB calcd for C19H22O4SNa ([M + Na]+) m/e 369.1137,
measured m/e 369.1155.
Phenyl 4-O-Benzoyl-2-O-benzyl-6-deoxy-1-thio-â-D-al-
lopyranoside (34). To a solution of 33 (0.55 g, 1.59 mmol) in
anhydrous CH2Cl2 (30 mL) was added DMAP (catalytic
amount), DIPEA (0.53 mL, 3.18 mmol), and BzCl (0.20 mL,
1.75 mmol) at -50°C. The reaction mixture was stirred and
allowed to warm to -10°C. Water was added to quench the
reaction. After removal of the solvent, the reaction mixture
was diluted with EtOAc. The organic layers were washed with
1 N HCl, saturated NaHCO3(aq), and brine, and then dried over
Na2SO4(s). Removal of the solvent followed by purification with
gradient column chromatography (hexanes/EtOAc ) 100:0 to
60:40) afforded the product (0.65 g, 1.44 mmol, 91%). 1H NMR
(400 MHz, CDCl3) δ 7.3-7.6 (m, 10H), 4.95 (d, J ) 9.8 Hz,
1H, H-1), 4.78 (d, J ) 11.4 Hz, 1H, PhCH2O), 4.62 (d, J )
11.4 Hz, 1H, PhCH2O), 4.18 (dd, J ) 3.0 Hz, J ) 3.0 Hz, 1H,
H-3), 3.70 (dq, J ) 9.4 Hz, J ) 6.2 Hz, 1H, H-5), 3.40 (dd, J )
9.8 Hz, J ) 3.0 Hz, 1H, H-2), 3.21 (dd, J ) 9.4 Hz, J ) 3.0 Hz,
1H, H-4), 1.33 (d, J ) 6.2 Hz, 3H, H-6); 13C NMR (100 MHz,
CDCl3) δ 166.2 (s), 137.3 (s), 133.56 (s), 133.49 (s), 132.5 (s),
130.0 (s), 129.9 (s), 129.1 (s), 128.79 (s), 128.68 (s), 128.44 (s),
128.34 (s), 127.81 (s), 83.5 (s), 76.9 (s), 74.5 (s), 72.8 (s), 70.1
(s), 67.2 (s), 18.0 (s); LRFAB m/e 473 ([M + Na]+); HRFAB
calcd for C26H26O5SNa ([M + Na]+) m/e 473.1400, measured
m/e 473.1380.
Phenyl 3-Azido-4-O-benzoyl-2-O-benzyl-3,6-dideoxy-1-
thio-â-D-glucopyranoside (35). To a solution of 34 (0.79 g,
1.76 mmol) and pyridine (0.31 mL, 3.86 mmol) in anhydrous
CH2Cl2 at 0 °C, Tf2O (0.53 mL, 3.17 mmol) was added slowly.
After the mixture was stirred for 1 h, TLC was performed. If
the reaction did not go to completion, more pyridine (0.15 mL,
1.90 mmol) and Tf2O (0.26 mL, 1.55 mmol) were added. After
completion of the reaction, the reaction mixture was diluted
with CH2Cl2, washed with water, saturated NaHCO3(aq), and
brine, and then dried over Na2SO4(s). The solution was filtered
through glass wool and transferred into a solution of NaN3
(1.14 g, 17.6 mmol) in DMF. The reaction mixture was stirred
overnight while the solvents were slowly evaporated with an
aspirator. After completion of the reaction, the reaction
mixture was diluted with EtOAc and filtered through Celite.
1
the product as a clear oil (5.19 g, 17.5 mmol, 95%). H NMR
(270 MHz, CDCl3) δ 7.4-7.5 (m, 2H), 7.2-7.3 (m, 3H), 4.40
(d, J ) 10.2 Hz, 1H, H-1), 4.01 (m, 2H, H-3, H-4), 3.85 (qd, J
) 6.6 Hz, J ) 1.3 Hz, 1H, H-5), 3.52 (dd, J ) 10.2 Hz, J ) 6.3
Hz, 1H, H-2), 1.41 (d, J ) 6.6 Hz, 3H, H-6), 1.41 (s, 3H), 1.32
(s, 3H); 13C NMR (68 MHz, CDCl3) δ 132.7 (s), 132.2 (s), 129.0
(s), 128.1 (s), 109.9 (s), 87.9 (s), 79.1 (s), 76.4 (s), 72.9 (s), 71.4
(s), 28.2 (s), 26.4 (s), 17.0 (s); LRFAB m/e 296 (M+); HRFAB
calcd for C15H20O4S (M+) m/e 296.1082, measured m/e 296.1078.
Phenyl 2-O-Benzyl-1-thio-â-D-fucopyranoside (31). To
a solution of compound 30 (5.19 g, 17.5 mmol) in anhydrous
THF (30 mL), BnBr (3.1 mL, 26.3 mmol), NaH (2.1 g, 87.5
mmol), and catalytic amount of TBAI were added. The reaction
mixture was stirred overnight. The excess BnBr was quenched
by addition of MeOH (2 mL). Then the reaction mixture was
slowly poured into a solution of ice and EtOAc. The aqueous
layer was extracted with EtOAc. The combined organic layers
were washed with 1 N HCl(aq), water, saturated NaHCO3(aq)
,
and brine, and then dried over Na2SO4(s). After removal of
solvents, the crude product was redissolved in an aqueous
solution (30 mL) of HOAc/TFA/H2O (80/1/20). The reaction
mixture was stirred at 60 °C for 1 h, and then the solvent was
removed with a rotovap. Water was added to the oily crude
product and removed again. Purification of the crude product
with gradient column chromatography (hexanes/EtOAc )
100:0 to 50:50) afforded the product as a clear oil (4.89 g, 14.1
1
mmol, 81%). H NMR (270 MHz, CDCl3) δ 7.2-7.6 (m, 10H),
4.94 (d, J ) 11.2 Hz, 1H, PhCH2O), 4.68 (d, J ) 11.2 Hz, 1H,
PhCH2O), 4.59 (d, J ) 9.6 Hz, 1H, H-1), 3.6-3.7 (m, 3H), 3.53
(dd, J ) 8.9 Hz, J ) 8.9 Hz, 1H), 1.33 (d, J ) 6.6 Hz, 3H,
H-6); 13C NMR (68 MHz, CDCl3) δ 138.3 (s), 134.2 (s), 132.0
(s), 129.1 (s), 128.8 (s), 128.5 (s), 128.3 (s), 127.7 (s), 87.6 (s),
78.3 (s), 75.5 (s, 2 carbons), 74.7 (s), 71.9 (s), 16.8 (s); LRFAB
m/e 345 ([M - H]+); HRFAB calcd for C19H21O4S ([M - H]+)
m/e 345.1160, measured m/e 345.1145.

Optimization of Aminoglycosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6279
Removal of the solvent followed by purification with gradient
(d, J ) 6.3 Hz, 3H, H-6′′); 13C NMR (100 MHz, CDCl3) δ 139.1
(s), 138.5 (s), 128.6 (s), 128.56 (s), 128.1 (s), 128.0 (s), 127.8
(s), 127.7 (s), 99.3 (s), 98.2 (s), 86.0 (s), 80.6 (s), 79.9 (s), 75.8
(s), 74.6 (s), 73.6 (s), 72.7 (s), 71.8 (s), 71.5 (s), 71.3 (s), 66.0
(s), 63.3 (s), 59.5 (s), 59.3 (s), 51.5 (s), 39.1 (s), 32.6 (s), 21.0
(s); MALDI calcd for C32H40N12O9Na ([M + Na]+) m/e 759.2933,
measured m/e 759.2977.
column chromatography (hexanes/EtOAc ) 100:0 to 65:35)
1
afforded the product (0.71 g, 1.49 mmol, 85%). H NMR (400
MHz, CDCl3) δ 8.0-8.1 (m, 2H), 7.3-7.6 (m, 13H), 4.97 (d, J
) 10.1 Hz, 1H, PhCH2O), 4.93 (dd, J ) 9.5 Hz, J ) 9.6 Hz,
1H, H-4), 4.79 (d, J ) 10.1 Hz, 1H, PhCH2O), 4.72 (d, J ) 9.7
Hz, 1H, H-1), 3.76 (dd, J ) 9.7 Hz, J ) 9.4 Hz, 1H, H-2), 3.65
(dq, J ) 9.6 Hz, J ) 6.2 Hz, 1H, H-5), 3.48 (dd, J ) 9.4 Hz, J
) 9.5 Hz, 1H, H-3), 1.30 (d, J ) 6.2 Hz, 3H, H-6); 13C NMR
(100 MHz, CDCl3) δ 165.2 (s), 137.5 (s), 133.7 (s), 132.5 (s),
130.0 (s), 129.44 (s), 129.26 (s), 128.80 (s), 128.75 (s), 128.68
(s), 128.4 (s), 128.1 (s), 88.0 (s), 79.7 (s), 75.6 (s), 75.2 (s), 73.7
(s), 68.6 (s), 18.0 (s); LRFAB m/e 498 ([M + Na]+); HRFAB
calcd for C26H25N3O4SNa ([M + Na]+) m/e 498.1463, measured
m/e 498.1453.
6-O-(2,3,4-Tri-O-benzyl-6-deoxy-r-D-glucopyranosyl)-
1,3,2′,6′-tetraazidoneamine (39). Refer to the general pro-
1
cedure for glycosylation and hydrolysis. H NMR (270 MHz,
CDCl3) δ 7.2-7.4 (m, 15H), 5.65 (d, J ) 3.6 Hz, 1H, H-1′), 4.96
(d, J ) 10.9 Hz, 1H, PhCH2O), 4.94 (d, J ) 4.3 Hz, 1H, H-1′′),
4.89 (d, J ) 10.9 Hz, 1H, PhCH2O), 4.78 (d, J ) 10.9 Hz, 1H,
PhCH2O), 4.76 (d, J ) 11.9 Hz, 1H, PhCH2O), 4.70 (d, J )
11.9 Hz, 1H, PhCH2O), 4.61 (d, J ) 10.9 Hz, 1H, PhCH2O),
3.9-4.1 (m, 3H), 3.1-3.7 (m, 12H), 2.30 (ddd, J ) 12.9 Hz, J
) 4.0 Hz, J ) 4.0 Hz, 1H, H-2eq), 1.48 (ddd, J ) 12.9 Hz, J )
12.5 Hz, J ) 12.5 Hz, 1H, H-2ax), 1.25 (d, J ) 5.9 Hz, 3H, H-6′′);
13C NMR (100 MHz, CDCl3) δ 138.9 (s), 138.2 (s, two carbons),
128.7 (s), 128.6 (s), 128.24 (s), 128.23 (s), 128.19 (s), 128.14
(s), 98.6 (s), 98.3 (s), 86.3 (s), 83.4 (s), 81.2 (s), 80.1 (s), 79.9
(s), 76.1 (s), 75.9 (s), 75.7 (s), 73.6 (s), 71.8 (s), 71.5 (s), 71.3
(s), 68.7 (s), 63.3 (s), 59.6 (s), 59.2 (s), 51.5 (s), 32.5 (s), 18.0
(s); MALDI calcd for C39H46N12O10Na ([M + Na]+) m/e 865.3352,
measured m/e 865.3340.
General Procedure for Glycosylation and Hydrolysis.
A solution of glycosyl donor, neamine derivative (1.2 equiv),
and activated powder 4 Å molecular sieves was stirred in
anhydrous Et2O and CH2Cl2 (Et2O, 4.5 mL; CH2Cl2, 1.5 mL)
at room temperature overnight. N-Iodosuccinimide (1.2 equiv)
was quickly added into the above solution, and the reaction
mixture was cooled to -70 °C. After the solution was warmed
to -40 °C, trifluoromethanesulfonic acid (0.15 equiv) was
added. The solution was stirred at low temperature till the
complete consumption of the glycosyl donor (∼4 h, monitored
by TLC, hexane/EtOAc ) 65: 35). The reaction was quenched
by the addition of triethylamine (3 mL). After being stirred
for 10 min, the reaction mixture was filtered through Celite
and the solvent was removed. The crude product was extracted
with EtOAc, washed with 10% aqueous Na2S2O3, saturated
NaHCO3(aq), and brine, and dried over Na2SO4(s). After removal
of the solvents, the crude product was purified by column
chromatography. The glycosylated compounds were often
mixed with inseparable impurities and were fully character-
ized after hydrolysis. The glycosylated product was dissolved
in tetrahydrofuran (1 mL) and methanol (5 mL), and sodium
methoxide (0.5 M in methanol, 1 mL) was added. The reaction
mixture was stirred at room temperature till the completion
of the reaction (∼2 h, monitored by TLC, EtOAc/hexane ) 50:
50). The reaction mixture was neutralized with Amberlite IR-
120 (H+) and filtered through Celite, and the solvent was
removed. The residue was purified via column chromatography
to provide the product as a colorless oil.
