Structure-activity relationships for antibacterial to antifungal conversion of kanamycin to amphiphilic analogues

Article abstract of DOI:10.1021/acs.joc.5b00248

Novel fungicides are urgently needed. It was recently reported that the attachment of an octyl group at the O-4″ position of kanamycin B converts this antibacterial aminoglycoside into a novel antifungal agent. To elucidate the structure-activity relation

Full text of DOI:10.1021/acs.joc.5b00248

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Article
Structure-Activity Relationships for Antibacterial to
Antifungal Conversion of Kanamycin to Amphiphilic Analogs
Marina Fosso, Madher N. AlFindee, Qian Zhang, Vincent de Paul N. Nziko, Yukie Kawasaki, Sanjib
K. Shrestha, Jeremiah Bearss, Rylee Gregory, Jon Y. Takemoto, and Cheng-Wei Tom Chang
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b00248 • Publication Date (Web): 31 Mar 2015
Downloaded from http://pubs.acs.org on April 5, 2015
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StructureꢀActivity Relationships for Antibacterial to
Antifungal Conversion of Kanamycin to Amphiphilic
Analogs
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Marina Fosso,† Madher N. AlFindee,† Qian Zhang,† Vincent de Paul Nzuwah Nziko,† Yukie Kawasaki,‡
Sanjib K. Shrestha,‡ Jeremiah Bearss,,† Rylee Gregory,† Jon Y. Takemoto,‡ and Cheng-Wei Tom
Chang†,*
† Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, Utah
84322ꢀ0300, U.S.A.
‡ Department of Biology, Utah State University, 5305 Old Main Hill, Logan, Utah 84322ꢀ5305, U.S.A.
tom.chang@usu.edu
* To whom correspondence should be addressed. Ph: (435) 797ꢀ3545. Fax: 435ꢀ797ꢀ3390.
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
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ABSTRACT. Novel fungicides are urgently needed. It was recently reported that the attachment of an
octyl group at the Oꢀ4" position of kanamycin B converts this antibacterial aminoglycoside into a novel
antifungal agent. To elucidate the structureꢀactivity relationship (SAR) for this phenomenon, a lead
compound FG03 with a hydroxyl group replacing the 3"ꢀNH2 group of kanamycin B was synthesized.
FG03’s antifungal activity and synthetic scheme inspired the synthesis of a library of kanamycin B
analogs alkylated at various hydroxyl groups. SAR studies of the library revealed that for antifungal
activity, the Oꢀ4" position is the optimal site for attaching a linear alkyl chain, and that the 3"ꢀNH2 and
6"ꢀOH groups of the kanamycin B parent molecule are not essential for antifungal activity. The
discovery of lead compound, FG03, is an example of reviving clinically obsolete drugs like kanamycin
by simple chemical modification and an alternative strategy for discovering novel antimicrobials.
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Introduction
Antifungal drug discovery is relatively neglected in medicine when compared to the investment in the
development of antibacterial, antiviral and antiꢀcancer therapeutics. And yet, pathogenic fungi, such as
Aspergillis fumigatus, Candida albicans, Cryptococcus neoformans, and Histoplasma capsulatum pose
serious threats to human health.1 Additionally, exposure to mycotoxins produced by molds presents
great challenges to health and food safety and security. Only a few new fungicides have been introduced
since the midꢀ1980s.2 Resistance to existing antifungal drugs is a major part of these problems. Many
fungicides used in agriculture are chemically synthesized heterocyclic compoundꢀbased, such as
triazoles and pyrimidines, and they highly resemble the antifungal drugs used for treatment of fungal
infections in human. As a consequence, resistant fungi found in agriculture and the environment have
counterparts that have evolved among pathogenic fungi found in humans.3 Thus, there is an urgent need
for the development of novel fungicides.
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Kanamycin is a class of aminoglycoside antibacterial agent.4 Nevertheless, it has become clinically
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obsolete due to the emergence of bacterial pathogens that are resistant to aminoglycoside antibiotics.5
Extensive efforts have been devoted to the chemical modification of kanamycin with the goal of
reviving its activities against resistant bacteria.6 In the past, these studies have focused primarily on the
structureꢀactivity relationship (SAR) of antibacterial activities. More recent discoveries, however, that
amphiphilic aminoglycosides can exert unexpected nonꢀbacterial antimicrobial activities have led to
new strategies for broadening the applications of kanamycin as well as other aminoglycosides.7.8
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We have previously reported the synthesis and antifungal investigation of a novel broadꢀspectrum
fungicide (FG08) (Figure 1).8 Plant leaf infection assays and greenhouse studies showed that FG08 is
capable of suppressing wheat fungal infections by Fusarium graminearum – the causative agent of
Fusarium Head Blight. FG08 can be viewed as a kanamycin derivative with three distinct structural
modifications on ring III: a linear octyl group at Oꢀ4" position, deoxygenation at Oꢀ6" position and the
replacement of 3"ꢀNH2 with OH. Among these modifications, the attachment of Oꢀ4" octyl group is
essential for converting the antibacterial kanamycin into an antifungal agent. Further investigation
confirms that FG08 exerts its antifungal activity by specifically increasing the permeability of the
fungal plasma membrane ꢀ a mechanism of action that differs from the antibacterial action of kanamycin
of binding ribosomal RNA and interfering with protein synthesis.8a,b Shortening the octyl chain to a
butyl group (FG01) or extending the chain length to a dodecyl group (FG02) diminished the antifungal
activity (Figure 1). These findings prompt further questions about the structural features that cause the
antibacterial to antifungal conversion. Major questions include: 1) Is substitution of 6"ꢀCH3 for the
kanamycin 6"ꢀOH on ring III as in FG08 needed for antifungal activity? 2) Is the attachment of the
octyl chain at the Oꢀ4" position required for optimal antifungal activity? and 3) Does the presence of the
3"ꢀNH2 group contribute to the antifungal activity of FG08? Herein, we report a comprehensive threeꢀ
stage SAR study to answers these questions. We designed FG03 to answer the first question. Since
multiple steps are required for the deoxygenation process that produces the 6"ꢀCH3, keeping the initial
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kanamycin 6"ꢀCH2OH moiety without losing antifungal activity will be crucial for simplifying and
scaling up the synthesis of lead antifungal compounds.
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Figure 1. Structures of Kanamycin and Its Amphiphilic Derivatives in the 1st Stage SAR Investigation
Results and Discussion
The synthesis of FG03 possessing 6"ꢀOH and 4"ꢀoctyl groups (Figure 1) started from the 1,3ꢀdiol 1.9
Tritylation of 1 selectively protected the primary alcohol, leaving a free hydroxyl group at position 4
(Scheme 1). Alkylation of the 4ꢀOH, followed by the acidꢀcatalyzed removal of the trityl group revealed
the 6ꢀOH in compound 2. Benzylation afforded the thiophenylglycoside, 3 as the glycosyl donor.
Glycosylation of 4 using the optimal condition14 developed in our group previously to ensure the
formation of α glycosidic bond, and followed by deacetylation, gave 5. Since reduction of azides and
hydrogenolysis of benzyl ether in oneꢀstep fashion can be problematic, we decided to adopt a twoꢀstep
process for the global deprotection.17 Staudinger reduction of compound 5 that converted the azido
groups into amines followed by hydrogenolysis of benzyl ethers, and ionꢀexchange, provided FG03 as a
chloride salt. The minimum inhibitory concentrations (MICs) of FG03 were determined (Table 1).
Despite possessing a 6"ꢀOH instead of 6"ꢀCH3 group, FG03 was found to be as effective as FG08 in
inhibiting the growth of a number of fungi including F. graminearum and lack antibacterial activity.
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Scheme 1.
