Design and synthesis of new aminoglycoside antibiotics containing neamine as an optimal core structure: Correlation of antibiotic activity with in vitro inhibition of translation

Article abstract of DOI:10.1021/ja9910356

The structure and activity of the pseudodisaccharide core found in aminoglycoside antibiotics was probed with a series of synthetic analogues in which the position of amino groups was varied around the glucopyranose ring. The naturally occurring structure

Full text of DOI:10.1021/ja9910356

J. Am. Chem. Soc. 1999, 121, 6527-6541
6527
Design and Synthesis of New Aminoglycoside Antibiotics Containing
Neamine as an Optimal Core Structure: Correlation of Antibiotic
Activity with in Vitro Inhibition of Translation
William A. Greenberg, E. Scott Priestley, Pamela S. Sears, Phil B. Alper,
Christoph Rosenbohm, Martin Hendrix, Shang-Cheng Hung, and Chi-Huey Wong*
Contribution from the Department of Chemistry and Skaggs Institute of Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
ReceiVed March 31, 1999
Abstract: The structure and activity of the pseudodisaccharide core found in aminoglycoside antibiotics was
probed with a series of synthetic analogues in which the position of amino groups was varied around the
glucopyranose ring. The naturally occurring structure neamine was the best in the series according to assays
for in vitro RNA binding and antibiotic activity. With this result in hand, neamine was used as a common core
structure for the synthesis of new antibiotics, which were evaluated for binding to models of the Escherichia
coli 16S A-site ribosomal RNA, in vitro protein synthesis inhibition, and antibiotic activity. Analysis of RNA
binding revealed some correlation between the relative affinity and specificity of RNA binding and antibacterial
efficacy. However, the correlation was not linear. This result led us to develop the in vitro translation assay
in an effort to better understand aminoglycoside-RNA interactions. A linear correlation between in vitro
translation inhibition and antibiotic activity was observed. In addition, IC50s in the protein synthesis assay
were typically lower than the Kds obtained for RNA binding, suggesting that binding of these compounds to
intact ribosomes is tighter in these cases than binding to the model RNA oligonucleotides. This reflects possible
differences in RNA conformation between intact ribosomes and the free RNA of the model system, or possible
high-affinity ribosomal binding sites in addition to the A-site RNA.
Introduction
As is the case for all classes of antibiotics, the emergence of
resistance has resulted in a need for new structures with anti-
biotic activity.12 Because of the unwieldy size and dense func-
tionality of aminoglycosides such as neomycin (Figure 1), an
important concern for the design of new compounds is the iden-
tification of smaller, simpler structures which retain the activity
of the larger parent structures. Analysis of the structural elements
of naturally occurring aminoglycosides reveals that the vast
majority contains the meso-1,3 diaminocyclitol 2-deoxystreptamine
(Figure 1), glycosylated at the 4-position, as well as the 5- (neo-
mycin class) or 6- (kanamycin-gentamicin class) position with
a variety of aminosaccharides. Several aminoglycosides (i.e.,
neomycin, ribostamycin, kanamycin B) share a common pseudo-
disaccharide core, 4-O-(2,6-diamino-2,6-dideoxy-R-D-glucopy-
ranosyl)-2-deoxystreptamine, commonly known as neamine (1,
Figure 1). Many other substitution patterns on the glucopyranose
are known, such as 2-amino-2-deoxyglucopyranose in paromo-
mycin, 6-amino-6-deoxyglucopyranose in kanamycin A, 4-amino-
4-deoxyglucopyranose in apramycin, and various deoxygenated
analogues, such as in tobramycin and gentamicin.
We sought to probe the structure of this pseudodisaccharide
core to find the optimal substitution pattern as a starting point
for synthesis of new potential antibiotics. A series of pseudo-
disaccharides with amino groups at variable positions (1-6)
was synthesized (Figure 2). These compounds were analyzed
for binding to models of the Escherichia coli 16S A-site
ribosomal RNA and for antibacterial activity. The optimal
structure, 1, was then incorporated as a core structure into a
series of analogues (7-15) appended with various polyamino,
The aminoglycosides1 are a group of clinically important
antibiotics which function through binding to specific sites in
prokaryotic ribosomal RNA and affecting the fidelity of protein
synthesis.2-4 They have served as a paradigm for studying small
molecule-RNA interactions in a rapidly growing number of
structural5-7 and chemical8-11 efforts.
(1) Umezawa, H., Hooper, I. R., Eds. Aminoglycoside Antibiotics;
Springer-Verlag: New York, Heidelberg, 1982.
(2) Cundliffe, E. Recognition sites for antibiotics within rRNA. In The
Ribosome: Structure, function, and eVolution; Hill, W. E., et al., Eds.;
American Society for Microbiology: Washington, DC, 1990; pp 479-490.
(3) Moazed, D.; Noller, H. F. Nature 1987, 327, 389-394.
(4) Woodcock, J.; Moazed, D.; Cannon, M.; Davies, J.; Noller, H. F.
EMBO J. 1991 10, 3099-3103.
(5) Fourmy, D.; Recht, M. I.; Blanchard, S. C.; Puglisi, J. D. Science
1996, 274, 1367-1371.
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347-362. (b) Fourmy, D.; Yoshizawa, S.; Puglisi, J. D. J. Mol. Biol. 1998,
277, 333-345.
(7) Yoshizawa, S.; Fourmy, D.; Puglisi, J. D. EMBO J. 1998, 17, 6437-
6448.
(8) (a) Hendrix, M.; Priestley, E. S.; Joyce, G. F.; Wong, C.-H. J. Am.
Chem. Soc. 1997, 119, 3641-3648. (b) Alper, P. B.; Hendrix, M.; Sears,
P.; Wong, C.-H. J. Am. Chem. Soc. 1998, 120, 1965-1978. (c) Wong, C.-
H.; Hendrix, M.; Priestley, E. S.; Greenberg, W. A. Chem. Biol 1998, 5,
397-406. (d) Wong, C.-H.; Hendrix, M.; Manning, D. D.; Rosenbohm,
C.; Greenberg, W. A. J. Am. Chem. Soc. 1998, 120, 8319-8327.
(9) (a)Wang, H.; Tor, Y. Angew. Chem., Int. Ed. Engl. 1998, 37, 109-
111. (b) Wang, H.; Tor, Y. J. Am. Chem. Soc. 1997, 119, 8734-8735.
(10) (a) Cho, J.; Hamasaki, K.; Rando, R. R. Biochemistry 1998, 37,
4985-4992. (b) Wang, Y.; Hamasaki, K.; Rando, R. R. Biochemistry 1997,
36, 768-779.
(11) Mei, H.-Y.; Cui, M.; Heldsinger, A.; Lemrow, S. M.; Loo, J. A.;
Sannes-Lowery, K. A.; Sharmeen, L.; Czarnik, A. W. Biochemistry 1998,
37, 14204-14212.
(12) Davies, J. Science 1994, 264, 375-382.
10.1021/ja9910356 CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/02/1999

6528 J. Am. Chem. Soc., Vol. 121, No. 28, 1999
Greenberg et al.
Scheme 1. Synthesis of Glycosyl Acceptors 17 and 20a
Figure 1. Structures of neomycin B, the diaminocyclitol 2-deoxy-
streptamine, which is found in most aminoglycoside antibiotics, and
the pseudodisaccharide neamine (1).
a Reagents and conditions: (a) TfN3, Et3N, CuSO4, MeOH/CH2Cl2.
(b) Ac2O, pyridine, 75% over two steps. (c) Candida antarctica lipase,
1:1 toluene/phosphate buffer, pH 6.2, 71% (95% based on recovered
starting material). (d) NaOMe, MeOH, 100%. (e) (i) Ley bis-
dihydropyran, CSA, CHCl3; (ii) Ac2O, DMAP, pyridine, 50% over two
steps. (f) NaOMe, MeOH/dioxane, 90%.
2-deoxystreptamine, to differentiate the 4-hydroxyl group from
the 5- and 6-hydroxyl groups. Two different procedures, one
enzymatic and the other chemical, were explored (Scheme 1).
The enzymatic approach relied on an enantioselective deacety-
lation using a resin-immobilized lipase. 2-Deoxystreptamine13
was converted in 75% overall yield to the diazido triacetyl
derivative 16 by Cu2+-catalyzed azido transfer with triflic
azide,14 followed by treatment with acetic anhydride and
4-DMAP in pyridine. Compound 16 was selectively deacetylated
with Novozym 435 (Candida antarctica lipase immobilized on
a macroporous acrylic resin; Novo Nordisk), providing 17 in
Figure 2. Structures of compounds 2-15.
amino alcohol, or aromatic substitutions which were designed
to improve RNA binding affinity and potentially antibacterial
activity. These compounds were tested for RNA binding and
antibacterial activity, as well as for their ability to inhibit protein
synthesis in vitro, using a luciferase-based assay. The results
of the latter assay correlate well in a qualitative sense with
antibacterial activity.
Results and Discussion
(13) Georgiadas, M. P.; Constantinou-Kokotou, V. J. Carbohydr. Chem.
1991, 10, 739-748.
(14) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett. 1996,
37, 6029-6032.
Synthesis of Glycosyl Acceptor. Synthesis of pseudodisac-
charides 2-6 required desymmetrization of the meso compound

New Aminoglycoside Antibiotics Containing Neamine
J. Am. Chem. Soc., Vol. 121, No. 28, 1999 6529
71% yield (95% based on recovered starting material). The
absolute stereochemistry of 17 was confirmed by showing that
synthetic paromamine (3) derived from 17 was identical in all
respects to paromamine obtained from the natural source (see
Experimental Section).
Scheme 2. Synthesis of Glycosyl Donors 22, 24, 29, and
33a
The second desymmetrization approach utilized the dispiro-
ketal protection/desymmetrization chemistry of Ley and co-
workers.15 Compound 16 was deacetylated (catalytic NaOMe,
MeOH, 100%) to provide 18. When treated with Ley’s (2R,2′R)-
PDHP reagent15 and catalytic camphorsulfonic acid in refluxing
chloroform, followed by acetylation, 19 was obtained as the
major isomer in 50% yield. The structure of 19 was confirmed
by solving its crystal structure (Scheme 1). Deacetylation
(catalytic NaOMe in MeOH/dioxane, 90%) provided 20, the
substrate for selective glycosylation at the 4-position. However,
attempts at glycosylation of 20 provided only the undesired â
anomer, likely due to the steric constraints of the dispiroketal
protecting group (data not shown). Therefore, glycosyl acceptor
17 was used for the synthesis of 2-6.
Synthesis of Glycosyl Donors. Scheme 2 outlines the
preparation of the glycosyl donors used in synthesizing 2-6.
Compound 22, the donor for preparing 3, was produced by
benzylation (BnBr, NaH, DMF, 60%) of thioglycoside 23.16
The 3-azido donor 24 was synthesized from known diaceto-
nide 2517 in five steps. Acetonide cleavage (90% TFA/H2O)
and peracetylation (Ac2O, NaOAc) provided glucopyranoside
26 in 59% yield. Conversion to the â-phenylthioglycoside 27
(thiophenol, BF3‚OEt2, CHCl3, 64%), acetate removal (NaOMe,
MeOH, 100%), and benzylation (BnBr, NaH, DMF, 85%)
provided donor 24.
The 4-azido donor 29 was synthesized from galactose
benzylidene acetal 30.18 Regioselective reduction of the ben-
zylidene acetal (BH3‚Me3N, AlCl3, THF, 85%)19 provided 31,
which was mesylated (CH3SO2Cl, pyridine, 73%). The mesylate
was displaced with sodium azide in DMF, providing 29 in 84%
yield. The 6-azido donor 33 was synthesized from 3420 by
tosylation of the primary 6-hydroxyl (TsCl, pyridine, 75%),
displacement with sodium azide in DMF (90%), and benzylation
(BnBr, NaH, DMF, 57%).
Synthesis of Pseudodisaccharides 2-6. 2-Deoxystreptamine
acceptor 17 was glycosylated with thioglycoside donors21 21,
22, 24, 29, and 33 (N-iodosuccinimide, triflic acid, Et2O/
CH2Cl2, -15 °C), providing pseudodisaccharides 37-41,
respectively, after deacetylation, in 58-100% yield (Scheme
3). The R/â selectivity of the glycosylation reactions was
typically in the range of 10:1 for these substrates. The use of
diethyl ether as cosolvent was crucial to this selectivity. In
dichloromethane at -60 °C, the R/â selectivity was only 2.5:1,
and the overall yield of 37 was poor. Reduction of azides and
benzyl ethers in a single step proved problematic, but complete
deprotection was effected in moderate to excellent yield over
two steps. The azides were reduced with hydrazine22 (H2NNH2,
a Reagents and conditions: (a) BnBr, NaH, DMF, 60%. (b) (i) 90%
TFA/H2O; (ii) Ac2O, NaOAc, 59% over two steps. (c) Thiophenol,
BF3‚OEt2, CHCl3, 64%. (d) NaOMe, MeOH, 100%. (e) BnBr, NaH,
DMF, 85%. (f) BH3‚Me3N, AlCl3, THF, 85%. (g) MsCl, pyridine, 73%.
(h) NaN3, DMF, 100 °C, 84%. (i) TsCl, pyridine, 75%. (j) NaN3, DMF,
80 °C, 90%. (k) BnBr, NaH, DMF, 57%.
20% Pd(OH)2/C, Degussa type, MeOH, reflux), followed by
benzyl ether cleavage (H2 (1 atm), 20% Pd(OH)2/C, Degussa
type, AcOH/H2O), providing 2-6.
Synthesis of Neamine Derivatives 7-15. Scheme 4 outlines
the synthesis of neamine derivatives 7-14. The best route to
the pseudodisaccharide building block 43, in which the 5-hy-
droxyl group of 2-deoxystreptamine is differentiated from the
other hydroxyl groups, begins from inexpensive neomycin.
Protection of the amino groups as azides using triflic azide14
was followed by peracetylation for the purpose of easier puri-
fication from byproducts of the azido transfer reaction and de-
acetylation. This material was benzylated (NaH, benzyl bromide,
tetrabutylammonium iodide, DMF, 80%), providing protected
neomycin 42 in 46% overall yield from neomycin sulfate. The
â-ribofuranosyl bond of neomycin was selectively cleaved with
1 N HCl in methanol/dioxane, providing building block 43 in
71% yield after recrystallization. Under these conditions, some
cleavage of the â-L-idopyranosyl bond occurs, but little or no
cleavage of the key R-L-glucopyranosyl bond occurred. The
methyl glycoside of the lower half of neomycin (neobiosamine)
was also obtained, albeit in lower yield due to the partial
cleavage of the aforementioned â-L-idose glycosidic bond.
(15) Downham, R.; Edwards, P. J.; Entwistle, D. A.; Kim, K. S.; Ley,
S. V. Tetrahedron: Asymmetry 1995, 6, 2403-2440.
(16) Buskas, T.; Garegg, P. J.; Konradsson, P.; Maloisel, J.-L. Tetrahe-
dron: Asymmetry 1994, 5, 2187-2194.
(17) Sleath, P. R.; Handlon, A. L.; Oppenheimer, N. J. J. Org. Chem.
1991, 56, 3608-3613.
(18) Liptak, A.; Jodal, I.; Harangi, J.; Nanasi, P. Acta Chim. Hung. 1983,
113, 415-422.
(19) Classon, B.; Garegg, P. J.; Helland, A.-C. J. Carbohydr. Chem. 1989,
8, 543.
(20) Nicolaou, K. C.; Winssinger, N.; Pastor, J.; DeRoose, F. J. Am.
Chem. Soc. 1997, 119, 449-450.
(21) Garegg, P. J. AdV. Carbohydr. Chem. Biochem. 1997, 52, 179.
(22) Malik, A. A.; Preston, J. B.; Archibald, T. G.; Cohen, M. P.; Baum,
K. Synthesis 1989, 450-451.