6-O-(2,3,4-Tri-O-benzyl-r-D-xylopyranosyl)-1,3,2′,6′-tet-
raazidoneamine (40). Refer to the general procedure for
glycosylation and hydrolysis. 1H NMR (270 MHz, CDCl3) δ
7.2-7.4 (m, 15H), 5.60 (d, J ) 3.6 Hz, 1H, H-1′), 5.01 (d, J )
3.3 Hz, 1H, H-1′′), 4.86 (d, J ) 11.2 Hz, 1H, PhCH2O), 4.80 (d,
J ) 11.2 Hz, 1H, PhCH2O), 4.7-4.8 (m, 3H, PhCH2O), 4.61
(d, J ) 11.5 Hz, 1H, PhCH2O), 3.8-4.2 (m, 4H), 3.2-3.7 (m,
12H), 2.31 (ddd, J ) 13.0 Hz, J ) 4.3 Hz, J ) 4.3 Hz, 1H,
H-2eq), 1.49 (ddd, J ) 13.0 Hz, J ) 12.6 Hz, J ) 12.6 Hz, 1H,
H-2ax); 13C NMR (100 MHz, CDCl3) δ 138.9 (s), 138.18 (s),
138.15 (s), 128.72 (s), 128.69 (s), 128.63 (s), 128.59 (s), 128.26
(s), 128.24 (s), 128.17 (s), 128.13 (s), 128.0 (s), 98.50 (s), 98.43
(s), 84.8 (s), 80.4 (s), 80.2 (s), 79.1 (s), 77.4 (s), 75.6 (s, two
carbons), 73.9 (s), 73.8 (s), 72.0 (s), 71.6 (s), 71.3 (s), 63.3 (s),
61.7 (s), 59.7 (s), 59.2 (s), 51.5 (s), 32.6 (s); MALDI calcd for
C38H44N12O10Na ([M + Na]+) m/e 851.3196, measured m/e
851.3158.
6-O-(3-Azido-2,6-di-O-benzyl-3-deoxy-r-D-galactopyra-
nosyl)-1,3,2′,6′-tetraazidoneamine (41). Refer to the general
procedure for glycosylation and hydrolysis. 1H NMR (400 MHz,
CDCl3) δ 7.3-7.4 (m, 10H), 5.47 (d, J ) 3.7 Hz, 1H, H-1′), 5.25
(d, J ) 2.9 Hz, 1H, H-1′′), 4.74 (s, 2H, PhCH2O), 4.61 (d, J )
12.1 Hz, 1H, PhCH2O), 4.57 (d, J ) 12.1 Hz, 1H, PhCH2O),
4.27 (dd, J ) 5.0 Hz, J ) 5.0 Hz, 1H), 3.9-4.1 (m, 4H), 3.3-
3.5 (m, 11H), 3.23 (dd, J ) 10.3 Hz, J ) 3.7 Hz, 1H), 2.33 (ddd,
J ) 12.9 Hz, J ) 4.2 Hz, J ) 4.2 Hz, 1H, H-2eq), 1.54 (ddd, J
) 12.9 Hz, J ) 12.8 Hz, J ) 12.8 Hz, 1H, H-2ax); 13C NMR
(100 MHz, CDCl3) δ 137.6 (s), 137.5 (s), 128.7 (s), 128.4 (s),
128.2 (s), 128.1 (s), 98.7 (s), 97.7 (s), 83.9 (s), 81.0 (s), 75.7 (s),
75.2 (s), 74.0 (s), 73.3 (s), 72.3 (s), 71.6 (s), 71.4 (s), 69.9 (s),
69.6 (s, two carbons), 63.4 (s), 61.5 (s), 59.8 (s), 59.3 (s), 51.4
(s), 32.3 (s); MALDI calcd for C32H39N15O10Na ([M + Na]+) m/e
816.2897, measured m/e 816.2889.
6-O-(2,3,4-Tri-O-benzyl-r-D-fucopyranosyl)-1,3,2′,6′-tet-
raazidoneamine (42). Refer to the general procedure for
glycosylation and hydrolysis. 1H NMR (270 MHz, CDCl3) δ
7.2-7.4 (m, 15H), 5.67 (d, J ) 3.9 Hz, 1H, H-1′), 5.01 (d, J )
4.6 Hz, 1H, H-1′′), 4.98 (d, J ) 11.2 Hz, 1H, PhCH2O), 4.86 (d,
J ) 12.2 Hz, 1H, PhCH2O), 4.80 (d, J ) 11.9 Hz, 1H, PhCH2O),
4.72 (d, J ) 12.2 Hz, 1H, PhCH2O), 4.71 (d, J ) 11.9 Hz, 1H,
PhCH2O), 4.63 (d, J ) 11.2 Hz, 1H, PhCH2O), 4.1-4.2 (m, 3H),
3.9-4.0 (m, 2H), 3.3-3.6 (m, 8H), 3.2-3.3 (m, 2H), 2.29 (ddd,
J ) 12.9 Hz, J ) 4.6 Hz, J ) 4.6 Hz, 1H, H-2eq), 1.48 (ddd, J
) 12.9 Hz, J ) 12.9 Hz, J ) 12.9 Hz, 1H, H-2ax), 1.11 (d, J )
6.6 Hz, 3H, H-6′′); 13C NMR (100 MHz, CDCl3) δ 139.2 (s),
138.57 (s), 138.55 (s), 128.6 (s), 128.5 (s), 128.2 (s), 127.9 (s),
127.7 (s), 99.1 (s), 98.1 (s), 86.1 (s), 79.8 (s), 78.7 (s), 77.9 (s),
76.1 (s), 75.9 (s), 75.1 (s), 73.8 (s), 73.6 (s), 71.8 (s), 71.6 (s),
71.2 (s), 68.6 (s), 63.4 (s), 59.5 (s), 59.3 (s), 51.5 (s), 32.6 (s),
6-O-(2,3,4,6-Tetra-O-benzyl-r-D-galactopyranosyl)-
1,3,2′,6′-tetraazidoneamine (37). Refer to the general pro-
1
cedure for glycosylation and hydrolysis. H NMR (270 MHz,
CDCl3) δ 7.2-7.4 (m, 20H), 5.56 (d, J ) 3.3 Hz, 1H, H-1′), 5.11
(d, J ) 3.3 Hz, 1H, H-1′′), 4.91 (d, J ) 11.6 Hz, 1H, PhCH2O),
4.84 (d, J ) 12.5 Hz, 1H, PhCH2O), 4.80 (d, J ) 12.2 Hz, 1H,
PhCH2O), 4.72 (d, J ) 12.2 Hz, 1H, PhCH2O), 4.71 (d, J )
11.5 Hz, 1H, PhCH2O), 4.53 (d, J ) 12.5 Hz, 1H, PhCH2O),
4.51 (d, J ) 12.2 Hz, 1H, PhCH2O), 4.39 (d, J ) 12.2 Hz, 1H,
PhCH2O), 4.1-4.2 (m, 3H), 3.8-3.9 (m, 3H), 3.2-3.7 (m, 10H),
3.09 (dd, J ) 10.6 Hz, J ) 3.6 Hz, 1H), 2.28 (ddd, J ) 13.0 Hz,
J ) 4.3 Hz, J ) 4.3 Hz, 1H, H-2eq) 1.47 (ddd, J ) 13.0 Hz, J
) 12.2 Hz, J ) 12.2 Hz, 1H, H-2ax); 13C NMR (100 MHz, CDCl3)
δ 139.0 (s), 138.56 (s), 138.54 (s), 137.9 (s), 128.60 (s), 128.56
(s), 128.48 (s), 128.2 (s), 128.0 (s), 127.9 (s), 127.8 (s), 99.3 (s),
98.3 (s), 85.8 (s), 79.9 (s), 78.6 (s), 76.6 (s), 76.0 (s), 75.3 (s),
74.8 (s), 73.8 (s), 73.7 (s), 73.6 (s), 71.8 (s), 71.7 (s), 71.2 (s),
71.1 (s), 69.3 (s), 63.1 (s), 59.8 (s), 59.4 (s), 51.4 (s), 32.5 (s);
MALDI calcd for C46H52N12O11Na ([M + Na]+) m/e 971.3771,
measured m/e 971.3808.
6-O-(2,3-Di-O-benzyl-4,6-dideoxy-r-D-xylo-hexopyrano-
syl)-1,3,2′,6′-tetraazidoneamine (38). Refer to the general
procedure for glycosylation and hydrolysis. 1H NMR (270 MHz,
CDCl3) δ 7.3-7.4 (m, 10H), 5.66 (d, J ) 3.9 Hz, 1H, H-1′), 5.00
(d, J ) 3.6 Hz, 1H, H-1′′), 4.81 (d, J ) 11.9 Hz, 1H, PhCH2O),
4.75 (d, J ) 11.9 Hz, 1H, PhCH2O), 4.72 (d, J ) 11.9 Hz, 1H,
PhCH2O), 4.66 (d, J ) 11.9 Hz, 1H, PhCH2O), 4.1-4.2 (m, 2H),
3.8-3.9 (m, 2H), 3.2-3.7 (m, 10H), 2.29 (ddd, J ) 13.5 Hz, J
) 3.9 Hz, J ) 3.9 Hz, 1H, H-2eq), 2.07 (m, 1H, H-4′′eq), 1.49
(ddd, J ) 13.5 Hz, J ) 12.9 Hz, J ) 12.9 Hz, 1H, H-2ax), 1.39
(ddd, J ) 13.2 Hz, J ) 10.9 Hz, J ) 10.9 Hz, 1H, H-4′′ax), 1.17

6280 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Wang et al.
16.7 (s); MALDI calcd for C39H46N12O10Na ([M + Na]+) m/e
865.3352, measured m/e 865.3317.
(d, J ) 3.6 Hz, 1H, H-1′), 5.01 (d, J ) 3.3 Hz, 1H, H-1′′), 4.23
(m, 1H), 4.0-4.1 (m, 4H), 3.85 (dd, J ) 9.2 Hz, J ) 8.9 Hz,
1H), 3.71 (dd, J ) 10.2 Hz, J ) 8.6 Hz, 1H), 3.4-3.6 (m, 6H),
3.29 (dd, J ) 13.5 Hz, J ) 6.9 Hz, 1H), 2.53 (d, J ) 12.5 Hz,
1H, H-2eq), 2.04 (m, 1H, H-4′′eq), 1.96 (ddd, J ) 12.5 Hz, J )
12.2 Hz, J ) 12.2 Hz, 1H, H-2ax), 1.37 (ddd, J ) 12.5 Hz, J )
11.9 Hz, J ) 11.9 Hz, 1H, H-4′′ax), 1.18 (d, J ) 6.4 Hz, 1H,
H-6′′); 13C NMR (100 MHz, D2O) (chloride salt) δ 102.8 (s),
96.1 (s), 83.7 (s), 77.7 (s), 74.3 (s), 73.7 (s), 70.9 (s), 69.5 (s),
68.4 (s), 67.2 (s), 66.7 (s), 53.7 (s), 50.1 (s), 48.6 (s), 40.4 (s),
39.7 (s), 28.2 (s), 20.1 (s); LRFAB m/e 453 ([M + H]+); HRFAB
calcd for C18H37N4O9 ([M + H]+) m/e 453.2561, measured m/e
453.2580.
6-O-(3-Azido-2-O-benzyl-3,6-dideoxy-r-D-glucopyrano-
syl)-1,3,2′,6′-tetraazidoneamine (43). Refer to the general
procedure for glycosylation and hydrolysis. 1H NMR (270 MHz,
CDCl3) δ 7.3-7.4 (m, 5H), 5.57 (d, J ) 3.6 Hz, 1H, H-1′), 5.01
(d, J ) 3.6 Hz, 1H, H-1′′), 4.72 (s, 2H, PhCH2O), 4.12 (m, 1H),
3.9-4.0 (m, 2H), 3.79 (dd, J ) 9.9 Hz, J ) 9.9 Hz, 1H), 3.2-
3.7 (m, 10H), 3.02 (dd, J ) 9.6 Hz, J ) 9.6 Hz, 1H), 2.33 (ddd,
J ) 13.0 Hz, J ) 4.3 Hz, J ) 4.3 Hz, 1H, H-2eq), 1.51 (ddd, J
) 13.0 Hz, J ) 12.5 Hz, J ) 12.5 Hz, 1H, H-2ax), 1.25 (d, J )
5.6 Hz, 3H, H-6′′); 13C NMR (100 MHz, CDCl3) δ 137.4 (s),
128.8 (s), 128.4 (s), 128.3 (s), 98.5 (s), 97.5 (s), 85.3 (s), 80.6
(s), 78.3 (s), 75.9 (s), 74.1 (s), 73.3 (s), 72.1 (s), 71.6 (s), 71.3
(s), 68.8 (s), 65.0 (s), 63.5 (s), 59.6 (s), 59.2 (s), 51.5 (s), 32.4
(s), 17.7 (s); MALDI calcd for C25H33N15O9Na ([M + Na]+) m/e
710.2478, measured m/e 710.2485.
