Selective deprotection of the Cbz amine protecting group for the facile synthesis of kanamycin A dimers linked at N-3″ position

Article abstract of DOI:10.1016/j.tet.2009.06.002

The optimal conditions for the regioselective deprotection of per-N-Cbz-kanamycin A and the reaction mechanism was investigated. We found that the Cbz group at N-3″ position was selectively deprotected under milder basic conditions and the cyclic carbamat

Full text of DOI:10.1016/j.tet.2009.06.002

Tetrahedron 65 (2009) 5922–5927
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Selective deprotection of the Cbz amine protecting group for the facile synthesis
of kanamycin A dimers linked at N-300 position
*
Gui-Hui Chen, Pan Pan, Ying Chen, Xiang-Bao Meng, Zhong-Jun Li
The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 April 2009
Received in revised form 26 May 2009
Accepted 1 June 2009
Available online 6 June 2009
The optimal conditions for the regioselective deprotection of per-N-Cbz-kanamycin A and the reaction
mechanism was investigated. We found that the Cbz group at N-300 position was selectively deprotected
under milder basic conditions and the cyclic carbamate is an intermediate of the deprotection reaction.
The selective deprotection of the amine protecting group enable us to synthesize several kanamycin A
dimers linked at N-300 position in a straightforward way.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Regioselective
Aminoglycoside
Kanamycin A
Dimer
1. Introduction
2. Results and discussion
Aminoglycosides are clinically important antibiotics to treat
infectious diseases caused by Gram-positive and Gram-negative
bacteria. These drugs bind to the decoding region of ribosome, in-
ducing codon misreading, inhibiting translocation, and eventually
leading to cell death.1–6 However, the appearance of aminoglyco-
side-resistant bacteria has significantly reduced their clinical ap-
plications.7–9 In the last several years, aminoglycoside dimers have
been synthesized by modifying the hydroxyl groups, such as the 50-
hydroxyl of kanamycin A and 5-hydroxyl of neamine, to enhance
the binding affinity to the RNA targets and/or to confer resistance to
aminoglycoside-modifying enzymes.10–12 Recently, the synthesis of
neamine dimers linked at the 40- or the 5-hydroxyl13a and at the
N-1 position13b have been reported, which are able to bind to
a specific sequence of HIV-1 RNA.
In a previous report, we have described a shorter route to the
synthesis of N-1 linked neamine dimer based on the selective for-
mation of cyclic carbamate.14 We report here the synthesis of new
kanamycin A dimers linked at the N-300 position via a more effective
route. When per-N-Cbz-kanamycin A was treated with NaOH under
carefully controlled conditions, per-N-Cbz-kanamycin A was se-
lectively deprotected at N-300 position in good yield. The depro-
tected product was used to synthesize several kanamycin A
dimmers.
2.1. Selective deprotection of the Cbz groups
There are four free amino groups in kanamycin A, and the
regioselective modification of the amines through a simple route is
always difficult. In a previous study, we have introduced the cyclic
carbamate group to protect the amines, and the carbamate groups
could be removed by different basic reagents, which afforded a se-
ries of analogs modified at the nitrogen positions of neamine and
kanamycin A.14 Since both the introduction and removal of the
cyclic carbamate protecting group could be achieved regiose-
lectively under basic condition, we explored the possibility to
remove the Cbz protecting group selectively by controlling the re-
action conditions. By careful adjustment of the basic deprotection
conditions, we succeeded in deprotecting the N-Cbz group at N-300
position of per-N-Cbz-kanamycin A specifically. Moreover, the re-
action mechanism was elucidated by the separation of the cyclic
carbamate intermediate in the deprotection process.
We initially observed that the reaction of per-N-Cbz-kanamycin
A (1) with a 2.5 M NaOH solution in dioxane at 50 ꢀC for 24 h led to
the selective deprotection of the Cbz groups at N-300 and N-60 po-
sitions and the formation of compound 5, the 1,3-di-Cbz-kanamy-
cin A, in 85% yield. It suggested that the vicinal hydroxyl groups
could aid the deprotection of the Cbz groups and the reaction oc-
curred possibly via the cyclic carbamate intermediate.
