Novel method for the synthesis of 3′,4′-dideoxygenated pyranmycin and kanamycin compounds, and studies of their antibacterial activity against aminoglycoside-resistant bacteria

Article abstract of DOI:10.1081/CAR-200059968

A novel protocol for converting a trans-diol to an alkene under mild conditions was developed. This method led to the synthesis of a 3′,4′-dideoxykanamycin (dibekacin) analog and a 3′,4′- dideoxypyranmycin that were found to be active against aminoglycosi

Full text of DOI:10.1081/CAR-200059968

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Novel Method for the Synthesis of
3′,4′‐Dideoxygenated Pyranmycin and
Kanamycin Compounds, and Studies
of Their Antibacterial Activity Against
Aminoglycoside‐Resistant Bacteria
Ravi Rai a , Hsiao‐Nung Chen a , Huiwen Chang a & Cheng‐Wei Tom
Chang a
a Department of Chemistry and Biochemistry , Utah State
University , Logan, Utah, USA
Published online: 16 Aug 2006.
To cite this article: Ravi Rai , Hsiao‐Nung Chen , Huiwen Chang & Cheng‐Wei Tom Chang (2005) Novel
Method for the Synthesis of 3′,4′‐Dideoxygenated Pyranmycin and Kanamycin Compounds, and Studies
of Their Antibacterial Activity Against Aminoglycoside‐Resistant Bacteria, Journal of Carbohydrate
Chemistry, 24:2, 131-143, DOI: 10.1081/CAR-200059968
To link to this article: http://dx.doi.org/10.1081/CAR-200059968
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Journal of Carbohydrate Chemistry, 24:131–143, 2005
Copyright # Taylor & Francis, Inc.
ISSN: 0732-8303 print
DOI: 10.1081/CAR-200059968
Novel Method for the
Synthesis of 30,40-
Dideoxygenated
Pyranmycin and Kanamycin
Compounds, and Studies of
Their Antibacterial Activity
Against Aminoglycoside-
Resistant Bacteria
Ravi Rai, Hsiao-Nung Chen, Huiwen Chang, and
Cheng-Wei Tom Chang
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah, USA
A novel protocol for converting a trans-diol to an alkene under mild conditions was
developed. This method led to the synthesis of a 30,40-dideoxykanamycin (dibekacin)
analog and a 30,40-dideoxypyranmycin that were found to be active against aminoglyco-
side-resistant bacteria.
Keywords Carbohydrate, Aminoglycoside antibiotics, Antibiotic, Drug resistance,
Drug design, Pyranmycin, Kanamycin, Dideoxygenation
Aminoglycoside antibiotics have long been used as bactericidal drugs.[1] Unlike
many antibiotics that are active only against gram-positive bacteria, aminogly-
cosides have broad-spectrum activity against both gram-positive and -negative
bacteria. However, their clinical usage has often been limited only to serious
infections due to the prevalence of aminoglycoside-resistant bacteria[1c,2] and
Received November 29, 2004; accepted January 10, 2005.
Address correspondence to Cheng-Wei Tom Chang, Department of Chemistry and
Biochemistry, Utah State University, 0300 Old Main Hill, Logan, Utah 84322-0300,
USA. Tel.: þ4357973545; Fax: þ4357973390; E-mail: chang@cc.usu.edu
131

R. Rai et al.
132
the high cytotoxicity of aminoglycosides.[3] In an effort to revive the effective-
ness of aminoglycoside antibiotics against resistant bacteria, we have been
working on modification and synthesis of neomycin[4] and kanamycin[5]
classes of aminoglycosides. We wish to report two novel pyranmycin and kana-
mycin compounds with activity against resistant strains equipped with amino-
glycoside-modifying enzymes.