6-O-(2,3,4-Tri-O-benzyl-r-L-fucopyranosyl)-1,3,2′,6′-tet-
raazidoneamine (44). Refer to the general procedure for
glycosylation and hydrolysis. 1H NMR (270 MHz, CDCl3) δ
7.2-7.4 (m, 15H), 5.67 (d, J ) 4.0 Hz, 1H, H-1′), 5.01 (d, J )
3.6 Hz, 1H, H-1′′), 4.94 (d, J ) 11.0 Hz, 1H, PhCH2O), 4.92 (d,
J ) 11.2 Hz, 1H, PhCH2O), 4.76 (s, 2H, PhCH2O), 4.74 (d, J )
11.0 Hz, 1H, PhCH2O), 4.64 (d, J ) 11.2 Hz, 1H, PhCH2O),
3.9-4.2 (m, 4H), 3.5-3.8 (m, 7H), 3.92-3.4 (m, 4H), 2.32 (ddd,
J ) 13.2 Hz, J ) 4.0 Hz, J ) 4.0 Hz, 1H, H-2eq), 1.51 (ddd, J
) 13.2 Hz, J ) 11.9 Hz, J ) 11.9 Hz, 1H, H-2ax), 1.16 (d, J )
6.3 Hz, 3H, H-6′′); 13C NMR (100 MHz, CDCl3) δ 138.66 (s),
138.55 (s), 137.4 (s), 128.9 (s), 128.7 (s), 128.44 (s), 128.38 (s),
127.90 (s), 127.87 (s), 127.6 (s), 102.0 (s), 97.7 (s), 85.0 (s), 80.2
(s), 78.6 (s), 77.5 (s), 76.9 (s), 76.0 (s), 75.1 (s), 75.0 (s), 72.7
(s), 71.7 (s), 71.6 (s), 71.2 (s), 67.9 (s), 63.2 (s), 59.5 (s,
two carbons), 51.5 (s), 32.7 (s), 16.8 (s); MALDI calcd for
C39H46N12O10Na ([M + Na]+) m/e 865.3352, measured m/e
865.3322.
6-O-(6-Deoxy-r-D-glucopyranosyl)neamine (18). Refer
to the general procedure for the synthesis of kanamycin B
1
analogues. H NMR (400 MHz, D2O) (chloride salt) δ 5.95 (d,
J ) 3.6 Hz, 1H, H-1′), 4.98 (s, 1H, H-1′′), 3.9-4.0 (m, 4H), 3.84
(dd, J ) 9.2 Hz, J ) 9.0 Hz, 1H), 3.71 (dd, J ) 9.8 Hz, J ) 9.2
Hz, 1H), 3.65 (m, 1H), 3.4-3.5 (m, 6H), 3.27 (dd, J ) 13.4 Hz,
J ) 7.1 Hz, 1H), 3.15 (dd, J ) 8.6 Hz, J ) 8.2 Hz, 1H), 2.49
(m, 1H, H-2eq), 1.91 (ddd, J ) 12.8 Hz, J ) 12.3 Hz, J ) 12.3
Hz, 1H, H-2ax), 1.22 (d, J ) 6.1 Hz, 3H, H-6′′); 13C NMR (100
MHz, D2O) (chloride salt) δ 102.0 (s), 96.0 (s), 83.9 (s), 78.0
(s), 74.9 (s), 74.4 (s), 72.8 (s), 72.2 (s), 71.0 (s), 69.4 (s), 69.2
(s), 68.6 (s), 53.8 (s), 50.2 (s), 48.6 (s), 40.4 (s), 28.5 (s), 17.0
(s); LRFAB m/e 469 ([M + H]+); HRFAB calcd for C18H37N4O10
([M + H]+) m/e 469.2510, measured m/e 469.2512.
6-O-(r-D-Xylopyranosyl)neamine (19). Refer to the gen-
eral procedure for the synthesis of kanamycin B analogues.
1H NMR (270 MHz, D2O) (chloride salt) δ 5.95 (s, 1H, H-1′),
5.01 (s, 1H, H-1′′), 3.9-4.0 (m, 3H), 3.86 (dd, J ) 8.6 Hz, J )
8.6 Hz, 1H), 3.4-3.8 (m, 11H), 3.28 (dd, J ) 13.2 Hz, J ) 6.9
Hz, 1H), 2.53 (d, J ) 12.2 Hz, 1H, H-2eq), 1.91 (m,1H, H-2ax);
13C NMR (100 MHz, D2O) (chloride salt) δ 102.1 (s), 96.2 (s),
83.9 (s), 77.8 (s), 74.4 (s), 73.1 (s), 71.9 (s), 70.9 (s), 69.4 (s),
69.1 (s), 68.5 (s), 62.7 (s), 53.7 (s), 50.0 (s), 48.5 (s), 40.4 (s),
28.2 (s); LRFAB m/e 455 ([M + H]+); HRFAB calcd for
C17H35N4O10 ([M + H]+) m/e 455.2353, measured m/e 455.2337.
General Procedure for the Synthesis of Kanamycin
B Analogues. To a starting material/THF solution in a
reaction vial equipped with a reflux condenser, 0.1 M NaOH(aq)
(0.5 mL) and PMe3 (1 M in THF, 5-7 equiv) were added. The
reaction mixture was stirred at 50 °C for 2 h. The product has
an Rf of 0 when eluted with an EtOAc/MeOH (9/1) solution
and has an Rf of 0.6 when eluted with i-PrOH/1 M NH4OAc
(2/1) solution. After completion of the reaction, the solvents
were removed, and the crude benzylated aminoglycoside was
added with a catalytic amount of Pd(OH)2/C (20% Degussa
type) and 5 mL of degassed HOAc/H2O (1/3). After being fur-
ther degassed, the reaction mixture was stirred at room
temperature under atmospheric H2 pressure. After being stir-
red for 1 day, the reaction mixture was filtered through Celite.
The residue was washed with water, and the combined solu-
tions were concentrated. The crude product was purified with
Amberlite CG50(NH4+) and was eluted with a gradient of NH4-
OH solution (0-20%). The final product with Cl- salt can be
prepared with an ion-exchange column packed with Dowex
1X8-200 (Cl- form) and eluting with water. After collection of
the desired fractions and removal of solvent, the final products
are subjected to a bioassay directly. The reported final products
6-O-(3-Amino-3-deoxy-r-D-galactopyranosyl)neamine
(20). Refer to the general procedure for the synthesis of
1
kanamycin B analogues. H NMR (400 MHz, D2O) (chloride
salt) δ 5.90 (d, J ) 3.6 Hz, 1H, H-1′), 5.14 (d, J ) 3.6 Hz, 1H,
H-1′′), 4.0-4.2 (m, 7H), 3.92 (dd, J ) 8.9 Hz, J ) 8.9 Hz, 1H),
3.82 (dd, J ) 9.9 Hz, J ) 8.6 Hz, 1H), 3.4-3.7 (m, 7H), 3.26
(dd, J ) 13.9 Hz, J ) 6.9 Hz, 1H), 2.53 (ddd, J ) 12.5 Hz, J )
4.3 Hz, J ) 4.3 Hz, 1H, H-2eq), 1.96 (ddd, J ) 12.5 Hz, J )
12.5 Hz, J ) 12.5 Hz, 1H, H-2ax); 13C NMR (100 MHz, D2O)
(chloride salt) δ 100.8 (s), 96.4 (s), 83.9 (s), 77.9 (s), 74.5 (s),
72.0 (s), 70.9 (s), 69.5 (s), 68.4 (s), 65.6 (s), 65.5 (s), 60.9 (s),
53.8 (s), 52.1 (s), 49.7 (s), 48.5 (s), 40.4 (s), 28.0 (s); LRFAB
m/e 484 ([M + H]+); HRFAB calcd for C18H38N5O10 ([M + H]+)
m/e 484.2619, measured m/e 484.2596.
6-O-(r-D-Fucopyranosyl)neamine (21). Refer to the gen-
eral procedure for the synthesis of kanamycin B analogues.
1H NMR (270 MHz, D2O) (chloride salt) δ 5.96 (d, J ) 3.3 Hz,
1H, H-1′), 5.00 (s, 1H, H-1′′), 4.26 (q, J ) 6.6 Hz, 1H, H-5′′),
3.8-4.1 (m, 7H), 3.71 (dd, J ) 9.9 Hz, J ) 8.9 Hz, 1H), 3.4-
3.6 (m, 5H), 3.28 (dd, J ) 13.5 Hz, J ) 6.9 Hz, 1H), 2.52 (m,
1H, H-2eq), 1.94 (ddd, J ) 12.5 Hz, J ) 12.5 Hz, J ) 12.5 Hz,
1H, H-2ax), 1.18 (d, J ) 6.6 Hz, 3H, H-6′′); 13C NMR (100 MHz,
D2O) (chloride salt) δ 102.3 (s), 96.1 (s), 83.6 (s), 77.7 (s), 74.3
(s), 71.9 (s), 70.9 (s), 69.59 (s), 69.47 (s), 68.66 (s), 68.45 (s),
68.3 (s), 53.7 (s), 50.1 (s), 48.6 (s), 40.4 (s), 28.2 (s), 15.7 (s);
LRFAB m/e 469 ([M + H]+); HRFAB calcd for C18H37N4O10 ([M
+ H]+) m/e 469.2510, measured m/e 469.2533.
1
are characterized by H and 13C NMR at this stage.
6-O-(r-D-Galactopyranosyl)neamine (16). Refer to the
general procedure for the synthesis of kanamycin B analogues.
1H NMR (270 MHz, D2O) (chloride salt) δ 5.91 (d, J ) 3.3 Hz,
1H, H-1′), 5.10 (d, J ) 3.3 Hz, 1H, H-1′′), 4.15 (J ) 6.3 Hz, J
) 5.9 Hz, 1H), 3.9-4.1 (m, 7H), 3.7-3.8 (m, 3H), 3.4-3.6 (m,
5H), 3.26 (dd, J ) 13.5 Hz, J ) 7.3 Hz, 1H), 2.52 (d, J ) 11.9
Hz, 1H, H-2eq), 1.96 (ddd, J ) 11.9 Hz, J ) 12.9 Hz, J ) 12.9
Hz, 1H, H-2ax); 13C NMR (100 MHz, D2O) (chloride salt) δ 101.7
(s), 96.3 (s), 83.9 (s), 78.0 (s), 74.4 (s), 72.7 (s), 71.0 (s), 69.44
(s), 69.40 (s), 69.28 (s), 68.8 (s), 68.5 (s), 61.4 (s), 53.8 (s), 49.8-
(s), 48.5 (s), 40.5 (s), 28.3 (s); LRFAB m/e 485 ([M + H]+);
HRFAB calcd for C18H37N4O11 ([M + H]+) m/e 485.2459,
measured m/e 485.2467.
6-O-(3-Amino-3,6-dideoxy-r-D-glucopyranosyl)neam-
ine (22). Refer to the general procedure for the synthesis of
1
kanamycin B analogues. H NMR (270 MHz, D2O) (chloride
salt) δ 6.01 (d, J ) 3.6 Hz, 1H, H-1′), 5.05 (d, J ) 3.3 Hz, 1H,
H-1′′), 3.9-4.1 (m, 6H), 3.81 (dd, J ) 9.6 Hz, J ) 8.9 Hz, 1H),
3.4-3.7 (m, 7H), 3.30 (dd, J ) 13.9 Hz, J ) 6.6 Hz, 1H), 2.55
(ddd, J ) 12.6 Hz, J ) 4.0 Hz, J ) 4.0 Hz, 1H, H-2eq), 1.98
(ddd, J ) 12.6 Hz, J ) 12.5 Hz, J ) 12.5 Hz, 1H, H-2ax), 1.28
(d, J ) 6.3 Hz, 3H, H-6′′); 13C NMR (100 MHz, D2O) (chloride
6-O-(4,6-Dideoxy-r-D-xylo-hexopyranosyl)neamine (17).
Refer to the general procedure for the synthesis of kanamycin
1
B analogues. H NMR (270 MHz, D2O) (chloride salt) δ 5.95

Optimization of Aminoglycosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6281
salt) δ 101.0 (s), 96.0 (s), 83.9 (s), 77.5 (s), 74.5 (s), 71.0 (s),
70.9 (s), 69.5 (s), 69.4 (s), 68.6 (s), 68.5 (s), 55.0 (s), 53.7(s),
50.0 (s), 48.6 (s), 40.4 (s), 28.1 (s), 16.7 (s); LRFAB m/e 468
([M + H]+); HRFAB calcd for C18H38N5O9 ([M + H]+) m/e
468.2670, measured m/e 468.2677.
0.30 mmol), Et3N (0.10 mL, 0.75 mmol), and DMAP (catalytic
amount) in anhydrous CH2Cl2 (5 mL), TsCl (0.12 g, 0.61 mmol)
was added slowly at 0 °C. The reaction mixture was stirred
overnight and allowed to warm to room temperature. After
the completion of the reaction, the reaction mixture was
diluted with EtOAc. The combined organic layers were washed
with 1 N HCl, saturated NaHCO3(aq), and brine, and then dried
over Na2SO4(s). After removal of solvent, the tosylated crude
product was dissolved in anhydrous DMF (5 mL), and NaN3
(0.20 g, 3.0 mmol) was added. The reaction mixture was stirred
at 0 °C overnight. After removal of the solvent, the residue
was diluted with EtOAc and filtered through Celite. Removal
of the solvent followed by purification with gradient column
chromatography (hexanes/EtOAc ) 100:0 to 65:35) afforded
the product (0.13 g, 0.25 mmol, 83%). 1H NMR (400 MHz,
CDCl3) δ 7.5-7.6 (m, 2H), 7.3-7.4 (m, 13H), 4.91 (d, J ) 10.3
Hz, 1H, PhCH2O), 4.90 (d, J ) 11.0 Hz, 1H, PhCH2O), 4.79
(d, J ) 11.0 Hz, 1H, PhCH2O), 4.73 (d, J ) 10.3 Hz, 1H,
PhCH2O), 4.67 (d, J ) 9.6 Hz, 1H, H-1), 3.87 (dt, J ) 9.3 Hz,
J ) 5.8 Hz, 1H), 3.67 (m, 1H), 3.58 (dd, J ) 8.9 Hz, J ) 8.9
Hz, 1H), 3.46 (dd, J ) 9.6 Hz, J ) 9.0 Hz, 1H, H-2), 3.37 (m,
1H), 3.33 (t, J ) 6.4 Hz, 2H), 3.02 (dd, J ) 9.2 Hz, J ) 9.2 Hz,
1H), 1.81 (m, 2H), 1.36 (d, J ) 6.2 Hz, 3H, H-6); 13C NMR
(100 MHz, CDCl3) δ 138.6 (s), 138.3 (s), 134.1 (s), 132.1 (s),
129.1 (s), 128.7 (s), 128.6 (s), 128.4 (s), 128.0 (s), 127.9 (s), 127.7
(s), 87.7 (s), 86.6 (s), 84.0(s), 81.5 (s), 75.88 (s), 75.76 (s), 75.62
(s), 70.1 (s), 48.6 (s), 29.9 (s), 18.4 (s); MALDI calcd for
C29H33N3O4SNa ([M + Na]+) m/e 542.2084, measured m/e
542.2066.