Since we have shown that there is selectivity between N-300 and
N-60 to form the corresponding cyclic carbamate groups, it is possible
to selectively deprotect the Cbz groups at these two positions by
* Corresponding author. Tel.: þ86 010 8280 1714; fax: þ86 010 8280 5496.
E-mail address: zjli@bjmu.edu.cn (Z.-J. Li).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.06.002

G.-H. Chen et al. / Tetrahedron 65 (2009) 5922–5927
5923
NH2
HO
HO
O
Cbz
O
HN
HO
O
HO
H
N
Cbz
OH
H
N
Cbz
O
2.5 M NaOH
50 °C, 24h.
O
HO
HO
HO
Cbz
O
OH
HN
H2N
HO
O
HO
H
N
Cbz
OH
5 (85%)
OH
H
N
O
Cbz
O
1.5 M NaOH
rt, 24h.
HO
Cbz NH
HO
HO
Cbz
HN
1
HO
O
HO
H
N
Cbz
OH
O
O
HO
OH
H2N
3 (62%)
Scheme 1. Selective deprotection of per-N-Cbz-kanamycin A.
carefully adjusting the reaction conditions. Indeed, when compound
1 was reacted with a 1.5 M NaOH solution in dioxane at room tem-
perature for 24 h, compound 3, the N-300 selectively deprotected tri-
Cbz-kanamycin A, was produced in 62% yield (Scheme 1).
There is only a few related reports on the deprotection of the Cbz
group under basic conditions.15,16 The mechanism via a cyclic car-
bamate intermediate has never been confirmed, because the cyclic
carbamate was unstable under basic conditions and generally could
not be separated and identified. In order to confirm the mechanism
of the deprotection reaction, the reaction intermediates were iso-
lated and characterized under various conditions.
When compound 1 was treated with a 1.5 M NaOH solution at
room temperature for 8 h, the intermediate 2 was detected and
separated as a by-product in 8% yield. Under this condition, com-
pound 3 was the major product and compound 1 was not con-
sumed completely. When the deprotection reaction was carried out
with a 2.5 M NaOH solution at 30 ꢀC for 12 h, multiple products
were formed including compound 3, compound 5 and another in-
termediate 4 (Scheme 3), which was obtained in 5% yield.
It’s difficult to determine the structures of compounds 3 and 5 di-
rectly through spectroscopic analysis. Because the structures of the
monocarbamate derivative 2 and the bicarbamate derivative 6 were
characterized previously,14 the formation of compounds 3 and 5 by
the basic treatment of compounds 2 and 6, respectively, confirmed
the structures of compounds 3 and 5 indirectly (Scheme 2).
The existence of carbamate intermediate indicated that the rate
of cyclic carbamate formation was faster than the rate of corre-
sponding ring opening. Additionally, the rate of the opening of the
five-membered cyclic carbamate was faster than the rate of the
formation of the six-membered cyclic carbamate; otherwise,
compound 3 could not be the main product. Based on the isolation
and characterization of the reaction intermediates and the reaction
products under various conditions, the deprotection mechanism of
Cbz was proposed in Scheme 3.
2.2. Synthesis of kanamycin A dimers
The derivatives obtained through the selective deprotection of
the Cbz protecting groups could be used in the selective modifi-
cation of aminoglycosides. A monomer that has a specific free
Intermediates 2 and 4 have been synthesized via different routes
in our previous work14 and their structures have been characterized.
H
H
N
Cbz
N
Cbz
HO
HO
O
HO
HO
O
Cbz
Cbz
O
HN
HN
HO
HO
1.5 M NaOH
rt, 8 h.