Overexpression of aminoglycoside-modifying enzymes from resistant
bacteria is the most commonly encountered mode of resistance.[2] Various ami-
noglycoside-modifying enzymes have been identified that catalyze a wide range
of modifications including acetylation, phosphorylation, and adenylation. For
example, one of the most prevalent modifying enzymes is APH(30), which cata-
lyzes phosphorylation at the 30-OH of both neomycin and kanamycin classes of
aminoglycosides, rendering the phosphorylated adduct incapable of binding
toward the ribosomal target (Fig. 1).
Aminoglycosides with deoxygenation at 30-OH have been demonstrated to
be effective against APH(30) as reported by Umezawa[1b,6] and others.[7] The
concept has led to the syntheses and discovery of tobramycin,[8] arbekacin,[9]
and other similar aminoglycosides (Fig. 2).[1b] Despite the fruitful results
from these studies, there are several shortcomings. First, most of the
research uses carbamate-type protecting groups for the protection of amino
groups on the aminoglycoside, resulting in the formation of polycarbamate
compounds with low solubility in organic media. The poor solubility of these
compounds poses difficulties in their purification and characterization.
Second, most of the syntheses begin with the kanamycin scaffold. There are
very few examples of deoxygenation on neomycin class antibiotics.[7d] Third,
the reported syntheses of both classes of antibiotics usually derive from kana-
mycin or neomycin, which limits the options for introducing novel structural
motifs at other desirable places of aminoglycosides.
In light of the existing deficiencies in the development of aminoglycosides
with 30-deoxygenation, we wish to report a novel method for convenient
synthesis of both kanamycin and neomycin classes of aminoglycosides with
Figure 1: Phosphorylation of kanamycin and neomycin classes aminoglycosides by APH(30).

Synthesis of 30,40-Dideoxygenated Pyranmycin and Kanamycin
133
Figure 2: Structure of kanamycin and neomycin classes aminoglycosides bearing 30 and/or 40
deoxygenation.
30,40-dideoxygenation. Our group has developed a new family of neomycin class
of aminoglycosides, pyranmycins, which bear a pyranose in the place of a
furanose at the ring III position.[4] A lead structure has been identified. We
have also developed an expedient method for preparation of kanamycin B
analogs.[5] Combining our results from these two families of aminoglycosides,
we intend to prepare the new aminoglycosides as indicated in Figure 3. Our
goal is to utilize neamine as the common core for the synthesis of 30,40-dideox-
ygenation products of both classes of aminoglycosides.
To avoid the solubility problem, we used azido groups as the surrogate for
amino groups. The synthesis of a key intermediate, 30,40-dideoxyneamine, began
from neamine. Neamine was obtained from acid-hydrolysis of neomycin, then
converted to tetraazidoneamine, 2, using TfN3 and CuSO4 (Sch. 1).[5] The
Figure 3: Proposed strategy for the synthesis of aminoglycoside with 30,40-dideoxygenation.

R. Rai et al.
134
Scheme 1: Synthesis of neamine acceptor. Conditions: (a) TfN3, CuSO4, H2O, CH2Cl2,
(b) Cyclohexone dimethyl ketal, TsOH-H2O, CH3CN, (c) (1) Tf2O, pyr., CH2Cl2; (2) Na2S2O3,
NaI, acetone, (d) BzCl, CH2Cl2, 2508C, (e) H2O, HOAc, dioxane, 658C.
selective protection of the diol was achieved using cyclohexanone dimethylketal.