Phenyl 2,3-Di-O-benzyl-6-deoxy-4-O-((R)-glycidyl)-1-
thio-â-D-glucopyranoside (61). To a solution of 566 (0.85 g,
1.95 mmol), NaH (0.31 g, 7.80 mmol, 60% dispersion in mineral
oil), and a couple of drops of DMF in anhydrous THF, 2R-(-
)-glycidyl tosylate (1.11 g, 4.87 mmol) was added. After the
mixture was stirred for 24 h, the reaction was quenched by
addition of NH4Cl (saturated) and the reaction mixture was
diluted with EtOAc. The combined organic layers were washed
with brine and dried over Na2SO4(s). Removal of the solvent
followed by purification with gradient column chromatography
(hexanes/EtOAc ) 100:0 to 70:30) afforded the product (0.83
g, 1.68 mmol, 86%). 1H NMR (400 MHz, CDCl3) δ 7.3-7.6 (m,
15H), 4.92 (d, J ) 10.3 Hz, 1H, PhCH2O), 4.91 (d, J ) 11.0
Hz, 1H, PhCH2O), 4.84 (d, J ) 11.0 Hz, 1H, PhCH2O), 4.74
(d, J ) 10.3 Hz, 1H, PhCH2O), 4.66 (d, J ) 9.8 Hz, 1H, H-1),
4.05 (dd, J ) 11.3 Hz, J ) 3.0 Hz, 1H), 3.62 (dd, J ) 8.9 Hz,
J ) 9.0 Hz, 1H), 3.54 (dd, J ) 11.3 Hz, J ) 6.6 Hz, 1H), 3.47
(dd, J ) 9.8 Hz, J ) 9.0 Hz, 1H, H-2), 3.40 (dq, J ) 9.4 Hz, J
) 6.2 Hz, 1H), 3.11 (dd, J ) 9.2 Hz, J ) 9.4 Hz, 1H), 3.09 (m,
1H), 3.76 (dd, J ) 5.1 Hz, J ) 4.4 Hz, 1H), 2.53 (dd, J ) 5.1
Hz, J ) 2.7 Hz, 1H), 1.40 (d, J ) 6.2 Hz, 3H, H-6); 13C NMR
(100 MHz, CDCl3) δ 138.9 (s), 138.7 (s), 134.1 (s), 132.1 (s),
131.9 (s), 129.1 (s), 128.64 (s), 128.61 (s), 128.4 (s), 128.3 (s),
128.11 (s), 128.0 (s), 127.9 (s), 127.7 (s), 87.7 (s), 86.6 (s), 84.3
(s), 81.5 (s), 75.9 (s), 75.7 (s), 75.6 (s), 74.6 (s), 51.1 (s), 44.5
(s), 18.3 (s); MALDI calcd for C29H32O5SNa ([M + Na]+) m/e
515.1863, measured m/e 515.1854.
6-O-(r-L-Fucopyranosyl)neamine (23). Refer to the gen-
eral procedure for the synthesis of kanamycin B analogues.
1H NMR (270 MHz, D2O) (chloride salt) δ 5.92 (d, J ) 3.6 Hz,
1H, H-1′), 5.27 (s, 1H, H-1′′), 4.23 (m, 1H), 3.8-4.1 (m, 8H),
3.4-3.6 (m, 5H), 3.28 (dd, J ) 13.5 Hz, J ) 6.9 Hz, 1H), 2.58
(ddd, J ) 12.9 Hz, J ) 4.6 Hz, J ) 4.6 Hz, 1H, H-2eq), 1.97
(ddd, J ) 12.9 Hz, J ) 12.5 Hz, J ) 12.5 Hz, 1H, H-2ax), 1.21
(d, J ) 6.6 Hz, 3H, H-6′′); 13C NMR (100 MHz, D2O) (chloride
salt) δ 100.4 (s), 96.3 (s), 81.8 (s), 77.8 (s), 74.8 (s), 71.8 (s),
71.0 (s), 69.43 (s), 69.36 (s), 68.48 (s), 68.32 (s), 68.18 (s), 53.8
(s), 48.63 (s), 48.46 (s), 40.4 (s), 28.4 (s), 15.8 (s); LRFAB m/e
469 ([M + H]+); HRFAB calcd for C18H37N4O10 ([M + H]+) m/e
469.2510, measured m/e 469.2485.
Phenyl 4-O-Allyl-2,3-di-O-benzyl-6-deoxy-1-thio-â-D-
glucopyranoside (57). To a solution of 566 (0.4 g, 0.92 mmol),
NaH (0.07 g, 1.83 mmol, 60% dispersion in mineral oil), and a
catalytic amount of TBAI in anhydrous THF, allyl bromide
(0.44 mL, 5.09 mmol) was added slowly at 0 °C. After the
mixture was stirred for 24 h, the excess allyl bromide was
quenched by addition of MeOH (1.0 mL). After removal of
solvent, the reaction mixture was diluted with EtOAc, washed
with 1 N HCl, water, saturated NaHCO3(aq), and brine, and
dried over Na2SO4(s). After removal of the solvent followed by
purification with gradient column chromatography (hexanes/
EtOAc )100:0 to 65:35), the product was obtained as a light-
1
yellowish solid (0.32 g, 0.67 mmol, 73%). H NMR (270 MHz,
CDCl3) δ 7.2-7.6 (m, 15H), 5.90 (ddd, J ) 17.5 Hz, J ) 10.2
Hz, J ) 5.9 Hz, 1H), 5.25 (d, J ) 17.5 Hz, 1H), 5.16 (d, J )
10.2 Hz, 1H), 4.88 (d, J ) 10.6 Hz, 1H, PhCH2O), 4.86 (d, J )
10.6 Hz, 1H, PhCH2O), 4.81 (d, J ) 10.6 Hz, 1H, PhCH2O),
4.73 (d, J ) 10.6 Hz, 1H, PhCH2O), 4.64 (d, J ) 9.6 Hz, 1H,
H-1), 4.32 (dd, J ) 12.2 Hz, J ) 5.9 Hz, 1H), 4.15 (dd, J )
12.2 Hz, J ) 5.9 Hz, 1H), 3.60 (dd, J ) 8.9 Hz, J ) 8.9 Hz,
1H), 3.44 (dd, J ) 8.9 Hz, J ) 9.6 Hz, 1H), 3.36 (m, 1H, H-5),
3.08 (dd, J ) 8.9 Hz, J ) 9.2 Hz, 1H), 1.35 (d, J ) 5.9 Hz, 3H,
H-6); 13C NMR (100 MHz, CDCl3) δ 138.7 (s), 138.3 (s), 134.9
(s), 134.2 (s), 132.1 (s), 129.1 (s), 128.62 (s), 128.5 (s), 128.4
(s), 128.1 (s), 128.0 (s), 127.9 (s), 127.6 (s), 117.4 (s), 87.7 (s),
86.7 (s), 83.4 (s), 81.4 (s), 76.0 (s), 75.9 (s), 75.6 (s), 74.3 (s),
18.4 (s); MALDI calcd for C29H32O4SNa ([M + Na]+) m/e
499.1914, measured m/e 499.1937.
Phenyl 2,3-Di-O-Benzyl-6-deoxy-4-O-(3-hydroxypropyl)-
1-thio-â-D-glucopyranoside (58). To a solution of 57 (0.31
g, 0.65 mmol) in anhydrous THF, borane/THF (0.98 mL, 1 M
solution) was added. The reaction mixture was stirred for 1 h
at room temperature. After completion of the reaction, H2O2
(0.30 mL, 30%) and a couple of drops of NaOH solution (3 M)
were added at 0 °C. After being stirred for 10 min, the reaction
mixture was diluted with EtOAc. The combined organic layers
were washed with brine and dried over Na2SO4(s). After
removal of the solvent followed by purification with gradient
column chromatography (hexanes/EtOAc ) 90:10 to 50:50), the
product was obtained (0.17 g, 0.34 mmol, 53%). 1H NMR (400
MHz, CDCl3) δ 7.3-7.6 (m, 15H), 4.90 (d, J ) 10.3 Hz, 1H,
PhCH2O), 4.90 (d, J ) 11.0 Hz, 1H, PhCH2O), 4.81 (d, J )
11.0 Hz, 1H, PhCH2O), 4.73 (d, J ) 10.3 Hz, 1H, PhCH2O),
4.64 (d, J ) 9.7 Hz, 1H, H-1), 3.99 (dt, J ) 9.0 Hz, J ) 5.6 Hz,
1H), 3.77 (dt, J ) 9.0 Hz, J ) 5.6 Hz, 1H), 3.73 (t, J ) 5.9 Hz,
2H), 3.58 (dd, J ) 8.9 Hz, J ) 8.9 Hz, 1H), 3.46 (dd, J ) 9.7
Hz, J ) 9.0 Hz, 1H), 3.36 (m, 1H, H-5), 3.03 (dd, J ) 9.2 Hz,
J ) 9.3 Hz, 1H), 1.81 (m, 2H), 1.38 (d, J ) 6.1 Hz, 3H, H-6);
13C NMR (100 MHz, CDCl3) δ 138.7 (s), 138.3 (s), 134.1 (s),
132.1 (s), 129.1 (s), 128.67 (s), 128.61 (s), 128.4 (s), 128.0 (s),
127.9 (s), 127.7 (s), 87.7 (s), 86.5 (s), 84.2 (s), 81.5 (s), 75.9 (s),
75.8 (s), 75.6 (s), 72.2 (s), 61.5 (s), 33.1 (s), 18.4 (s); MALDI
calcd for C29H34O5SNa ([M + Na]+) m/e 517.2019, measured
m/e 517.2030.
Phenyl 4-O-((R)-2-Acetoxyl-3-azidopropyl)-2,3-di-O-
benzyl-6-deoxy-1-thio-â-D-glucopyranoside (62). For the
first step of the procedure, refer to the synthesis of 67. To a
solution of 67 (0.15 g, 0.28 mmol) in anhydrous CH2Cl2 (5 mL)
were added DMAP (catalytic amount), Et3N (0.12 mL, 0.84
mmol), and Ac2O (0.053 mL, 0.56 mmol) at room temperature.
After the reaction was completed (∼4 h), the reaction was
quenched by addition of NaHCO3 (saturated). After removal
of solvent, the reaction mixture was diluted with EtOAc. The
organic layer was washed with brine and dried over Na2SO4(s)
.
Removal of the solvent followed by purification with gradient
column chromatography (hexanes/EtOAc ) 100:0 to 65:35)
afforded the product (0.13 g, 0.23 mmol, 80%, two-step yield:
52%). 1H NMR (400 MHz, CDCl3) δ 7.3-7.6 (m, 15H), 5.01
(m, 1H), 4.92 (d, J ) 10.3 Hz, 1H, PhCH2O), 4.90 (d, J ) 11.1
Hz, 1H, PhCH2O), 4.79 (d, J ) 11.1 Hz, 1H, PhCH2O), 4.71
(d, J ) 10.3 Hz, 1H, PhCH2O), 4.64 (d, J ) 9.7 Hz, 1H, H-1),
3.93 (dd, J ) 10.2 Hz, J ) 4.8 Hz, 1H), 3.74 (dd, J ) 10.2 Hz,
Phenyl 4-O-(3-Azidopropyl)-2,3-di-O-benzyl-6-deoxy-1-
thio-â-D-glucopyranoside (59). To a solution of 58 (0.15 g,

6282 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Wang et al.
J ) 5.6 Hz, 1H), 3.58 (dd, J ) 8.1 Hz, J ) 8.1 Hz, 1H), 3.3-
3.5 (m, 4H), 3.04 (dd, J ) 9.2 Hz, J ) 9.2 Hz, 1H), 2.01 (s, 3H,
CH3CO2), 1.36 (d, J ) 6.2 Hz, 3H, H-6); 13C NMR (100 MHz,
CDCl3) δ 170.3 (s, CH3CO2), 138.6 (s), 138.2 (s), 134.1 (s), 132.1
(s), 129.1 (s), 128.7 (s), 128.6 (s), 128.4 (s), 128.1 (s), 128.0 (s),
127.8 (s), 127.7 (s), 87.7 (s), 86.5 (s), 84.3 (s), 81.5 (s), 75.8 (s),
75.6 (s), 75.5 (s), 71.8 (s), 71.7 (s), 50.9 (s), 21.0 (s, CH3CO2),
18.3 (s); MALDI calcd for C31H35N3O6SNa ([M + Na]+) m/e
600.2139, measured m/e 600.2144.