O
HO
O
HO
H
N
H
N
Cbz
OH
Cbz
O
OH
O
O
HO
HO
O
O
OH
H2N
HN
2
3
O
NH2
O
N
H
HO
HO
O
Cbz
O
O
HN
HO
HO
HO
Cbz
2.5 M NaOH
50 °C, 6h.
HN
O
H
HO
O
HO
HO
N
Cbz
OH
H
N
Cbz
O
OH
O
OH
O
HO
H2N
O
HN
5
6
O
Scheme 2. The ring-opening reaction of the cyclic carbamate.

5924
G.-H. Chen et al. / Tetrahedron 65 (2009) 5922–5927
H
N
H
H
N
Cbz
O
N
Cbz
O
Cbz
O
HO
HO
HO
HO
HO
HO
Cbz
Cbz
O
Cbz
O
HN
HN
HN
HO
HO
HO
O
O
H
N
H
N
O
H
N
HO
Cbz
OH
HO
Cbz
HO
Cbz
OH
O
OH
OH
O
O
O
HO
HO
O
O
OH
HO
Cbz NH
H2N
HN
2 (intermediate)
3
1
O
O
NH2
O
N
H
HO
HO
O
Cbz
HN
HO
Cbz
O
HN
HO
O
HO
H
O
H
HO
N
Cbz
OH
HO
N
Cbz
OH
O
OH
O
HO
O
OH
HO
H2N
H2N
4 (intermediate)
5
Scheme 3. The mechanism via a carbamate intermediate for the selective deprotection of aminoglycosides.
O
O
O
H2
3
R1
O
O
R2
OH
O
R1
R2
R1
R1H
Pd/C
EDC, DMAP
pyridine
O
O
O
3
(94%)
7
(87%)
(95%)
8
9
O
O
O
O
O
H
H
N
7
H2
Pd/C
NH2
N
R1
N
H
R1
N
H
R1
R2
N
H
R2
EDC, DMAP
O
O
(93%)
10
12
(87%)
11
O
O
O
H
N
NH2
HN
N
H
N
H
R1
HN
O
7
EDC, DMAP
HO
HO
O
HO
O
Cbz
Cbz
OH
HN
HO
HN
O
O
Cbz NH
O
H
N
H
N
H
N
Cbz NH
O
HO
NH2
HO
N
H
N
R1
OH
HN
H
O
O
13
14 (92%)
Pd/C
H2
O
H
N
Cbz
O
HO
HO
O
Cbz
O
H
N
R1
=
HO
O
N
R2
HN
Cbz
OH
HO
NH
O
H
O
O
HO
HO
O
H2N
O
HO
OH
H
N
H2N
O
HN
HO
N
H
N
H
R2
NH2
O
OH
O
HO
HO
(89%)
15
R2
=
H2N
HO
O
HO
NH2
O
OH
O
HO
OH
HN
Scheme 4. Synthesis of dimers of kanamycin A.

G.-H. Chen et al. / Tetrahedron 65 (2009) 5922–5927
5925
amino group, such as compound 3, is useful for the synthesis of
various dimers linked at the nitrogen positions.
Preparative column chromatography was performed on silica gel
60. Chemical reagents and starting materials were purchased from
Aldrich Chemical Co. or Acros Chemical Co. and were used without
purification unless otherwise noted. Pyridine was dried over CaH2
and kept under nitrogen.
Specifically, compound 3 was converted to the corresponding
carboxylic acid 7 through the reaction with succinic anhydride in
94% yield (Scheme 4). The coupling of the carboxylic acid 7 with
compounds 3, 10, and 1314 in the presence of EDC/DMAP followed
by deprotection with catalytic hydrogenolysis led to the formation
of corresponding dimers 9, 12, and 15 in 83%, 81%, and 82% yields,
respectively.
The new dimer analogs were tested for their ability to suppress
the growth of different bacteria, including pathogenic and resistant
strains, by measuring the inhibition rate at three concentrations
4.1.1. General procedure for hydrogenolysis
To a solution or suspension of glycoside (0.1 mmol) in MeOH/
H2O (10 mL/2 mL), an HCl solution (1 M, 1 mL), and 10% Pd/C
(50 mg) was added. The reaction mixture was treated with H2 for
8 h under pressure (5.06 MPa) at room temperature. The catalyst
was removed by filtration and the filtrate was concentrated in
vacuum to obtain the desired product.