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Table 1: MIC (ꢀg/mL) Values of FG08 and FG03a
Organism
FG08
FG03
Bacteria
Escherichia coli TG1a
Staphylococcus aureus (ATCC25923)b
Filamentous fungi
125ꢀ250
250
>500
NDc
Fusarium graminearum Bꢀ4ꢀ5A
Pythium ultimum
Curvularia brachyspora
Bortrytis cinerea
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Rhodotorula pilimanae (ATCC26423)
Candida albicans (ATCC10231)
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a Gramꢀnegative bacteria, b Gramꢀpositive bacteria, c Not determined
The observed antifungal activities of FG03 led to the designs of other kanamycin derivatives with 6"ꢀ
OH and the 2nd stage SAR investigation. In this stage, we planned to determine if octylation at other
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hydroxyl groups will enable the antibacterial to antifungal conversion (Figure 2). The design for FG05
is twoꢀcarbon shorter than FG06 since the position of alkylation is Oꢀ6" which is considered extended
as compared to the octyl group at Oꢀ4" position (FG03). The designs of FG10 and FG11 were intended
to help understand the role of 3"ꢀNH2 as well as the effect of the octyl group at the Oꢀ5 position.
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Figure 2. Structures of Amphiphilic Kanamycin Derivatives in the 2nd Stage SAR Investigation
The synthesis of FG05 and FG06 commenced with regioselective ring opening of the known compound
610 to obtain 711 with a free 6ꢀOH (Scheme 2). Alkylation using n-hexyl bromide and nꢀoctyl bromide
provided the phenylthioglycosides as the glycosyl donors, 8a and 8b, respectively. Glycosylation
followed by deacetylation gave 9a and 9b. Staudinger reaction, hydrogenation, and ionꢀexchange
afforded FG05 and FG06, with C6 and C8 alkyl chain at the Oꢀ6" position, respectively.
Scheme 2.
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The synthesis of FG07 started with the alkylation of diacetoneꢀDꢀglucose 10 using octyl bromide to give
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the known compound 1112 (Scheme 3). Acidꢀcatalyzed hydrolysis of compound 11 followed by
acetylation provided 12. Treatment of 12 with thiophenol in the presence of BF3ꢀOEt2 gave 13 as a pure
βꢀanomer. Deprotection of the acetyl groups of 13, followed by benzylation, afforded the
phenylthioglycoside, 14 as the glycosyl donor. Glycosylation of 4 using 14 in the presence of NIS and
TMSOTf, followed by deacetylation, gave compound 15. Staudinger reaction, hydrogenation, and ion
exchange afforded FG07 which has a C8 alkyl chain at the 3" position.
Scheme 3.
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The synthesis of FG09 started from the known compound 1613 (Scheme 4). Alkylation of the 2ꢀOH
gave 17, which upon treatment with Ac2O/AcOH/H2SO4 provided 18. Reaction with thiophenol in the
presence of BF3ꢀOEt2 gave 19. Deacetylation, followed by benzylation gave 20. Following the same
glycosylation and global deprotection process as described previously, FG09 with the C8 alkyl chain at
position 2" was prepared as a chloride salt.
Scheme 4.
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The synthesis of FG10 and FG11 began with a benzylation at the 3´ and 4´ positions of the neamine
derivative 22,14 and was followed by the acidꢀcatalyzed cleavage of the cyclohexylidene protecting
group, which gave the glycosyl acceptor 2315 (Scheme 5). Glycosylation of 23 with the known
thiophenyl donors 24a16 and 24b17 gave the compounds 25a and 25b, respectively. Both compounds
have a free hydroxyl group at Oꢀ5, which was alkylated directly without purification to provide 26a and
26b, respectively. Staudinger reaction, hydrogenation, and ionꢀexchange afforded FG10 and FG11
respectively, with the C8 alkyl chain at Oꢀ5 position. FG10 has a free hydroxyl (OH) group at the 3ʺ
position, while FG11 has an amino (NH2) group at this position. FG10 is thus an analog of FG08, and
FG11 more closely resembles kanamycin B.
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Scheme 5.
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The preparation of FG12 and FG13 started with selective benzylation of 2218 affording a mixture of
regioisomers (27a and 27b) (Scheme 6). The regioisomer 27a has a Bn group at the Oꢀ4´ position
whereas the regioisomer 27b has the Bn group at the Oꢀ3´ position. Attempts to separate 27a and 27b
were unsuccessful. That mixture of 27a and 27b was then used as so. Alkylation of the free hydroxyl
group in each regioisomer, followed by the acidꢀcleavage of the cyclohexylidene protecting group gave
compounds 28a and 28b as an inseparable mixture. Glycosylation of 28a and 28b with the donor 24a
afforded 29a and 29b, which upon Staudinger reduction, hydrogenolysis, and ion exchange gave a
mixture of FG12 and FG13.
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Scheme 6.
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The synthesized kanamycin B analogs were again tested for growth inhibitory activities against the
filamentous fungus F. graminearum, Gꢀ bacterium E. coli (ATCC25922) and G+ bacterium S. aureus
(ATCC25923, G+) (Table 2). From the MIC's, all of the amphiphilic kanamycin B analogs, regardless
of the positions of alkylation, were inactive against bacteria (MIC>32 ꢀg/mL). For antifungal activity,
Oꢀ4" appeared to be the optimal site for antifungal activity while Oꢀ2" was the next best site. Alkylation
at Oꢀ3" and Oꢀ6" positions of ring III with octyl groups enabled only moderate antifungal activities.
Interestingly, shortening the chain length to a hexyl group (entry 5, Table 2) reduced the antifungal
activity dramatically. Finally, alkylation at ring I (Oꢀ3' or Oꢀ4') abolishes both antifungal and
antibacterial activities.
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Table 2: MIC Values of FG Compounds
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MIC (µg/mL)
E. colia
≥250
Entry Alkylation site
Compound
F. graminearum
S. aureusb
≥250
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ND
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≥250
≥250
≥250
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≥250
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≥500
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≥500
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FG07
FG03 (6''ꢀOH)
FG08 (6''ꢀH)
FG05
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ND
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≥250
≥250
≥250
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Oꢀ6'' (C8H17)
Oꢀ5
FG06
FG10 (3''ꢀOH)
FG11 (3''ꢀNH2)
FG12 & FG13
kanamycin
Oꢀ5
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a: ATCC25922; b: ATCC25923
By comparing the results from FG10 and FG11, the presence of 3"ꢀNH2 appears to enhance both
antifungal and antibacterial activities (entries 7 and 8, Table 2). To further analyze the effect of 3"ꢀNH2,
a 3rd stage SAR investigation was pursued with synthesis of more analogs bearing the 3"ꢀNH2 group
(Figure 3). Additionally, since the presence of an octyl group at the Oꢀ2'' or Oꢀ6'' positions of ring III
also promoted modest antifungal activity, we also designed analogs incorporated with two octyl groups
at ring III.
Figure 3. Structures of Amphiphilic Kanamycin Derivatives in the 3rd Stage SAR Investigation
The syntheses of the needed glycosyl donors for these 3"ꢀNH2 containing kanamycin analogs began
with the preparation of phenyl 3ꢀazidoꢀ3ꢀdeoxyꢀ1ꢀthioꢀβꢀD
ꢀglucopyranoside, 3019 and its derivatives,
3119 and 3219 (Scheme 6). Using a diversion approach and similar synthetic methodologies, compounds
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33 and 34 were prepared with Bn and octyl group incorporated at Oꢀ4 position, respectively. Alkylation
of 33 and acetylation of 34 led to the formation of glycosyl donors, 37c and 37e, respectively. Using the
common methods reported in the literature,14,19 glycosyl donor, 37a was synthesized from 31 whereas
glycosyl donors, 37d were prepared from 32. Following the glycosylation and deacetylation,
compounds were subjected to Staudinger reduction and hydrogenolysis, and the desired kanamycin
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analogs were purified by column chromatography using either CG50 (NH4 ) resin or silica gel (Scheme
8). After ionꢀexchange, these analogs were obtained as chloride salts.
Scheme 7.
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Scheme 8.