Phenyl 2,3-di-O-Benzyl-6-deoxy-4-O-((R)-2,3-diazidopro-
pyl)-1-thio-â-D-glucopyranoside (66). Refer to the synthesis
of 63. 1H NMR (400 MHz, CDCl3) δ 7.3-7.6 (m, 15H), 4.95 (d,
J ) 10.8 Hz, 2H, PhCH2O), 4.75 (d, J ) 10.8 Hz, 1H, PhCH2O),
4.73 (d, J ) 10.8 Hz, 1H, PhCH2O), 4.66 (d, J ) 9.8 Hz, 1H,
H-1), 3.91 (dd, J ) 9.6 Hz, J ) 4.1 Hz, 1H), 3.6-3.7 (m, 2H),
3.5-3.6 (m, 2H), 3.40 (m, 1H), 3.29 (dd, J ) 12.7 Hz, J ) 4.9
Hz, 1H), 3.22 (dd, J ) 12.7 Hz, J ) 6.9 Hz, 1H), 3.03 (dd, J )
9.2 Hz, J ) 9.1 Hz, 1H), 1.38 (d, J ) 6.1 Hz, 3H, H-6); 13C
NMR (100 MHz, CDCl3) δ 138.6 (s), 138.1 (s), 134.1 (s), 132.0
(s), 129.1 (s), 128.7 (s), 128.6 (s), 128.4 (s), 128.1 (s), 128.0 (s),
127.96 (s), 127.7 (s), 87.7 (s), 86.6 (s), 84.2 (s), 81.7 (s), 75.9
(s), 75.6 (s), 75.4 (s), 73.3 (s), 61.3 (s), 51.7 (s), 18.4 (s); LRFAB
m/e 583 ([M + Na]+); HRFAB calcd for C29H32N6O4SNa ([M +
Na]+) m/e 583.2103, measured m/e 583.2085.
Phenyl 2,3-di-O-Benzyl-6-deoxy-4-O-((S)-2,3-diazidopro-
pyl)-1-thio-â-D-glucopyranoside (63). For the first step of
the procedure, refer to the synthesis of 67. To a solution of 67
(0.20 g, 0.37 mmol) and pyridine (0.048 mL, 0.60 mmol) in
anhydrous CH2Cl2 at 0 °C, Tf2O (0.088 mL, 0.52 mmol) was
added slowly. After the mixture was stirred for a half hour,
TLC was performed. After completion of the reaction, the
reaction mixture was diluted with CH2Cl2, washed with water,
saturated NaHCO3(aq), and brine, and then dried over Na2-
SO4(s). The solution was filtered through glass wool and
transferred into a solution of NaN3 (0.20 g, 3.0 mmol) in DMF.
The reaction mixture was stirred overnight while the solvents
were slowly evaporated with aspirator. After most of the
solvent was removed, the reaction mixture was diluted with
EtOAc and filtered through Celite. Removal of the solvent
followed by purification with gradient column chromatography
(hexanes/EtOAc ) 100:0 to 65:35) afforded the product (0.13
g, 0.23 mmol, 62%, two-step yield: 40%). 1H NMR (400 MHz,
CDCl3) δ 7.3-7.6 (m, 15H), 4.96 (d, J ) 10.6 Hz, 2H, PhCH2O),
4.77 (d, J ) 11.0 Hz, 1H, PhCH2O), 4.74 (d, J ) 10.3 Hz, 1H,
PhCH2O), 4.66 (d, J ) 9.8 Hz, 1H, H-1), 3.94 (dd, J ) 9.7 Hz,
J ) 6.0 Hz, 1H), 3.69 (dd, J ) 9.7 Hz, J ) 5.2 Hz, 1H), 3.62
(dd, J ) 8.9 Hz, J ) 8.8 Hz, 1H), 3.54 (m, 1H), 3.49 (dd, J )
9.6 Hz, J ) 8.9 Hz, 1H), 3.39 (dq, J ) 9.4 Hz, J ) 6.2 Hz, 1H),
3.3-3.4 (m, 2H), 3.04 (dd, J ) 9.2 Hz, J ) 9.2 Hz, 1H), 1.38
(d, J ) 6.2 Hz, 3H, H-6); 13C NMR (100 MHz, CDCl3) δ 138.5
(s), 138.1 (s), 134.1 (s), 132.1 (s), 129.1 (s), 128.8 (s), 128.6 (s),
128.4 (s), 128.2 (s), 128.1 (s), 128.0 (s), 127.7 (s), 87.7 (s), 86.4
(s), 84.1 (s), 81.7 (s), 75.8 (s), 75.6 (s), 75.4 (s), 72.6 (s), 61.0
(s), 51.7 (s), 18.4 (s); MALDI calcd for C29H32N6O4SNa ([M +
Na]+) m/e 583.2098, measured m/e 583.2084.
Phenyl 2,3-Di-O-benzyl-6-deoxy-4-O-((S)-glycidyl)-1-
thio-â-D-glucopyranoside (64). Refer to the synthesis of 61.
1H NMR (400 MHz, CDCl3) δ 7.3-7.6 (m, 15H), 4.93 (d, J )
10.2 Hz, 1H, PhCH2O), 4.90 (d, J ) 11.0 Hz, 1H, PhCH2O),
4.86 (d, J ) 11.0 Hz, 1H, PhCH2O), 4.76 (d, J ) 10.2 Hz, 1H,
PhCH2O), 4.67 (d, J ) 9.8 Hz, 1H, H-1), 3.88 (dd, J ) 11.1 Hz,
J ) 3.3 Hz, 1H), 3.77 (dd, J ) 11.1 Hz, J ) 6.2 Hz, 1H), 3.63
(dd, J ) 8.9 Hz, J ) 9.0 Hz, 1H), 3.48 (dd, J ) 8.9 Hz, J ) 9.7
Hz, 1H), 3.40 (m, 1H), 3.1-3.2 (m, 2H), 2.79 (dd, J ) 4.6 Hz,
J ) 4.9 Hz, 1H), 2.57 (dd, J ) 4.9 Hz, J ) 2.6 Hz, 1H), 1.40 (d,
J ) 6.1 Hz, 3H, H-6); 13C NMR (100 MHz, CDCl3) δ 138.6 (s),
138.3 (s), 134.1 (s), 132.1 (s), 131.9 (s), 129.1 (s), 128.6 (s), 128.4
(s), 128.2 (s), 127.9 (s), 127.7 (s), 87.7 (s), 86.5 (s), 84.3 (s), 81.4
(s), 76.0 (s), 75.7 (s, two carbons), 74.2 (s), 50.8 (s), 44.7 (s),
18.3 (s); LRFAB m/e 515 ([M + Na]+); HRFAB calcd for
C29H32O5S Na ([M + Na]+) m/e 515.1868, measured m/e
515.1871.
Phenyl 4-O-((R)-3-Azido-2-hydroxypropyl)-2,3-Di-O-
benzyl-6-deoxy-1-thio-â-D-glucopyranoside (67). To a so-
lution of 64 (0.1 g, 0.20 mmol), CeCl3‚7H2O (0.04 g, 0.10 mmol)
in CH3CN/H2O (4.5 mL/0.5 mL), NaN3 (0.02 g, 0.22 mmol) was
added. The reaction mixture was stirred for 24 h under reflux
till the completion of the reaction. After removal of solvent,
the residue was diluted with EtOAc. The organic layer was
washed with 1 N HCl, water, saturated NaHCO3(aq), and brine
and then dried over Na2SO4(s). After removal of the solvent
followed by purification with gradient column chromatography
(hexanes/EtOAc ) 100:0 to 60:40), the product was obtained
1
(0.07 g, 0.13 mmol, 65%). H NMR (400 MHz, CDCl3) δ 7.3-
7.5 (m, 15H), 4.97 (d, J ) 11.1 Hz, 1H, PhCH2O), 4.96 (d, J )
10.3 Hz, 1H, PhCH2O), 4.76 (d, J ) 11.1 Hz, 1H, PhCH2O),
4.72 (d, J ) 10.3 Hz, 1H, PhCH2O), 4.65 (d, J ) 9.7 Hz, 1H,
H-1), 3.7-3.8 (m, 2H), 3.67 (m, 1H), 3.61 (dd, J ) 8.9 Hz, J )
9.0 Hz, 1H), 3.50 (dd, J ) 8.9 Hz, J ) 9.6 Hz, 1H), 3.37 (dq, J
) 9.3 Hz, J ) 6.2 Hz, 1H), 3.22 (m, 2H), 3.09 (dd, J ) 9.2 Hz,
J ) 9.2 Hz, 1H), 1.37 (d, J ) 6.2 Hz, 3H, H-6); 13C NMR (100
MHz, CDCl3) δ 138.1 (s), 138.0 (s), 134.0 (s), 132.1 (s), 129.1
(s), 128.7 (s), 128.6 (s), 128.4 (s), 128.1 (s), 128.0 (s), 127.8 (s),
87.8 (s), 86.0 (s), 84.3 (s), 81.7 (s), 75.9 (s), 75.8 (s), 75.5 (s),
74.5 (s), 70.2 (s), 53.3 (s), 18.4 (s); MALDI calcd for C29H32N3O5-
SNa ([M + Na]+) m/e 558.2033, measured m/e 558.2024.
Phenyl 4-O-((R)-3-Azido-2-((R)-glycidyl)propyl)-2,3-di-
O-benzyl-6-deoxy-1-thio-â-D-glucopyranoside (68). Refer
1
to the synthesis of 61. H NMR (270 MHz, CDCl3) δ 7.2-7.5
(m, 15H), 4.90 (d, J ) 11.2 Hz, 1H, PhCH2O), 4.88 (d, J )
10.2 Hz, 1H, PhCH2O), 4.76 (d, J ) 11.2 Hz, 1H, PhCH2O),
4.69 (d, J ) 10.2 Hz, 1H, PhCH2O), 4.62 (d, J ) 9.6 Hz, 1H,
H-1), 3.8-3.9 (m, 2H), 3.3-3.7 (m, 8H), 3.09 (m, 1H), 3.00 (dd,
J ) 9.2 Hz, J ) 9.2 Hz, 1H), 2.73 (dd, J ) 4.9 Hz, J ) 4.0 Hz,
1H), 2.51 (dd, J ) 4.9 Hz, J ) 3.0 Hz, 1H), 1.35 (d, J ) 6.3 Hz,
3H, H-6); 13C NMR (68 MHz, CDCl3) δ 138.5 (s), 138.0 (s), 133.9
(s), 131.9 (s), 129.0 (s), 128.54 (s), 128.50 (s), 128.3 (s), 127.95
(s), 127.86 (s), 127.6 (s), 87.5 (s), 86.3 (s), 84.1 (s), 81.3 (s), 78.7
(s), 75.6 (s), 75.5 (s, two carbons), 72.9 (s), 71.5 (s), 51.9 (s),
50.9 (s), 44.2 (s), 18.3 (s); LRFAB m/e 614 ([M + Na]+); HRFAB
calcd for C32H37N3O6SNa ([M + Na]+) m/e 614.2300, measured
m/e 614.2293.
Phenyl 4-O-((R)-2-((R)-2-Acetoxyl-3-azidopropyl)-3-azi-
dopropyl)-2,3-di-O-benzyl-6-deoxy-1-thio-â-D-glucopyra-
noside (69). Refer to the synthesis of 62. 1H NMR (400 MHz,
CDCl3) δ 7.3-7.6 (m, 15H), 5.02 (m, 1H), 4.93 (d, J ) 11.2 Hz,
1H, PhCH2O), 4.91 (d, J ) 10.2 Hz, 1H, PhCH2O), 4.76 (d, J
) 11.2 Hz, 1H, PhCH2O), 4.71 (d, J ) 10.2 Hz, 1H, PhCH2O),
4.65 (d, J ) 9.7 Hz, 1H, H-1), 3.82 (dd, J ) 9.9 Hz, J ) 4.8 Hz,
1H), 3.6-3.7 (m, 3H), 3.58 (dd, J ) 8.9 Hz, J ) 8.9 Hz, 1H),
3.4-3.5 (m, 4H), 3.37 (m, 1H), 3.2-3.3 (m, 2H), 2.98 (dd, J )
9.1 Hz, J ) 9.1 Hz, 1H), 2.08 (s, 3H, CH3CO2), 1.36 (d, J ) 6.1
Hz, 3H, H-6); 13C NMR (100 MHz, CDCl3) δ 170.3 (s, CH3CO2),
138.7 (s), 138.2 (s), 134.1 (s), 132.1 (s), 129.1 (s), 128.7 (s), 128.6
(s), 128.4 (s), 128.1 (s), 127.9 (s), 127.7 (s), 87.7 (s), 86.5 (s),
84.3 (s), 81.6 (s), 79.4 (s), 75.7 (s), 75.5 (s), 73.2 (s), 71.4 (s),
69.0 (s), 52.0 (s), 50.9 (s), 21.1 (s, CH3CO2), 18.4 (s); MALDI
calcd for C34H40N6O7SNa ([M + Na]+) m/e 699.2571, measured
m/e 699.2541.