The key transformation is the elimination of diol to alkene. To our surprise, we
were unable to locate didexoygenation methods that are compatible with the
presence of the azido group and the acid-labile glycoside bond despite
numerous documentations. In general, the reported methods for dideoxygena-
tion often require reductive or harsh conditions, for example, the presence of
Zn, NaI, and heating from dimesylated compound (Tipson-Cohen method),[10]
acid-catalyzed elimination from a diol using ethyl orthoformate (Crank-
Eastwood method),[11] LiAlH4/TiCl4 (McMurry-Fleming method),[12] dipho-
sphorous tetraiodide from diol (Kuhn-Winterstein reaction),[13] SnCl2/HCl,[14]
and PPh3 and I2.[15]
Among these reported methods, the method involving mesylated compound
and Zn-mediated elimination appeared to be the most suitable one of being
modified to meet our needs. Since a triflated hydroxyl group is more reactive
than a mesylated hydroxyl group, we expected that the ditriflate could be
replaced with a trans diiodide, in which the two iodides are in an antiparallel
configuration (Fig. 4). Such a configuration can induce a facile elimination
under the catalysis of I2, producing the desired alkene and I2. To avoid compli-
cation from the possible addition reaction between the alkene and I2, Na2S2O3
was added to reduce I2 into I2, allowing I2/I2 to function as a catalyst. We
were pleased to discover that the elimination occurred smoothly as expected,
providing compounds 4 and 5. Compound 5 was converted to 4, giving an
overall yield of ꢀ80%.

Synthesis of 30,40-Dideoxygenated Pyranmycin and Kanamycin
135
Figure 4: Proposed mechanism for elimination.
Regioselective glycosylation of 4 was obtained in a similar fashion as
reported in our previous work generating compound 8 (Sch. 2). The glycosyl
donor, 7, was selected based on the ease of synthesis and the higher antibacter-
ial activity from the same donor in our work on kanamycin B analogs.
Compound 8 was subjected to Staudinger reaction, hydrogenation, and ion
exchange, and the final product, RT501 (a/b ¼ 7/1), was obtained as a
chloride salt.
Regioselective protection of the C-6 hydoxyl group using benzoyl chloride
furnished compound 6 in excellent yield (Sch. 3). Compound 6 was glycosylated
Scheme 2: Synthesis of 30,40-dideoxy kanamycin B analog. Conditions: (a) NIS, TfOH,
Et2O:CH2Cl2 (3:1), (b) (i) PMe3, NaOH, THF, (ii) Pd(OH)2/C, HOAc, H2O, (iii) Dowex 1X8-200
(Cl2 form).

R. Rai et al.
136
Scheme 3: Synthesis of 30,40-dideoxy pyranmycin. Conditions: (a) (i) BF3-OEt2, CH2Cl2,
(ii) NaOMe, MeOH, (b) (i) PMe3, THF, (ii) Pd(OH)2/C, HOAc, H2O, (iii) Dowex 1X8-200 (Cl2 form).
with the corresponding trichloroacetimidate donor, 9, from the lead structure
of our previous work, generating the 30,40-dideoxy pyranmycin adduct RR501.
After the synthesis was completed, both representative aminoglycosides
were assayed against aminoglycoside-susceptible and -resistant strains of
Escherichia coli using amikacin, kanamycin, ribostamycin, and butirosin as
the controls. One resistant strain was equipped with the pTZ19U-3 plasmid
encoded for APH(30)-I, which rendered resistance to kanamycin, neomycin, livi-
domycin, paromomycin, and ribostamycin. The other resistant strain was
equipped with the pSF815 plasmid encoded for AAC(60) and APH(200), which
produced a bifunctional enzyme that catalyzed acetylation of amino group at
C-60 and phosphorylation of hydroxyl group at C-200 position. This bifunctional
enzyme enables bacteria to acquire resistance against gentamycin, tobra-
mycin, netilmicin, and kanamycin.