(1 mm, 10 mm, and 100 mm). Aminoglycoside susceptible Escherichia
coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were
used as the standard reference strains and E. coli (440) was used as
the resistant strain. And kanamycin A was used as the control.
4.1.2. 1,3,60-Tri-N-benzyloxycarbonyl-300,200-N,O-carbonyl-
kanamycin A (2) and 1,3,60-tri-N-benzyloxycarbonyl-
The results showed that the dimer analogs at 100
tration were active against the two standard strains, but they were
not active against the resistant E. coli strain (Table 1).
m
m concen-
kanamycin A (3)
To a solution of compound 114 (1 g, 0.98 mmol) in dioxane/H2O
(100 mL/30 mL), a 1.5 M NaOH solution (30 mL) was added. The
resulting mixture was stirred for 24 h at room temperature. After
completion of the reaction (monitored by TLC, CHCl3/MeOH/
NH3$H2O¼4: 3: 1), an HCl solution (1 M) was added to adjust the pH
to 2. After removal of the solvent, ice water was added and the solid
was filtered. An NaOH solution (1 M) was added to the filtrate to
adjust the pH to 9, and the resulting solid was filtered and separated
by silica column chromatography (CHCl3/MeOH/NH3$H2O¼12:2:0.2
and CHCl3/MeOH/NH3$H2O¼5:2:0.2) to afford compound 3 as
a white solid (0.54 g, 62%).
Table 1
The bacteria growth inhibition rates of the aminoglycosides at 100
mm
Entry Compound E. coli (ATCC 25922) S. aureus (ATCC 25923) E. coli (440)
1
2
3
4
Kanamycin A 88.0
94.0
93.5
28.8
93.4
d
d
d
d
9
88.7
87.8
87.8
12
15
When the reacting time was shortened to 8 h, compound 2
could be detected and separated by silica column chromatography
(CHCl3/MeOH/NH3$H2O¼12:2:0.2) in a low yield (8.2%).
Although the prepared dimmers did not show any activity against
Compound 2, 1H NMR (DMSO-d6, 300 MHz)
d 7.89 (s, 1H,
the drug resistant E. coli strain 440 in our initial study, their activity
against other drug resistant strains still needs to be determined.
Clearly, there is a need to better understand the structural and
functional requirements for the antibacterial activity of kanamycin A
dimers. Studies toward this goal are in progress in our laboratories.
NHCO2), 7.33–7.30 (m, 17H, Ph and NHCO2), 6.85 (br s, 1H, NHCO2),
5.56 (br s, 1H), 5.41 (d, J¼6.0 Hz, 1H), 5.37 (s, 1H), 5.30 (s, 1H), 5.09–
4.85 (m, 9H), 4.35 (s, 1H), 3.81 (s, 2H), 3.56–3.15 (m, 6H), 3.07 (br s,
1H), 1.70 (br s, 1H, H-2eq), 1.49–1.37 (m, 1H, H-2ax); 13C NMR
(DMSO-d6, 75 MHz)
d 159.4, 156.6, 155.7, 155.6, 137.2, 137.0, 128.4,
127.9, 127.8, 127.3, 101.3, 94.3, 84.7, 80.1, 76.8, 74.9, 74.8, 72.7, 72.3,
70.7, 70.5, 68.9, 65.4, 59.4, 56.8, 50.7, 49.8, 41.7, 34.8; ESI-HRMS
Calcd for C43H53N4O18 ([MþH]þ) m/z 913.3360, found 913.3366.