6530 J. Am. Chem. Soc., Vol. 121, No. 28, 1999
Scheme 3. Synthesis of Pseudodisaccharides 2-6a
Greenberg et al.
a Reagents and conditions: (a) NIS, TfOH, Et2O/CH2Cl2, -15 °C, 30 min. (b) NaOMe, MeOH. (c) H2NNH2, 20% Pd(OH)2/C, MeOH, reflux.
(d) H2, 20% Pd(OH)2/C, AcOH/H2O.
The 5-hydroxyl group of 43 was alkylated (NaH, allyl
bromide, tetrabutylammonium iodide, DMF, 99%), giving 44,
which was subjected to ozonolysis and reductive amination of
the resultant aldehyde 45, providing compounds 46-53. For
substrates in which more than one amine was available to react
during reductive amination, all but one amine was protected as
the benzyl carbamate. In the case of 46, the secondary amine
formed in the reductive amination step was protected as a benzyl
carbamate before proceeding with the next step. This step was
found to be unnecessary and was not performed on the other
compounds in this series. One-step deprotection of all protecting
groups by catalytic hydrogenation was problematic, as was
observed with compounds 2-6. In this case, the best depro-
tection protocol relied on first reducing the azides by a
Staudinger reaction (PMe3, THF/H2O), followed by benzyl ether
and benzyl carbamate reduction with sodium in ammonia,
providing compounds 7-14 after cation-exchange chromatog-
raphy.
Because the deprotection procedure described in Scheme 4
did not allow introduction of aromatic substituents, a new
procedure was sought which would efficiently reduce at least
four azides and three benzyl ethers. After much investigation,
the conditions shown in Scheme 5 were developed and used to
synthesize benzimidazole-containing neamine derivative 15.
Benzimidazole has been used as an arginine mimic.23 Protected
compound 54 was subjected to transfer hydrogenation (H2NNH2,
Raney Ni, EtOH),22 followed by catalytic hydrogenolysis under
acidic conditions (H2 (1 atm), Pd(OH)2/C (Degussa type),
AcOH/H2O), to provide 15.
is implicated for the antibiotic activity of this class of molecules.
By studying the interactions of aminoglycosides with small
model RNA sequences, it is expected that a better understanding
of the molecular basis of their antibiotic activity will be gained,
thereby allowing the design of antibiotics with improved profiles
with respect to toxicity and resistance. Compounds 1-6 were
screened on a streptavidin-modified SPR chip for RNA binding
against three related 5′-biotinylated RNA oligonucleotides which
have served as A-site model sequences (Figure 3) in our
previous studies.8 AS-wt contains the wild-type sequence from
E. coli, U1406A contains a single-base mutation which improves
binding of aminoglycosides, and U1495A contains a single-
base mutation which is deleterious to binding. The improved
binding to the U1406A mutant is rationalized by forming an
A-U base pair with U1495, which forms a critical hydrogen
bond to the aminoglycoside paromomycin according to the NMR
structure.5 In the wild-type sequence, U1495 is mispaired to
U1406 and likely is less conformationally well-defined. The
U1495 mutation is deleterious to binding because the same
U1495 that forms a hydrogen bond to the aminoglycoside has
been replaced by an adenine.
Data obtained from the SPR screen are illustrated in Figure
4 and summarized in Table 1. Neamine was found to be the
most effective and specific A-site binder, although the 2- and
6-amino derivatives 3 and 6 did have quite good affinity for
the U1406A mutant. More importantly, neamine was the most
active antibiotic. In fact, of compounds 2-6, only 6 showed
any activity in the Kirby-Bauer disk assay24 against E. coli,
Pseudomonas aeruginosa, or Staphylococcus aureus. Its mini-
mum inhibitory concentration (MIC)25 was less than that of
RNA Binding and Antibiotic Activity of 2-6. For several
years, we have used surface plasmon resonance (SPR) to probe
the interactions of aminoglycosides and synthetic analogues with
RNA.8 In particular, we have been interested in specific binding
to prokaryotic 16S ribosomal A-site RNA, the binding site which
(24) Phillips, I.; Williams, D. In Laboratory Methods in Antimicrobial
Chemotherapy; Garrod, L., Ed.; Churchill Livingstone Press: Edinburgh,
1978; pp 3-30.
(25) (a) Waterworth, P. M. In Laboratory Methods in Antimicrobial
Chemotherapy; Garrod, L., Ed.; Churchill Livingstone Press: Edinburgh,
1978; pp 31-40. (b) Barry, A. L. The Antimicrobic Susceptibility Test:
Principles and Practice; Lea and Febiger: Philadelphia, PA, 1976.
(23) Haubner, R.; Finsinger, D.; Kessler, H. Angew. Chem., Int. Ed. Engl.
1997, 36, 1374-1389.

New Aminoglycoside Antibiotics Containing Neamine
J. Am. Chem. Soc., Vol. 121, No. 28, 1999 6531
Scheme 4. Synthesis of Neamine Derivatives 7-14a
a Reagents and conditions: (a) TfN3, Et3N, CuSO4, MeOH/H2O/CH2Cl2. (b) Ac2O, pyridine. (c) NaOMe, MeOH, 58% from neomycin sulfate.
(d) BnBr, NaH, Bu4NI, DMF, 80%. (e) 1 M HCl, MeOH/dioxane, 71%. (f) allyl bromide, Bu4NI, DMF, 99%. (g) O3, CH2Cl2/MeOH, then Me2S.
(h) RNH2, NaCNBH3, AcOH/MeOH, 33-63% over two steps. (i) PMe3, THF/H2O. (j) Na, NH3/THF, 13-80% over two steps.
Scheme 5. Synthesis of Aromatic Neamine Derivative 15a
a Reagents and conditions: (a) Raney nickel, hydrazine, EtOH. (b)
H2 (1 atm), 20% Pd(OH)2/C, AcOH/H2O, 32% over two steps.
neamine (1) (Table 1). With these results, it was concluded that
neamine (1) was the most promising pseudodisaccharide core
for further development.
RNA Binding and Antibiotic Activity of 7-15. The
diamine, triamine, amino alcohol, and aromatic substitutions on
neamine derivatives 7-15 were chosen for several reasons. The
Figure 3. A-Site model RNA sequences screened by surface plasmon
encouraging results on simplified neomycin analogues8b sug-
resonance for aminoglycoside binding.
gested that linear alkyl diamines might be effective replacements
for more complex aminosaccharides. We were also interested
in probing the effect of neighboring hydroxyl groups, in keeping
RNA binding data obtained by SPR and antibiotic activity
for compounds 7-15 are summarized in Table 2. As expected,
addition of amines to the neamine core typically resulted in a
with the identification of 1,2- and 1,3-amino alcohols as an
effective phosphate-binding motif.26 Finally, the structural
10-20-fold enhancement in RNA binding affinity compared
simplicity of this series, relative to the natural products and
to the parent pseudodisaccharide. Compounds 7-10, 12, and
previously synthesized analogues,8b meant that their synthesis
13 all bound in the submicromolar range. Specificity was slightly
would be easier and more amenable to rapid development of
analogues.
degraded, being in the 2-3-fold range, compared to 4-6-fold
for neamine. There is only a weak correlation between RNA
binding affinity and antibacterial activity. Despite binding more
strongly than neamine, several analogues were weaker antibiot-
(26) Hendrix, M.; Alper, P. B.; Priestley, E. S.; Wong, C.-H. Angew.
Chem., Int. Ed. Engl. 1997, 36, 95-98.

6532 J. Am. Chem. Soc., Vol. 121, No. 28, 1999
Greenberg et al.
Figure 5. Schematic of the in vitro translation assay. A plasmid-borne
luciferase reporter gene is combined with E. coli cytoplasmic S-30
extract, containing transcription and translation machinery, and incu-
bated with necessary cofactors and amino acids for 30 min. Reactions
are then assayed for production of active luciferase.
Figure 4. Representative SPR binding data (equivalents bound as a
function of concentration) for compounds 1-6 to AS-wt RNA.
Table 1. RNA Binding to Variants of the Decoding Region A-Site
and Antibacterial Activity for Compounds 1-6a
MIC, E. coli
ATCC 25922
glycosides.8c Many compounds which are known to be effective
antibiotics, such as gentamicin and ribostamycin, appeared to
be relatively poor A-site binders with respect to both affinity
and specificity. In an effort to understand this discrepancy, we
reasoned that studies on inhibition of whole ribosomes, using a
cell-free protein synthesis assay, could be informative. Direct
study in vitro on the effect of these compounds on translation,
the acknowledged mechanism of antibiotic action, should
provide a more accurate model than the indirect study solely
of RNA binding.
The coupled transcription/protein synthesis assay is illustrated
in Figure 5. A cytoplasmic extract of E. coli (which includes
ribosomes and transcription machinery) is combined with all
amino acids and required cofactors, a plasmid containing the
reporter gene luciferase, and varying concentrations of the
aminoglycoside of interest. After induction with IPTG, the
reaction proceeds for 30 min and is then assayed for expression
of enzymatically active luciferase.
Protein synthesis inhibition data were collected for a series
of compounds of interest, including the aminoglycosides neo-
mycin, gentamicin, and ribostamycin, previously reported
synthetic derivatives 55 and 56,8b neamine, and compounds 12
and 13 from this work (Figure 6). Dose-dependent inhibition
by these compounds is illustrated in Figure 7. The data are
collected and compared to MIC values against E. coli and Kd
values on the AS-wt RNA in Table 3.
The natural products neomycin and gentamicin are equally
potent antibiotics, with measured MICs of 1.6 µM, although
the AS-wt binding data are very different (low nanomolar versus
low micromolar). When IC50 values in the protein synthesis
assay are compared, however, the two compounds appear
similar. Both inhibit translation and, by inference, bind to the
A-site of intact ribosomes at 20-30 nM concentration. The
synthetic derivatives 55 and 56 also inhibit translation in this
range, which is consistent with their exceptional antibiotic
activity, despite their apparently weaker A-site binding compared
to neomycin in the SPR assay. The new translation inhibition
data explain why the synthetic molecule 56 is as potent an
antibiotic as most active naturally occurring aminoglycosides.
Ribostamycin curiously has a MIC (12.5 µM) that is lower than
its Kd against AS-wt (25 µM). However, it inhibits translation
at 100 nM, suggesting that it binds to the intact ribosome at a
250-fold lower concentration than it binds to the A-site model
RNA. Likewise, neamine inhibits translation at a 20-fold lower
concentration than it binds to AS-wt. Compounds 12 and 13
compd
AS-wt
U1406A
U1495A
1
2
3
4
5
6
7.8
>300
50
80
140
80
5.5
>300
15
80
110
20
31
>300
60
70
120
70
50
no activity
no activity
no activity
no activity
>250
a Dissociation constants and minimum inhibitory concentrations are
in units of micromoles per liter.
Table 2. RNA Binding to Variants of the Decoding Region A-Site
and Antibacterial Activity for Compounds 7-15a
MIC, E. coli
ATCC 25922
compd
AS-wt
U1406A
U1495A
7
8
9
10
11
12
13
14
15
0.7
0.5
0.8
0.4
7.0
0.8
1.0
4.0
4.9
0.4
0.4
0.5
0.25
8.0
0.7
0.7
4.0
3.1
1.5
1.0
1.0
0.7
10
1.6
1.8
6.0
3.7
100
50
100
100
>100
25
25
100
>100
aDissociation constants and minimum inhibitory concentrations are
in units of micromoles per liter.
ics. Similar antibiotic profiles were observed against S. aureus,
but none of the synthetic compounds (or neamine) were active
against P. aeruginosa.
The isomers 12 and 13 present an interesting case. The
addition of hydroxymethyl groups to the diaminoalkyl substitu-
ent had negligible effect on RNA binding (compare 12 and 13
to 7) but did have a beneficial effect on antibiotic activity. The
simplest explanation for the improvement is that the hydroxyl
group improves cell permeability. Another possible factor is
that in vitro binding to these model RNA sequences does not
exactly mimic the binding event on whole ribosomes in vivo.
This issue is discussed in detail in the following section. The
fact that the isomers 12 and 13 behaved identically both in RNA
binding and in antibiotic activity assays suggests that the
substituent at the 5-position of the 2-deoxystreptamine ring
remains flexible upon binding; that is, both isomers can adopt
productive conformations when bound to RNA.
In Vitro Inhibition of Translation. The lack of a close
correlation for these compounds between effectiveness as RNA
binders and as antibiotics is in line with similar observations
made previously for many of the naturally occurring amino-

New Aminoglycoside Antibiotics Containing Neamine
J. Am. Chem. Soc., Vol. 121, No. 28, 1999 6533
Figure 6. Structures of the aminoglycosides and synthetic derivatives that were tested in the in vitro translation assay. Gentamicin was tested as
the commercially available gentamicin C complex.
somal RNA is the most prevalent RNA in the cell, and at low
inhibitor concentrations, a tight ribosome-binding inhibitor is
titrating only a fraction of the very large number of ribosomes.
Only at higher concentrations are all ribosomes saturated and
protein synthesis significantly impaired. More potent ribosome-
targeting antibiotics could be envisioned if they were designed
to be catalytic inhibitors, as is the case with the extremely toxic
proteins ricin27 and diphtheria toxin.28
The apparent differences in aminoglycoside binding affinity
for the 16S A-site RNA, whether isolated in a model oligo-
nucleotide or in the context of the ribosome, may be due to a
number of factors. It is possible that aminoglycosides have other
high-affinity binding sites on the ribosome besides the A-site.
However, no such sites were identified in Noller’s footprinting
experiments on intact ribosomes.3,4 It is possible that additional
stabilizing contacts are made with neighboring ribosomal
proteins or with RNA which is distal in primary sequence but
proximal in space. It is also possible that neighboring proteins
or RNA affect binding indirectly by affecting the conformation
of the A-site RNA so that it adopts a different structure, more
amenable to aminoglycoside binding, than it does as an isolated
Figure 7. Semilogarithmic plot of in vitro inhibition of translation,
measured as the yield of active luciferase as a function of concentration
for neomycin, ribostamycin, neamine, and compounds 13 and 56. The
data points represent the average of 1-3 independent experiments.
system such as the A-site model oligonucleotides. The in vitro
Table 3. Comparison of Results for in Vitro Translation
Inhibition, Minimum Inhibitory Concentration, and A-Site RNA
Binding for Aminoglycosides Shown in Figure 6a
translation assay provides a good correlation between in vitro
activity and antibiotic activity and represents a reliable new
method of understanding the relative efficacy of synthetic
aminoglycoside analogues. A clearer understanding of ami-
noglycoside interactions with the ribosome on a molecular level
awaits high-resolution structural data on the ribosome, a
formidable challenge due to its size (several megadaltons) and
complexity, but one which we hope will be solved soon.29
in vitro translation
IC50
MIC, E. coli
ATCC 25922
compd
Kd (AS-wt)
gentamicin
56 (ref 8b)
neomycin
55 (ref 8b)
ribostamycin
13
0.020
0.020
0.028
0.025
0.10
1.6
1.6
1.6
3.1
12.5
25
1.7
0.26
0.020
1.7
25
1.0
Experimental Section
0.33
12
neamine
0.42
0.41
25
50
0.8
7.8
General. All reactions were carried out in oven-dried glassware
under a positive pressure of argon. Tetrahydrofuran and diethyl ether
were distilled from sodium metal/benzophenone ketyl. Acetonitrile,
toluene, and dichloromethane were distilled from calcium hydride.
Reactions were monitored by analytical thin-layer chromatography
(TLC) on EM silica gel 60 F254 plates (0.25 mm), visualized by
ultraviolet light and/or by staining with ceric ammonium molybdate
a All values are concentrations in micromoles per liter.
had an order of magnitude improvement over neamine in AS-
wt binding but had very similar IC50 values, which is consistent
with their similar MICs.
An overall review of Table 3 shows a close qualitative
correlation between in vitro translation IC50 values and MIC
values against E. coli. In general, MICs are at about 100-fold
higher concentrations than IC50s. The differences in these values
likely reflect two major factors: cell permeability and high
ribosome concentration. The latter is of particular note. Ribo-
1
or ninhydrin. H NMR spectra were obtained on a Bruker AMX-400
(27) Endo, Y.; Mitsui, K.; Motizuki, M.; Tsurugi, K. J. Biol. Chem. 1987,
262, 5908-5912.
(28) Ward, W. H. Trends Biochem. Sci. 1987, 12, 28-31.
(29) Ban, N.; Freeborn, B.; Nissen, P.; Penczek, P.; Grassucci, R. A.;
Sweet, R.; Frank, J.; Moore, P. B.; Steitz, T. A. Cell 1998, 93, 1105-
1115.