Phenyl 4-O-((S)-2-Acetoxyl-3-azidopropyl)-2,3-di-O-
benzyl-6-deoxy-1-thio-â-D-glucopyranoside (65). Refer to
the synthesis of 62. 1H NMR (400 MHz, CDCl3) δ 7.3-7.6 (m,
15H), 5.05 (m, 1H), 4.93 (d, J ) 10.2 Hz, 1H, PhCH2O), 4.91
(d, J ) 11.0 Hz, 1H, PhCH2O), 4.77 (d, J ) 11.0 Hz, 1H,
PhCH2O), 4.72 (d, J ) 10.2 Hz, 1H, PhCH2O), 4.65 (d, J ) 9.7
Hz, 1H, H-1), 3.99 (dd, J ) 10.1 Hz, J ) 5.1 Hz, 1H), 3.73 (dd,
J ) 10.1 Hz, J ) 5.3 Hz, 1H), 3.58 (dd, J ) 9.9 Hz, J ) 8.9
Hz, 1H), 3.3-3.5 (m, 4H), 3.03 (dd, J ) 9.2 Hz, J ) 9.2 Hz,
1H), 2.08 (s, 3H, CH3CO2), 1.36 (d, J ) 6.1 Hz, 3H, H-6); 13C
NMR (100 MHz, CDCl3) δ 170.2 (s, CH3CO2), 138.4 (s), 138.2
(s), 134.1 (s), 132.1 (s), 129.1 (s), 128.7 (s), 128.6 (s), 128.4 (s),
128.1 (s), 127.7 (s), 87.7 (s), 86.4 (s), 84.2 (s), 81.5 (s), 75.8 (s),
75.6 (s), 75.5 (s), 71.6 (s), 71.4 (s), 50.9 (s), 21.1 (s, CH3CO2),
18.3 (s); LRFAB m/e 600 ([M + Na]+); HRFAB calcd for
C31H35N3O6SNa ([M + Na]+) m/e 600.2144, measured m/e
600.2168.
6-O-(4-O-(3-Azidopropyl)-2,3-di-O-benzyl-6-deoxy-r-D-
glucopyranosyl)-1,3,2′,6′-tetraazidoneamine (70). Refer to

Optimization of Aminoglycosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6283
the general procedure for glycosylation and hydrolysis. 1H
NMR (400 MHz, CDCl3) δ 7.3-7.4 (m, 10H), 5.70 (d, J ) 3.7
Hz, 1H, H-1′), 4.95 (d, J ) 11.0 Hz, 1H, PhCH2O), 4.94 (d, J )
3.6 Hz, 1H, H-1′′), 4.76 (d, J ) 11.8 Hz, 1H, PhCH2O), 4.74 (d,
J ) 11.0 Hz, 1H, PhCH2O), 4.70 (d, J ) 11.8 Hz, 1H, PhCH2O),
4.17 (m, 1H), 3.9-4.0 (m, 3H), 3.3-3.7 (m, 14H), 2.96 (dd, J )
9.3 Hz, J ) 9.3 Hz, 1H), 2.34 (ddd, J ) 13.0 Hz, J ) 4.4 Hz,
J ) 4.4 Hz, 1H, H-2eq), 1.7-1.8(m, 2H), 1.53 (ddd, J ) 13.0
Hz, J ) 12.8 Hz, J ) 12.8 Hz, 1H, H-2ax), 1.27 (d, J ) 6.1 Hz,
3H,H-6′′); 13C NMR (100 MHz, CDCl3) δ 138.9 (s), 138.2 (s),
128.7 (s), 128.6 (s), 128.1 (s), 127.9 (s), 98.6 (s), 98.3 (s), 86.5
(s), 85.1 (s), 80.9 (s), 80.1 (s), 79.8 (s), 76.1 (s), 75.7 (s), 73.6
(s), 71.8 (s), 71.6 (s), 71.3 (s), 70.2 (s), 68.7 (s), 63.3 (s), 59.5
(s), 59.3 (s), 51.5 (s), 48.5 (s), 32.5 (s), 29.9 (s), 18.0 (s); MALDI
calcd for C35H45N15O10Na ([M + Na]+) m/e 858.3366, measured
m/e 858.3392.
1H NMR (400 MHz, CDCl3) δ 7.3-7.5 (m, 10H), 5.70 (d, J )
3.8 Hz, 1H, H-1′), 4.99 (d, J ) 11.2 Hz, 1H, PhCH2O), 4.94 (d,
J ) 3.6 Hz, 1H, H-1′′), 4.76 (d, J ) 11.8 Hz, 1H, PhCH2O),
4.72 (d, J ) 11.2 Hz, 1H, PhCH2O), 4.71 (d, J ) 11.8 Hz, 1H,
PhCH2O), 4.18 (m, 1H), 3.9-4.0 (m, 3H), 3.68 (dd, J ) 8.9 Hz,
J ) 8.9 Hz, 1H), 3.4-3.6 (m, 10H), 3.2-3.3 (m, 4H), 2.96 (dd,
J ) 9.2 Hz, J ) 9.4 Hz, 1H), 2.55 (ddd, J ) 13.3 Hz, J ) 4.0
Hz, J ) 4.0 Hz, 1H, H-2eq), 1.49 (ddd, J ) 13.3 Hz, J ) 12.6
Hz, J ) 12.6 Hz, 1H, H-2ax), 1.29 (d, J ) 6.2 Hz, 3H, H-6′′);
13C NMR (100 MHz, CDCl3) δ 138.8 (s), 138.0 (s), 128.72 (s),
128.66 (s), 128.51 (s), 128.24 (s), 128.17 (s), 127.98 (s), 98.5
(s), 98.3 (s), 86.6 (s), 84.0 (s), 80.9 (s), 80.2 (s), 79.9 (s), 76.1
(s), 75.7 (s), 73.6 (s), 73.3 (s), 71.8 (s), 71.6 (s), 71.2 (s), 68.5
(s), 63.4 (s), 61.3 (s), 59.5 (s), 59.3 (s), 51.7 (s), 51.5 (s), 32.5
(s), 18.0 (s); MALDI calcd for C35H44N18O10Na ([M + Na]+) m/e
899.3380, measured m/e 899.3387.
6-O-(4-O-((R)-3-Azido-2-hydroxypropyl)-2,3-di-O-benzyl-
6-deoxy-r-D-glucopyranosyl)-1,3,2′,6′-tetraazidoneam-
ine (71). Refer to the general procedure for glycosylation and
hydrolysis. 1H NMR (400 MHz, CDCl3) δ 7.3-7.4 (m, 10H),
5.69 (d, J ) 3.7 Hz, 1H, H-1′), 5.01 (d, J ) 11.1 Hz, 1H,
PhCH2O), 4.95 (d, J ) 3.5 Hz, 1H, H-1′′), 4.74 (d, J ) 11.7 Hz,
1H, PhCH2O), 4.72 (d, J ) 11.1 Hz, 1H, PhCH2O), 4.70 (d, J
) 11.7 Hz, 1H, PhCH2O), 4.19 (m, 1H), 3.9-4.0 (m, 3H), 3.2-
3.8 (m, 15H), 3.03 (dd, J ) 9.3 Hz, J ) 9.4 Hz, 1H), 2.35 (ddd,
J ) 13.0 Hz, J ) 4.5 Hz, J ) 4.5 Hz, 1H, H-2eq), 1.52 (ddd, J
) 13.0 Hz, J ) 12.5 Hz, J ) 12.5 Hz, 1H, H-2ax), 1.29 (d, J )
6.2 Hz, 3H, H-6′′); 13C NMR (100 MHz, CDCl3) δ 138.1 (s),
137.7 (s), 128.54 (s), 128.49 (s), 128.2 (s), 128.1 (s), 128.0 (s),
127.9 (s), 98.2 (s), 98.1 (s), 86.3 (s), 84.2 (s), 80.2 (s, two
carbons), 79.7 (s), 75.9 (s), 75.6 (s), 74.4 (s), 73.4 (s), 71.6 (s),
71.4 (s), 71.1 (s), 70.0 (s), 68.6 (s), 63.2 (s), 59.4 (s), 59.0 (s),
53.0 (s), 51.3 (s), 32.3 (s), 17.8 (s); MALDI calcd for C35H45N15O11-
Na ([M + Na]+) m/e 874.3315, measured m/e 874.3298.
6-O-(2,3-Di-O-benzyl-6-deoxy-4-O-((S)-2,3-diazidopropyl)-
r-D-glucopyranosyl)-1,3,2′,6′-tetraazidoneamine (72). Re-
fer to the general procedure for glycosylation and hydrolysis.
1H NMR (400 MHz, CDCl3) δ 7.3-7.6 (m, 10H), 5.69 (d, J )
3.7 Hz, 1H, H-1′), 4.99 (d, J ) 11.3 Hz, 1H, PhCH2O), 4.95 (d,
J ) 3.5 Hz, 1H, H-1′′), 4.75 (d, J ) 12.2 Hz, 1H, PhCH2O),
4.72 (d, J ) 12.2 Hz, 1H, PhCH2O), 4.71 (d, J ) 11.3 Hz, 1H,
PhCH2O), 4.18 (m, 1H), 3.9-4.0 (m, 3H), 3.3-3.7 (m, 15H),
2.97 (dd, J ) 9.4 Hz, J ) 9.1 Hz, 1H), 2.35 (ddd, J ) 13.0 Hz,
J ) 4.3 Hz, J ) 4.3 Hz, 1H, H-2eq), 1.52 (ddd, J ) 13.0 Hz, J
) 12.6 Hz, J ) 12.6 Hz, 1H, H-2ax), 1.28(d, J ) 6.2 Hz, 3H,
H-6′′); 13C NMR (100 MHz, CDCl3) δ 138.7 (s), 138.0 (s), 128.72
(s), 128.69 (s), 128.26 (s), 128.19 (s), 128.0 (s), 98.4 (s), 98.3
(s), 86.5 (s), 84.0 (s), 80.7 (s), 80.2 (s), 79.9 (s), 76.1 (s), 75.7
(s), 73.6 (s), 72.6 (s), 71.8 (s), 71.6 (s), 71.3 (s), 68.4 (s), 63.4
(s), 60.9 (s), 59.5 (s), 59.3 (s), 51.8 (s), 51.5 (s), 32.5 (s), 18.0
(s); MALDI calcd for C35H44N18O10Na ([M + Na]+) m/e 899.3380,
measured m/e 899.3353.
6-O-(4-O-((S)-3-Azido-2-hydroxypropyl)-2,3-di-O-benzyl-
6-deoxy-r-D-glucopyranosyl)-1,3,2′,6′-tetraazidoneam-
ine (73). Refer to the general procedure for glycosylation and
hydrolysis. 1H NMR (400 MHz, CDCl3) δ 7.3-7.5 (m, 10H),
5.70 (d, J ) 3.8 Hz, 1H, H-1′), 5.00 (d, J ) 11.1 Hz, 1H,
PhCH2O), 4.93 (d, J ) 3.6 Hz, 1H, H-1′′), 4.75 (d, J ) 11.1 Hz,
1H, PhCH2O), 4.74 (d, J ) 11.6 Hz, 1H, PhCH2O), 4.70 (d, J
) 11.6 Hz, 1H, PhCH2O), 4.19 (m, 1H), 3.9-4.0 (m, 3H), 3.80
(m, 1H), 3.5-3.7 (m, 10H), 3.39 (m, 1H), 3.2-3.3 (m, 3H), 3.11
(dd, J ) 9.4 Hz, J ) 9.4 Hz, 1H), 2.35 (ddd, J ) 13.3 Hz, J )
4.5 Hz, J ) 4.5 Hz, 1H, H-2eq), 1.52 (ddd, J ) 13.3 Hz, J )
12.6 Hz, J ) 12.6 Hz, 1H, H-2ax), 1.28 (d, J ) 6.3 Hz, 3H, H-6′′);
13C NMR (100 MHz, CDCl3) δ 138.1 (s), 137.9 (s), 128.74 (s),
128.67 (s), 128.54 (s), 128.28 (s), 128.22 (s), 128.17 (s), 98.5
(s), 98.3 (s), 86.7 (s), 84.5 (s), 80.3 (s, two carbons), 79.9 (s),
76.1 (s), 76.0 (s), 75.5 (s), 73.6 (s), 71.8 (s), 71.6 (s), 71.2 (s),
70.8 (s), 68.9 (s), 63.4 (s), 59.6 (s), 59.2 (s), 53.1 (s), 51.5 (s),
32.5 (s), 17.9 (s); MALDI calcd for C35H45N15O11Na ([M + Na]+)
m/e 874.3315, measured m/e 874.3293.