3. Conclusion
In conclusion, we developed a novel and effective approach for
the synthesis of new kanamycin A dimers and other derivatives at
the N-300 position through the selective deprotection of Cbz-pro-
tected aminoglycosides. The deprotection mechanism was in-
vestigated through the separation and characterization of cyclic
carbamate intermediates. Two homodimers of kanamycin A and
one heterotrimer of neamine and kanamycin A were prepared with
different dicarbonyl linker at the N-300 position.
Compound 3, 1H NMR (DMSO-d6, 300 MHz)
d 8.07 (br s, 2H),
7.34 (s, 16H, Ph and NHCO2), 7.04 (br s, 1H, NHCO2), 5.31 (s, 2H),
5.13–4.89 (m, 11H), 4.35 (br s,1H), 3.97 (br s, 1H), 3.81–3.73 (m, 2H),
3.30–2.92 (m, 7H), 1.83 (br s, 1H, H-2eq), 1.53 (br s, 1H, H-2ax); 13C
NMR (DMSO-d6, 75 MHz)
d 156.7, 155.9, 155.6, 137.3, 137.1, 128.3,
127.7, 99.9, 97.2, 82.5, 80.5, 74.3, 73.0, 72.4, 72.0, 70.0, 68.3, 67.0,
65.3, 65.0, 60.2, 56.4, 50.0, 49.5, 34.4; ESI-HRMS Calcd for
C42H55N4O17 ([MþH]þ) m/z 887.3556, found 887.3561.
4. Experimental
4.1. General
4.1.3. 1,3-Di-N-benzyloxycarbonyl-60,40-N,O-carbonyl-kanamycin A
(4) and 1,3-di-N-benzyloxycarbonyl-kanamycin A (5)
To a solution of compound 1 (1 g, 0.98 mmol) in dioxane/H2O
(100 mL/30 mL), a 2.5 M NaOH solution (30 mL) was added. The
resulting mixture was stirred for 24 h at 50 ꢀC. After completion of
the reaction (monitored by TLC, CHCl3/MeOH/NH3$H2O¼4:3:1), an
HCl solution (1 M) was added to adjust the pH to 10. After removal
of the solvent, ice water was added. The resulting solid was filtered
and separated by silica column chromatography (CHCl3/MeOH/
NH3$H2O¼8:2:0.2 and CHCl3/MeOH/NH3$H2O¼6:3:1) to afford
compound 5 as a white solid (0.63 g, 85%).
Proton magnetic resonance spectra (including 1H–1H COSY,
HMQC, and HMBC) were recorded using a Jeol 300 spectrometer.
Chemical shifts were reported as parts per million (ppm) downfield
from tetramethylsilane in d unit, and coupling constants were given
in cycles per second (Hz). 13C spectra (including DEPT) were
obtained using the Jeol 300 spectrometer at 75 Hz. Routine 13C
NMR spectra were fully decoupled by broad-band waltz decou-
pling. All NMR spectra were recorded at ambient temperature.
High-resolution electronspray (ESI-HRMS) mass spectra were
obtained from Bruker APEX IV-FTMS 7.0T mass spectrometer. Re-
actions were monitored by thin layer chromatography (TLC) on
silica gel 60 F254. Visualization was performed by ultraviolet light
and/or by staining with ceric ammonium molybdate or ninhydrin.
When the reaction was carried out at 30 ꢀC for 12 h, compound 4
could be detected and separated by silica column chromatography
(CHCl3/MeOH/NH3$H2O¼8:2:0.2) as a white solid in a low yield (5%).