6534 J. Am. Chem. Soc., Vol. 121, No. 28, 1999
Greenberg et al.
(400 MHz), AMX-500 (500 MHz), or DRX-600 (600 MHz) spectrom-
eter at ambient temperature. Data were reported as follows: chemical
shift on the δ scale (using either TMS or residual protio solvent as
internal standard), multiplicity (br ) broad, s ) singlet, d ) doublet,
t ) triplet, q ) quartet, m ) multiplet), coupling constant(s) in hertz,
and integration. 13C NMR spectra were obtained with proton decoupling
on a Bruker AMX-400 (100 MHz), AMX-500 (125 MHz), or DRX-
600 (150 MHz) spectrometer and were reported in ppm with residual
solvent for internal standard (77.0 for CDCl3).
bis-dihydropyran15 (0.208 g, 0.652 mmol) in CHCl3 (10 mL, distilled
from P2O5), and the resulting mixture was refluxed for 18 h. The
reaction mixture was cooled to room temperature, diluted with ethyl
acetate, and washed with saturated aqueous sodium bicarbonate and
brine. The aqueous layers were backwashed with ethyl acetate, and
the combined organics were dried (MgSO4) and concentrated under
reduced pressure. The residue was coevaporated twice from anhydrous
toluene. Acetic anhydride (0.140 mL, 1.48 mmol) was added dropwise
to a solution of the residue and 4-(dimethylamino)pyridine (3.1 mg,
0.025 mmol) in pyridine (5 mL) at ambient temperature. The reaction
mixture was stirred for 24 h and then quenched by addition of methanol
(3 mL). The reaction mixture was diluted with ethyl acetate and washed
with saturated aqueous sodium bicarbonate and brine. The aqueous
layers were backwashed with ethyl acetate and the combined organics
dried (Na2SO4) and concentrated under reduced pressure. The residue
was chromatographed on silica gel with a gradient of 0 to 4% ethyl
acetate in hexanes to provide 19 (0.146 g, 50%) as a slightly yellow
solid: Rf 0.29 (10% ethyl acetate/hexanes); 1H NMR (500 MHz, CDCl3)
δ 7.41-7.34 (m, 8H), 7.32-7.27 (m, 2H), 4.98 (dd, J ) 9.5, 9.5, 1H),
4.76 (dd, J ) 2.0, 12.0, 1H), 4.62 (dd, J ) 2.0, 12.0, 1H), 3.72 (dd, J
) 9.5, 9.5, 1H), 3.68 (dd, J ) 9.5, 9.5, 1H), 3.52 (ddd, J ) 4.5, 10.0,
12.5, 1H), 3.42 (ddd, J ) 4.5, 10.0, 12.5, 1H), 2.18 (s, 3H), 2.13 (ddd,
J ) 4.5, 4.5, 13.0, 1H), 2.09-2.01 (m, 2H), 1.91-1.64 (m, 8H), 1.53-
1.45 (m, 2H), 1.36 (ddd, J ) 13.0, 13.0, 13.0, 1H); 13C NMR (125
MHz, CDCl3) δ 169.5, 142.8, 142.7, 128.4, 128.2, 127.5, 127.4, 125.9,
98.3, 97.9, 73.0, 72.4, 72.3, 72.0, 68.6, 58.5, 57.2, 33.5, 33.4, 32.4,
28.0, 20.7, 18.9 (x2); HRMS (FAB) m/e calcd for C30H34CsN6O6 (M
+ Cs+) 707.1594, found 707.1615.
Dispiroketal (20). A suspension of acetylated dispiroketal 19 (0.146
g, 0.254 mmol) in methanol (5 mL) and ether (1 mL) was treated with
sufficient sodium methoxide (approximately 1 M in methanol) to give
a “pH” of ∼10. After the solution was stirred for 21 h at ambient
temperature, TLC indicated incomplete reaction. 1,4-Dioxane (3 mL)
was added, and all solid dissolved. After 3 h, the reaction was di-
luted with ethyl acetate and washed with 5% ammonium chloride and
brine. The organic phase was dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by chromatography on silica
gel with a gradient of 0 to 10% ethyl acetate in hexanes (loaded as a
dichloromethane solution) to provide 20 (0.121 g, 90%) as a color-
less solid: Rf 0.22 (10% ethyl acetate in hexanes); 1H NMR (400
MHz, CDCl3) δ 4.76 (dd, J ) 2.5, 11.7, 1H), 4.73 (dd, J ) 2.5, 11.7,
1H), 3.64-3.50 (m, 4H), 3.38-3.33 (m, 1H), 2.57 (br s, 1H, OH),
2.17-1.99 (m, 5H), 1.92-1.85 (m, 2H), 1.80-1.70 (m, 4H), 1.53-
1.47 (m, 2H), 1.25 (app q, J ) 10.1, 1H); 13C NMR (125 MHz, CDCl3)
δ 142.8 (×2), 128.3, 127.4 (×2), 125.9 (×2), 98.1, 97.9, 73.7, 72.4,
72.2, 71.7, 70.4, 60.1, 57.4, 33.4, 32.4, 28.1, 28.0, 18.9, 18.8; HRMS
(FAB) m/e calcd for C28H32CsN6O5 (M + Cs+) 665.1489, found
665.1469.
2-Deoxy-1,3-diazido-4,5,6-tri-O-acetylstreptamine (15). Triflic
azide solution (ca. 0.5 M in dichloromethane, 80 mL, 40 mmol) was
added slowly dropwise to a mixture of 2-deoxystreptamine13 (3.24 g,
20.0 mmol), triethylamine (29.0 mL, 208 mmol), and CuSO4 (193 mg,
1.21 mmol) in methanol (200 mL). The reaction was stirred for 21 h
at room temperature and concentrated under reduced pressure to a green
oil. (Caution: Do not concentrate completely, as triflic azide has been
reported to be explosiVe in the absence of solVent.) The residue was
purified by chromatography on silica gel with a gradient from 80%
ethyl acetate/1% triethylamine/19% hexanes to 20% methanol/1%
triethylamine/79% ethyl acetate. This material (4.3 g) was dissolved
in anhydrous pyridine (40 mL), cooled to 0 °C, and treated with acetic
anhydride (8.5 mL, 90 mmol). The reaction was allowed to warm to
room temperature and stirrred for 19 h. 4-(Dimethylamino)pyridine (114
mg, 0.933 mmol) was added, followed 3 h later by an additional aliquot
of acetic anhydride (3.0 mL, 32 mmol). After 40 min, the reaction was
quenched by addition of methanol (20 mL) and concentrated under
reduced pressure. The residue was dissolved in ethyl acetate (100 mL),
washed successively with 1 N HCl, saturated sodium bicarbonate, and
brine, dried (Na2SO4), filtered, and concentrated under reduced pressure.
To remove residual triflic amide, the residue was dissolved in ethyl
acetate (100 mL), washed with 1 N NaOH (twice) and brine, dried
(Na2SO4), filtered, and concentrated under reduced pressure. This
material was chromatographed with a gradient of 15 to 30% ethyl
acetate in hexanes to give 15 (5.11 g, 75%) as a colorless amorphous
solid: Rf 0.31 (25% ethyl acetate/hexanes); 1H NMR (400 MHz, CDCl3)
δ 5.08-5.01 (m, 3H), 3.65-3.51 (m, 2H), 2.33 (ddd, J ) 4.5, 4.5,
13.5, 1H), 2.09 (s, 6H), 2.01 (s, 3H), 1.56 (ddd, J ) 12.7, 12.7, 13.5,
1H); 13C NMR (100 MHz, CDCl3) δ 169.9, 169.5, 73.5, 71.5, 58.0,
31.8, 20.5, 20.4; HRMS (FAB) m/e calcd for C12H17N6O6 (M + H+)
341.1210, found 341.1217.
2-Deoxy-1,3-diazido-5,6-di-O-acetylstreptamine (17). Novozym
435 (Candida antarctica lipase immobilized on a macroporous acrylic
resin, Novo Nordisk, 0.329 g) was added to 16 (0.340 g, 1.00 mmol)
in a well-stirred mixture of toluene (7 mL) and 0.1 M sodium phosphate
buffer, pH 6.2 (7 mL), at ambient temperature. After 72 h, TLC
indicated no further conversion, and the reaction mixture was filtered
and the retained enzyme washed with ethyl acetate and water. The
aqueous layer of the combined filtrate and washings was extracted twice
with ethyl acetate. The combined organics were dried (Na2SO4), filtered,
and concentrated under reduced pressure. The residue was chromato-
graphed on silica gel with 10% acetone in toluene to give 17 (0.211 g,
71%) as a colorless solid, along with recovered 16 (0.085 g, 25%).
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-1-phenylthio-â-D-glucopy-
ranoside (22). Sodium hydride (0.203 g, 8.46 mmol) was added in
one portion to a solution of thioglycoside 2315 (0.782 g, 2.63 mmol)
and benzyl bromide (1.2 mL, 10 mmol) in dimethylformamide (20 mL)
at 0 °C. The reaction was allowed to slowly warm to ambient
temperature. After 2 h, the reaction was quenched by addition of
methanol (3 mL). This material was combined with an identically
processed reaction on 201 mg of thioglycoside 23. The combined
quenched reaction mixtures were concentrated under reduced pressure.
The residue was dissolved in ethyl acetate, washed with saturated
sodium bicarbonate and brine, dried (Na2SO4), filtered, and concentrated
under reduced pressure. The residue was recrystallized from ethanol
to give 22 (0.895 g, 60%) as colorless needles: Rf 0.62 (25% ethyl
acetate/hexanes); 1H NMR (500 MHz, CDCl3) δ 7.60 (m, 2H), 7.35-
7.19 (m, 18H), 4.86 (d, J ) 10.5, 1H), 4.83 (d, J ) 10.5, 1H), 4.79 (d,
J ) 11.0, 1H), 4.62 (d, J ) 12.0, 1H), 4.58 (d, J ) 10.5, 1H), 4.54 (d,
J ) 12.0, 1H), 4.41 (d, J ) 10.0, 1H), 3.78 (dd, J ) 2.0, 11.0, 1H),
3.74 (dd, J ) 4.0, 11.0, 1H), 3.61 (dd, J ) 9.5, 9.5, 1H), 3.51 (dd, J
) 9.5, 9.5, 1H), 3.47 (ddd, J ) 2.0, 4.0, 9.5, 1H), 3.34 (dd, J ) 9.5,
9.5, 1H); 13C NMR (125 MHz, CDCl3) δ 138.2, 137.8, 137.6, 133.6,
131.1, 129.0, 128.5, 128.4, 128.3, 128.2, 128.0, 127.9, 127.8, 127.6,
127.5, 85.9, 85.0, 79.3, 77.5, 75.9, 75.0, 73.4, 68.7, 65.0; HRMS (FAB)
m/e calcd for C33H33CsN3O4S (M + Cs+) 700.1246, found 700.1273.
1
Compound 17: Rf 0.17 (10% acetone/toluene); H NMR (500 MHz,
CDCl3) δ 4.99-4.94 (m, 2H), 3.62-3.56 (m, 2H), 3.52-3.46 (m, 1H),
2.82 (br s, 1H, OH), 2.30 (ddd, J ) 4.5, 4.5, 13.0, 1H), 2.10 (s, 3H),
2.09 (s, 3H), 1.46 (ddd, J ) 12.5, 13.0, 13.0, 1H); 13C NMR (125
MHz, CDCl3) δ 170.7, 169.9, 74.7, 73.8, 73.5, 60.3, 58.2, 31.8, 20.7,
20.6; HRMS (FAB) m/e calcd for C10H15N6O5 (M + H+) 299.1104,
found 299.1115.
2-Deoxy-1,3-diazidostreptamine (18). Sodium methoxide (ap-
proximately 1 M solution in methanol) was added dropwise to 16 (2.47
g, 7.24 mmol) in methanol (30 mL) to give a “pH” of 11. After being
stirred for 2.5 h at ambient temperature, the reaction was neutralized
with Amberlite IR-120 (H+). The solution was concentrated under
reduced pressure to give 18 (1.55 g, 100%) as a white solid: Rf 0.24
1
(75% ethyl acetate/hexanes); H NMR (500 MHz, CD3OD) δ 3.38-
3.29 (m, 2H), 3.23-3.20 (m, 3H), 2.08 (ddd, J ) 4.4, 4.4, 13.0, 1H),
1.22 (ddd, J ) 12.3, 12.3, 13.0, 1H); 13C NMR (125 MHz, CD3OD) δ
77.6, 77.0, 62.3, 33.4; MS (ESI neg) m/e 213 ([M - H+]-).
Acetylated Dispiroketal (19). Camphorsulfonic acid (0.013 g, 0.055
mmol) was added to a solution of 16 (0.109 g, 0.511 mmol) and Ley’s