The minimum inhibitory concentration (MIC) results are summarized in
Table 1. As expected, both kanamycin and ribostamycin are either inactive or
much less active against aminoglycoside-resistant bacteria. Kanamycin-class
antibiotics are, however, less effective than neomycin-class antibiotics
against bacteria equipped with AAC60/APH200. Incorporation of an (S)-4-
amino-2-hydroxybutyryl (AHB) group at the N-1 position appears to be the
superior design against both strains of resistant bacteria. Although both
RR501 and RT501 contain no AHB group at N-1, they are both active
against resistant bacteria equipped with APH(30). They are, however, less
active against bacteria equipped with AAC60/APH2”. RR501 is slightly more
active than RT501 against both resistant strains, which is consistent with
the results obtained from commercially available aminoglycosides. By

Synthesis of 30,40-Dideoxygenated Pyranmycin and Kanamycin
Table 1: Minimum inhibitory concentration of synthesized aminoglycosidesa
Strains
137
E. coli (TG1)
E. coli (TG1)
Compounds
E. coli (TG1)
(pSF815)b
(pTZ19U-3)c
Amikacin
Kanamycin B
Ribostamycin
Butirosin
1
4
2
0.5
8
1
Inactive
16
0.5
32
Inactive
0.5
4
0.5
4
RR501
RT501
8
Inactive
4
aUnit: mg/mL.
bPlasmid encoded for AAC60/APH200.
cPlasmid encoded for APH(30)-I.
summarizing the information from MIC values, we believe a better design
should be a neomycin class with the AHB group at N-1 and 30,40-dideoxygena-
tion (or 30-deoxygenation). Nevertheless, the problem of the acid-labile glycosi-
dic bond between rings II and III is an obstacle that remains to be overcome.
Therefore, our design of RR501 that has better stability in acidic media
could be valuable for designing new aminoglycosides against a broad
spectrum of aminoglycoside-resistant bacteria.
In conclusion, we have developed a novel protocol for transforming a trans-
diol to an alkene under mild conditions with high efficiency. Our method can be
very useful for chemical modification of compounds with functional groups sen-
sitive to reduction, acid, or heating. We have further demonstrated the efficacy
of our method by synthesizing 30,40-dideoxykanamycin (dibekacin) analog and
30,40-dideoxypyranmycin. Antibacterial activity has been shown. Further work
of synthesizing other potent analogs of these compounds is being carried out.
EXPERIMENTAL SECTION
5,6-Cyclohexylidene-1,3,20,60-tetraazido-30,40-dideoxy-30-enoneamine (5).
To a solution of starting material (0.85 g, 1.67 mmol) in anhydrous CH2Cl2
(10 mL) and pyridine (0.76 mL, 9.3 mmol) at 08C, triflic anhydride (1.36 mL,
8.0 mmol) was added slowly. The reaction mixture was stirred at 08C for
45 min monitored by TLC (Hexane:EtOAc ¼ 3:1). After completion of the
reaction, the reaction is quenched by adding a few drops of water and diluted
with CH2Cl2. The organic layer was washed with water, NaHCO3, and brine,
then dried over Na2SO4. After concentration, the crude product (1.06 g,
1.51 mmol) was dissolved in acetone, and then a catalytic amount of NaI and
Na2S2O3 (0.9 g, 5.95 mmol) was added. After being stirred overnight at rt, the
reaction mixture was diluted with EtOAc. The organic layer was washed with

R. Rai et al.
138
1 N Na2S2O3, water, and brine, then dried over Na2SO4. Removal of solvents and
purification with column chromatography afforded the desired product (0.39 g,
1
0.76 mmol, 49%). H NMR (270 MHz, CDCl3) d 5.91 (d, J ¼ 10.5 Hz, 1H), 5.87
(d, J ¼ 10.8 Hz, 1H), 5.69 (d, J ¼ 3.9 Hz, 1H, H-10), 4.59 (m, 1H, H-50), 4.1
(m, 1H), 3.86 (m, 1H, H-4), 3.80 (m, 1H, H-1), 3.2–3.7 (m, 5H), 2.3 (m, 1H,
H-2eq), 1.2–1.6 (m, 11H).
1,3,20,60-Tetraazido-30,40-dideoxy-30-enoneamine (4). A solution of
starting material (2.58 g, 5.4 mmol) in a mixture of water (25 mL), 1,4
dioxane (25 mL), and acetic acid (36 mL) was stirred at 658C for 24 hr. After
completion of the reaction (monitored with TLC [Hexane:EtOAc ¼ 65:35]),
the reaction mixture was diluted with EtOAc and saturated NaHCO3(aq)
.