Compound 4, 1H NMR (DMSO-d6, 300 MHz)
d 7.34 (s, 12H, Ph
and NHCO2), 5.57–5.51 (m, 1H), 5.14–4.80 (m, 6H), 4.23 (br s, 1H),
3.86 (br s, 1H), 3.70 (br s, 2H), 3.49–3.04 (m, 6H), 2.79–2.72 (m, 1H),

5926
G.-H. Chen et al. / Tetrahedron 65 (2009) 5922–5927
1.88–1.83 (m, 1H, H-2eq), 1.23–1.17 (m, 1H, H-2ax); 13C NMR (DMSO-
d6, 75 MHz) 155.9, 155.5, 152.4, 137.1, 136.9, 128.3, 127.8, 101.2,
1H NMR (DMSO-d6, 300 MHz)
d 7.94 (br s, 1H, NHCO), 7.70 (br s,
d
1H, NHCO), 7.32–7.30 (m, 34H, Ph and NHCO2), 7.02 (d, J¼8.7 Hz,
1H, NHCO2), 6.87 (br s, 1H, NHCO2), 5.08–4.74 (m, 32H), 3.82 (br s,
2H), 3.55–3.26 (m, 13H), 3.05 (br s, 4H), 2.73 (br s, 1H), 2.30 (br s,
4H), 1.84 (br s, 2H, H-2eq), 1.45 (br s, 2H, H-2ax); 13C NMR (DMSO-
97.5, 84.9, 80.1, 77.9, 74.6, 72.7, 72.0, 69.9, 66.3, 65.2, 61.4, 60.3, 55.0,
50.0, 49.2, 42.6, 34.4; ESI-HRMS Calcd for C35H47N4O16 ([MþH]þ)
m/z 779.2992, found 779.2990.
Compound 5, 1H NMR (DMSO-d6, 300 MHz)
d
7.34 (s, 11H, Ph),
d6, 75 MHz) d 172.7, 171.7, 160.3, 156.7, 156.6, 155.9, 155.8, 155.6,
7.19 (br s, 1H, NHCO2), 5.52 (br s, 1H), 4.94–4.81 (m, 7H), 4.28–4.24
(m, 2H), 3.72 (br s, 1H), 3.05 (s, 4H), 2.79 (br s, 2H),1.92–1.89 (m, 1H,
H-2eq), 1.40–1.33 (br s, 1H, H-2ax); 13C NMR (DMSO-d6, 75 MHz)
137.3, 137.2, 137.0, 128.4, 127.8, 127.6, 101.3, 101.1, 97.2, 84.6, 84.3,
80.2, 74.4, 74.1, 72.9, 72.7, 72.3, 70.7, 70.5, 70.2, 68.7, 67.0, 65.3,
65.0, 60.1, 56.6, 55.7, 50.2, 50.0, 49.6, 42.1, 41.7, 34.5, 30.4; ESI-
HRMS Calcd for C91H117N10O37 ([MþH]þ) m/z 1941.7586, found
1941.7558.
d
155.8, 155.6, 137.2, 136.9, 128.3, 127.8, 127.5, 127.1, 101.2, 97.5, 84.6,
80.1, 74.8, 72.9, 72.8, 72.7, 72.6, 72.5, 71.2, 69.5, 65.4, 65.2, 60.5, 55.1,
50.2, 49.5, 42.7, 34.9; ESI-HRMS Calcd for C34H49N4O15 ([MþH]þ)
m/z 753.3189, found 753.3183.
4.1.8. The dimer of kanamycin A linked with nine atoms (12)
The title compound was prepared from compound 11 according
to the general procedure (12 h) as solid hexahydrochloride (93%).
4.1.4. 1,3,60-Tri-N-benzyloxycarbonyl-kanamycin A-300-N-
succinamide (7)
1H NMR (D2O, 300 MHz)
d
5.39 (s, 1H), 5.29 (s, 1H), 4.92 (s, 2H),
3.76–3.07 (m, 37H), 2.71 (br s, 1H), 2.36 (s, 6H), 1.79–1.75 (m, 2H, H-
2ax); 13C NMR (75 MHz, D2O)
175.9, 175.5, 161.3, 101.6, 100.9, 98.5,
To a solution of compound 3 (400 mg, 0.45 mmol) in pyridine/
methanol (100 mL/20 mL), succinic anhydride (120 mg, 1.2 mmol)
was added. The resulting mixture was stirred for 8 h at room
temperature. After removal of the solvent, ice water (50 mL), and
1 M HCl (5 mL) was added. The resulting solid was filtered, washed
with 0.02 M aqueous HCl to afford compound 7 as a white solid
(418 mg, 94%).