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The MIC's of the 3rd stage amphiphilic kanamycins are summarized in Table 3. It is clear that the
presence of 3"ꢀNH2 does not increase the antifungal activity (FG14 vs. FG09 and FG15 vs. FG08,
entries 1 and 2). Again, attachment of the octyl group at the Oꢀ4" position yielded optimal antifungal
activity as compared to other sites at ring III. Compounds bearing with two octyl groups (FG17 and
FG18, entries 4 and 5) showed no improvement in antifungal activities, but they had enhanced activities
against S. aureus (ATCC25923) (Table 3). Perhaps, as the lipophilicity increases, these amphiphilic
aminoglycosides are less fungalꢀspecific and behave like other amphiphilic aminoglycosides that
contain longer alkyl chain (ex. hexadecyl) and show strong antibacterial activities.7a,b
Table 3: MIC Values of FG Compounds
MIC (µg/mL)
Entry Alkylation site
Compound
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E. colia
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125
S. aureusb
125ꢀ250
125
1
2
Oꢀ2''
Oꢀ4''
62.5
15.6
FG14
FG15
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Oꢀ6''
Oꢀ4" and Oꢀ6"
Oꢀ2" and Oꢀ4"
-
31.3
31.3
31.3
>500
64ꢀ125
125
125
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125
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32
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FG16
FG17
FG18
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kanamycin
a: ATCC25922; b: ATCC25923
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Conclusion
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We have shown that Oꢀ4" position is the optimal site for attaching a linear alkyl chain that will enable
the conversion of antibacterial kanamycin into an antifungal agent. One octyl group at the Oꢀ4" position
of ring III is the best design for inducing antifungal activity. Octylation at the hydroxyl groups on ring I
or II causes loss of antibacterial activity and no gain in antifungal activity. We have shown that a 3"ꢀ
NH2 group has no or even a negative role in generating antifungal activity. We have also revealed that
the site for attaching the linear alkyl chain is essential for the selective antifungal activity. The fact that
FG03 has the same level of antifungal activity as FG08 indicates that deoxygenation of 6"ꢀOH is not
necessary. There is a growing interest in the antibacterial activities of amphiphilic aminoglycosides. Our
work here not only offers a new application of amphiphilic aminoglycosides but also detailed SAR that
maximizes the antifungal activity. These SAR investigations could lead to the development of new
antifungal kanamycin derivative that can be produced in large quantity.20 Finally, while traditional drug
discovery is often laborious and longꢀterm, reviving old drug with simple chemical modifications and
new applications may serve as an improved and alternative strategy for new drug development.
Experimental Section
General Procedures.
All chemicals were purchased from the commercially available resources without any further
purification. Dry solvents like DMF, DMSO, and THF were dried over molecular sieves.
Dichloromethane was dried by distillation over calcium hydride. Mass spectrometry was taken by high
resolution mass spectrometry (HRMS) using a TOF mass spectrometer. Two NMR instruments were
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1
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used 300 or 400 MHz for the H and C Nuclei. CDCl3, CD3OD, and D2O were used as solvents. The
parts per million (ppm) were used to express the chemical shifts on δ scale. The peaks splitting pattern
were expressed as (s; for the singlet), (d; doublet), (t; triplet), (q; quadrate), (m; multiplet), and (ddd;
doublet of doublets of doublets). Coupling constants J were measured in Hertz (Hz).
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General Procedure for OꢀAlkylation of Carbohydrates. To a solution of starting material in
anhydrous DMF, octyl bromide or benzyl bromide (2.0 equivalents), NaH (5.0 equivalents), and
catalytic amount of TBAI were added. The reaction was stirred overnight. When complete, the reaction
was quenched by addition of MeOH (5 mL) and was slowly poured into a mixture of ice and EtOAc.
The aqueous layer was extracted with EtOAc. The combined organic layers were washed with 1 N
aqueous HCl, water, saturated aqueous NaHCO3 and brine, and then dried over solid Na2SO4. After
removal of the solvent and purification with gradient column chromatography (hexane:EtOAc = 100:0
to 60:40), the product was obtained.
General Procedure for the Glycosylation using Phenylthioglycosyl donor and Deacetylation. A
solution of glycosyl donor, neamine derivative (1.2 equivalents), and activated powder 4 Å molecular
sieve was stirred at room temperature for 2 hours in 12 mL of a mixed anhydrous solution Et2O:CH2Cl2
= 3:1. The mixture was cooled to ꢀ70°C and Nꢀiodosuccinimide (1.2 equivalents) was quickly added.
After the temperature has warmed up to ꢀ40°C, trifluoromethanesulfonic acid (0.15 equivalents) was
added. The solution was stirred at low temperature till the complete consumption of the glycosyl donor.
The reaction mixture was quenched by addition of solid NaHCO3, Na2S2O3, and Na2SO4. After being
stirred for 15 minutes, the reaction mixture was filtered through Celite. The residue was washed
thoroughly with EtOAc. After removal of the solvents, the crude product was purified with gradient
column chromatography. The glycosylated compounds were often mixed with inseparable impurities,
and were therefore fully characterized after hydrolysis. The glycosylated product was dissolved in THF
(1 mL) and MeOH (5 mL), and 1M NaOMe in MeOH (0.5 mL) was added. The mixture was stirred at
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room temperature until TLC analysis indicated completion of the reaction (about 30 minutes). The
reaction was neutralized with Amberlite IRꢀ120 (H+), and filtered through Celite. After removal of the
solvents, the crude product was purified with gradient column chromatography (Hexane:EtOAc = 100:0
to 50:50) to afford the expected product.
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General procedure for the synthesis of kanamycin B analogs: To a solution of starting material
and THF in a reaction vial equipped with a reflux condenser, 0.1 M NaOH(aq) (0.5 mL ) and PMe3 (1M
in THF, 5 ꢀ 7 equivalents) were added. The reaction mixture was stirred at 50ºC for 2 hrs. The product
has a Rf of 0 when eluted with EtOAc/MeOH (9/1) solution and a Rf of 0.6 when eluted with iPrOH/1M
NH4OAc (2/1) solution. After completion of the reaction, the solvents were removed, and the crude
benzylated aminoglycoside was added with a catalytic amount of Pd(OH)2/C (20% Degussa type) and 5
mL of degassed HOAc/H2O (1/3). After being further degassed, the reaction mixture was stirred at room
temperature under atmospheric H2 pressure. After being stirred for 1 day, the reaction mixture was
filtered through Celite. The residue was washed with water, and the combined solutions were
+
concentrated. The crude product was purified with Amberlite CG50(NH4 ) eluted with a gradient of
NH4OH solution (0% – 20%). The final product was obtained as an HCl salt after elution with water
through an ionꢀexchange column packed with Dowex 1X8ꢀ200 (Clꢀ form). After collection of the
1
desired fractions and removal of solvent, the final products were characterized by H and 13C NMR
before being subjected to bioassay.
Phenyl 2,3ꢀdiꢀOꢀbenzylꢀ4ꢀOꢀnꢀoctylꢀ1ꢀthioꢀβꢀDꢀglucopyranoside (2). To a solution of 1 (1.80
g, 3.98 mmol) in anhydrous CH2Cl2 were added TrCl (1.77 g, 6.36 mmol), Et3N (1.12 mL, 7.95 mmol)
and a catalytic amount of DMAP. The reaction mixture was stirred overnight at room temperature.
When complete, the reaction was quenched by addition of MeOH (5 mL). Then the mixture was washed
with water, saturated aqueous NaHCO3, and brine, dried over Na2SO4 and concentrated. The tritylated
crude product was then dissolved in anhydrous DMF, and octyl bromide (1.7 mL, 9.79 mmol), NaH
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The Journal of Organic Chemistry
(0.39 g, 9.79 mmol) and a catalytic amount of TBAI were added. The reaction was stirred overnight.