New Aminoglycoside Antibiotics Containing Neamine
J. Am. Chem. Soc., Vol. 121, No. 28, 1999 6535
1,2,4,6-Tetra-O-acetyl-3-azido-3-deoxy-r/â-D-glucopyranoside (26).
Diacetonide 2517 (0.77 g, 2.7 mmol) was treated with 90% trifluoro-
acetic acid/water (10 mL). After 1 h, the reaction mixture was
concentrated under reduced pressure and coevaporated twice with water.
The residue was dried under oil pump vacuum. This material was
dissolved in hot acetic anhydride (2 mL) and added dropwise to a
suspension of sodium acetate (0.502 g, 6.1 mmol) in refluxing acetic
anhydride (2 mL). After 10 min, the reaction mixture was cooled to
ambient temperature, poured into a well-stirred dichloromethane/water
mixture, and neutralized with sodium carbonate. The organic phase
was separated and washed with brine, and the aqueous phases were
backwashed with ethyl acetate. The combined organic layers were dried
(Na2SO4), filtered, and concentrated under reduced pressure. The residue
was purified by chromatography on silica gel with a gradient of 25 to
33% ethyl acetate in hexanes to give 26 (0.596 g, 59%) as a colorless
oil: Rf 0.36 (33% ethyl acetate/hexanes); 1H NMR (500 MHz, CDCl3,
3:1 mixture of â and R anomers), â-anomer δ 5.68 (d, J ) 8.0, 1H),
5.05 (dd, J ) 8.0, 10.0, 1H), 5.03 (dd, J ) 10.0, 10.0, 1H), 4.25 (d, J
) 4.5, 12.5, 1H), 4.10 (d, J ) 2.5, 12.5, 1H), 3.79 (ddd, J ) 2.5, 4.5,
10.0, 1H), 3.70 (dd, J ) 10.0, 10.0, 1H), 2.13 (s, 3H), 2.12 (s, 3H),
2.12 (s, 3H), 2.09 (s, 3H), R-anomer δ 6.31 (d, J ) 3.5, 1H), 5.03 (m,
1H), 4.96 (dd, J ) 4.0, 10.5, 1H), 4.22 (dd, J ) 4.0, 13.0, 1H), 4.10
(m, 1H), 4.04 (m, 1H), 3.97 (dd, J ) 10.5, 10.5, 1H), 2.19 (s, 3H),
2.14 (s, 3H), 2.10 (s, 3H), 2.10 (s, 3H); 13C NMR (125 MHz, CDCl3,
3:1 mixture of â and R anomers), â-anomer δ 169.1, 169.0, 168.9,
91.8, 73.5, 70.0, 67.7, 64.2, 61.4, 20.8, 20.7, 20.6 (×2), R-anomer δ
170.6, 169.3, 169.2, 88.7, 70.0, 69.9, 67.7, 61.4, 60.8, 30.9, 20.8, 20.6,
20.4; HRMS (FAB) m/e calcd for C14H19CsN3O9 (M + Cs+) 506.0176,
found 506.0159.
dissolved in ethyl acetate, washed with saturated sodium bicarbonate
solution and brine, dried (Na2SO4), filtered, and concentrated under
reduced pressure. The residue was purified by chromatography on silica
gel with a gradient of 0 to 10% ethyl acetate in hexane (compound
was loaded in dichloromethane) to provide 25 (0.476 g, 85%) as a
slightly off-white solid: Rf 0.27 (10% ethyl acetate/hexanes); 1H NMR
(500 MHz, CDCl3) δ 7.58-7.56 (m, 2H), 7.47-7.45 (m, 2H), 7.39-
7.24 (m, 16H), 4.90 (d, J ) 10.0, 1H), 4.80 (d, J ) 10.5, 1H), 4.74 (d,
J ) 10.0, 1H), 4.63 (d, J ) 9.5, 1H), 4.62 (d, J ) 12.0, 1H), 4.56 (d,
J ) 9.5, 1H), 4.54 (d, J ) 12.0, 1H), 3.75 (dd, J ) 2.0, 11.0, 1H),
3.72 (dd, J ) 3.5, 11.0, 1H), 3.60 (dd, J ) 9.0, 9.0, 1H), 3.49 (dd, J
) 9.0, 9.0, 1H), 3.45 (m, 1H), 3.33 (dd, J ) 9.5, 9.5, 1H); 13C NMR
(125 MHz, CDCl3) δ 138.0, 137.4, 137.3, 133.4, 132.0, 129.0, 128.6,
128.5, 128.4, 128.2, 128.1, 128.0, 127.8, 127.7, 127.6, 87.7, 79.3, 79.2,
76.1, 75.3, 74.9, 73.5, 70.6, 68.6; HRMS (FAB) m/e calcd for C33H33-
NaN3O4S (M + Na+) 590.2089, found 590.2073.
2,3,6-Tri-O-benzyl-1-phenylthio-â-D-galactopyranoside (31). A
solution of acetal 3018 (0.203 g, 0.375 mmol) in THF (4 mL) was
stirred for 10 min over 4-Å molecular sieves. Borane-trimethylamine
complex (0.178 g, 2.44 mmol) was added in one portion, followed by
aluminum chloride (0.307 g, 2.30 mmol). After 4.5 h, additional
borane-trimethylamine complex (0.111 g, 1.52 mmol) and aluminum
chloride (0.158 g, 1.18 mmol) were added, and the reaction was stirred
overnight at ambient temperature. The reaction mixture was filtered
through Celite, neutralized with IR-120(H+), filtered again through
Celite, and concentrated under reduced pressure. The residue was
coevaporated four times with methanol, dissolved in ethyl acetate,
filtered, and concentrated. This material was purified by chromatography
on silica gel with a gradient of 0 to 5% ethyl acetate in toluene to give
31 (173 mg, 85%) as a cloudy oil: Rf 0.16 (5% ethyl acetate/toluene);
1H NMR (500 MHz, CDCl3) δ 7.58-7.56 (m, 2H), 7.41-7.39 (m,
2H), 7.37-7.28 (m, 12H), 7.25-7.23 (m, 4H), 4.83 (d, J ) 10.0, 1H),
4.74 (d, J ) 10.5, 1H), 4.72 (d, J ) 12.0, 1H), 4.68 (d, J ) 12.0, 1H),
4.64 (d, J ) 10.0, 1H), 4.56 (s, 2H), 4.10 (m, 1H), 3.82-3.73 (m,
3H), 3.61-3.56 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 138.1, 137.8,
137.6, 133.8, 131.7, 128.8, 128.4, 128.3(x2), 128.2, 127.9, 127.8, 127.7
(×2), 127.2, 87.6, 82.5, 76.9 (×2), 75.6, 73.6, 72.0, 69.4, 66.8; HRMS
(FAB) m/e calcd for C33H34CsO5S (M + Cs+) 675.1181, found
675.1161.
3-Azido-3-deoxy-2,4,6-tri-O-acetyl-1-phenylthio-â-D-glucopy-
ranoside (27). Boron trifluoride diethyl etherate (1.0 mL, 7.9 mmol)
was added dropwise to a solution of glycosyl acetate 26 (0.593 g, 1.59
mmol) and thiophenol (175 µL, 1.70 mmol) in chloroform (3 mL,
distilled from phosphorus pentoxide). The reaction mixture was stirred
at ambient temperature for 22 h and then quenched by addition of
saturated sodium bicarbonate (several milliliters). The layers were
separated, and the aqueous layer was backwashed with dichloromethane.
The combined organic phases were dried (Na2SO4), filtered, and
concentrated under reduced pressure. The residue was purified by
chromatography on silica gel with a gradient of 3 to 4% acetone in
toluene to provide 27 (0.431 g, 64%) as a colorless, amorphous solid:
2,3,6-Tri-O-benzyl-4-O-methanesulfonyl-1-phenylthio-â-D-galac-
topyranoside (32). Methanesulfonyl chloride (25 µL, 0.32 mmol) was
added dropwise to a solution of alcohol 31 (0.138 g, 0.254 mmol) in
pyridine (3 mL) at 0 °C. The reaction was allowed to slowly warm to
ambient temperature. Additional portions of methanesulfonyl chloride
were added until TLC indicated that the reaction was complete (7 h).
The reaction mixture was diluted with ethyl acetate, washed with 1 N
HCl, saturated sodium bicarbonate, and brine, dried (Na2SO4), filtered,
and concentrated under reduced pressure. The residue was purified by
chromatography on silica gel with 14% ethyl acetate in hexanes to
provide 32 (116 mg, 73%) as a colorless oil: Rf 0.27 (5% ethyl acetate/
1
Rf 0.17 (5% acetone/toluene); H NMR (500 MHz, CDCl3) δ 7.51-
7.47 (m, 2H), 7.33-7.30 (m, 3H), 4.94 (dd, J ) 10.0, 10.0, 1H), 4.92
(dd, J ) 10.0, 10.0, 1H), 4.66 (d, J ) 10.0, 1H), 4.21-4.15 (m, 2H),
3.68 (m, 1H), 3.67 (dd, J ) 10.0, 10.0, 1H), 2.18 (s, 3H), 2.12 (s, 3H),
2.08 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 170.6, 169.2, 169.0, 132.8,
131.9, 128.9, 128.3, 86.2, 76.3, 70.0, 68.2, 65.8, 62.2, 20.8, 20.7, 20.6;
HRMS (FAB) m/e calcd for C18H21CsN3O7S (M + Cs+) 556.0155,
found 556.0173.
3-Azido-3-deoxy-1-phenylthio-â-D-glucopyranoside (28). Sodium
methoxide (0.4 M in methanol, 100 µL, 40 µmol) was added to a
suspension of triacetate 27 (0.431 g, 1.02 mmol) in methanol (10 mL).
The reaction was stirred for 16 h at ambient temperature. TLC indicated
that the reaction was incomplete, and additional sodium methoxide
solution (3.0 mL, 1.2 mmol) was added. After 1.5 h, the reaction was
quenched with IR-120(H+), filtered, and concentrated under reduced
pressure to give 28 (0.303 g, quantitative) as a colorless solid: Rf 0.14
(25% acetone/toluene); 1H NMR (500 MHz, CD3OD) δ 7.56-7.54 (m,
2H), 7.32-7.26 (m, 3H), 4.62 (d, J ) 9.5, 1H), 3.84 (dd, J ) 2.0,
12.0, 1H), 3.65 (dd, J ) 5.8, 12.0, 1H), 3.35 (m, 1H), 3.33 (dd, J )
9.0, 9.0, 1H), 3.27 (dd, J ) 9.5, 9.5, 1H), 3.21 (dd, J ) 9.5, 9.5, 1H);
13C NMR (125 MHz, CD3OD) δ 134.9, 132.9, 129.9, 128.5, 89.6, 82.4,
73.0, 72.8, 70.0, 62.5; HRMS (FAB) m/e calcd for C12H15NaN3O4S
(M + Na+) 320.0681, found 320.0691.
3-Azido-3-deoxy-2,4,6-tri-O-benzyl-1-phenylthio-â-D-glucopy-
ranoside (24). Sodium hydride (0.085 g, 3.5 mmol) was added in one
portion to a solution of thioglycoside 28 (0.293 g, 0.985 mmol) and
benzyl bromide (420 µL, 3.53 mmol) in dimethylformamide (5 mL) at
0 °C. The reaction was allowed to slowly warm to ambient temperature.
After 4 h, the reaction was quenched with methanol (l mL). The reaction
mixture was concentrated under reduced pressure. The residue was
1
hexanes); H NMR (400 MHz, CDCl3) δ 7.57-7.54 (m, 2H), 7.40-
7.23 (m, 18H), 5.34 (d, J ) 2.0, 1H), 4.84 (d, J ) 10.5, 1H), 4.81 (d,
J ) 10.3, 1H), 4.75 (d, J ) 10.3, 1H), 4.66 (m, 1H), 4.63 (d, J ) 11.2,
1H), 4.59 (d, J ) 10.7, 1H), 4.48 (d, J ) 11.2, 1H), 3.78-3.71 (m,
3H), 3.65-3.62 (m, 2H), 2.98 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
137.7, 137.5, 137.0, 133.3, 132.1, 128.9, 128.5, 128.4 (×2), 128.2 (×2),
128.1, 127.9 (×2), 127.6, 87.8, 80.9, 76.5, 76.0, 75.7, 75.4, 73.8, 72.9,
67.9, 39.0; HRMS (FAB) m/e calcd for C34H36CsO7S2 (M + Cs+)
753.0957, found 753.0941.
4-Azido-4-deoxy-2,3,6-tri-O-benzyl-1-phenylthio-â-D-glucopy-
ranoside (29). Sodium azide (0.326 g, 5.01 mmol) was added to a
solution of mesylate 32 (0.608 g, 0.979 mmol) in dimethylformamide
(10 mL). The reaction was stirred at 100 °C for 13 h, cooled to ambient
temperature, and concentrated under reduced pressure. The residue was
dissolved in ethyl acetate and washed with saturated sodium bicarbonate
and brine. The aqueous layers were backwashed with ethyl acetate,
and the combined organic phases were dried (Na2SO4), filtered, and
concentrated under reduced pressure. The residue was purified by
chromatography on silica gel in toluene to give 29 (0.468 g, 84%) as
1
a colorless, amorphous solid: Rf 0.23 (toluene); H NMR (500 MHz,
CDCl3) δ 7.57-7.55 (m, 2H), 7.40-7.23 (m, 18H), 4.91 (d, J ) 11.0,

6536 J. Am. Chem. Soc., Vol. 121, No. 28, 1999
Greenberg et al.
1H), 4.88 (d, J ) 11.0, 1H), 4.83 (d, J ) 10.5, 1H), 4.71 (d, J ) 10.5,
1H), 4.63 (d, J ) 11.5, 1H), 4.62 (d, J ) 9.0, 1H), 4.57 (d, J ) 12.0,
1H), 3.80 (dd, J ) 2.0, 10.5, 1H), 3.72 (dd, J ) 4.5, 10.5, 1H), 3.66
(dd, J ) 9.8, 9.8, 1H), 3.55 (dd, J ) 9.0, 9.0, 1H), 3.51 (dd, J ) 9.0,
9.0, 1H), 3.32 (ddd, J ) 2.0, 4.5, 10.5, 1H); 13C NMR (125 MHz,
CDCl3) δ 138.0, 137.8, 137.6, 133.4, 132.1, 128.9, 128.5, 128.4, 128.2,
128.0, 127.7, 87.6, 84.9, 80.5, 78.0, 75.8, 75.4, 73.5, 69.2, 61.9; HRMS
(FAB) m/e calcd for C33H33CsN3O4S (M + Cs+) 700.1246, found
700.1271.
1-Phenylthio-6-O-p-toluenesulfonyl-â-D-glucopyranoside (35). A
solution of p-toluenesulfonyl chloride in pyridine (0.57 M, 15 mL, 8.6
mmol) was added dropwise to thioglycoside 34 (1.84 g, 6.76 mmol) in
pyridine (20 mL) at 0 °C. The transfer was quantitated with pyridine
(5 mL). After 4 h at 0 °C, the reaction was allowed to slowly warm to
ambient temperature and stirred for an additional 16 h. The reaction
was then quenched by addition of methanol (3 mL) and concentrated
under reduced pressure. The residue was dissolved in dichloromethane
and washed sequentially with saturated sodium bicarbonate, 1 N HCl,
and saturated sodium bicarbonate. The aqueous layers were backwashed
with dichloromethane. The organic phases were combined, dried
(MgSO4), decanted, and concentrated under reduced pressure. The
residue was chromatographed on silica gel with a gradient of 50% ethyl
acetate/hexanes to 100% ethyl acetate to provide 35 (2.17 g, 75%) as
a colorless foam: Rf 0.37 (1% methanol/ethyl acetate); 1H NMR (400
MHz, CDCl3) δ 7.79 (m, 1H), 7.77 (m, 1H), 7.44-7.41 (m, 1H), 7.29
(m, 1H), 7.27 (m, 1H), 7.24-7.19 (m, 3H), 4.46 (d, J ) 9.5, 1H), 4.26
(m, 2H), 3.84 (br s, 3H, OH), 3.55-3.38 (m, 3H), 3.28 (dd, J ) 9.5,
9.0, 1H), 2.38 (s, 3H); HRMS (FAB) m/e calcd for C19H22NaO7S2 (M
+ Na+) 449.0705, found 449.0718.
4-O-(2,3,4,6-Tetra-O-benzyl-r-D-glucopyranosyl)-2-deoxy-
streptamine (37). Donor 21 (0.349 g, 0.551 mmol) and acceptor 17
(0.125 g, 0.419 mmol) were coevaporated twice from dry toluene and
further dried under high vacuum overnight. Diethyl ether (8.0 mL) and
dichloromethane (2.0 mL) were added, and the reaction was cooled to
-30 °C. Trifluoromethanesulfonic acid (7.0 µL) was added to a solution
of N-iodosuccinimide (0.184 g, 0.818 mmol) in diethyl ether (6.0 mL)
and dichloromethane (6.0 mL). A portion (8.0 mL) of the resulting
faint orange solution was added slowly dropwise to the solution of 21
and 17, which was maintained at -30 °C. After 30 min, the reaction
mixture was diluted with diethyl ether and washed with 10% aqueous
sodium bisulfite, saturated sodium bicarbonate, and brine. The aqueous
layers were back extracted with diethyl ether. The combined organic
phases were dried (Na2SO4), filtered, and concentrated under reduced
pressure. The residue was purified by chromatography on silica gel
with a gradient of 2 to 10% ethyl acetate in toluene to provide a slightly
yellow oil (0.311 g, Rf 0.14 in 5% ethyl acetate/toluene). This material
was dissolved in tetrahydrofuran (1.0 mL) and methanol (9.0 mL), and
sodium methoxide (0.5 M in methanol, 200 µL, 0.1 mmol) was added.
The reaction mixture was stirred at ambient temperature for 16 h. The
solution was neutralized with Amberlite IR-120(H+), filtered, and
evaporated under reduced pressure. The residue was purified by
chromatography on silica gel with a gradient of 5 to 7% acetone in
toluene to give 37 (0.244 g, 79%) as a colorless oil: Rf 0.21 (10%
acetone/toluene); 1H NMR (500 MHz, CDCl3) δ 7.37-7.26 (m, 18H),
7.14-7.12 (m, 2H), 4.96 (d, J ) 4.0, 1H, H1′), 4.93 (m, 2H), 4.89 (d,
J ) 12.0, 1H), 4.81 (d, J ) 10.5, 1H), 4.79 (br s, 1H, OH), 4.74 (d, J
) 11.5, 1H), 4.65 (d, J ) 12.0, 1H), 4.51 (d, J ) 10.5, 1H), 4.47 (d,
J ) 12.5, 1H), 4.04 (dd, J1 ) J2 ) 9.5, 1H), 4.03 (m, 1H) 3.81 (dd, J
) 3.0, 10.5, 1H), 3.76 (dd, J1 ) J2 ) 9.5, 1H), 3.69 (dd, J ) 1.8, 10.5,
1H), 3.48-3.37 (m, 3H), 3.24 (m, 1H), 3.18 (dd, J1 ) J2 ) 9.0, 1H),
2.98 (br s, 1H, OH), 2.28 (ddd, J ) 4.5, 4.5, 13.5, 1H), 1.46 (ddd, J )
12.0, 12.0, 13.5, 1H); 13C NMR (125 MHz, CDCl3) δ 138.3, 138.0,
137.7, 136.8, 128.7, 128.5, 128.4, 128.3, 128.0, 127.8, 127.7, 127.6,
101.3, 85.4, 82.3, 79.1, 77.7, 75.6, 75.3, 75.1, 74.5, 73.5, 71.4, 68.0,
59.6, 59.4, 32.3; HRMS (FAB) m/e calcd for C40H44CsN6O8 (M + Cs+)
869.2275, found 869.2247.
6-Azido-6-deoxy-1-phenylthio-â-D-glucopyranoside (36). Sodium
azide (0.397 g, 6.11 mmol) was added to thioglycoside 35 (2.17 g,
5.09 mmol) in dimethylformamide (20 mL). This suspension was placed
in an 80 °C oil bath and stirred for 5 h. The reaction mixture was
cooled to ambient temperature and concentrated under reduced pres-
sure. The residue was dissolved in ethyl acetate and washed with
saturated sodium bicarbonate and brine. The aqueous layers were
backwashed with ethyl acetate, and the combined organic phases were
dried (Na2SO4), decanted, and concentrated under reduced pressure.
The crude material was purified by chromatography on silica gel with
ethyl acetate to give 36 (1.36 g, 90%) as a slightly yellow oil: Rf 0.42
4-O-(2-Azido-2-deoxy-3,4,6-tri-O-benzyl-r-D-glucopyranosyl)-2-
deoxystreptamine (38). Donor 22 (148 mg, 0.261 mmol) and acceptor
17 (60 mg, 0.201 mmol) were coevaporated twice from dry toluene
and further dried under high vacuum overnight. Diethyl ether (4.0 mL)
and dichloromethane (1.0 mL) were added, and the reaction was cooled
to -10 °C. Trifluoromethanesulfonic acid (5.0 µL, 56 µmol) was added
to a solution of N-iodosuccinimide (118 mg, 0.524 mmol) in diethyl
ether (3.0 mL) and dichloromethane (3.0 mL). A portion (5.5 mL) of
the resulting faint orange solution was added slowly dropwise to the
solution of 22 and 17, which was maintained at -10 °C. After 30 min,
the reaction mixture was diluted with diethyl ether and washed with
10% aqueous sodium bisulfite, saturated sodium bicarbonate, and brine.
The aqueous layers were back extracted with diethyl ether. The
combined organic phases were dried (Na2SO4), filtered, and concen-
trated under reduced pressure. The residue was purified by chroma-
tography on silica gel with a gradient of 3 to 5% ethyl acetate in toluene
to provide a slightly yellow oil (90 mg, Rf 0.15 in 5% acetone/toluene).
This material was dissolved in tetrahydrofuran (0.2 mL) and methanol
(1.8 mL), and sodium methoxide (0.6 M in methanol, 100 µL, 60 µmol)
was added. The reaction mixture was stirred at ambient temperature
for 19 h. The solution was neutralized with Amberlite IR-120(H+),
filtered, and evaporated under reduced pressure. The residue was
purified by chromatography on silica gel with 7% acetone in toluene
to give 38 (78 mg, 58%) as a colorless oil: Rf 0.10 (10% acetone/
1
(1% methanol/ethyl acetate); H NMR (500 MHz, CDCl3) δ 7.54-
7.52 (m, 2H), 7.31-7.29 (m, 3H), 4.92 (br s, 1H, OH), 4.51 (d, J )
10.0, 1H), 4.27 (br s, 1H, OH), 3.98 (br s, 1H, OH), 3.56-3.37 (m,
5H), 3.34 (dd, J ) 9.0, 9.5, 1H); 13C NMR (125 MHz, CDCl3) δ 133.1,
131.2, 129.1, 128.5, 87.8, 78.4, 77.5, 71.8, 70.1, 51.4; HRMS (FAB)
m/e calcd for C12H15NaN3O4S (M + Na+) 320.0681, found 320.0692.
6-Azido-6-deoxy-2,3,4-tri-O-benzyl-1-phenylthio-â-D-glucopy-
ranoside (33). Sodium hydride (0.376 g, 15.7 mmol) was added in
one portion to a solution of thioglycoside 36 (1.34 g, 4.5 mmol) and
benzyl bromide (1.9 mL, 16 mmol) in dimethylformamide (20 mL) at
0 °C. After 1 h, the reaction mixture was allowed to slowly warm to
ambient temperature. After 3 h, additional portions of benzyl bromide
(0.55 mL, 4.6 mmol) and sodium hydride (88 mg, 3.7 mmol) were
added. TLC indicated no change after an additional 1.5 h, and the
reaction was quenched with methanol (3 mL). The reaction was
concentrated under reduced pressure. The residue was dissolved in ethyl
acetate, washed with 5% ammonium chloride solution and brine, dried
(Na2SO4), filtered, and concentrated under reduced pressure. The residue
was purified by chromatography on silica gel with a gradient of 0 to
10% ethyl acetate in hexane (compound was loaded in dichloromethane)
to provide 33 (1.47 g, 57%) as a colorless solid (an analytically pure
sample was obtained by recrystallization from ethanol): Rf 0.24 (10%
1
toluene); H NMR (500 MHz, CDCl3) δ 7.35-7.25 (m, 13H), 7.16-
1
ethyl acetate/hexanes); H NMR (500 MHz, CDCl3) δ 7.59 (m, 2H),
7.14 (m, 2H), 5.15 (d, J ) 3.6, 1H, H1′), 4.92 (d, J ) 10.5, 1H), 4.85
(d, J ) 10.5, 1H), 4.78 (d, J ) 10.8, 1H), 4.64 (d, J ) 12.0, 1H), 4.54
(d, J ) 10.8, 1H), 4.49 (d, J ) 12.0, 1H), 4.20 (d, J ) 2.0, 1H, OH),
7.42-7.25 (m, 18H), 4.93 (d, J ) 10.5, 2H), 4.88 (d, J ) 11.0, 1H),
4.85 (d, J ) 11.0, 1H), 4.76 (d, J ) 10.0, 1H), 4.67 (d, J ) 10.0, 1H),
4.61 (d, J ) 11.0, 1H), 3.71 (dd, J ) 9.0, 9.0, 1H), 3.56-3.51 (m,
3H), 3.46 (m, 1H), 3.35 (dd, J ) 13.0, 5.5, 1H); 13C NMR (125 MHz,
CDCl3) δ 138.1, 137.9, 137.6, 132.9, 132.7, 129.0, 128.5, 128.5, 128.4,
128.2, 128.0 (×2), 127.9 (×2), 127.8 (×2) 87.7, 86.5, 80.8, 78.1, 78.0,
75.8, 75.5, 75.2, 51.4; HRMS (FAB) m/e calcd for C33H33CsN3O4S
(M + Cs+) 700.1246, found 700.1220.
4.09 (m, 1H), 3.98 (dd, J )J2) 9.1, 1H), 3.82-3.76 (m, 2H) 3.68
1
(dd, J ) 2.0, 10.8, 1H), 3.64 (dd, J ) 3.6, 10.2, 1H), 3.45-3.38 (m,
3H), 3.26-3.24 (m, 2H), 2.94 (d, J ) 2.0, 1H, OH), 2.30 (m, 1H),
1.48 (m, 1H); 13C NMR (150 MHz, CDCl3) δ 137.8, 137.7, 137.5,
128.5, 128.4 (×2), 128.0 (×2), 127.9, 127.8 (×2), 127.7, 99.5, 83.9,
81.0, 78.1, 75.8, 75.6, 75.3, 75.0, 73.6, 71.7, 68.0, 64.2, 59.6, 58.7,