After being stirred overnight, the organic layer was washed with NaHCO3
and brine, then dried over Na2SO4. Removal of solvents and purification
with column chromatography afforded the desired product (2.02 g, 5.2 mmol,
95%). 1H NMR (270 MHz, CDCl3) d 5.95 (d, J ¼ 10.5 Hz, 1H), 5.90
(d, J ¼ 10.8 Hz, 1H), 5.54 (d, J ¼ 3.9 Hz, 1H, H-10), 4.72 (m, 1H, H-50), 4.09
(m, 1H), 3.2–3.6 (m, 7H), 2.3 (m, 1H, H-2eq), 1.57 (d, J ¼ 3.2 Hz 1H, H-2ax);
13C NMR (68 MHz, CDCl3) d 129.5 (s), 122.6 (s), 97.5 (s), 81.6 (s), 77.5 (s),
77.1 (s), 76.6 (s), 76.4 (s), 75.7 (s), 68.8 (s), 59.9 (s), 59.3 (s), 55.5 (s), 53.8 (s),
32.2 (s), 29.7 (s); HRESI Calcd for C12H16N12O4Na [M þ Na]þm/e 415.1315;
measure m/e 415.1314.
5-O-(400-Azido-400,600-dideoxy-b-D-glucopyranosyl)-1,3,20,60-tetra-
azido-30,40-dideoxy-30-enoneamine (10). A solution of glycosyl donor
(0.17 g, 0.39 mmol) and the acceptor (0.17 g, 0.33 mmol) and activated powder
˚
4 A molecular sieve was stirred in anhydrous CH2Cl2 (8 mL) at rt for 1 hr and
then cooled to 2508C. To this solution, BF3.OEt2 (0.05 mL) was added. After
being stirred for 45 min, the reaction mixture was quenched by the addition of
powder NaHCO3. After being stirred for 15 min, the reaction mixture was
filtered through Celite. The residue was washed thoroughly with EtOAc.
Removal of solvent and purification with column chromatography provided
desired product mixed with inseparable impurities. The crude product was dis-
solved in anhydrous methanol, and then a catalytic amount of NaOMe (2 M in
MeOH) was added. After being stirred overnight at rt, the reaction mixture
was added with Amberlite IR-120 (Hþ) then filtered through Celite. The
residue was washed thoroughly with MeOH. The combined solution was concen-
trated, then redissolved in EtOAc, and then washed with water and brine and
dried over Na2SO4. Removal of solvents and purification with column chromato-
graphy afforded the desired product (0.16 g, 0.28 mmol, 86%). 1H NMR
(270 MHz, CDCl3) d 5.96 (d, J ¼ 10.5 Hz, 1H), 5.91 (d, J ¼ 10.8 Hz, 1H), 5.70
(d, J ¼ 3.9 Hz, 1H, H-10), 5.6 (d, J ¼ 3.9 Hz, 1H, H-10), 4.26 (ddd, J ¼ 9.9 Hz,
J ¼ 4.8 Hz, J ¼ 2.2 Hz, 1H), 4.0–4.1 (dd, J ¼ 7.1 Hz, J ¼ 6.9 Hz, 1H), 3.2–3.7

Synthesis of 30,40-Dideoxygenated Pyranmycin and Kanamycin
139
(m, 11H), 3.09 (dq, J ¼ 9.9 Hz, J ¼ 6.3 Hz, 1H, H-500), 2.32 (ddd, J ¼ 13.2 Hz,
J ¼ 4.5 Hz, J ¼ 4.5 Hz, 1H, H-2eq), 1.36 (ddd, J ¼ 13.2 Hz, J ¼ 12.5 Hz,
J ¼ 12.5 Hz, 1H, H-2ax), 1.21 (d, J ¼ 6.3 Hz, H-600); 13C NMR (68 MHz, CDCl3)
d 129.0 (s), 127.9 (s), 127.2 (s), 122.9 (s), 122.0 (s), 94.9 (s), 94.0 (s), 86.1 (s),
85.7 (s), 80.7(s), 79.1 (s), 76.8 (s), 76.5 (s), 76.1 (s), 75.9 (s), 75.8 (s), 75.1 (s),
74.4 (s), 74.3 (s), 70.8 (s), 69.2 (s), 68.9 (s), 66.9 (s), 59.7 (s), 59.1 (s), 58.8 (s),
54.0 (s), 53.5 (s), 53.2 (s), 51.0 (s), 45.8 (s), 31.7 (s), 29.1 (s), 17.9 (s); HRESI
Calcd for C18H25N15O7Na [M þ Na]þm/e 586.1959; measure m/e 586.1979.