6-O-(4-O-((R)-3-Azido-2-((R)-3-azido-2-hydroxypropyl)-
propyl)-2,3-di-O-benzyl-6-deoxy-r-D-glucopyranosyl)-
1,3,2′,6′-tetraazidoneamine (75). Refer to the general pro-
1
cedure for glycosylation and hydrolysis. H NMR (400 MHz,
CDCl3) δ 7.3-7.4 (m, 10H), 5.69 (d, J ) 3.7 Hz, 1H, H-1′), 5.00
(d, J ) 11.4 Hz, 1H, PhCH2O), 4.97 (d, J ) 3.6 Hz, 1H, H-1′′),
4.74 (d, J ) 11.4 Hz, 1H, PhCH2O), 4.71 (d, J ) 11.4 Hz, 2H,
PhCH2O), 4.18 (m, 1H), 3.8-4.0 (m, 6H), 3.69 (dd, J ) 9.0 Hz,
J ) 8.8 Hz, 1H), 3.4-3.6 (m, 10H), 3.2-3.4 (m, 6H), 2.97 (dd,
J ) 9.3 Hz, J ) 9.3 Hz, 1H), 2.34 (ddd, J ) 12.7 Hz, J ) 4.5
Hz, J ) 4.5 Hz, 1H, H-2eq), 1.51 (ddd, J ) 12.7 Hz, J ) 12.7
Hz, J ) 12.7 Hz, 1H, H-2ax), 1.28 (d, J ) 6.3 Hz, 3H, H-6′′);
13C NMR (100 MHz, CDCl3) δ 138.9 (s), 138.0 (s), 128.7 (s),
128.6 (s), 128.20 (s), 128.16 (s), 128.0 (s), 127.9 (s), 127.6 (s),
98.3 (s, two carbons), 86.2 (s), 84.1 (s), 80.8 (s), 80.1 (s), 80.0
(s), 79.7 (s), 76.0 (s), 75.6 (s), 73.5 (s), 73.4 (s), 72.3 (s), 71.8
(s), 71.6 (s), 71.3 (s), 70.1 (s), 68.3 (s), 63.4 (s), 59.5 (s), 59.2
(s), 53.4 (s), 52.2 (s), 51.5 (s), 32.5 (s), 18.1 (s); MALDI calcd
for C38H50N18O12Na ([M + Na]+) m/e 973.3748, measured m/e
973.3740.
6-O-(3-Azido-4-O-((S)-3-azido-2-hydroxypropyl)-2-O-
benzyl-3,6-dideoxy-r-D-glucopyranosyl)-1,3,2′,6′-tetraazi-
doneamine (76). Refer to the general procedure for glycosy-
1
lation and hydrolysis. H NMR (400 MHz, CDCl3) δ 7.4-7.5
(m, 5H), 5.60 (d, J ) 3.6 Hz, 1H, H-1′), 4.98 (d, J ) 3.5 Hz,
1H, H-1′′), 4.76 (d, J ) 11.7 Hz, 1H, PhCH2O), 4.73 (d, J )
11.7 Hz, 1H, PhCH2O), 4.36 (d, J ) 2.0 Hz, 1H), 4.17 (m, 1H),
3.9-4.0 (m, 3H), 3.87 (dd, J ) 10.0 Hz, J ) 10.0 Hz, 1H), 3.7-
3.8 (m, 2H), 3.65 (m, 1H), 3.3-3.6 (m, 9H), 3.27 (dd, J ) 9.4
Hz, J ) 9.5 Hz, 1H), 2.83 (dd, J ) 9.7 Hz, J ) 9.7 Hz, 1H),
2.36 (ddd, J ) 13.5 Hz, J ) 4.2 Hz, J ) 4.2 Hz, 1H, H-2eq),
1.53 (ddd, J ) 13.5 Hz, J ) 12.9 Hz, J ) 12.9 Hz, 1H, H-2ax),
1.27 (d, J ) 6.3 Hz, 3H, H-6′′); 13C NMR (100 MHz, CDCl3) δ
137.3 (s), 128.8 (s), 128.5 (s), 128.4 (s), 98.5 (s), 97.2 (s), 85.2
(s), 83.2 (s), 80.7 (s), 78.3 (s), 75.8 (s), 75.0 (s), 73.4 (s), 72.1
(s), 71.6 (s), 71.3 (s), 70.4 (s), 68.3 (s), 64.2 (s), 63.5 (s), 59.6
(s), 59.1 (s), 53.4 (s), 51.5 (s), 32.4 (s), 18.0 (s); MALDI calcd
for C28H38N18O10Na ([M + Na]+) m/e 809.2911, measured m/e
809.2929.
6-O-(4-O-(3-Aminopropyl)-6-deoxy-r-D-glucopyranosyl)-
neamine (49). Refer to the general procedure for the synthesis
1
kanamycin B analogues. H NMR (400 MHz, D2O) (chloride
salt) δ 5.96 (d, J ) 3.1 Hz, 1H, H-1′), 4.96 (d, J ) 2.8 Hz, 1H,
H-1′′), 3.8-4.0 (m,8H), 3.4-3.7 (m, 9H), 3.26 (dd, J ) 13.6 Hz,
J ) 6.9 Hz, 1H), 3.10 (dd, J ) 7.3 Hz,, J ) 7.0 Hz, 2H), 3.04
(dd, J ) 9.6 Hz, J ) 9.4 Hz, 1H), 2.50 (ddd, J ) 12.3 Hz, J )
4.2 Hz, J ) 4.2 Hz, 1H, H-2eq), 1.92 (m, 1H, H-2ax), 1.25 (d, J
) 5.9 Hz, 3H, H-6′′); 13C NMR (100 MHz, D2O) (chloride salt)
δ 101.8 (s), 95.9 (s), 83.8 (s), 83.7 (s), 77.6 (s), 74.3 (s), 72.5 (s),
72.2 (s), 70.9 (s), 70.6 (s), 69.5 (s), 68.5 (s), 68.3 (s), 53.7 (s),
50.2 (s), 48.6 (s), 40.4 (s), 37.9 (s), 28.3 (s), 27.4 (s), 17.3 (s);
LRFAB m/e 526 ([M + H]+); HRFAB calcd for C21H44N5O10 ([M
+ H]+) m/e 526.3088, measured m/e 526.3065.
6-O-(4-O-((R)-3-Amino-2-hydroxylpropyl)-6-deoxy-r-D-
glucopyranosyl)neamine (50). Refer to the general proce-
dure for the synthesis kanamycin B analogues. 1H NMR (400
MHz, D2O) (chloride salt) δ 5.99 (d, J ) 3.9 Hz, 1H, H-1′), 4.98
(d, J ) 3.8 Hz, 1H, H-1′′), 4.0-4.1 (m, 5H), 3.7-3.9 (m, 6H),
6-O-(2,3-Di-O-benzyl-6-deoxy-4-O-((R)-2,3-diazidopro-
pyl)-r-D-glucopyranosyl)-1,3,2′,6′-tetraazidoneamine (74).
Refer to the general procedure for glycosylation and hydrolysis.

6284 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Wang et al.
3.5-3.6 (m, 5H), 3.28 (dd, J ) 13.6 Hz, J ) 6.9 Hz, 1H), 3.19
(dd, J ) 13.2 Hz, J ) 3.3 Hz, 1H), 3.09 (dd, J ) 9.5 Hz, J )
9.4 Hz, 1H), 3.03 (dd, J ) 13.2 Hz, J ) 9.2 Hz, 1H), 2.53 (ddd,
J ) 12.6 Hz, J ) 4.0 Hz, J ) 4.0 Hz, 1H, H-2eq), 1.97 (ddd, J
) 12.6 Hz, J ) 12.6 Hz, J ) 12.6 Hz, 1H, H-2ax), 1.27 (d, J )
6.2 Hz, 3H, H-6′′); 13C NMR (100 MHz, D2O) (chloride salt) δ
101.8 (s), 95.9 (s), 84.1 (s), 83.8 (s), 77.4 (s), 74.3 (s, two
carbons), 72.6 (s), 72.2 (s), 70.9 (s), 69.5 (s), 68.5 (s), 68.3 (s),
67.1 (s), 53.7 (s), 50.2 (s), 48.7 (s), 42.2 (s), 40.5 (s), 28.1 (s),
17.3 (s); LRFAB m/e 542 ([M + H]+); HRFAB calcd for
C21H44N5O11 ([M + H]+) m/e 542.3037, measured m/e 542.3055.
general procedure for the synthesis kanamycin B analogues.
1H NMR (400 MHz, D2O) (chloride salt) δ 5.98 (d, J ) 3.9 Hz,
1H, H-1′), 5.03 (d, J ) 3.7 Hz, 1H, H-1′′), 3.9-4.2 (m, 6H),
3.8-3.9 (m, 2H), 3.79 (dd, J ) 10.1 Hz, J ) 9.1 Hz, 1H), 3.70
(dd, J ) 10.5 Hz, J ) 6.5 Hz, 1H), 3.4-3.6 (m, 6H), 3.37 (dd,
J ) 9.9 Hz, J ) 10.0 Hz, 1H), 3.30 (dd, J ) 13.6 Hz, J ) 6.6
Hz, 1H), 3.17 (dd, J ) 13.3 Hz, J ) 9.9 Hz, 1H), 3.01 (dd, J )
13.3 Hz, J ) 9.1 Hz, 1H), 2.53 (ddd, J ) 12.6 Hz, J ) 4.2 Hz,
J ) 4.2 Hz, 1H, H-2eq), 1.96 (ddd, J ) 12.6 Hz, J ) 12.6 Hz, J
) 12.6 Hz, 1H, H-2ax), 1.32 (d, J ) 6.3 Hz, 3H, H-6′′); 13C NMR
(100 MHz, D2O) (chloride salt) δ 100.8 (s), 96.0 (s), 83.9 (s),
80.0 (s), 77.4 (s), 74.5 (s), 73.6 (s), 70.8 (s), 69.5 (s), 68.52 (s),
68.44 (s, 2 carbons), 67.1 (s), 53.9 (s), 53.7 (s), 50.0 (s), 48.6
(s), 41.8 (s), 40.3 (s), 28.1 (s), 17.3 (s); LRFAB m/e 541 ([M +
H]+); HRFAB calcd for C21H45N6O10 ([M + H]+) m/e 541.3197,
measured m/e 541.3191; LRFAB m/e 541 ([M + H]+); HRFAB
calcd for C21H45N6O10 ([M + H]+) m/e 541.3197, measured m/e
541.3191.
6-O-(6-Deoxy-4-O-((S)-2,3-diaminopropyl)-r-D-glucopy-
ranosyl)neamine (51). Refer to the general procedure for the
1
synthesis kanamycin B analogues. H NMR (400 MHz, D2O)
(chloride salt) δ 5.98 (d, J ) 3.7 Hz, 1H, H-1′), 4.98 (d, J ) 3.6
Hz, 1H, H-1′′), 4.0-4.1 (m, 7H), 3.7-3.9 (m, 4H), 3.3-3.6 (m,
7H), 3.29 (dd, J ) 13.5 Hz, J ) 7.0 Hz, 1H), 3.14 (dd, J ) 9.4
Hz, J ) 9.4 Hz, 1H), 2.53 (ddd, J ) 12.7 Hz, J ) 3.9 Hz, J )
3.9 Hz, 1H, H-2eq), 1.97 (ddd, J ) 12.7 Hz, J ) 12.5 Hz, J )
12.5 Hz, 1H, H-2ax), 1.27 (d, J ) 6.2 Hz, 3H, H-6′′); 13C NMR
(100 MHz, D2O) (chloride salt) δ 101.9 (s), 95.9 (s), 83.81 (s),
83.78 (s), 77.4 (s), 74.3 (s), 72.7 (s), 72.2 (s), 70.9 (s), 69.5 (s,
two carbons), 68.4 (s), 68.2 (s), 53.7 (s), 50.2 (s), 49.2 (s), 48.7
(s), 40.4 (s), 38.8 (s), 28.1 (s), 17.4 (s); LRFAB m/e 541 ([M +
H]+); HRFAB calcd for C21H45N6O10 ([M + H]+) m/e 541.3197,
measured m/e 541.3192.
Acknowledgment. This work is supported by DAR-
PA (Grant DAAD 19-03-1-0050). We also acknowledge
Utah State University (CURI Grant), National Founda-
tion for Infectious Diseases (New Investigator Matching
Grant), and Frontier Scientific Inc. for generous finan-
cial support. We also thank Prof. Mobashery from Notre
Dame University for the generous gift of the pTZ19U-3
and pSF815 plasmids.
6-O-(4-O-((S)-3-Amino-2-hydroxylpropyl)-6-deoxy-r-D-
glucopyranosyl)neamine (52). Refer to the general proce-
dure for the synthesis kanamycin B analogues. 1H NMR (400
MHz, D2O) (chloride salt) δ 5.93 (d, J ) 3.9 Hz, 1H, H-1′), 4.96
(d, J ) 3.7 Hz, 1H, H-1′′), 3.9-4.1 (m, 5H), 3.7-3.8 (m, 6H),
3.4-3.5 (m, 5H), 3.26 (dd, J ) 13.6 Hz, J ) 6.9 Hz, 1H), 3.18
(dd, J ) 13.2 Hz, J ) 3.3 Hz, 1H), 3.06 (dd, J ) 9.4 Hz, J )
9.4 Hz, 1H), 3.02 (dd, J ) 13.2 Hz, J ) 8.8 Hz, 1H), 2.47 (ddd,
J ) 12.6 Hz, J ) 3.9 Hz, J ) 3.9 Hz, 1H, H-2eq), 1.88 (ddd, J
) 12.6 Hz, J ) 12.6 Hz, J ) 12.6 Hz, 1H, H-2ax), 1.26 (d, J )
6.3 Hz, 3H, H-6′′); 13C NMR (100 MHz, D2O) (chloride salt) δ
101.8 (s), 96.0 (s), 84.0 (s), 83.9 (s), 78.1 (s), 74.4 (s), 74.3 (s),
72.6 (s), 72.1 (s), 70.9 (s), 69.4 (s), 68.6 (s), 68.3 (s), 66.9 (s),
53.8 (s), 50.2 (s), 48.6 (s), 42.2 (s), 40.4 (s), 28.6 (s), 17.3 (s);
LRFAB m/e 542 ([M + H]+); HRFAB calcd for C21H44N5O11 ([M
+ H]+) m/e 542.3037, measured m/e 542.3018.