New Aminoglycoside Antibiotics Containing Neamine
J. Am. Chem. Soc., Vol. 121, No. 28, 1999 6537
32.0; HRMS (FAB) m/e calcd for C33H37CsN9O7 (M + Cs+) 804.1870,
found 804.1838.
(×2), 101.1, 85.2, 80.4, 78.9, 75.7, 75.5, 75.4, 74.5, 73.6, 70.6, 68.2,
61.8, 59.6, 59.3, 32.2; HRMS (FAB) m/e calcd for C33H37CsN9O7 (M
+ Cs+) 804.1870, found 804.1896.
4-O-(3-Azido-3-deoxy-2,4,6-tri-O-benzyl-r-D-glucopyranosyl)-2-
deoxystreptamine (39). Donor 24 (138 mg, 0.244 mmol) and acceptor
17 (60 mg, 0.202 mmol) were coevaporated twice from dry toluene
and further dried under high vacuum overnight. Diethyl ether (4.0 mL)
and dichloromethane (1.0 mL) were added, and the reaction was cooled
to -15 °C. Trifluoromethanesulfonic acid (5.0 µL, 56 µmol) was added
to a solution of N-iodosuccinimide (110 mg, 0.489 mmol) in diethyl
ether (3.0 mL) and dichloromethane (3.0 mL). A portion (3.0 mL) of
the resulting faint orange solution was added slowly dropwise to the
solution of 24 and 17, which was maintained at -15 °C. After 30 min,
the reaction mixture was diluted with diethyl ether and washed with
10% aqueous sodium bisulfite, saturated sodium bicarbonate, and brine.
The aqueous layers were back extracted with diethyl ether. The
combined organic phases were dried (Na2SO4), filtered, and concen-
trated under reduced pressure. The residue was purified by chroma-
tography on silica gel with 5% ethyl acetate in toluene to provide a
slightly yellow oil (Rf 0.46 in 10% acetone/toluene). This material was
dissolved in tetrahydrofuran (0.4 mL) and methanol (1.6 mL), and
sodium methoxide (0.5 M in methanol, 100 µL, 50 µmol) was added.
The reaction mixture was stirred at ambient temperature for 20 h. An
additional portion of sodium methoxide (0.5 M in methanol, 200 µL,
100 µmol) was added. After being stirred a further 1 h, the solution
was neutralized with Amberlite IR-120(H+), filtered, and evaporated
under reduced pressure. The residue was purified by chromatography
on silica gel with 10% acetone in toluene to give 39 (139 mg, 100%)
as a colorless oil: Rf 0.22 (10% acetone/toluene); 1H NMR (500 MHz,
CDCl3) δ 7.35-7.25 (m, 13H), 7.16-7.14 (m, 2H), 5.15 (d, J ) 3.6,
1H, H1′), 4.92 (d, J ) 10.6, 1H), 4.85 (d, J ) 10.6, 1H), 4.78 (d, J )
10.8, 1H), 4.64 (d, J ) 12.0, 1H), 4.54 (d, J ) 10.8, 1H), 4.49 (d, J )
12.0, 1H), 4.20 (d, J ) 2.3, 1H, OH), 4.09 (m, 1H), 3.98 (dd, J1 ) J2
) 9.1, 1H), 3.82-3.76 (m, 2H) 3.68 (dd, J ) 1.8, 10.8, 1H), 3.64 (dd,
J ) 3.6, 10.2, 1H), 3.45-3.38 (m, 3H), 3.26-3.24 (m, 2H), 2.94 (d,
J ) 1.8, 1H, OH), 2.30 (m, 1H), 1.48 (m, 1H); 13C NMR (125 MHz,
CDCl3) δ 137.5, 137.3, 136.3, 128.8, 128.7, 128.6, 128.4, 128.2 (×2),
128.0, 127.9, 100.2, 85.2, 77.2, 76.2, 75.5, 75.3, 74.9, 74.3, 73.6, 71.0,
67.8, 65.9, 59.5, 59.2, 32.2; HRMS (FAB) m/e calcd for C33H37CsN9O7
(M + Cs+) 804.1870, found 804.1840.
4-O-(6-Azido-6-deoxy-2,3,4-tri-O-benzyl-r-D-glucopyranosyl)-2-
deoxystreptamine (41). Donor 33 (273 mg, 0.481 mmol) and acceptor
17 (60 mg, 0.202 mmol) were coevaporated twice from dry toluene
and further dried under high vacuum overnight. Diethyl ether (8.0 mL)
and dichloromethane (2.0 mL) were added, and the reaction was cooled
to -15 °C. Trifluoromethanesulfonic acid (10.0 µL, 113 µmol) was
added to a solution of N-iodosuccinimide (216 mg, 0.960 mmol) in
diethyl ether (6.0 mL) and dichloromethane (6.0 mL). A portion (6.0
mL) of the resulting faint orange solution was added slowly dropwise
to the solution of 33 and 17, which was maintained at -15 °C. After
30 min, the reaction mixture was diluted with diethyl ether and washed
with 10% aqueous sodium bisulfite, saturated sodium bicarbonate, and
brine. The aqueous layers were back extracted with diethyl ether. The
combined organic phases were dried (Na2SO4), filtered, and concen-
trated under reduced pressure. The residue was purified by chroma-
tography on silica gel with 4% ethyl acetate in toluene to provide a
colorless oil (331 mg, Rf 0.55 in 10% acetone/toluene). This material
was dissolved in tetrahydrofuran (0.8 mL) and methanol (3.2 mL), and
sodium methoxide (0.9 M in methanol, 400 µL, 377 µmol) was added.
The reaction mixture was stirred at ambient temperature for 19 h, and
the solution was then neutralized with Amberlite IR-120(H+), filtered,
and evaporated under reduced pressure. The residue was purified by
chromatography on silica gel with 5% acetone in toluene to give 41
1
(222 mg, 82%) as a colorless oil: Rf 0.30 (10% acetone/toluene); H
NMR (500 MHz, CDCl3) δ 7.35-7.26 (m, 15H), 4.96 (d, J ) 4.0, 1H,
H1′), 4.92 (m, 2H), 4.81 (d, J ) 11.0, 1H), 4.88 (d, J ) 12.0, 1H),
4.73 (d, J ) 11.5, 1H), 4.62 (m, 2H), 4.06 (m, 1H), 4.03 (dd, J ) 9.2,
9.2, 1H), 3.60-3.54 (m, 3H), 3.45-3.36 (m, 4H), 3.26 (m, 1H), 3.17
(m, 1H), 3.02 (s, 1H), 2.27 (ddd, J ) 4.5, 4.5, 13.5, 1H), 1.45 (ddd, J
) 12.5, 12.5, 12.5, 1H); 13C NMR (125 MHz, CDCl3) δ 138.1, 137.7,
136.7, 128.7, 128.5, 128.4, 127.9, 127.6, 100.8, 85.2, 82.0, 79.3, 78.2,
75.5, 75.3, 75.2, 74.6, 71.0, 59.6, 59.3, 51.0, 32.2; HRMS (FAB) m/e
calcd for C33H37CsN9O7 (M + Cs+) 804.1870, found 804.1896.
4-O-(r-D-Glucopyranosyl)-2-deoxystreptamine (2). A solution of
pseudodisaccharide 37 (91 mg, 0.12 mmol) in methanol (6 mL) was
degassed by evacuating the flask and backfilling with argon three times.
Hydrazine (25 µL, 0.80 mmol) was added, followed immediately by
20% Pd(OH)2 on carbon (∼10 mg, Degussa type), and the reaction
mixture was heated at reflux for 16 h. The solution was cooled to
ambient temperature, filtered through Celite, and concentrated under
reduced pressure to afford a colorless oil (80 mg). This material was
dissolved in a mixture of acetic acid (2.5 mL) and water (2.5 mL), and
the solution was degassed by evacuating the flask and backfilling with
argon three times. Next, 20% Pd(OH)2 on carbon (∼10 mg, Degussa
type) was added, and the solution was purged with hydrogen. The
reaction mixture was stirred at ambient temperature under hydrogen
gas (∼1 atm, balloon) for 22 h (an additional portion of catalyst was
added after 6 h). The solution was filtered through Celite and
concentrated under reduced pressure. The residue was purified by
chromatography on Amberlite CG-50 (NH4+, 1.5-cm-diameter × 10.5-
cm column) with a gradient of 0 to 1.2% concentrated ammonium
hydroxide in water. Fractions containing product were pooled and
concentrated under reduced pressure. The residue was dissolved in water
(several milliliters), acidified with acetic acid, and lyophilized to provide
2 (57 mg, 100%) as its oily diacetate salt: 1H NMR (600 MHz, D2O)
δ 5.28 (d, J ) 4.0, 1H, H1′), 3.80 (dd, J ) 2.2, 12.0, 1H), 3.71 (m,
1H), 3.67 (app t, J ) 9.7, 1H), 3.63-3.58 (m, 3H), 3.54 (dd, J ) 4.0,
10.0, 1H), 3.47-3.42 (m, 2H), 3.29 (app t, J ) 9.7, 1H), 3.22 (m,
1H), 2.38 (ddd, J ) 4.2, 4.2, 12.5, 1H), 1.83 (s, 6H), 1.73 (ddd, J )
12.5, 12.5, 12.5, 1H); 13C NMR (150 MHz, D2O) δ 181.5, 99.5,81.6,
74.8, 73.9, 73.4, 73.1, 72.0, 70.2, 61.4, 50.5, 49.4, 29.0, 23.6; MS (ESI)
325 (MH+).
4-O-(4-Azido-4-deoxy-2,3,6-tri-O-benzyl-r-D-glucopyranosyl)-2-
deoxystreptamine (40). Donor 29 (137 mg, 0.241 mmol) and acceptor
17 (60 mg, 0.202 mmol) were coevaporated twice from dry toluene
and further dried under high vacuum overnight. Diethyl ether (4.0 mL)
and dichloromethane (1.0 mL) were added, and the reaction was cooled
to -15 °C. Trifluoromethanesulfonic acid (6.0 µL, 68 µmol) was added
to a solution of N-iodosuccinimide (112 mg, 0.498 mmol) in diethyl
ether (3.0 mL) and dichloromethane (3.0 mL). A portion (3.0 mL) of
the resulting faint orange solution was added slowly dropwise to the
solution of 29 and 17, which was maintained at -15 °C. After 30 min,
the reaction mixture was diluted with diethyl ether and washed with
10% aqueous sodium bisulfite, saturated sodium bicarbonate, and brine.
The aqueous layers were back extracted with diethyl ether. The
combined organic phases were dried (Na2SO4), filtered, and concen-
trated under reduced pressure. The residue was purified by chroma-
tography on silica gel with 5% ethyl acetate in toluene to provide a
colorless oil (154 mg, Rf 0.54 in 10% acetone/toluene). This material
was dissolved in tetrahydrofuran (0.4 mL) and methanol (1.6 mL), and
sodium methoxide (0.7 M in methanol, 200 µL, 140 µmol) was added.
The reaction mixture was stirred at ambient temperature for 19 h, and
the solution was then neutralized with Amberlite IR-120(H+), filtered,
and evaporated under reduced pressure. The residue was purified by
chromatography on silica gel with 5% acetone in toluene to give 40
1
(125 mg, 92%) as a colorless oil: Rf 0.07 (5% acetone/toluene); H
NMR (500 MHz, CDCl3) δ 7.42-7.26 (m, 15H), 4.95 (d, J ) 3.5, 1H,
H1′), 4.90 (s, 2H), 4.86 (d, J ) 12.0, 1H), 4.70 (d, J ) 11.5, 1H), 4.65
(d, J ) 12.0, 1H), 4.59 (d, J ) 1.0, 1H, OH), 4.49 (d, J ) 12.0, 1H),
3.87 (app t, J ) 9.8, 1H), 3.84 (m, 1H), 3.76-3.70 (m, 2H) 3.66 (dd,
J ) 1.8, 10.8, 1H), 3.60 (dd, J ) 3.5, 9.5, 1H), 3.47-3.36 (m, 3H),
3.26 (m, 1H), 3.17 (m, 1H), 2.88 (d, J ) 1.0, 1H, OH), 2.26 (ddd, J )
4.4, 4.4, 13.5, 1H), 1.44 (app q, J ) 12.5, 1H); 13C NMR (125 MHz,
CDCl3) δ 137.6, 136.5, 128.7, 128.5 (×2), 128.4, 128.1, 128.0, 127.8
Paromamine (3). A solution of pseudodisaccharide 38 (73 mg, 0.11
mmol) in methanol (6 mL) was degassed by evacuating the flask and
backfilling with argon three times. Hydrazine (85 µL, 2.7 mmol) was
added, followed immediately by 20% Pd(OH)2 on carbon (∼10 mg,
Degussa type), and the reaction mixture was heated at reflux for 90 h.
The solution was cooled to ambient temperature, filtered through Celite,