d
95.8, 84.3, 84.0, 79.7, 78.5, 73.7, 73.4, 73.1, 72.9, 72.5, 71.8, 71.6, 71.3,
70.7, 68.6, 68.3, 65.8, 61.1, 60.2, 55.4, 55.2, 50.3, 50.2, 48.8, 48.0,
40.8, 39.9, 39.6, 31.5, 28.0; ESI-HRMS Calcd for C43H82N10O25
([Mþ2H]2þ) m/z 569.2715, found 569.2720.
1H NMR (DMSO-d6, 300 MHz)
d
7.73 (br s, 1H, NHCO), 7.33 (s,
4.1.9. The protected dimer of kanamycin A linked with neamine (14)
To a solution of compounds 3 (116 mg, 0.12 mmol) and 1314
(45 mg, 0.059 mmol) in pyridine (20 mL), DMAP (30 mg,
0.24 mmol), and EDC (140 mg, 0.89 mmol) was added. The result-
ing mixture was stirred for 12 h at room temperature. After removal
of the solvent, ice water was added. The resulting solid was filtered,
washed with 0.02 M aq HCl, dissolved in DMF and crashed with
H2O to afford compound 14 as a white solid (146 mg, 92%).
17H, Ph and NHCO2), 7.03 (br s, 1H, NHCO2), 5.50 (br s, 1H), 5.00–
4.94 (m, 8H), 3.85 (br s, 1H), 3.55–3.30 (m, 14H), 2.99 (br s, 1H), 2.35
(s, 4H), 1.85 (br s, 1H, H-2eq), 1.48 (br s, 1H, H-2ax); 13C NMR (DMSO-
d6, 75 MHz)
d 175.4, 172.8, 156.7, 155.9, 155.6, 137.3, 137.1, 136.9,
128.3, 127.7, 127.6, 101.3, 97.2, 84.5, 80.2, 74.1, 72.9, 72.4, 70.9, 70.2,
67.0, 65.3, 65.0, 60.1, 56.6, 50.0, 49.6, 34.5, 30.5; ESI-HRMS Calcd for
C46H59N4O20 ([MþH]þ) m/z 987.3717, found 987.3751.
1H NMR (DMSO-d6, 300 MHz)
d 7.93 (br s, 2H, NHCO), 7.69 (br s,
4.1.5. The protected dimer of kanamycin A linked with succinic
acid (8)
2H, NHCO), 7.32 (s, 44H, Ph and NHCO2), 7.03 (br s, 2H, NHCO2), 6.85
(br s, 2H, NHCO2), 4.99–4.92 (m, 20H), 4.48 (br s, 22H), 3.82 (s, 2H),
3.55–3.29 (m, 18H), 3.02 (s, 8H), 2.71 (br s, 2H), 2.29 (br s, 8H), 1.84
(br s, 2H, H-2eq), 1.45 (br s, 2H, H-2ax); 13C NMR (DMSO-d6, 75 MHz)
To a solution of compounds 3 (72 mg, 0.081 mmol) and 7 (80 mg,
0.081 mmol) in pyridine (20 mL), DMAP(20 mg, 0.16 mmol), and
EDC (100 mg, 0.64 mmol) was added. The resulting mixture was
stirred for 8 h at room temperature. After removal of the solvent, ice
water was added. The resulting solid was filtered, washed with
0.02 M aq HCl and recrystallized in CHCl3/MeOH to afford compound
8 as a white solid (131 mg, 87%).
d
172.6, 171.5, 159.1, 158.3, 156.6, 156.2 (s, NHCO2), 155.9, 155.6,
137.3, 137.1, 137.0, 128.3, 127.8, 127.6, 127.5, 101.3, 98.9, 97.1, 84.6,
81.9, 80.2, 76.8, 75.0, 74.1, 72.9, 72.5, 71.2, 70.8, 70.3, 70.1, 67.0, 65.3,
65.2, 65.0, 60.1, 56.5, 50.2, 50.0, 49.5, 42.1, 35.5, 35.0, 34.5, 30.8,
30.4; ESI-HRMS Calcd for C126H162N16O50 ([Mþ2H]2þ
) m/z
1H NMR (DMSO-d6, 300 MHz)
d
7.71 (br s, 2H, NHCO), 7.32 (s,
1350.5385, found 1350.5548.