When complete, the reaction was quenched by addition of MeOH (5 mL) and was slowly poured into a
mixture of ice and EtOAc. The aqueous layer was extracted with EtOAc. The combined organic layers
were washed with water, saturated aqueous NaHCO3 and brine, and then dried over Na2SO4. After
removal of the solvent, the obtained crude product was dissolved in 50 mL of a mixed solution of
CH2Cl2:MeOH = 1:1 and pꢀtoluenesulfonic acid monohydrate (0.61 g, 3.20 mmol) was added. The
resulting mixture was stirred at room temperature overnight. When complete, the reaction mixture was
quenched with Et3N (1.35 mL) and extracted with EtOAc. The organic layer was washed with 1N
aqueous HCl, water, saturated aqueous NaHCO3, brine, and dried over Na2SO4. After removal of the
solvent and purification with a gradient column chromatography (hexane:EtOAc = 100:0 to 40:60), 2
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was obtained as a white solid, mp 98ꢀ99 °C (1.84 g, 3.26 mmol, 84%). H NMR (CDCl3, 300 MHz) δ
7.5 (m, 2H), 7.2 – 7.4 (m, 13H), 4.88 (d, J = 10.3 Hz, 1H), 4.85 (s, 1H), 4.84 (s, 1H), 4.74 (d, J = 10.3
Hz, 1H), 4.70 (d, J = 10.0 Hz, 1H), 3.9 (m, 1H), 3.5 – 3.8 (m, 4H), 3.43 (t, J = 9.6 Hz, 1H), 3.3 (m, 2H),
1.94 (t, J = 6.9 Hz, 1H, OH), 1.5 (m, 2H), 1.2 (m, 10H), 0.87 (t, J = 6.9 Hz, 3H); 13C NMR (CDCl3, 75
MHz) δ 138.5, 138.0, 133.5, 131.9 (2 carbons), 129.1 (2 carbons), 128.5 (4 carbons), 128.3 (2 carbons),
128.0, 127.9, 127.82 (2 carbons), 127.75, 87.5, 86.5, 81.0, 79.5, 78.2, 75.9, 75.6, 73.6, 62.3, 31.9, 30.5,
29.6, 29.3, 26.2, 22.7, 14.2; ESI/APCI calcd for C34H44O5SNa ([M+Na]+) m/z 587.2802; measured m/z
587.2803.
Phenyl 2,3,6ꢀtriꢀOꢀbenzylꢀ4ꢀOꢀnꢀoctylꢀ1ꢀthioꢀβꢀDꢀglucopyranoside (3). To a solution of 2
(1.15 g, 2.04 mmol) in DMF (40 mL) were added BnBr (0.49 mL, 4.07 mmol) and a catalytic amount of
TBAI. The mixture was then transferred in an iceꢀwater bath and NaH (0.16g, 4.07 mmol) was slowly
added. When TLC analysis performed the following day indicated completion, the reaction was
quenched with MeOH (2 mL) and poured over ice. The mixture was extracted with EtOAc. The organic
layer was washed with 1N aqueous HCl, saturated aqueous NaHCO3, water and brine, and then dried
over Na2SO4. After removal of the solvent and purification with a gradient column chromatography
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(hexane:EtOAc = 100:0 to 50:50), 3 was obtained as a yellowish solid, mp 87ꢀ88 °C (1.26 g, 1.92
mmol, 95%). 1H NMR (CDCl3, 300 MHz) δ 7.6 (m, 2H), 7.2 – 7.4 (m, 18H), 4.9 (m, 3H), 4.6 – 4.8 (m,
4H), 3.7 – 3.9 (m, 3H), 3.4 – 3.7 (m, 5H), 1.5 (m, 2H), 1.3 (m, 10H), 0.91 (t, J = 7.2 Hz, 3H); 13C NMR
(CDCl3, 75 MHz) δ 138.6, 138.5, 138.2, 134.0, 132.0 (2 carbons), 129.2 (2 carbons), 128.5 (4 carbons),
128.4 (2 carbons), 128.3 (2 carbons), 127.9 (2 carbons), 127.8, 127.7 (3 carbons), 127.6, 127.5, 87.5,
86.8, 80.8, 79.4, 78.2, 75.9, 75.6, 73.5, 73.4, 69.2, 32.0, 30.5, 29.6, 29.4, 26.3, 22.8, 14.3; ESI/APCI
calcd for C41H50O5SNa ([M+Na]+) m/z 677.3271; measured m/z 677.3280.
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6ꢀOꢀ(2,3,6ꢀTriꢀOꢀbenzylꢀ4ꢀOꢀnꢀoctylꢀαꢀDꢀglucopyranosyl)ꢀ1,3,2´,6´ꢀtetraazidoneamine (5).
Please refer to the general procedure for glycosylation and deacetylation. Compound 5 was obtained as
a light yellowish oil (0.16 g, 0.17 mmol, 47%). 1H NMR (CDCl3, 300 MHz) δ 7.2 – 7.4 (m, 15H), 5.63
(d, J = 3.4 Hz, 1H), 5.02 (d, J = 3.8 Hz, 1H), 4.92 (d, J = 11.0 Hz, 1H), 4.75 (d, J = 12.4 Hz, 1H), 4.72
(m, 2H), 4.64 (d, J = 12.0 Hz, 1H), 4.51 (d, J = 12.4 Hz, 1H), 4.1 – 4.2 (m, 1H), 4.0 – 4.1 (m, 1H), 3.96
(d, J = 10.3 Hz, 1H), 3.89 (d, J = 10.0 Hz, 1H), 3.2 – 3.8 (m, 18H), 2.31 (ddd, J = 13.1, 4.5, 4.1 Hz, 1H),
13
1.51 (ddd, J = 13.0, 12.4, 12.4 Hz, 1H), 1.4 – 1.5 (m, 2H), 1.2 (m, 10H), 0.88 (t, J = 6.9 Hz, 3H); C
NMR (CDCl3, 75 MHz) δ 138.8, 138.1, 137.8, 128.55 (2 carbons), 128.49 (2 carbons), 128.4 (2
carbons), 128.13 (2 carbons), 128.06 (2 carbons), 128.0 (3 carbons), 127.9, 127.7, 98.6, 98.2, 86.3, 86.1,
81.4, 79.6, 78.0, 75.9, 75.7, 73.7, 73.5 (2 carbons), 71.6 (2 carbons), 71.4, 71.1, 68.5, 62.9, 59.6, 59.2,
51.3, 32.4, 31.9, 30.4, 29.6, 29.3, 26.2, 22.8, 14.2; ESI/APCI calcd for C47H62N12O11Na ([M+Na]+) m/z
993.4553; measured m/z 993.4563.
6ꢀOꢀ(4ꢀOꢀnꢀOctylꢀDꢀglucopyranosyl)neamine (FG03). Please refer to the general procedure
for the synthesis of kanamycin B analogs. FG03 was obtained (0.06 g, 0.11 mmol, 42%) as a chloride
salt. 1H NMR (D2O, 300 MHz) (chloride salt) δ 5.81 (d, J = 3.8 Hz, 1H), 4.93 (d, J = 3.8 Hz, 1H), 3.3 ꢀ
4.0 (m, 17H), 3.1 – 3.2 (m, 2H), 2.4 (m, 1H), 1.7 ꢀ 1.9 (m, 1H), 1.4 – 1.5 (m, 2H), 1.1 ꢀ 1.2 (m, 10H),
0.71 (t, J = 6.9 Hz, 3H); 13C NMR (D2O, 100 MHz) (chloride salt) δ 101.7, 96.1, 83.8, 77.8, 77.6, 74.3,
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73.7, 72.9, 72.3, 71.8, 70.9, 69.4, 68.4, 60.4, 53.7, 49.9, 48.5, 40.3, 31.3, 29.4, 28.7, 28.6, 28.2, 25.4,
22.2, 13.7; ESI/APCI calcd for C26H53N4O11+ ([M+H]+) m/z 597.3705; measured m/z 597.3708
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Phenyl 2,3,4ꢀtriꢀOꢀbenzylꢀ6ꢀOꢀnꢀhexylꢀ1ꢀthioꢀβꢀDꢀglucopyranoside (8a). Please refer to the
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general procedure for Oꢀalkylation of sugars except hexyl bromide has been used. Compound 8a was
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obtained as a light yellowish oil (0.69 g, 1.1 mmol, 99%). H NMR (CDCl3, 300 MHz) δ 7.6 – 7.7 (m,
2H), 7.2 – 7.5 (m, 18H), 4.9 – 5.0 (m, 4H), 4.77 (d, J = 10.0 Hz, 1H), 4.70 (d, J = 10.0 Hz, 2H), 3.6 –
3.8 (m, 4H), 3.4 – 3.6 (m, 4H), 1.6 (m, 2H), 1.2 – 1.5 (m, 6H), 0.93 (t, J = 7.1 Hz, 3H); 13C NMR
(CDCl3, 75 MHz) δ 138.6, 138.3, 138.2, 134.1, 132.0 (2 carbons), 129.0 (2 carbons), 128.60 (5
carbons), 128.57 (2 carbons), 128.4 (2 carbons), 128.03 (2 carbons), 127.97 (3 carbons), 127.9, 127.5,
87.6, 86.9, 81.0, 79.3, 78.0, 76.0, 75.6, 75.2, 71.9, 69.7, 31.9, 30.0, 26.0, 22.3, 14.3; ESI/APCI calcd for
C39H46O5SNa ([M+Na]+) m/z 649.2958, measured m/z 649.2971.