6538 J. Am. Chem. Soc., Vol. 121, No. 28, 1999
Greenberg et al.
and concentrated under reduced pressure to afford a colorless solid (68
mg). This material was dissolved in a mixture of acetic acid (2 mL)
and water (2 mL), and the solution was degassed by evacuating the
flask and backfilling with argon three times. Next, 20% Pd(OH)2 on
carbon (∼10 mg, Degussa type) was added, and the solution was purged
with hydrogen. The reaction mixture was stirred at ambient temperature
under hydrogen gas (∼1 atm, balloon) for 21 h (an additional portion
of catalyst was added after 5 h). The solution was filtered through Celite
and concentrated under reduced pressure. The residue was purified by
chromatography on Amberlite CG-50 (NH4+, 1.5-cm-diameter × 10.5-
cm column) with a gradient of 0 to 2.5% concentrated ammonium
hydroxide in water. Fractions containing product were pooled and
concentrated under reduced pressure. The residue was dissolved in water
(3 mL), acidified with acetic acid, and lyophilized to provide 3 (45
mg, 82%) as its oily triacetate salt. All spectral data for synthetic and
naturally derived paromamine were identical: [R]D ) +68° (c ) 1,
H2O) (natural paromamine), [R]D ) +67° (c ) 1, H2O) (synthetic
paromamine); Rf 0.11 (10% concentrated ammonium hydroxide/
was purified by chromatography on Amberlite CG-50 (NH4+, 1.5-cm-
diameter × 10.5-cm column) with a gradient of 0 to 2.5% concentrated
ammonium hydroxide in water. Fractions containing product were
pooled and concentrated under reduced pressure. The residue was
dissolved in water (3 mL), acidified with acetic acid, and lyophilized
to provide 5 (42 mg, 45%) as its oily triacetate salt: Rf 0.07 (10%
1
concentrated ammonium hydroxide/methanol); H NMR (600 MHz,
D2O) δ 5.40 (d, J ) 3.9, 1H, H1′), 4.04 (m, 1H), 3.82 (app t, J ) 10.0,
1H), 3.77 (dd, J ) 3.4, 12.3, 1H), 3.72-3.67 (m, 2H), 3.64-3.60 (m,
2H), 3.49-3.43 (m, 2H), 3.21 (m, 1H), 3.14 (app t, J ) 10.0, 1H),
2.38 (ddd, J ) 4.2, 4.2, 12.5, 1H), 1.83 (s, 9H), 1.73 (ddd, J ) 12.5,
12.5, 12.5, 1H); 13C NMR (150 MHz, D2O) δ 181.8, 98.1, 80.9, 74.3,
73.5, 71.9, 70.5, 69.6, 61.1, 52.8, 50.5, 49.0, 29.0, 23.8; MS (ESI) 324
(MH+).
4-O-(6-Amino-6-deoxy-r-D-glucopyranosyl)-2-deoxystrept-
amine (6). A solution of pseudodisaccharide 41 (106 mg, 0.16 mmol)
in methanol (5 mL) was degassed by evacuating the flask and back-
filling with argon three times. Hydrazine (19 µL, 0.60 mmol) was added,
followed immediately by 20% Pd(OH)2 on carbon (∼10 mg, Degussa
type), and the reaction mixture was heated at reflux for 19 h. The
solution was cooled to ambient temperature, filtered through Celite,
and concentrated under reduced pressure to afford a waxy solid (95
mg). This material was dissolved in a mixture of acetic acid (2.5 mL)
and water (2.5 mL), and the solution was degassed by evacuating the
flask and backfilling with argon three times. Next, 20% Pd(OH)2 on
carbon (∼20 mg, Degussa type) was added, and the solution was purged
with hydrogen. The reaction mixture was stirred at ambient temperature
under hydrogen gas (∼1 atm, balloon) for 22 h. The solution was filtered
through Celite and concentrated under reduced pressure. The residue
was purified by chromatography on Amberlite CG-50 (NH4+, 1.5-cm-
diameter × 10.5-cm column) with a gradient of 0 to 4% concentrated
ammonium hydroxide in water. Fractions containing product were
pooled and concentrated under reduced pressure. The residue was
dissolved in water (3 mL), acidified with acetic acid, and lyophilized
to provide 6 (79 mg, 99%) as its oily triacetate salt: Rf 0.12 (20%
1
methanol); H NMR (600 MHz, D2O) δ 5.54 (d, J ) 3.9, 1H, H1′),
3.85-3.79 (m, 2H), 3.76 (m, 1H), 3.71 (app t, J ) 9.5, 1H), 3.66 (dd,
J ) 6.4, 12.2, 1H), 3.56 (app t, J ) 9.2, 1H), 3.47 (app t, J ) 9.5, 1H),
3.42-3.36 (m, 2H), 3.33 (dd, J ) 3.9, 10.9, 1H), 3.21 (m, 1H) 2.37
(ddd, J ) 4.3, 4.3, 12.6, 1H), 1.83 (s, 9H), 1.71 (ddd, J ) 12.6, 12.6,
12.6, 1H); 13C NMR (150 MHz, D2O) δ 182.1, 98.0, 82.0, 75.6, 74.4,
73.4, 70.2, 70.0, 61.1, 54.9, 50.6, 49.6, 29.7, 23.9; MS (ESI) 324 (MH+).
4-O-(3-Amino-3-deoxy-r-D-glucopyranosyl)-2-deoxystrept-
amine (4). A solution of pseudodisaccharide 39 (139 mg, 0.20 mmol)
in methanol (10 mL) was degassed by evacuating the flask and
backfilling with argon three times. Hydrazine (63 µL, 2.0 mmol) was
added, followed immediately by 20% Pd(OH)2 on carbon (∼10 mg,
Degussa type), and the reaction mixture was heated at reflux for 46 h
(an additional portion of catalyst was added after 17 h). The solution
was cooled to ambient temperature, filtered through Celite, and
concentrated under reduced pressure to afford a colorless solid (130
mg). This material was dissolved in a mixture of acetic acid (2.5 mL)
and water (2.5 mL), and the solution was degassed by evacuating the
flask and backfilling with argon three times. Next, 20% Pd(OH)2 on
carbon (∼10 mg, Degussa type) was added, and the solution was purged
with hydrogen. The reaction mixture was stirred at ambient temperature
under hydrogen gas (∼1 atm, balloon) for 46 h (an additional portion
of catalyst was added after 23 h). The solution was filtered through
Celite and concentrated under reduced pressure. The residue was
purified by chromatography on Amberlite CG-50 (NH4+, 1.5-cm-
diameter × 10.5-cm column) with a gradient of 0 to 2.5% concentrated
ammonium hydroxide in water. Fractions containing product were
pooled and concentrated under reduced pressure. The residue was
dissolved in water (3 mL), acidified with acetic acid, and lyophilized
to provide 4 (52 mg, 50%) as its oily triacetate salt: Rf 0.10 (10%
1
concentrated ammonium hydroxide/methanol); H NMR (600 MHz,
D2O) δ 5.43 (d, J ) 3.9, 1H, H1′), 3.90 (m, 1H), 3.74 (dd, J ) 10.0,
10.0, 1H), 3.66 (dd, J ) 9.5, 9.5, 1H), 3.62 (dd, J ) 9.5, 9.5, 1H),
3.55 (dd, J ) 3.9, 10.0, 1H), 3.48 (m, 1H), 3.44 (dd, J ) 9.5, 9.5, 1H),
3.31 (dd, J ) 3.1, 13.3, 1H), 3.26 (dd, J ) 9.5, 9.5, 1H), 3.20 (m, 1H),
3.07 (dd, J ) 8.0, 13.4, 1H), 2.38 (m, 1H), 1.83 (s, 9H), 1.75 (ddd, J
) 12.5, 12.5, 12.5, 1H); 13C NMR (150 MHz, D2O) δ 181.5, 96.5,
79.2, 74.0, 73.8, 73.0, 71.6(x2), 69.5, 50.6, 48.5, 41.1, 28.9, 23.7; MS
(ESI) 324 (MH+).
Hexaazido-hepta-O-benzyl Neomycin (42). A freshly prepared
dichloromethane solution of triflic azide14 (180 mL, ∼1.5 M) was added
slowly to a mixture of neomycin trisulfate hydrate (25.0 g, 26.2 mmol),
triethylamine (34.5 mL, 250 mmol), and CuSO4‚5H2O (411 mg, 1.65
mmol) in 150 mL of H2O plus 400 mL of methanol. The mixture was
stirred at room temperature. When TLC analysis showed incomplete
reaction after 8 h (Rf ) 0.6, EtOAc), an additional portion of triflic
azide (90 mL, ∼1.5 M in dichloromethane) was added, and the mixture
was stirred for 10 h. The solvent was then removed under reduced
pressure, and the residue was redissolved in 200 mL of ethyl acetate.
The solution was extracted with saturated aqueous NaHCO3 and
concentrated to a green foam. For ease of purification, the product was
peracetylated: the crude green foam was dissolved in 30 mL of pyridine
and 20 mL of acetic anhydride, and 4-(dimethylamino)pyridine (168
mg, 5 mol %) was added. The mixture was stirred for 3 h at room
temperature, concentrated under reduced pressure, redissolved in 200
mL of ethyl acetate, and extracted twice with 100 mL of 1 M aqueous
HCl. The organic layer was concentrated and passed through a plug of
silica, eluting with 2:1 EtOAc/hexanes, and then concentrated to a light
yellow gum. This was dissolved in 100 mL of methanol, and sodium
methoxide in methanol was added to a pH of 11. The solution was
stirred for 2 h and then neutralized with Amberlite IR-120 (H+), filtered,
and concentrated under reduced pressure, providing an off-white foam
(11.72 g, 15.2 mmol, 58%) which was directly benzylated: the foam
was dissolved in 80 mL of DMF, and tetrabutylammonium iodide was
added (2.8 g, 7.5 mmol). Benzyl bromide was added (31.6 mL, 266
mmol), followed by sodium hydride (3.65 g, 152 mmol) in ∼100-mg
1
concentrated ammonium hydroxide/methanol); H NMR (600 MHz,
D2O) δ 5.58 (d, J ) 3.9, 1H, H1′), 3.79-3.69 (m, 4H), 3.64-3.58 (m,
2H), 3.54 (app t, J ) 10.0, 1H), 3.45-3.39 (m, 2H), 3.36 (app t, J )
10.6, 1H), 3.19 (m, 1H), 2.36 (ddd, J ) 4.3, 4.3, 12.6, 1H), 1.80 (s,
9H), 1.71 (ddd, J ) 12.6, 12.6, 12.6, 1H); 13C NMR (150 MHz, D2O)
δ 182.0, 98.1, 81.0, 74.9, 73.7, 73.4, 68.6, 66.5, 60.8, 55.6, 50.5, 49.4,
29.2, 23.9; MS (ESI) 324 (MH+).
4-O-(4-Amino-4-deoxy-r-D-glucopyranosyl)-2-deoxystrept-
amine (5). A solution of pseudodisaccharide 40 (124 mg, 0.18 mmol)
in methanol (10 mL) was degassed by evacuating the flask and
backfilling with argon three times. Hydrazine (30 µL, 0.96 mmol) was
added, followed immediately by 20% Pd(OH)2 on carbon (∼10 mg,
Degussa type), and the reaction mixture was heated at reflux for 21 h
(an additional portion of catalyst was added after 17 h). The solution
was cooled to ambient temperature, filtered through Celite, and
concentrated under reduced pressure to afford a waxy solid (111 mg).
This material was dissolved in a mixture of acetic acid (2.5 mL) and
water (2.5 mL), and the solution was degassed by evacuating the flask
and backfilling with argon three times. Next, 20% Pd(OH)2 on carbon
(∼20 mg, Degussa type) was added, and the solution was purged with
hydrogen. The reaction mixture was stirred at ambient temperature
under hydrogen gas (∼1 atm, balloon) for 15 h. The solution was filtered
through Celite and concentrated under reduced pressure. The residue