6-O-Benzyol-1,3,20,60-tetraazido-30,40-dideoxy-30-enoneamine (6). To
a solution of starting material (1.13 g, 2.88 mmol) and DIPEA (0.60 mL,
3.45 mmol) in anhydrous CH2Cl2, at -508C, BzCl (0.33 mL, 2.87 mmol) was
slowly added. After being stirred overnight, the reaction was quenched by
adding a few drops of water and then diluted with EtOAc. The reaction
mixture was washed with NaHCO3 and brine, then dried over Na2SO4.
Removal of solvents and purification with column chromatography afforded
the desired product (1.21 g, 2.44 mmol, 85%). 1H NMR (270MHz, CDCl3) d
7.3–8.0 (m, 5H) 5.95 (d, J ¼ 10.5 Hz, 1H), 5.90 (d, J ¼ 10.8 Hz, 1H), 5.64
(d, J ¼ 3.9 Hz, 1H, H-10), 5.17 (t, J ¼ 9.8 Hz, 1H, H-6), 4.70 (m, 1H, H-50), 4.0–
4.1 (dd, J ¼ 7.1 Hz, J ¼ 6.9 Hz, 1H), 3.89 (m, 1H, H-20), 3.80 (m, 1H, H-5), 3.69
(m, 1H, H-4), 3.58 (m, 1H, H-1), 3.38 (m, 1H, H-3), 3.2 (m, 1H, H-60), 2.34
(m, 1H, H-2eq), 1.69 (d, J ¼ 3.2 Hz 1H, H-2ax). 13C NMR (68 MHz, CDCl3) d
171.3 (s), 166.2 (s), 133.6 (s), 130.0 (s), 129.8 (s), 129.5 (s), 129.7 (s), 129.5 (s),
129.4 (s), 129.2 (s), 128.5 (s), 122.9 (s), 97.8 (s), 81.2 (s), 77.8 (s), 77.6 (s), 77.2
(s), 76.7 (s), 75.6 (s), 68.8 (s), 60.4 (s), 59.2 (s), 58.3 (s), 55.3 (s), 53.8 (s), 32.2
(s), 21.0 (s), 14.2 (s).
6-O-(400-Azido-400,600-dideoxy-a-D-glucopyranosyl)-1,3,20,60-tetraazido-
30,40-dideoxy-30-enoneamine (8).