Supporting Information Available: HPLC and mass
spectrometric results for kanamycin B analogues. This mate-
rial is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Reviews: (a) Haddad, J.; Kotra, L. P.; Mobashery, S. In Glyco-
chemistry: Principles, Synthesis, and Applications; Wang, P. G.,
Bertozzi, C. R., Ed.; Marcel Dekker: New York, 2001; p307-
424. (b) Kondo, S. Development of arbekacin and synthesis of
new derivatives stable to enzymatic modifications by methicillin-
resistant Staphyloccus aureus. Jpn. J. Antibiot. 1994, 47, 561-
574. (c) Vakulenko, S. B.; Mobashery, S. Versatility of aminogly-
cosides and prospects for their future. Clin. Microbiol. Rev. 2003,
16, 430-450. (d) Hooper, I. R. Aminoglycoside Antibiotics;
Springer-Verlag: New York, 1982.
(2) (a) Mingeot-Leclercq, M.-P.; Glupczynski, Y.; Tulkens, P. M.
Aminoglycosides: activity and resistance. Antimicrob. Agents
Chemother. 1999, 43, 727-737. (b) Kotra, L. P.; Haddad, J.;
Mobashery, S. Aminoglycosides: Perspectives on mechanisms
of action and resistance and strategies to counter resistance.
Antimicrob. Agents Chemother. 2000, 44, 3249-3256. (c) Wright,
G. D. Aminoglycoside-modifying enzymes. Curr. Opin. Microbiol.
1999, 2, 499-503.
(3) (a) Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D.
Structure of the A Site of Escherichia coli 16S ribosomal RNA
complexed with an aminoglycoside antibiotic. Science 1996, 274,
1367-1371. (b) Ma, C.; Baker, N. A.; Joseph, S.; McCammon, J.
A. Binding of aminoglycoside antibiotics to the small ribosomal
subunit: A continuum electrostatics investigation. J. Am. Chem.
Soc. 2002, 124, 1438-1442. (c) Fourmy, D.; Recht, M. I.; Puglisi,
J. D. Binding of neomycin-class aminoglycoside antibiotics to the
A-site of 16S rRNA. J. Mol. Biol. 1998, 277, 347-362.
(4) (a) Fong, D. H.; Berghuis, A. M. Substrate promiscuity of an
aminoglycoside antibiotic resistance enzyme via target mimicry.
EMBO J. 2002, 21, 2323-2331. (b) Owston, M. A.; Serpersu, E.
H. Cloning overexpression, and purification of aminoglycoside
antibiotic 3-acetyltransferase-IIIb: conformational studies with
bound substrates. Biochemistry 2002, 41, 10764-10770. (c)
Pedersen, L. C.; Benning, M. M.; Holden, H. M. Structural
investigation of the antibiotic and ATP-binding sites in kana-
mycin nucleotidyltransferase. Biochemistry 1995, 34, 13305-
13311. (d) Cox, J. R.; McKay, G. A.; Wright, G. D.; Serpersu, E.
H. Arrangement of substrates at the active site of an aminogly-
coside antibiotic 3′-pohosphotransferase as determined by NMR.
J. Am. Chem. Soc. 1996, 118, 1295-1301. (e) Sakon, J.; Liao,
H. H.; Kanikula, A. M.; Benning, M. M.; Rayment, I.; Holden,
H. M. Molecular structure of kanamycin nucleotidyltransferase
determined to 3.0 Å resolution. Biochemistry 1993, 32, 11977-
11984. (f) DiGiammarino, E. L.; Draker, K.-a.; Wright, G. D.;
Serpersu, E. H. Solution studies of isepamicin and conforma-
tional comparisons between isepamicin and buritiroson A when
6-O-(6-Deoxy-4-O-((R)-2,3-diaminopropyl)-r-D-glucopy-
ranosyl)neamine (53). Refer to the general procedure for the
1
synthesis kanamycin B analogues. H NMR (400 MHz, D2O)
(chloride salt) δ 5.97 (d, J ) 3.9 Hz, 1H, H-1′), 4.96 (d, J ) 3.7
Hz, 1H, H-1′′), 4.0-4.1 (m, 6H), 3.8-3.9 (m, 3H), 3.7-3.8 (m,
2H), 3.5-3.6 (m, 2H), 3.4-3.5 (m, 5H), 3.26 (dd, J ) 13.6 Hz,
J ) 7.0 Hz, 1H), 3.13 (dd, J ) 9.4 Hz, J ) 9.4 Hz, 1H), 2.50
(ddd, J ) 12.6 Hz, J ) 3.9 Hz, J ) 3.9 Hz, 1H, H-2eq), 1.96
(ddd, J ) 12.6 Hz, J ) 12.6 Hz, J ) 12.6 Hz, 1H, H-2ax), 1.25
(d, J ) 6.2 Hz, 3H, H-6′′); 13C NMR (100 MHz, D2O) (chloride
salt) δ 101.8 (s), 95.8 (s), 83.8 (s, two carbons), 77.4 (s), 74.3
(s), 72.4 (s), 72.2 (s), 70.9 (s), 69.6 (s), 69.5 (s), 68.5 (s), 68.1
(s), 53.8 (s), 50.2 (s), 49.2 (s), 48.7 (s), 40.5 (s), 39.1 (s), 28.2
(s), 17.4 (s); LRFAB m/e 541 ([M + H]+); HRFAB calcd for
C21H45N6O10 ([M + H]+) m/e 541.3197, measured m/e 541.3190.
6-O-(4-O-((R)-3-Amino-2-((R)-3-amino-2-hydroxylpropyl)-
propyl)-6-deoxy-r-D-glucopyranosyl)neamine (54). Refer
to the general procedure for the synthesis kanamycin B
1
analogues. H NMR (400 MHz, D2O) (chloride salt) δ 5.95 (d,
J ) 3.9 Hz, 1H, H-1′), 4.94 (d, J ) 3.8 Hz, 1H, H-1′′), 3.9-4.0
(m, 6H), 3.7-3.9 (m, 8H), 3.5-3.6 (m, 2H), 3.2-3.3 (m, 4H),
3.0-3.1 (m, 2H), 2.49 (ddd, J ) 12.6 Hz, J ) 4.1 Hz, J ) 4.1
Hz, 1H, H-2eq), 1.92 (ddd, J ) 12.6 Hz, J ) 12.6 Hz, J ) 12.6
Hz, 1H, H-2ax), 1.22 (d, J ) 6.2 Hz, 3H, H-6′′); 13C NMR (100
MHz, D2O) (chloride salt) δ 102.8 (s), 95.9 (s), 83.8 (s), 77.4
(s), 75.4 (s), 74.3 (s), 72.5 (s), 72.3 (s), 71.1 (s), 70.9 (s), 70.8
(s), 69.5 (s), 68.4 (s), 68.1 (s), 66.9 (s), 53.7 (s), 50.1 (s), 48.6
(s), 42.0 (s, two carbons), 40.9 (s), 40.5 (s), 28.1 (s), 17.4 (s);
LRFAB m/e 615 ([M + H]+); HRFAB calcd for C24H51N6O12 ([M
+ H]+) m/e 615.3565, measured m/e 615.3589.
6-O-(3-Amino-4-O-((S)-3-amino-2-hydroxylpropyl)-3,6-
dideoxy-r-D-glucopyranosyl)neamine (55). Refer to the

Optimization of Aminoglycosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6285
bound to an aminoglycoside 6′-N-acetyltransferase determined
by NMR spectroscopy. Biochemistry 1998, 37, 3638-3644. (g)
Burk, D. L.; Hon, W. C.; Leung, A. K.-W.; Berghuis, A. M.
Structural analyses of nucleotide binding to an aminoglycoside
phosphotransferase. Biochemistry 2001, 40, 8756-8764.
43, 6496-6500. (k) Agnelli, F.; Sucheck, S. J.; Marby, K. A.;
Rabuka, D.; Yao, S.-L.; Sears, P. S.; Liang, F.-S.; Wong, C.-H.
Dimeric aminoglycosides as antibiotics. Angew. Chem., Int. Ed.
2004, 43, 1562-1566. (l) Bryan, M. C.; Wong, C.-H. Aminogly-
coside array for the high-throughput analysis of small molecule-
RNA interactions. Tetrahedron Lett. 2004, 45, 3639-3642.
(6) Elchert, B.; Li, J.; Wang, J.; Hui, Y.; Rai, R.; Ptak, R.; Ward, P.;
Takemoto, J. Y.; Bensaci, M.; Chang, C.-W. T. Application of the
synthetic aminosugars for glycodiversification: Synthesis and
antimicrobial studies of pyranmycin. J. Org. Chem. 2004, 69,
1513-1523.
(7) Li, J.; Wang, J.; Czyryca, P. G.; Chang, H.; Orsak, T. W.;
Evanson, R.; Chang, C.-W. T. Application of glycodiversifica-
tion: Expedient synthesis and antibacterial evaluation of a
library of kanamycin B analogs. Org. Lett. 2004, 6, 1381-1384.
(8) Phenylthioglycopyranosides can be prepared from treating the
corresponding acetyl glycopyranosides with HSPh and BF3-
OEt3. For reference, see the following. Tai, C.-A.; Kulkarni, S.
S.; Hung, S.-C. Facile Cu(OTf)2-catalyzed preparation of per-O-
acetylated hexopyranoses with stoichiometric acetic anhydride
and sequential one-pot anomeric substitution to thioglycosides
under solvent-free conditions. J. Org. Chem. 2003, 68, 8719-
8722.
(9) Garegg, P. J. In Preparative Carbohydrate Chemistry; Hanessian,
S., Ed.; Marcel Dekker: New York, 1997; pp 53-67.
(10) Methods for Dilution Antimicrobial Susceptibility Testing for
Bacteria That Grow Aerobically (Approved Standard M7-A5) and
Performance Standards for Antimicrobial Disk Susceptibility
Tests (Approved Standard M2-A7); National Committee for
Clinical Laboratory Standards: Wayne, PA.
(11) One resistant strain is E. coli (TG1) equipped with the pTZ19U-3
plasmid encoded for APH(3′)-I. The other resistant strain is E.
coli (TG1) equipped with the pSF815 plasmid encoded for AAC-
(6′) and APH(2′′).
(12) The structure is based on PDB 1L8U with bound neomycin B
and ADP: Fong, D. H.; Berghuis, A. M. Substrate promiscuity
of an aminoglycoside antibiotic resistance enzyme via target
mimicry. EMBO J. 2002, 21, 2323-2331.
(5) For examples, see the following. (a) Tanaka, H.; Nishida, Y.;
Furuta, Y.; Kobayashi, K. A convenient synthesis pathway for
multivalent assembly of aminoglycoside antibiotics starting from
amikacin. Bioorg. Med. Chem. Lett. 2002, 12, 1723-1726. (b)
Hanessian, S.; Tremblay, M.; Swayze, E. E. Tobramycin analogs
with C-5 aminoalkyl ether chains intended to mimic rings III
and IV of paromomycin. Tetrahedron 2003, 59, 983-993. (c)
Hanessian, S.; Kornienko, A.; Swayze, E. E. Probing the
functional requirements of the L-haba side-chain of amikacin-
synthesis, 16S A-site rRNA binding, and antibacterial activity.
Tetrahedron 2003, 59, 995-1007. (d) Seeberger, P. H.; Baumann,
M.; Zhang, G.; Kanemitsu, T.; Swayze, E. E.; Hofstadler, S. A.;
Griffey, R. H. Synthesis of neomycin analogs to investigate
aminoglycoside-RNA interactions. Synlett 2003, 1323-1326. (e)
Chou, C.-H.; Wu, C.-S.; Chen, C.-H.; Lu, L.-D.; Kulkarni, S. S.;
Wong, C.-H.; Hung, S.-C. Regioselective glycosylation of neamine
core: a facile entry to kanamycin B related analogues. Org. Lett.
2004, 6, 585-588. (f) Ding, Y.; Hofstadler, S. A.; Swayze, E. E.;
Risen, L.; Griffey, R. H. Design and Synthesis of paromomycin-
related heterocycle-substituted aminoglycoside mimetics based
on a mass spectrometry RNA-binding assay. Angew. Chem., Int.
Ed. 2003, 42, 3409-3412. (g) Francois, B.; Szychowski, J.;
Adhikari, S. S.; Pachamuthu, K.; Swayze, E. E.; Griffey, R. H.;
Migawa, M. T.; Westhof, E.; Hanessian, S. Antibacterial ami-
noglycosides with a modified mode of binding to the ribosomal-
RNA decoding site. Angew. Chem., Int. Ed. 2004, 43, 6735-6738.
(h) Fridman, M.; Belakhov, V.; Lee, L. V.; Liang, F.-S.; Wong,
C.-H.; Baasov, T. Dual effect of synthetic aminoglycosides:
antibacterial activity against Bacillus anthracis and inhibition
of anthrax lethal factor. Angew. Chem., Int. Ed. 2005, 44, 447-
452. (i) Fridman, M.; Belakhov, V.; Yaron, S.; Baasov, T. A new
class of branched aminoglycosides: Pseudo-pentasaccharide
derivatives of neomycin B. Org, Lett. 2003, 5, 3575-3578. (j)
Liang, F.-S.; Wang, S.-K.; Nakatani, T.; Wong, C.-H. Targeting
RNAs with tobramycin analogues. Angew. Chem., Int. Ed. 2004,
JM050368C