New Aminoglycoside Antibiotics Containing Neamine
J. Am. Chem. Soc., Vol. 121, No. 28, 1999 6539
portions every 3-5 min over 2 h. After a total of 12 h, the solvent was
removed in vacuo, and the residue was dissolved in 200 mL of ethyl
acetate, extracted with 200 mL of water, washed with 100 mL of brine,
and concentrated. The product was purified by silica gel chromatog-
raphy, eluting first with hexanes to remove excess benzyl bromide and
then with 4:1 hexanes/EtOAc, providing 42 as a straw-colored gum
(17.2 g, 12.1 mmol, 80%, 46% from neomycin sulfate): Rf 0.5 (4:1
hexanes/EtOAc); 1H NMR (400 MHz, CDCl3) δ 7.15-7.37 (m, 35H),
6.16 (d, J ) 3.8, 1H), 5.66 (d, J ) 5.5, 1H), 4.69-4.99 (m, 6H), 4.39-
4.66 (m, 8H), 4.21-4.34 (m, 2H), 4.12 (m, 1H), 4.03 (dd, J1 ) 11, J2
) 8, 1H), 3.92-3.99 (m, 2H), 3.74-3.83 (m, 3H), 3.61-3.70 (m, 2H),
3.56 (dd, J1 ) 10, J2 ) 3, 1H), 3.40-3.50 (m, 4H), 3.30-3.38 (m,
2H), 3.20-3.30 (m, 3H), 3.11 (m, 1H), 2.99 (dd, J1 ) 10, J2 ) 3.5,
1H), 2.86 (dd, J1 ) 13.5, J2 ) 3.5, 1H), 2.25 (ddd, J1 ) 12.5, J2 ) J3
) 4.5, 1H), 1.42 (ddd, J1 ) J2 ) J3 ) 12.5, 1H); 13C NMR (100 MHz,
CDCl3) δ 138.3, 137.9, 137.7, 137.0, 136.9, 128.7, 128.5, 128.4, 128.3,
128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 127.5, 127.4, 106.4, 98.6,
95.6, 84.2, 82.5, 82.0, 81.7, 79.9, 78.6, 75.5, 75.3, 75.1, 74.8, 74.4,
73.3, 72.8, 72.3, 71.7, 71.4, 70.9, 70.2, 63.1, 60.3, 59.9, 57.2, 51.3,
51.0, 32.5; HRMS (FAB) for C72H76N18O13 (M + Cs+) calcd 1533.4894,
found 1533.4998.
1,3,2′,6′-Tetraazido-6,3′,4′-tri-O-benzyl Neamine (43). Compound
42 (3.87 g, 2.76 mmol) was dissolved in 120 mL of 1 M HCl in 5:5:1
methanol/dioxane/water and stirred under reflux for 24 h. The acid was
quenched with solid NaHCO3, and the solution was filtered and
concentrated in vacuo. The residue was chromatographed on silica (75:
15:10 hexanes/EtOAc/toluene), providing an oil, which was further
purified by recrystallization from 8:1:1 hexanes/EtOAc/toluene, produc-
ing 43 as colorless long needles (1.03 g). The mother liquor and
remaining column fractions containing starting material were resubjected
to the reaction conditions for 16 h, providing an additional 330 mg of
43 after chromatography and recrystallization. Total yield of 43 was
1.36 g (1.96 mmol, 71%): Rf 0.4 (5:1 hexanes/EtOAc); 1H NMR (400
MHz, CDCl3) δ 7.25-7.43 (m, 15H), 5.25 (d, J ) 3.0, 1H), 4.85-
4.92 (m, 6H), 4.63 (d, J ) 9.5, 1H), 4.14 (m, 1H), 3.98 (dd, J1 ) 8.5,
J2 ) 7.5, 1H), 3.70 (br, 1H), 3.47-3.67 (m, 4H), 3.45 (m, 1H), 3.40
(dd, J1 ) 10.5, J2 ) 3.5, 1H), 3.23-3.32 (m, 3H), 2.30 (ddd, J1 )
10.5, J2 ) J3 ) 4.5, 1H), 1.48 (ddd, J1 ) J2 ) J3 ) 10.5, 1H); 13C
NMR (100 MHz, CDCl3) δ 137.7, 137.6, 137.4, 128.6, 128.5, 128.3,
128.1, 127.8, 99.3, 83.6, 82.9, 80.6, 78.7, 76.7, 75.7, 75.4, 75.2, 71.3,
64.1, 59.8, 58.7, 50.9, 32.3; HRMS (FAB) for C33H36N12O6 (M + Cs+)
calcd 829.1935, found 829.1968.
4.0, 1H), 4.82-4.93 (m, 4H), 4.64 (app. q, J ) 10.5, 1H), 4.50 (m,
2H), 4.27 (ddd, J1 )10, J2 )4.0, J3 )2.5, 1H), 3.94 (dd, J1 ) 10.5, J2
) 9.0, 1H), 3.52-3.60 (m, 2H), 3.50 (dd, J1 ) 13.0, J2 ) 2.5, 1H),
3.35-3.43 (m, 6H), 2.32 (ddd, J1 ) 12.5, J2 ) J3 ) 4.5, 1H), 1.51
(ddd, J1 ) J2 ) J3 ) 12.5, 1H); 13C NMR (125 MHz, CDCl3) δ 197.8,
137.5, 137.5, 137.0, 128.6, 128.5, 128.2, 128.1, 128.0, 127.9, 127.8,
98.1, 85.1, 84.3, 80.0, 79.3, 78.7, 78.3, 75.9, 75.4, 75.1, 71.2, 63.3,
60.2, 58.8, 50.8, 32.0; HRMS (FAB) for C35H38N12O7 (M + Cs+) calcd
871.2041, found 871.2071.
General Procedure for Reductive Aminations (46-54). Crude
aldehyde 45 (∼100 mg, 0.14 mmol) was dissolved in 0.90 mL of
methanol. Three equivalents of the appropriate protected amine30 (0.40
mmol) was added as a 1 M solution in methanol (0.40 mL), followed
by 0.43 mL of 1 M solution of acetic acid in methanol. The pH was
checked and adjusted to about 6 with acetic acid, if necessary.
NaCNBH3 was added as a freshly prepared 0.3 M solution in methanol
(0.19 mL), and the mixture was stirred at room temperature for 2 h.
The reaction was quenched with a few drops of water and concentrated
under reduced pressure, and the product was purified by silica
chromatography (1-5% MeOH/0.5-1% triethylamine/dichloromethane).
In the case of 46, the secondary amine arising from the reductive
amination was protected as a benzyl carbamate (benzyl chloroformate,
pyridine) before proceeding to the next step. Reported yields are
combined for the ozonolysis and reductive amination steps.
Compound 46: yield 48 mg, 39%; 1H NMR (500 MHz, CDCl3) δ
7.25-7.40 (m, 25H), 5.50 (m, 1H), 5.43 (m, 1H), 5.01-5.13 (m, 4H),
4.80-4.87 (m, 4H), 4.73 (m, 1H), 4.60-4.65 (m, 2H), 4.24 (m, 1H),
3.96-4.00 (m, 3H), 3.85-3.89 (m, 1H), 3.05-3.54 (m, 13H), 2.26
(m, 1H), 1.63 (m, 2H), 1.42 (m, 1H); 13C NMR (125 MHz, CDCl3) δ
156.6, 155.8, 137.7, 137.6, 137.4, 136.7, 136.5, 129.6, 129.2, 128.8,
128.4, 128.3, 128.0, 127.9, 127.8, 127.5, 97.6, 84.7, 84.5, 84.4, 79.9,
79.6, 78.6, 75.7, 75.4, 75.2, 75.1, 71.8, 71.1, 67.4, 66.5, 62.7, 60.1,
59.1, 50.9, 47.7, 46.5, 45.3, 44.6, 38.0, 37.4, 32.1, 27.8, 25.4; HRMS
(FAB) for C54H60N14O10 (M + Cs+) calcd 1197.3671, found 1197.3738.
Compound 47: yield 78 mg, 60%; 1H NMR (400 MHz, CDCl3) δ
7.25-7.42 (m, 20H), 5.71 (m, 1H), 5.59 (d, J ) 2.9, 1H), 5.07 (s,
2H), 4.83-4.88 (m, 5H), 4.61 (m, 1H), 4.26 (m, 1H), 3.90-4.03 (m,
3H), 3.19-3.56 (m, 11H), 2.81 (m, 2H), 2.59-2.73 (m, 2H), 2.38 (m,
4H), 2.26 (m, 1H), 2.15-2.18 (m, 4H), 1.62-1.65 (m, 4H), 1.45 (m,
1H); 13C NMR (100 MHz, CDCl3) δ 137.6, 137.4, 136.7, 136.5, 128.5,
128.4, 128.1, 128.0, 127.9, 127.8, 97.6, 84.8, 84.4, 79.8, 78.7, 77.5,
75.6, 75.4, 75.1, 71.1, 66.5, 63.1, 60.2, 59.1, 56.5, 55.6, 50.9, 49.3,
48.7, 41.6, 39.6, 37.4, 32.1, 26.7; HRMS (FAB) for C50H63N15O8 (M
+ Cs+) calcd 1134.4038, found 1134.4099.
5-O-Allyl-1,3,2′,6′-tetraazido-6,3′,4′-tri-O-benzyl Neamine (44).
Compound 43 (810 mg, 1.21 mmol) was dissolved in 10 mL of DMF.
Tetrabutylammonium iodide (425 mg, 1.14 mmol) was added, followed
by sodium hydride (44 mg, 1.73 mmol). Allyl bromide was added (303
µL, 3.5 mmol), and the mixture was stirred at room temperature for 2
h. The reaction was quenched with 1 mL of methanol, the solvent was
removed under reduced pressure, and the residue was redissolved in
30 mL of ethyl acetate and extracted with brine. After the organic layer
was concentrated, the product was purified by silica chromatography
(10:1 hexanes/EtOAc), providing 44 as a colorless gum (890 mg,
Compound 48: yield 65 mg, 52%; 1H NMR (400 MHz, CDCl3) δ
7.25-7.38 (m, 25H), 5.56 (d, J ) 3.8, 1H), 5.28 (m, 2H), 5.02-5.09
(m, 4H), 4.81-4.90 (m, 4H), 4.71 (m, 1H), 4.59 (m, 1H), 4.25 (m,
1H), 4.02 (m, 1H), 3.91 (m, 1H), 3.76 (m, 1H), 3.04-3.56 (m, 13H),
2.64 (m, 2H), 2.51 (m, 4H), 2.19 (m, 1H), 1.37 (m, 1H); 13C NMR
(100 MHz, CDCl3) δ 156.5, 137.6, 137.6, 137.6, 128.7, 128.7, 128.6,
128.6, 128.6, 128.5, 128.5, 128.4, 128.4, 128.4, 128.3, 128.3, 128.3,
128.2, 128.1, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 97.6,
84.6, 84.4, 79.5, 78.7, 75.3, 75.1, 71.7, 71.1, 66.6, 63.0, 60.0, 59.2,
54.0, 53.9, 50.9, 45.7, 38.9, 31.9; HRMS (FAB) for C55H63N15O10 (M
+ H+) calcd 1094.4961, found 1094.4917.
1
99%): Rf 0.55 (10:1 hexanes/EtOAc); H NMR (500 MHz, CDCl3) δ
7.26-7.41 (m, 15H), 5.96 (m, 1H) 5.59 (d, J ) 4.0, 1H), 5.30 (m,
1H), 5.19 (m, 1H), 4.86-4.90 (m, 3H), 4.81 (m, 2H), 4.63 (m, 1H),
4.45 (m, 1H), 4.38 (m, 1H), 4.27 (ddd, J1 ) 10, J2 ) 4.5, J3 ) 3.0,
1H), 3.99 (dd, J1 ) 10.5, J2 ) 9.0, 1H), 3.32-3.59 (m, 9H), 2.27 (ddd,
J1 ) 13.0, J2 ) J3 ) 4.5, 1H), 1.44 (ddd, J1 ) J2 ) J3 ) 13.0, 1H);
13C NMR (125 MHz, CDCl3) δ 137.7, 137.6, 137.3, 128.5, 128.4, 128.3,
128.0, 127.9, 127.7, 116.4, 97.6, 84.4, 84.1, 80.0, 78.6, 77.5, 75.9, 75.1,
70.9, 63.5, 60.0, 59.1, 50.9, 32.3; HRMS (FAB) for C36H40N12O6 (M
+ Cs+) calcd 869.2248, found 869.2214.
Aldehyde 45. Compound 44 (290 mg, 0.39 mmol) was dissolved
in 15 mL of 1:1 dichloromethane/methanol and cooled to -78 °C.
Ozone was bubbled into the solution until it turned blue (∼ 20 s), at
which time the solution was flushed with O2. Dimethyl sulfide (300
µL, 4.1 mmol) was added, and the mixture was warmed to room
temperature and stirred for 1 h. The solvent was removed under reduced
pressure, providing ∼290 mg of crude 45 as a colorless oil, which was
used without further purification: Rf 0.1 (6:1 hexanes/EtOAc); 1H NMR
(500 MHz, CDCl3) δ 9.55 (s, 1H), 7.26-7.38 (m, 15H), 5.49 (d, J )
Compound 49: yield 81 mg, 63%; 1H NMR (400 MHz, CDCl3) δ
7.27-7.38 (m, 20H), 5.68 (d, J ) 3.8, 1H), 5.28 (m, 1H), 5.12 (m,
4H), 4.84-4.91 (m, 4H), 4.63 (m, 1H), 4.28 (m, 1H), 3.88-4.10 (m,
3H), 3.25-3.58 (m, 11H), 2.85 (m, 2H), 2.70 (m, 1H), 2.23-2.54 (m,
9H), 1.44 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 156.3, 137.6, 137.4,
136.8, 136.7, 128.5, 128.5, 128.3, 128.2, 128.2, 128.1, 128.0, 127.9,
127.8, 97.7, 84.6, 84.5, 79.7, 78.7, 77.6, 75.7, 75.5, 75.1, 71.0, 67.3,
67.1, 66.7, 63.3, 59.1, 57.5, 57.0, 52.6, 50.9, 49.8, 46.3, 45.8, 43.7,
37.4, 32.2; HRMS (FAB) for C49H59N15O8 (M + Cs+) calcd 1118.3725,
found 1118.3789.
Compound 50: yield 61 mg, 53%; 1H NMR (400 MHz, CDCl3) δ
7.25-7.35 (m, 15H), 5.69 (d, J ) 2.9, 1H), 4.83-4.89 (m, 6H), 4.63
(m, 1H), 4.28 (m, 1H), 3.99-4.06 (m, 3H), 3.91 (m, 1H), 3.68 (m,
(30) (a) Atwell, G. J.; Denny, W. A. Synthesis 1984, 1032-1033. (b)
Adamczyk, M.; Fishpaugh, J. R.; Heuser, K. J. Org. Prep. Proced. Int.
1998, 30, 339-348.