Phenyl 2,3,4ꢀtriꢀOꢀbenzylꢀ6ꢀOꢀnꢀoctylꢀ1ꢀthioꢀβꢀDꢀglucopyranoside (8b). Please refer to the
general procedure for Oꢀalkylation of sugars. Compound 8b was obtained as a light yellowish oil (0.70
g, 1.07 mmol, 97%). 1H NMR (CDCl3, 300 MHz) δ 7.6 – 7.7 (m, 2H), 7.2 – 7.5 (m, 18H), 4.9 – 5.0 (m,
4H), 4.79 (d, J = 10.3 Hz, 1H), 4.71 (d, J = 10.0 Hz, 2H), 3.7 – 3.8 (m, 4H), 3.4 – 3.6 (m, 4H), 1.6 (m,
13
2H), 1.2 – 1.5 (m, 10H), 0.94 (t, J = 6.9 Hz, 3H); C NMR (CDCl3, 100 MHz) δ 138.6, 138.4, 138.2,
134.2, 132.0 (2 carbons), 129.0 (2 carbons), 128.63 (5 carbons), 128.64 (2 carbons), 128.4 (2 carbons),
128.1 (2 carbons), 128.0 (3 carbons), 127.9, 127.5, 87.7, 86.9, 81.0, 79.4, 78.0, 76.0, 75.6, 75.2, 71.9,
69.8, 32.1, 30.1, 29.7, 29.5, 26.4, 22.9, 14.3; ESI/APCI calcd for C41H50O5SNa ([M+Na]+) m/z
677.3271, measured m/z 677.3280.
6ꢀOꢀ(2,3,4ꢀTriꢀOꢀbenzylꢀ6ꢀOꢀnꢀhexylꢀαꢀDꢀglucopyranosyl)ꢀ1,3,2´,6´ꢀtetraazidoneamine (9a).
Please refer to the general procedure for glycosylation and deacetylation. Compound 9a was obtained as
1
a light yellowish oil (0.37 g, 0.39 mmol, 40%). H NMR (CDCl3, 300 MHz) δ 7.2 ꢀ 7.4 (m, 15H), 5.69
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(d, J = 3.8 Hz, 1H), 5.05 (d, J = 3.8 Hz, 1H), 4.97 (d, J = 11.0 Hz, 1H), 4.88 (d, J = 10.7 Hz, 1H), 4.82
(d, J = 11.0 Hz, 1H), 4.75 (s, 1H), 4.74 (s, 1H), 4.59 (d, J = 10.7 Hz, 1H), 4.48 (d, J = 2.4 Hz, 1H), 4.1 –
4.2 (m, 1H), 3.9 – 4.1 (m, 3H), 3.1 – 3.7 (m, 15H), 2.97 (d, J = 3.4 Hz, 1H), 2.92 (d, J = 4.1 Hz, 1H),
2.32 (ddd, J = 13.1, 4.5, 4.1 Hz, 1H), 1.6 (m, 2H), 1.50 (ddd, J = 13.1, 12.7, 12.7 Hz, 1H), 1.2 – 1.4 (m,
6H), 0.88 (t, J = 6.9 Hz, 3H); 13C NMR (CDCl3, 75 MHz) δ 138.8, 138.16, 138.07, 128.61 (2 carbons),
128.58 (2 carbons), 128.51 (2 carbons), 128.16 (2 carbons), 128.10 (4 carbons), 128.04 (2 carbons),
127.8, 98.6, 98.2, 85.9, 81.5, 79.7, 79.6, 75.8 (2 carbons), 75.4, 73.5, 71.9 (2 carbons), 71.7, 71.6, 71.4,
71.1, 69.1, 63.0, 59.6, 59.2, 51.3, 32.4, 31.7, 29.4, 25.8, 22.7, 14.2 ; ESI/APCI calcd for
C45H58N12O11Na ([M+Na]+) m/z 965.4240; measured m/z 965.4255.
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6ꢀOꢀ(2,3,4ꢀTriꢀOꢀbenzylꢀ6ꢀOꢀnꢀoctylꢀαꢀDꢀglucopyranosyl)ꢀ1,3,2´,6´ꢀtetraazidoneamine (9b).
Please refer to the general procedure for glycosylation and deacetylation. Compound 9b was obtained as
1
a light yellowish oil (0.36 g, 0.37 mmol, 38%). H NMR (CDCl3, 300 MHz) δ 7.2 ꢀ 7.4 (m, 15H), 5.69
(d, J = 3.8 Hz, 1H), 5.05 (d, J = 3.8 Hz, 1H), 4.97 (d, J = 11.0 Hz, 1H), 4.88 (d, J = 10.7 Hz, 1H), 4.82
(d, J = 11.0 Hz, 1H), 4.75 (s, 1H), 4.74 (s, 1H), 4.59 (d, J = 10.7 Hz, 1H), 4.48 (d, J = 2.4 Hz, 1H), 4.1 –
4.2 (m, 1H), 3.9 – 4.1 (m, 3H), 3.1 – 3.7 (m, 15H), 2.97 (d, J = 3.4 Hz, 1H), 2.92 (d, J = 4.1 Hz, 1H),
2.32 (ddd, J = 13.1, 4.5, 4.1 Hz, 1H), 1.6 (m, 2H), 1.50 (ddd, J = 13.1, 12.7, 12.7 Hz, 1H), 1.2 – 1.4 (m,
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6H), 0.88 (t, J = 6.9 Hz, 3H); C NMR (CDCl3, 100 MHz) δ 138.9, 138.3, 138.2, 128.7 (4 carbons),
128.6 (2 carbons), 128.23 (6 carbons), 128.22 (2 carbons), 127.9, 98.7, 98.3, 85.8, 81.6, 79.8 (2
carbons), 77.7, 75.9 (2 carbons), 75.5, 73.6, 72.0, 71.8, 71.7, 71.6, 71.3, 69.2, 63.2, 59.7, 59.3, 51.4,
32.5, 32.1, 29.7, 29.6, 29.5, 26.3, 22.9, 14.3 ; ESI/APCI calcd for C47H62N12O11Na ([M+Na]+) m/z
993.4553; measured m/z 993.4578.
6ꢀOꢀ(6ꢀOꢀnꢀHexylꢀDꢀglucopyranosyl)neamine (FG05). Please refer to the general procedure
for the synthesis of kanamycin B analogs. FG05 was obtained (0.12 g, 0.21 mmol, 86%) as a chloride
salt. 1H NMR (D2O, 300 MHz) (chloride salt) δ 5.85 (d, J = 4.1 Hz, 1H), 4.89 (d, J = 3.5 Hz, 1H), 3.2 ꢀ
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4.0 (m, 18H), 3.1 (m, 1H), 2.4 (m, 1H), 1.8 (m, 1H), 1.3 – 1.5 (m, 2H), 1.0 ꢀ 1.2 (m, 6H), 0.69 (t, J = 6.9
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Hz, 3H); C NMR (D2O, 100 MHz) (chloride salt) δ 101.9, 95.5, 83.8, 77.0, 74.3, 72.9, 72.11, 72.07,
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71.7, 70.8, 69.4, 69.3, 68.8, 68.4, 53.6, 49.9, 48.4, 40.4, 31.1, 28.6, 28.1, 25.0, 22.1, 13.6; ESI/APCI
calcd for C24H49N4O11 ([M+H]+) m/z 569.3392; measured m/z 569.3408.