A
solution of glycosyl donor (0.6 g,
˚
1.29 mmol), acceptor (0.56 g, 1.42 mmol), and activated 4 A molecular sieves in
anhydrous Et2O (12 mL) and CH2Cl2 (4mL) was stirred overnight. The reaction
mixture was cooled to 2788C, and NIS (0.17 g, 0.75 mmol) was added. The
reaction mixture was allowed to warm up to 2458C, and then TfOH (28.3 mL,
0.31 mmol) was added. After completion of the reaction (monitored by TLC
[hexane/EtOAc ¼ 3/1]), the reaction was quenched by adding a few drops of
triethyl amine. After being stirred for several minutes, the reaction mixture
was concentrated, diluted with EtOAc, and then filtered through Celite. The
residue was washed with EtOAc. The combined organic solution was washed
with 1 N Na2S2O3, water, and brine, then dried over Na2SO4. Removal of
solvents and purification with column chromatography afforded the desired
product (0.69 g, 0.93 mmol, 72%). 1H NMR (270 MHz, CDCl3) d 7.2–7.4
(m, 10H), 6.2 (d, J ¼ 3.2 Hz, 1H, H-10), 5.9 (d, J ¼ 10.5 Hz, 1H), 5.86
(d, J ¼ 1.3 Hz, 1H), 5.83 (d, J ¼ 3.6 Hz, 1H, H-100), 4.94 (d, J ¼ 10.9 Hz, 1H,

R. Rai et al.
140
PhCH2O), 4.80 (d, J ¼ 10.9 Hz, 1H, PhCH2O), 4.77 (d, J ¼ 11.9 Hz, 1H, PhCH2O),
4.70 (d, J ¼ 11.9 Hz, 1H, PhCH2O), 4.38 (d, J ¼ 1.6 Hz, 1H), 4.13 (m, 1H), 3.8–4.0
(m, 2H), 3.3–3.7 (m, 10H), 3.11 (dd, J ¼ 9.9 Hz, J ¼ 9.9 Hz, 1H), 2.33 (ddd,
J ¼ 13.0 Hz, J ¼ 4.3 Hz, J ¼ 4.3 Hz, 1H, H-2eq), 1.50 (ddd, J ¼ 13.0 Hz,
J ¼ 12.2 Hz, J ¼ 12.2 Hz, 1H, H-2ax), 1.27 (d, J ¼ 5.9 Hz, 3H, H-600); 13C NMR
(68 MHz, CDCl3) d 171.3 (s), 166.2 (s), 137.9 (s), 137.7 (s), 129.1 (s), 128.6 (s),
128.4 (s), 128.1 (s), 128.0 (s), 127.9 (s), 123.4 (s), 98.23 (s), 96.9 (s), 86.2 (s), 79.7
(s), 77.5 (s), 77.1 (s), 76.6 (s), 75.9 (s), 60.4 (s), 59.3 (s), 50.0 (s), 53.9 (s), 32.6 (s),
29.7 (s), 18.4 (s), 14.2 (s).
General procedure for Staudinger reaction and hydrogena-
tion. To the trisaccharides (8 or 10)/THF solution in a reaction flask
(or vial) equipped with a reflux condenser, 0.1 M NaOH(aq) (0.5 mL) and PMe3
(1M in THF, 6 equivalents) were added. The reaction mixture was stirred
at 508C for 2 hr. The product has an Rf of 0 when eluted with EtOAc/MeOH
(9/1) solution and an Rf of 0.9 when eluted with iPrOH/1 M NH4OAc (2/1)
solution. After completion of the reaction, the reaction mixture was cooled to
rt and loaded to a short column (5 cm in height) packed with a layer of silica
gel on top of a layer of Celite. The column was eluted with a series of solutions
as follows: THF, THF/MeOH, MeOH, and MeOH/conc. NH4OH (from 0% to
28% of conc. NH4OH). The fractions containing the desired product were
analyzed by TLC and collected. After removal of solvents, the crude perbenzy-
lated aminoglycoside was added with a catalytic amount of Pd(OH)2/C (20%
Degussa type) and 5 mL of degassed HOAc/H2O (1/1). After being further
degassed, the reaction mixture was stirred at rt under atmospheric H2
pressure. After being stirred for 1 d, the reaction mixture was filtered
through Celite. The residue was washed with water, and the combined sol-
utions were concentrated, affording pure final product as an acetate salt.