6540 J. Am. Chem. Soc., Vol. 121, No. 28, 1999
Greenberg et al.
4H), 3.33-3.57 (m, 8H), 2.84 (m, 2H), 2.70 (m, 2H), 2.40 (m, 6H),
2.27 (m, 1H), 1.43 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 137.6,
137.4, 136.8, 128.5, 128.5, 128.4, 128.2, 128.0, 128.0, 128.0, 127.9,
127.8, 97.6, 84.6, 79.8, 78.7, 77.5, 75.7, 75.5, 75.1, 73.1, 71.0, 66.9,
63.3, 60.1, 59.2, 58.7, 53.7, 50.9, 50.0, 46.3, 32.2; HRMS (FAB) for
C41H52N14O7 (M + Cs+) calcd 985.3198, found 985.3226.
96.3, 84.6, 76.8, 74.3, 72.0, 71.0, 70.7, 67.7, 55.0, 51.6, 50.3, 49.1,
46.2, 41.6, 38.1, 30.6, 25.2; LRMS (ESI) for C17H38N6O6 (M + H+)
calcd 423, found 423, (M + Cl-), calcd 457, found 457.
1
Compound 8: yield 34 mg, 58%; H NMR (400 MHz, D2O) δ
5.84 (d, J ) 3.5, 1H), 4.18-4.24 (m, 2H), 4.04-4.14 (m, 2H), 3.94
(m, 1H), 3.88 (t, J ) 9.5, 1H), 3.80 (t, J ) 9.5, 1H), 3.30-3.60 (m,
12H), 3.21 (t, J ) 7.8, 2H), 3.10 (t, J ) 7.8, 2H), 2.94 (s, 3H), 2.46
(ddd, J1 ) J2 ) J3 ) 12.5, 1H), 2.11-2.24 (m, 4H), 1.91 (ddd, J1 )
Compound 51: yield 59 mg, 44%; 1H NMR (400 MHz, CDCl3) δ
7.27-7.41 (m, 20H), 5.63 (d, J ) 3.8, 1H), 5.08 (s, 2H), 4.82-4.88
(m, 6H), 4.61 (d, J ) 11, 1H), 4.26 (m, 1H), 3.97-4.02 (m, 2H), 3.88
(m, 1H), 3.16-3.59 (m, 13H), 2.86 (m, 1H), 2.74 (m, 1H), 2.56 (m,
1H), 2.28 (m, 1H), 1.27-1.50 (m, 6H); 13C NMR (100 MHz, CDCl3)
δ 153.3, 137.5, 137.4, 128.6, 128.5, 128.5, 128.5, 128.4, 128.1, 128.1,
128.0, 127.9, 127.8, 97.6, 87.8, 84.5, 79.7, 78.7, 77.4, 75.7, 77.1, 77.1,
77.1, 75.7, 75.1, 63.2, 60.1, 59.2, 58.8, 50.9, 46.5, 40.7, 32.2, 31.0,
30.3, 30.1, 23.0; LRMS (ESI) for C49H60N14O9 (M + H+) calcd 990,
found 990.
Compound 52: yield 75 mg, 56%; 1H NMR (400 MHz, CDCl3) δ
7.26-7.41 (m, 20H), 5.65 (d, J ) 3.8, 1H), 5.08 (s, 2H), 4.80-4.88
(m, 6H), 4.61 (d, J ) 11.4, 1H), 4.27 (m, 1H), 3.97-4.07 (m, 2H),
3.84 (m, 1H), 3.16-3.58 (m, 13H), 2.87 (m, 1H), 2.72 (m, 1H), 2.55
(m, 1H), 2.28 (m, 1H), 1.28-1.50 (m, 6H); 13C NMR (100 MHz,
CDCl3) δ 153.3, 137.6, 137.4, 128.5, 128.5, 128.1, 128.1, 128.0, 128.0,
127.8, 97.7, 84.5, 79.7, 78.7, 77.5, 75.7, 75.5, 75.1, 73.3, 71.1, 66.6,
63.2, 62.5, 60.1, 59.1, 58.8, 50.9, 46.8, 40.7, 32.2, 31.0, 30.3, 30.1,
23.0; HRMS (FAB) for C49H60N14O9 (M + Cs+) calcd 1121.3722, found
1121.3772.
12.5, J2 ) J3 ) 4.0, 1H), 1.64-1.77 (m, 4H), 1.41-1.51 (m, 2H); 13
C
NMR (100 MHz, D2O) δ 99.0, 87.8, 86.5, 79.0, 77.4, 74.9, 70.7, 59.0,
55.7, 55.5, 55.3, 53.3, 52.3, 51.3, 50.3, 42.4, 42.1, 38.9, 30.6, 29.4,
25.8, 24.5; LRMS (ESI) for C21H47N7O6 (M + H+) calcd 494, found
494, (M + Cl-) calcd 528, found 528.
1
Compound 9: yield 39 mg, 44%; H NMR (400 MHz, D2O) δ
5.88 (d, J ) 3.5, 1H), 4.20 (t, J ) 9, 1H), 3.92- -4.14 (m, 4H), 3.77-
3.86 (m, 2H), 3.34-3.64 (m, 8H), 3.21 (t, J ) 6.5, 4H), 3.03 (m, 4H),
2.49 (ddd, J1 ) J2 ) J3 ) 12.5, 1H), 1.93 (ddd, J1 ) 12.5, J2 ) J3 )
4.0, 1H); 13C NMR (100 MHz, D2O) δ 95.7, 87.9, 85.6, 75.5, 74.9,
72.6, 72.5, 70.6, 55.6, 55.4, 52.9, 52.4, 51.3, 42.5, 38.1, 30.3; LRMS
(ESI) for C18H41N7O6 (M + H+) calcd 452, found 452, (M + Cl-)
calcd 486, found 486.
1
Compound 10: yield 8 mg, 13%; H NMR (400 MHz, D2O) δ
5.84 (d, J ) 3.8, 1H), 4.21 (m, 1H), 4.04-4.13 (m, 2H), 3.93 (m, 1H),
3.85 (t, J ) 9.5, 1H), 3.79 (t, J )9, 1H), 3.26-3.62 (m, 13H), 2.80-
2.90 (m, 8H), 2.48 (ddd, J1 ) J2 ) J3 ) 12.5, 1H), 1.91 (ddd, J1 )
12.5, J2 ) J3 ) 4.0, 1H); 13C NMR (100 MHz, D2O) δ 102.7, 87.9,
86.7, 81.0, 73.3, 69.5, 68.2, 56.8, 56.0, 55.5, 54.8, 51.5, 51.0, 50.0,
45.5, 45.2, 35.1, 34.2, 30.3; LRMS (ESI) for C20H43N7O6 (M + H+)
calcd 478, found 478, (M + Cl-) calcd 512, found 512.
Compound 53: yield 66 mg, 62%; 1H NMR (500 MHz, CDCl3) δ
7.26-7.42 (m, 15H), 5.70 (d, J ) 3.5, 1H), 4.84-4.90 (m, 6H), 4.63
(m, 1H), 4.26 (m, 1H), 3.99-4.08 (m, 2H), 3.88 (m, 1H), 3.33-3.68
1
(m, 13H), 2.84 (m, 2H), 2.69 (m, 1H), 2.31 (m, 3H), 1.46 (m, 1H); 13
C
Compound 11: yield 34 mg, 68%; H NMR (400 MHz, D2O) δ
NMR (125 MHz, CDCl3) δ 137.5, 137.4, 134.6, 128.5, 127.9, 127.8,
97.5, 84.6, 84.5, 79.6, 78.7, 77.5, 75.6, 75.4, 75.1, 73.0, 71.6, 63.8,
62.6, 60.6, 60.0, 59.4, 50.4, 46.9, 46.1, 32.0; HRMS (FAB) for
C38H47N13O8 (M + Cs+) calcd 946.2725, found 946.2696.
5.87 (d, J ) 3.5, 1H), 4.24 (m, 2H), 4.07-4.16 (m, 2H), 3.79-3.97
(m, 6H), 3.35-3.65 (m, 17H), 2.50 (ddd, J1 ) J2 ) J3 ) 12.5, 1H),
1.94 (ddd, J1 ) 12.5, J2 ) J3 ) 4.0, 1H); 13C NMR (100 MHz, D2O)
δ 96.0, 87.8, 85.3, 75.9, 73.2, 72.5, 70.5, 68.4, 66.6, 55.5, 54.9, 54.5,
52.3, 51.2, 50.7, 44.1, 42.3, 37.8, 30.4; LRMS (ESI) for C20H42N6O7
(M + H+) calcd 479, found 479, (M + Cl-) calcd 513, found 513.
Compound 54: yield 62 mg, 62%; 1H NMR (400 MHz, CDCl3) δ
7.20-7.53 (m, 19H), 5.70 (d, J ) 3.8, 1H), 4.77-4.91 (m, 6H), 4.62
(m, 1H), 4.29 (m, 1H), 3.99-4.11 (m, 4H), 3.90 (m, 1H), 3.25-3.58
(m, 8H), 2.81 (m, 2H), 2.28 (m, 1H), 1.45 (m, 1H); 13C NMR (100
MHz, CDCl3) δ 153.7, 137.6, 137.6, 137.5, 128.6, 128.6, 128.5, 128.5,
128.4, 128.1, 128.1, 128.0, 127.9, 127.9, 127.8, 122.3, 97.7, 84.6, 84.4,
79.4, 78.6, 77.5, 75.7, 75.4, 75.1, 73.3, 71.1, 63.1, 60.1, 59.2, 51.0,
49.5, 47.7, 32.2; HRMS (FAB) for C43H47N15O6 (M + Cs+) calcd
1002.2888, found 1002.2844.
1
Compound 12: yield 21 mg, 60%; H NMR (400 MHz, D2O) δ
5.81 (d, J ) 3.8, 1H), 4.10-4.23 (m, 3H), 4.03 (t, J ) 9, 1H), 3.88-
3.96 (m, 2H), 3.71-3.86 (m, 3H), 3.32-3.58 (m, 9H), 2.99 (t, J )
7.8, 2H), 2.45 (ddd, J1 ) J2 ) J3 ) 12.5, 1H), 1.89 (ddd, J1 ) 12.5,
J2 ) J3 ) 4.0, 1H), 1.64-1.77 (m, 4H), 1.41-1.51 (m, 2H); 13C NMR
(100 MHz, D2O) δ 94.0, 87.8, 85.5, 75.0, 72.6, 72.5, 70.6, 68.7, 61.9,
60.4, 55.6, 52.8, 52.3, 51.3, 47.5, 42.4, 41.5, 30.5, 29.1, 29.0, 24.4;
LRMS (ESI) for C20H44N6O7 (M + H+) calcd 481, found 481, (M +
Cl-) calcd 515, found 515.
General Procedure for Deprotection of 46-53 (7-14). The
starting material was dissolved in 4.5 mL of THF and 0.5 mL of 0.1
M aqueous NaOH. Then, 4.5 equiv of PMe3 was added as a 1 M
solution in THF, and the mixture was stirred at room temperature (or
50 °C if the reaction was proceeding slowly) for 2 h. When complete,
the mixture was concentrated and passed through a short silica column
(10:1 MeOH/NH3(aqueous). The crude amine product was then
dissolved in 2 mL of 1:1 EtOH/THF and added in small portions to a
blue solution of sodium metal (∼80 mg) in liquid ammonia (25 mL)
and THF (10 mL). After all starting material had been added, the
mixture was stirred until the blue color returned and was stable for 15
min. At this time, the reaction was quenched with ammonium formate
(500 mg in 1 mL of water), the solvent was evaporated off, and the
product mixture was lyophilized. Products were purified by ion-
exchange chromatography (Amberlite CG-50, NH4+ form) with a linear
gradient of aqueous ammonia. Gradients of 0-10, 0-15, or 0-20%
concentrated aqueous ammonia were used. Fractions (3 mL) were
collected with an automated fraction collector, and those containing
product were pooled and lyophilized and then converted to their
hydrochloride salts by dissolving in an excess of 0.1 M aqueous HCl
and lyophilizing again. Yields are reported for the HCl salts, combined
for the two-step deprotection.
1
Compound 13: yield 34 mg, 80%; H NMR (400 MHz, D2O) δ
5.85 (d, J ) 3.5, 1H), 4.24 (m, 2H), 4.05-4.15 (m, 2H), 3.80-3.95
(m, 4H), 3.75 (m, 1H), 3.35-3.65 (m, 9H), 3.00 (t, J ) 7.6, 2H), 2.49
(ddd, J1 ) J2 ) J3 ) 12.5, 1H), 1.94 (ddd, J1 ) 12.5, J2 ) J3 ) 4.0,
1H), 1.65-1.75 (m, 4H), 1.43-1.49 (m, 2H); 13C NMR (100 MHz,
D2O) δ 95.8, 87.9, 85.3, 75.8, 74.8, 72.5, 70.6, 68.4, 61.6, 60.6, 55.6,
52.3, 51.3, 46.9, 42.4, 41.6, 30.3, 29.0, 28.9, 24.4; LRMS (ESI) for
C20H44N6O7 (M + H+) calcd 481, found 481, (M + Cl-) calcd 515,
found 515.
1
Compound 14: yield 11 mg, 76%; H NMR (500 MHz, D2O) δ
5.82 (d, J ) 3.5, 1H), 4.25 (m, 1H), 4.18 (t, J ) 9.2, 1H), 4.13 (m,
1H), 4.04 (t, J ) 9.5, 1H), 3.76-3.92 (m, 8H), 3.75 (m, 1H), 3.51-
3.59 (m, 2H), 3.38-3.48 (m, 6H), 2.46 (ddd, J1 ) J2 ) J3 ) 12.5,
1H), 1.89 (ddd, J1 ) 12.5, J2 ) J3 ) 4.0, 1H); 13C NMR (100 MHz,
D2O) δ 102.5, 87.9, 85.4, 75.1, 72.3, 70.9, 68.6, 62.6, 59.9, 59.8, 58.6,
55.6, 52.3, 51.2, 47.6, 42.3, 32.8; HRMS (FAB) for C17H37N5O8 (M +
H+) calcd. 440.2720, found 440.2704.
Compound 15. Protected compound 54 (60 mg, 0.069 mmol) was
dissolved in 2 mL of ethanol. Hydrazine (11 µL, 0.35 mmol) was added,
followed by Raney nickel (∼30 mg). The mixture was stirred at room
temperature for 2 h and then filtered through Celite, concentrated, and
redissolved in 2 mL of 1:1 acetic acid/water. Palladium hydroxide (20
mg, 20% on C, Degussa type) was added, and the mixture was stirred
under a hydrogen balloon for 12 h. The product was filtered, lyophilized,
and purified by ion-exchange chromatography (Amberlite CG-50, NH4+
form) with a linear gradient (0-10%) of aqueous ammonia. Fractions
1
Compound 7: yield 19 mg, 65%; H NMR (600 MHz, D2O) δ
5.69 (d, J ) 3.6, 1H), 4.03 (m, 1H), 3.86-3.98 (m, 3H), 3.76 (m, 1H),
3.68 (dd, J1) J2 ) 9.0, 1H), 3.44 (dd, J1 ) J2 ) 9.0, 1H), 3.40 (dd, J1
) 13.5, J2 ) 3.5, 1H), 3.24-3.39 (m, 6H), 3.14 (m, 2H), 3.05 (m,
2H), 2.33 (ddd, J1 ) J2 ) J3 ) 12.5, 1H), 2.02-2.10 (m, 2H), 1.42
(ddd, J1 ) 12.5, J2 ) J3 ) 4.0, 1H); 13C NMR (150 MHz, D2O) δ

New Aminoglycoside Antibiotics Containing Neamine
J. Am. Chem. Soc., Vol. 121, No. 28, 1999 6541
(3 mL) were collected with an automated fraction collector, and those
containing product were pooled and lyophilized and then converted to
the hydrochloride salt by dissolving in an excess of 0.1 M aqueous
Final concentrations in the translation mixture were as follows: Tris-
acetate (pH 7.4), 70 mM; dithiothreitol, 2.2 mM; ATP (sodium salt),
1.5 mM; UTP, GTP, CTP (sodium salts), 1 mM each; phophoenolpyru-
vate (potassium salt), 33 mM; PEG-8000, 2.4% (w/v); folinic acid, 42
µg/mL; pyridoxine hydrochloride, 33 µg/mL; NADP, 33 µg/mL; FAD,
33 µg/mL; p-aminobenzoic acid, 13.6 µg/mL; tRNA (from E. coli, type
XXI from Sigma), 0.2 mg/mL; potassium acetate, 88 mM; ammonium
acetate, 44 mM; calcium acetate, 4 mM; magnesium acetate, 8 mM;
IPTG (isopropylthiogalactoside, induces lac expression), 1 mM; amino
acids (all 20), 0.1 mM of each; 8 µL of S-30 extract and 1 µg of DNA
template per 25 µL of translation solution. The translation assays were
performed by mixing all of the reagents, varying amounts of the
compounds to be tested, and the DNA template in a RNase-free
microcentrifuge tube. The S-30 extract was always added last, and the
reaction was maintained at 21 ( 1 °C in a water bath. The reaction
was terminated after 30 min by diluting the reaction 10-fold with
luciferase dilution reagent (25 mM Tris-phosphate, pH 7.8; 2 mM
DTT; 2 mM 1,2-diaminocyclohexane N,N,N′,N′-tetraacetate; 10%
glycerol; 1% Triton X-100; and 1 mg/mL bovine serum albumin).
Translation yield was determined by mixing 10 µL of the diluted
reaction mixture with 50 µL of luciferase assay reagent (20 mM Tricine,
pH 7.8; 15 mM MgSO4; 0.1 mM EDTA; 33.3 mM DTT; 270 µM
coenzyme A; 470 µM luciferin; and 530 µM ATP) and monitoring the
luminescence with a Turner Designs luminometer. For each assay,
points were collected in duplicate, and the full assays were performed
at least three times for each compound.
1
HCl and lyophilizing again: yield 16 mg, 32%; H NMR (400 MHz,
CDCl3) δ 7.83 (m, 2H), 7.61 (m, 2H), 5.86 (d, J ) 3.8, 1H), 4.18-
4.34 (m, 3H), 4.08 (t, J ) 9.8, 1H), 3.81-3.96 (m, 3H), 3.31-3.64
(m, 10H), 2.49 (m, 1H), 1.95 (app q, J ) 12.7, 1H); 13C NMR (100
MHz, CDCl3) δ 202.9, 135.1, 129.0, 117.2, 95.9, 85.4, 75.8, 74.9, 72.6,
72.4, 70.5, 68.5, 55.5, 52.3, 51.3, 50.7, 44.4, 42.4, 30.3; HRMS (FAB)
for C22H37N7O6 (M + H+) calcd 496.2884, found 496.2889.
SPR Binding Studies. 5′-Biotinylated A-site RNA was prepared
exactly as described previously.8 Binding experiments and curve fitting
were also performed as previously described.8 Data from 2-4
independent experiments for each compound were averaged to obtain
the reported dissociation constants.
Antibiotic Testing. Minimal inhibitory concentrations (MICs) were
determined according to published protocols.25 Briefly, E. coli ATCC
25922 was grown in Mueller-Hinton broth (cation-adjusted, BBL
Microbiology Systems) to an optical density (OD600) of ∼ 0.5, and
then diluted to OD600) 0.1. Samples of antibiotic were prepared in
Mueller-Hinton broth over the desired concentration range. For each
concentration, 50 mL of diluted culture was combined with 1 mL of
antibiotic sample and incubated at 37 °C for 4-6 h. The control sample
(no antibiotic) typically would have an OD600 of 1.2-1.5 at this time.
The absorbance of each sample was read, and the MIC was considered
to be the lowest concentration of antibiotic at which OD600 was less
than 1% of the control.
In Vitro Translation Assay. A coupled transcription-translation
assay was performed with luciferase DNA to determine the extent of
translational inhibition in the presence of the various aminoglycosides.
The DNA template was constructed by cleaving the luciferase gene
from the vector pBestluc (Promega) and placing it into the vector
pBluescript SK- (Stratagene) behind the lac promoter. The transcription/
translation mixture, or S-30 extract, and the reaction buffers were
prepared as recommended by Ellman et al.31 with slight modifications.
Magnesium and calcium concentrations were optimized for the extract.
Acknowledgment. We gratefully acknowledge support from
the National Institutes of Health and additional funding from
Novartis. We thank Dr. Raj Chadha for determining the crystal
structure of compound 18.
JA9910356
(31) Ellman, J.; Mendel, D.; Anthony-Cahill, S.; Noren, C. J.; Schultz,
P. G. Methods Enzymol. 1991, 202, 301-337.