34H, Ph and NHCO2), 7.06 (br s, 2H, NHCO2), 5.00–4.90 (m,16H), 4.47
(br s,13H), 3.84 (br s, 2H), 3.55–3.31 (m,16H), 3.00 (br s, 2H), 2.38 (s,
4H), 1.86 (br s, 2H, H-2eq), 1.48 (br s, 2H, H-2ax); 13C NMR (DMSO-d6,
4.1.10. The dimer of kanamycin A linked with neamine (15)
The title compound was prepared from compound 14 according
75 MHz)
d
172.6, 156.7, 155.9, 155.6, 137.3, 137.1, 136.9, 128.3, 127.7,
to the general procedure (24 h) as solid octahydrochloride (89%).
127.6, 101.3, 97.2, 84.5, 80.2, 74.2, 72.9, 72.4, 70.9, 70.1, 67.0, 65.7,
65.4, 65.0, 60.1, 56.5, 50.0, 49.6, 42.2, 34.5, 30.4; ESI-HRMS Calcd for
C88H111N8O36 ([MþH]þ) m/z 1855.7095, found 1855.7159; Calcd for
C88H110N8O36Na ([MþNa]þ) m/z 1877.6915, found 1877.6912.
1H NMR (300 MHz, D2O)
d 5.50 (s, 1H), 5.29 (s, 2H), 4.93 (s, 2H),
3.72–3.34 (m, 43H), 3.05 (s, 8H), 2.70 (s, 2H), 2.36 (s, 10H), 2.36 (s,
10H), 2.10 (br s, 1H), 1.82–1.74 (m, 2H), 1.53–1.42 (m, 1H); 13C NMR
(75 MHz, D2O) d 175.9,175.5,161.1,160.4,101.0, 98.6, 97.3, 84.0, 80.5,
79.7, 76.1, 74.8, 73.5, 73.2, 73.0, 72.8, 72.6, 71.8, 71.6, 70.8, 70.7, 69.3,
68.7, 65.8, 60.2, 55.5, 54.4, 50.2, 49.6, 48.8, 43.3, 40.5, 40.0, 39.7,
31.8, 31.4, 28.2; ESI-HRMS Calcd for C62H116N16O34 ([Mþ2H]2þ) m/z
814.3909, found 814.3872.
4.1.6. The dimer of kanamycin A linked with succinic acid (9)
The title compound was prepared from compound 8 according
to the general procedure (12 h) as solid hexahydrochloride (95%).
1H NMR (D2O, 300 MHz)
d
5.14 (s, 2H), 4.90 (s, 2H), 3.76–3.08 (m,
30H), 2.90 (br s, 2H), 2.37 (s, 4H), 2.06–2.02 (m, 1H, H-2eq), 1.37–
1.33 (m, 1H, H-2ax); 13C NMR (D2O, 75 MHz)
175.6, 100.6, 100.1,
Acknowledgments
d
85.6, 85.3, 74.3, 72.9, 72.8, 72.0, 71.7, 71.1, 69.5, 66.7, 60.2, 55.4, 50.8,
49.4, 40.3, 32.1, 31.1; ESI-HRMS Calcd for C40H75N8O24 ([MþH]þ)
m/z 1050.4888, found 1050.4877.
We acknowledge the financial support from the National Basic
Research Program (973 Program, Grant No. 2004CB518904).
References and notes
4.1.7. The protected dimer of kanamycin A linked with nine
atoms (11)
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G.-H. Chen et al. / Tetrahedron 65 (2009) 5922–5927
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