1
Most of the reported final products are characterized by H and 13C NMR at
this stage. The product has an Rf of 0 when eluted with iPrOH/1 M NH4OAc
(2/1) solution and an Rf of 0.1–0.2 when eluted with conc. NH4OH/MeOH
(2/5) solution. The final product with Cl2 salt can be prepared with an ion-
exchange column packed with Dowex 1X8–200 (Cl2 form) and eluting with
water. After collection of desired fractions and removal of solvent, the final
products are subjected to bioassay directly.
5-O-(400-Amino-400,600-dideoxy-b-D-glucopyranosyl)-30,40-dideoxynea-
mine (RR501) contains about 10% inseparable byproduct. 1H NMR
(400 MHz, D2O) (chloride salt), d 5.73 (d, J ¼ 3.4 Hz, 1H, H-10), 5.2
(d, J ¼ 8.0 Hz, 1H, H-100), 4.0–4.1 (m, 1H), 3.8–4.0 (m, 4H), 3.4–3.8 (m, 4H),
3.36 (dd, J ¼ 13.5 Hz, J ¼ 3.5 Hz, 1H), 3.1–3.2 (m, 2H), 3.0 (dd, J ¼ 10.2 Hz,
J ¼ 9.9 Hz, 1H), 2.2 (m, 1H, H-2eq), 2.0–2.2 (m, 4H), 1.96 (dd, J ¼ 12.6 Hz,
J ¼ 12.6 Hz, 1H, H-2ax), 1.48 (dd, J ¼ 6.9 Hz, J ¼ 6.6 Hz, 3H); 13C NMR

Synthesis of 30,40-Dideoxygenated Pyranmycin and Kanamycin
141
(68 MHz, D2O) (chloride salt) d 102.6 (s, C-100), 95.4 (s, C-10), 81.0 (s), 76.3 (s),
73.5 (s), 73.0 (s), 71.7 (s), 69.6 (s), 66.2 (s), 56.5 (s), 49.7 (s), 48.9 (s), 48.7 (s),
42.4 (s), 28.3 (s), 25.2 (s), 20.4 (s), 16.7 (s). HRESI Calcd for C18H42N5O7Na
[M þ Na]þ m/e 458.2593; measure m/e 458.2590.
6-O-(400-Amino-400,600-dideoxy-a-D-glucopyranosyl)-30,40-dideoxynea-
1
mine (RT501) (a/b ¼ 7/1, only the a epimer reported) H NMR (400 MHz,
D2O) (Chloride salt) 5.90 (d, J ¼ 2.7 Hz, 1H, H-10), 5.10 (d, J ¼ 2.7 Hz, 1H,
H-100), 4.4 (m, 1H), 3.8–4.1 (m, 7H), 3.5–3.7 (m, 5H), 3.36 (d, J ¼ 5.2 Hz,
1H), 3.35 (d, J ¼ 5.2 Hz, 1H), 3.2 (dd, J ¼ 7.8 Hz, 1H), 3.1 (t, J ¼ 9.1 Hz,
1H), 2.6 (m, 1H, H-2eq), 2.0 (ddd, J ¼ 9.1 Hz, J ¼ 9.1 Hz, J ¼ 9.1 Hz, 1H,
H-2ax), 1.42 (d, J ¼ 6.3 Hz, 3H, H-600); 13C NMR (100 MHz, D2O) (chloride
salt) 104.6 (s), 97.7 (s), 86.6 (s), 79.5 (s), 76.9 (s), 74.5 (s), 71.6 (s), 68.8 (s),
68.6 (s), 59.5 (s), 52.9 (s), 51.5 (s), 51.4(s), 45.3 (s), 30.8 (s), 28.2 (s), 23.3
(s), 19.6 (s); HRESI Calcd for C18H38N5O7 [MH]þm/e 436.2771; measure
m/e 436.2751.
ACKNOWLEDGEMENT
We acknowledge DARPA (DAAD 19-03-1-0050) for generous financial support.
We also thank Prof. Mobashery from Notre Dame University for the generous
gift of the pTZ19U-3 and pSF815 plasmids.
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