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6ꢀOꢀ(6ꢀOꢀnꢀOctylꢀDꢀglucopyranosyl)neamine (FG06). Please refer to the general procedure
for the synthesis of kanamycin B analogs. FG06 was obtained (0.09 g, 0.16 mmol, 82%) as a chloride
salt. 1H NMR (D2O, 300 MHz) (chloride salt) δ 5.92 (d, J = 4.1 Hz, 1H), 4.95 (d, J = 3.8 Hz, 1H), 3.3 ꢀ
4.0 (m, 18H), 3.2 (m, 1H), 2.4 – 2.5 (m, 1H), 1.9 (m, 1H), 1.4 – 1.5 (m, 2H), 1.1 ꢀ 1.2 (m, 10H), 0.74 (t,
13
J = 7.2 Hz, 3H); C NMR (D2O, 100 MHz) (chloride salt) δ 102.0, 95.6, 83.8, 76.9, 74.3, 72.9, 72.2,
72.1, 71.8, 70.9, 69.5, 69.3, 68.8, 68.4, 53.7, 49.9, 48.6, 40.4, 31.3, 28.8, 28.7, 28.6, 28.0, 25.4, 22.2,
13.7; ESI/APCI calcd for C26H53N4O11 ([M+H]+) m/z 597.3705; measured m/z 597.3708.
1,2:5,6ꢀDiꢀOꢀisopropylideneꢀ3ꢀOꢀnꢀoctylꢀαꢀD
ꢀglucopyranose (11).12 Please refer to the general
procedure for Oꢀalkylation of sugars. Compound 11 was obtained as a light yellowish oil (3.76 g, 10.10
mmol, 99%).
1,2,4,6ꢀTetraꢀOꢀacetylꢀ3ꢀOꢀnꢀoctylꢀ
D
ꢀglucopyranose (12). A solution of 11 (4.40 g, 11.8
o
mmol) in 150 mL of a mixed solution of AcOH/TFA/H2O (80/1/19) was stirred at 55 C overnight.
When TLC analysis indicated completion of the reaction, the solvents were removed. After being dried
in vacuo for a few hours, the crude product was dissolved in Ac2O (50 mL) and TFA (5 mL), and the
mixture was stirred at room temperature overnight. Solid NaHCO3 was then added to neutralize the
excess acid. EtOAc was added to dilute the solution and the organic layer was washed with water,
saturated aqueous NaHCO3 (3 times), and brine. The organic layer was then dried over Na2SO4, filtered,
and concentrated. Purification by gradient column chromatography (hexane:EtOAc = 100:0 to 40:60)
provided 12 (4.75 g, 10.3 mmol, 87%) as a mixture of α/β anomers in a 1/2 ratio. The obtained
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1
compound was isolated as yellowish oil mixed with inseparable impurities so only H NMR
characterization was performed. 1H NMR (CDCl3, 300 MHz) (α and β anomers) δ 6.27 (d, J = 3. 8 Hz,
1H, Hꢀ1α), 5.62 (d, J = 8.3 Hz, 1H, Hꢀ1β), 4.9 – 5.5.1 (m, 4H), 3.9 – 4.2 (m, 4H), 3.4 – 3.8 (m, 8H), 2.0
– 2.1 (m, 24H), 1.1 – 1.3 (m, 24H), 0.86 (t, J = 6.9 Hz, 6H).
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Phenyl 2,4,6ꢀtriꢀOꢀacetylꢀ3ꢀOꢀnꢀoctylꢀ1ꢀthioꢀβꢀDꢀglucopyranoside (13). A solution of 12 (4.04
g, 8.77 mmol) and thiophenol (3.4 mL, 33.3 mmol) in anhydrous CH2Cl2 (50 mL) was cooled down to 0
oC and BF3ꢀOEt2 was slowly added. The reaction was stirred for 2 days at room temperature till
completion. Solid NaHCO3, Na2SO4 and some few drops of water were then added and the mixture was
stirred for 1 hour. The solution was then filtered through a Fritz funnel and the collected solids were
washed with EtOAc. After removal of the solvents, purification by gradient column chromatography
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afforded 13 as a light yellowish oil (2.91 g, 5.70 mmol, 65%). H NMR (CDCl3, 300 MHz) δ 7.4 (m,
2H), 7.1 – 7.2 (m, 3H), 4.89 (dd, J = 9.6, 9.6 Hz, 1H), 4.88 (dd, J = 9.6, 9.6 Hz, 1H), 4.56 (d, J = 10.3
Hz, 1H), 3.9 – 4.1 (m, 3H), 3.5 – 3.6 (m, 1H), 3.4 – 3.5 (m, 2H), 2.03 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H),
13
1.3 – 1.4 (m, 2H), 1.1 – 1.2 (m, 10H), 0.77 (t, J = 6.9 Hz, 3H); C NMR (CDCl3, 100 MHz) δ 170.6,
169.4, 169.2, 132.9, 132.5 (2 carbons), 129.0 (2 carbons), 128.1, 86.2, 81.9, 76.1, 72.9, 71.5, 69.8, 62.7,
31.9, 30.4, 29.5, 29.4, 26.1, 22.7, 21.1, 20.9, 20.8, 14.2 ; ESI/APCI calcd for C26H38O8SNa ([M+Na]+)
m/z 533.2180; measured m/z 533.2187.
Phenyl 2,3,6ꢀtriꢀOꢀbenzylꢀ4ꢀOꢀnꢀoctylꢀ1ꢀthioꢀβꢀDꢀglucopyranoside (14). To a solution of 13
(2.91 g, 5.70 mmol) in anhydrous MeOH (40 mL), 0.5 mL of a 1M solution of NaOMe in MeOH was
added and the mixture was stirred at room temperature for 1 hour. When complete, the reaction was
quenched by adding amberlite IR 120 H+ resin to the mixture, followed by filtration through Celite and
concentration of the filtrate. The obtained crude product was dissolved in DMF (40 mL), and BnBr
(10.0 mL, 84.0 mmol) and a catalytic amount of TBAI were added. The mixture was then transferred in
an iceꢀwater bath and NaH (3.36 g, 84.0 mmol) was slowly added. When TLC analysis performed the
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following day indicated completion, the reaction was quenched with MeOH (5 mL) and poured over ice.
The mixture was diluted with EtOAc, extracted with 1N aqueous HCl, saturated aqueous NaHCO3,
water and brine, and dried over Na2SO4. After removal of the solvents, purification by gradient column
chromatography (Hexane:EtOAc = 100:0 to 50:50) gave 14 as a yellowish solid, mp 79ꢀ81 °C (2.00 g,
3.05 mmol, 44%). 1H NMR (CDCl3, 300 MHz) δ 7.2 – 7.8 (m, 20H), 5.0 (m, 2H), 4.86 (d, J = 10.3 Hz,
1H), 4.6 – 4.8 (m, 4H), 3.8 – 4.0 (m, 4H), 3.5 – 3.7 (m, 4H), 1.7 – 1.8 (m, 2H), 1.3 ꢀ 1.5 (m, 10H), 1.01
(t, J = 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 138.7, 138.65, 138.55, 134.3, 132.3 (2 carbons),
129.2 (2 carbons), 128.75 (4 carbons), 128.67 (2 carbons), 128.5 (2 carbons), 128.3 (2 carbons), 128.1
(2 carbons), 128.0 (2 carbons), 127.9, 127.7, 87.7, 87.1, 81.2, 79.4, 78.1, 75.7, 75.3, 74.4, 73.7, 69.4,
32.2, 31.0, 29.9, 29.6, 26.7, 23.0, 14.5 ; ESI/APCI calcd for C41H50O5SNa ([M+Na]+) m/z 677.3271;
measured m/z 677.3270.
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6ꢀOꢀ(2,4,6ꢀTriꢀOꢀbenzylꢀ3ꢀOꢀnꢀoctylꢀαꢀDꢀglucopyranosyl)ꢀ1,3,2´,6´ꢀtetraazidoneamine (15).
Please refer to the general procedure for glycosylation and deacetylation. Compound 15 was obtained as
a light yellowish oil (0.21 g, 0.22 mmol, 59%). 1H NMR (CDCl3, 300 MHz) δ 7.1 – 7.5 (m, 15H), 5.60
(d, J = 3.8 Hz, 1H), 5.02 (d, J = 3.8 Hz, 1H), 4.83 (d, J = 10.7 Hz, 1H), 4.76 (d, J = 12.0 Hz, 1H), 4.70
(d, J = 11.7 Hz, 1H), 4.61 (d, J = 12.4 Hz, 1H), 4.54 (s, 1H), 4.53 (s, 1H), 4.49 (d, J = 12.4 Hz, 1H),
4.45 d, J = 10.7 Hz, 1H), 4.0 – 4.1 (m, 2H), 3.0 – 4.0 (m, 18H), 2.30 (ddd, J = 13.1, 4.5, 4.1 Hz, 1H),
1.6 – 1.7 (m, 2H), 1.49 (ddd, J = 12.7, 12.7, 12.7 Hz, 1H), 1.2 ꢀ1.4 (m, 10H), 0.88 (t, J = 6.9 Hz, 3H);
13C NMR (CDCl3, 100 MHz) δ 138.4, 138.1, 137.9, 128.7 (6 carbons), 128.32 (2 carbons), 128.25 (2
carbons), 128.1 (4 carbons), 128.0, 98.7, 98.4, 85.8, 81.5, 79.9, 79.6, 77.8, 75.9, 75.4, 74.0, 73.6 (2
carbons), 71.7, 71.5 (2 carbons), 71.3, 68.5, 63.0, 59.7, 59.3, 51.4, 32.5, 32.1, 30.9, 29.8, 29.5, 26.5,
22.9, 14.3; ESI/APCI calcd for C47H62N12O11Na ([M+Na]+) m/z 993.4553; measured m/z 993.4564.
6ꢀOꢀ(3ꢀOꢀnꢀOctylꢀDꢀglucopyranosyl)neamine (FG07). Please refer to the general procedure
for the synthesis of kanamycin B analogs. FG07 was obtained (0.08 g, 0.14 mmol, 42%) as a chloride
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salt. 1H NMR (D2O, 300 MHz) (chloride salt) δ 5.81 (d, J = 4.1 Hz, 1H), 4.93 (d, J = 4.0 Hz, 1H), 3.3 ꢀ
4.0 (m, 18H), 3.16 (dd, J = 13.7, 6.9 Hz, 1H), 2.42 (ddd, J = 12.4, 4.1, 4.1 Hz, 1H), 1.87 (ddd, J = 12.7,
12.4, 12.4 Hz, 1H), 1.4 – 1.5 (m, 2H), 1.0 ꢀ 1.3 (m, 10H), 0.71 (t, J = 6.9 Hz, 3H); 13C NMR (D2O, 100
MHz) (chloride salt) δ 101.8, 96.2, 83.8, 81.3, 77.7, 74.2, 73.4, 73.2, 71.3, 70.8, 69.4, 68.9, 68.4, 60.5,
53.6, 49.9, 48.4, 40.3, 31.3, 29.5, 28.7, 28.6, 28.1, 25.3, 22.2, 13.6; ESI/APCI calcd for C26H53N4O11
([M+H]+) m/z 597.3705; measured m/z 597.3720.
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Methyl 3ꢀOꢀbenzylꢀ4,6ꢀOꢀbenzylideneꢀ2ꢀOꢀnꢀoctylꢀαꢀDꢀglucopyranoside (17). Please refer to
the general procedure for Oꢀalkylation of sugars. Compound 17 was obtained as a white solid, mp 68ꢀ69
°C (2.37 g, 4.89 mmol, 90%). 1H NMR (CDCl3, 300 MHz) δ 7.5 (m, 2H), 7.2 – 7.4 (m, 8H), 5.58 (s,
1H), 4.89 (d, J = 11.3 Hz, 1H), 4.84 (d, J = 3.8 Hz, 1H), 4.82 (d, J = 11.3 Hz, 1H), 4.30 (dd, J = 9.6, 4.1
Hz, 1H), 3.98 (dd, J = 9.3, 8.9 Hz, 1H), 3.6 – 3.9 (m, 6H), 3.46 (s, 3H), 1.6 – 1.7 (m, 2H), 1.2 – 1.4 (m,
13
10H), 0.90 (t, J = 6.9 Hz, 3H); C NMR (CDCl3, 100 MHz) δ 139.0, 137.7, 129.1, 128.5 (2 carbons),
128.4 (2 carbons), 128.2 (2 carbons), 127.7, 126.3 (2 carbons), 101.5, 99.2, 82.2, 80.7, 78.6, 75.5, 72.4,
69.3, 62.6, 55.2, 32.1, 30.3, 29.7, 29.5, 26.2, 22.9, 14.4 ; ESI/APCI calcd for C29H40O6Na ([M+Na]+)
m/z 507.2717; measured m/z 507.2723.
1,3,4,6ꢀTetraꢀOꢀacetylꢀ2ꢀOꢀnꢀoctylꢀDꢀglucopyranose (18). Please refer to the synthesis of 12.
Compound 18 was obtained as a light yellowish oil (1.50 g, 3.26 mmol, 76%) in a mixture of α/β
anomers in a 6/1 ratio. 1H NMR (αꢀanomer) (CDCl3, 300 MHz) δ 6.25 (d, J = 3.8 Hz, 1H), 5.21 (dd, J =
10.0, 9.6 Hz, 1H), 4.94 (dd, J = 10.3, 9.6 Hz, 1H), 4.17 (dd, J = 13.0, 4.1 Hz, 1H), 3.9 – 4.0 (m, 2H), 3.4
– 3.6 (m, 2H), 3.3 (m, 1H), 2.05 (s, 3H), 1.95 (s, 3H), 1.92 (s, 3H), 1.91 (s, 3H), 1.3 – 1.4 (m, 2H), 1.1
(m, 10H), 0.75 (t, J = 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 170.5, 170.1, 169.7, 169.0, 89.3,
76.7, 71.6 (2 carbons), 69.8, 68.1, 61.7, 31.9, 29.8, 29.3 (2 carbons), 25.9, 22.7, 21.0, 20.8, 20.72, 20.66,
14.1; ESI/APCI calcd for C22H36O10Na ([M+Na]+) m/z 483.2201; measured m/z 483.2192.
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Phenyl 3,4,6ꢀtriꢀOꢀacetylꢀ2ꢀOꢀnꢀoctylꢀ1ꢀthioꢀDꢀglucopyranoside (19). Please refer to the
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synthesis of 13. Compound 19 was obtained as a light yellowish oil (0.78 g, 1.53 mmol, 56%) in a
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mixture of α/β anomers in a 3/1 ratio. The obtained compound was isolated with inseparable impurities
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so only H NMR characterization was performed. H NMR (αꢀanomer) (CDCl3, 300 MHz) δ 7.5 (m,
2H), 7.3 (m, 3H), 5.77 (d, J = 5.5 Hz, 1H), 5.30 (dd, J = 9.6, 9.6 Hz, 1H), 5.01 (dd, J = 10.3, 9.3 Hz,
1H), 4.54 (ddd, J = 10.3, 5.2, 2.1 Hz, 1H), 4.29 (dd, J = 12.0, 5.2 Hz, 1H), 3.99 (dd, J = 12.4, 2.1 Hz,
1H), 3.6 – 3.8 (m, 2H), 3.3 – 3.5 (m, 1H), 2.04 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.5 (m, 2H), 1.2 – 1.4
(m, 10H), 0.87 (t, J = 6.9 Hz, 3H).
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Phenyl 3,4,6ꢀtriꢀOꢀbenzylꢀ2ꢀOꢀnꢀoctylꢀ1ꢀthioꢀαꢀ
Dꢀglucopyranoside (20). Please refer to the
synthesis of 14. Compound 20 was obtained as a light yellowish oil (1.21 g, 1.85 mmol, 95%). 1H NMR
(CDCl3, 300 MHz) (α anomer) δ 7.5 – 7.7 (m, 2H), 7.2 ꢀ7.5 (m, 18H), 5.86 (d, J = 4.8 Hz, 1H), 5.10 (d,
J = 11.0 Hz, 1H), 4.95 (d, J = 10.7 Hz, 1H), 4.87 (d, J = 10.7 Hz, 1H), 4.68 (d, J = 11.7 Hz, 1H), 4.59
(d, J = 11.0 Hz, 1H), 4.50 (d, J = 12.1 Hz, 1H), 3.5 – 4.0 (m, 8H), 1.6 – 1.8 (m, 2H), 1.3 – 1.5 (m, 10H),
13
0.95 (t, J = 7.2 Hz, 3H); C NMR (CDCl3, 100 MHz) δ 139.2, 138.6, 138.3, 135.1, 132.0, 131.8 (2
carbons), 129.2 (2 carbons), 128.7 (5 carbons), 128.3 (3 carbons), 128.2 (3 carbons), 128.0 (2 carbons),
127.9, 127.3, 87.2, 82.8, 81.0, 77.2, 76.0, 75.4, 73.7, 71.5, 70.7, 68.9, 32.2, 30.4, 29.8, 29.6, 26.5, 23.0,
14.5 ; ESI/APCI calcd for C41H50O5NaS ([M+Na]+) m/z 677.3271; measured m/z 677.3277.
6ꢀOꢀ(3,4,6ꢀTriꢀOꢀbenzylꢀ2ꢀOꢀnꢀoctylꢀαꢀDꢀglucopyranosyl)ꢀ1,3,2´,6´ꢀtetraazidoneamine (21).
Please refer to the general procedure for glycosylation and deacetylation. Compound 21 was obtained as
1
a light yellowish oil (0.10 g, 0.11 mmol, 59%). H NMR (CDCl3, 300 MHz) δ 7.1 – 7.5 (m, 15H), 5.60
(d, J = 3.8 Hz, 1H), 5.02 (d, J = 3.8 Hz, 1H), 4.83 (d, J = 10.7 Hz, 1H), 4.76 (d, J = 12.0 Hz, 1H), 4.70
(d, J = 11.7 Hz, 1H), 4.61 (d, J = 12.4 Hz, 1H), 4.54 (s, 1H), 4.53 (s, 1H), 4.49 (d, J = 12.4 Hz, 1H),
4.45 d, J = 10.7 Hz, 1H), 4.0 – 4.1 (m, 2H), 3.0 – 4.0 (m, 18H), 2.30 (ddd, J = 13.1, 4.5, 4.1 Hz, 1H),
1.6 – 1.7 (m, 2H), 1.49 (ddd, J = 12.7, 12.7, 12.7 Hz, 1H), 1.2 ꢀ1.4 (m, 10H), 0.88 (t, J = 6.9 Hz, 3H);
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13C NMR (CDCl3, 100 MHz) δ 139.0, 138.1, 137.9, 128.64 (6 carbons), 128.58, 128.26, 128.19, 128.1
(4 carbons), 128.0, 127.8, 98.5, 98.2, 86.5, 81.4, 80.9,, 79.6,, 75.9, 76.4, 76.0, 75.8, 75.4, 73.6, 71.9,
71.7, 71.6, 71.2, 68.5, 63.1, 59.7, 59.3, 51.4, 32.5, 32.0, 30.4, 29.7, 29.5, 26.1, 22.9, 14.3 ; ESI/APCI
calcd for C47H62N12O11Na ([M+Na]+) m/z 993.4553; measured m/z 993.4570.
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6ꢀOꢀ(2ꢀOꢀnꢀOctylꢀDꢀglucopyranosyl)neamine (FG09). Please refer to the general procedure
for the synthesis of kanamycin B analogs. FG09 was obtained (0.04 g, .07 mmol, 52%) as a chloride
salt. 1H NMR (D2O, 300 MHz) (chloride salt) δ 5.81 (d, J = 3.8 Hz, 1H), 5.06 (d, J = 3.4 Hz, 1H), 3.0 ꢀ
13
4.0 (m, 19H), 2.4 (m, 1H), 1.4 – 1.5 (m, 3H), 1.1 ꢀ 1.2 (m, 10H), 0.72 (t, J = 6.5 Hz, 3H); C NMR
(D2O, 100 MHz) (chloride salt) δ 100.3, 96.4, 83.9, 80.1, 78.0, 74.4, 73.3, 73.2, 72.7, 70.8, 69.5, 69.4,
68.4, 60.7, 53.7, 49.8, 48.4, 40.3, 31.3, 29.3, 28.7, 28.5, 28.0, 25.1, 22.2, 13.6; ESI/APCI calcd for
C26H53N4O11 ([M+H]+) m/z 597.3705; measured m/z 597.3701.
3´,4´ꢀDiꢀOꢀbenzylꢀ1,3,2´,6´ꢀtetraazidoneamine (23).15 To a solution of 2218 (3.60 g, 7.11
mmol) in DMF (40 mL) were added BnBr (3.40 mL, 28.5 mmol) and a catalytic amount of TBAI. The
mixture was then transferred in an iceꢀwater bath and NaH (1.14 g, 28.5 mmol) was slowly added.
When TLC analysis performed the following day indicated completion, the reaction was quenched with
MeOH (2 mL) and poured over ice. The mixture was extracted with EtOAc. The organic layer was
washed with 1N aqueous HCl, saturated aqueous NaHCO3, water and brine, and dried over Na2SO4.
After removal of the solvent, a brownish crude product was obtained, to which 80 mL of mixed solution
of dioxane: H2O = 1:1 was added, followed by 35 mL glacial acetic acid. The resulting mixture was
o
refluxed at 60~65 C overnight. When complete, the reaction mixture was quenched with saturated
aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed with 1N aqueous HCl,
water, saturated aqueous NaHCO3, brine, and dried over Na2SO4. After removal of the solvent followed
by purification with a gradient column chromatography (pure hexane to hexane: EtOAc = 40:60), 23
was obtained as a light yellowish oil (2.03 g, 6.62 mmol, 42%).
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6ꢀOꢀ(2,3,4,6ꢀTetraꢀOꢀbenzylꢀαꢀDꢀglucopyranosyl)ꢀ3´,4´ꢀOꢀdibenzylꢀ1,3,2´,6´ꢀ
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tetraazidoneamine (25a). A solution of 23 (0.20 g, 0.33 mmol), 25a (0.25 g, 0.40 mmol), and activated
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powder 4Å molecular sieve was stirred at room temperature for 2 hours in 12 mL of a mixed anhydrous
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solution Et2O:CH2Cl2 = 3:1. The mixture was cooled to ꢀ70 C and Nꢀiodosuccinimide (0.09 g, 0.40
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mmol) was quickly added. After the temperature has warmed up to ꢀ40 C, trifluoromethanesulfonic
acid (0.05 mL) was added. The solution was stirred at low temperature till the complete consumption of
the glycosyl donor. The reaction mixture was quenched by addition of solid NaHCO3, Na2S2O3 and
Na2SO4. After being stirred for 15 minutes, the reaction mixture was filtered through Celite. The residue
was washed thoroughly with EtOAc. After removal of the solvents, the crude product was purified with
gradient column chromatography (Hexane:EtOAc = 100:0 to 50:50) to afford 25a as a yellowish oil.
Because it was mixed with inseparable impurities, it was used as so in the next step.
6ꢀOꢀ(3ꢀAzidoꢀ3ꢀdeoxyꢀ2,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀglucopyranosyl)ꢀ3´,4´ꢀOꢀdibenzylꢀ1,3,2´,6´ꢀ
tetraazidoneamine (25b). Please refer to the synthesis of 25a. Compound 25b was also obtained as a
yellowish oil mixed with inseparable impurities and was then used as so in the next step.
6ꢀOꢀ(2,3,4,6ꢀTetraꢀOꢀbenzylꢀαꢀDꢀglucopyranosyl)ꢀ3´,4´ꢀOꢀdibenzylꢀ5ꢀOꢀnꢀoctylꢀ1,3,2´,6´ꢀ
tetraazidoneamine (26a). Please refer to the general procedure for Oꢀalkylation of sugars. Compound
26a was obtained as a light yellowish oil (0.20 g, 0.16 mmol, 57%). 1H NMR (CDCl3, 300 MHz) δ 7.2 ꢀ
7.5 (m, 30H), 5.72 (d, J = 3.4 Hz, 1H), 5.62 (d, J = 3.8 Hz, 1H), 4.8 – 5.0 (m, 8H), 4.67 (d, J = 11.3 Hz,
1H), 4.65 (d, J = 12.0 Hz, 1H), 4.53 (d, J = 11.3 Hz, 1H), 4.47 (d, J = 12.0 Hz, 1H), 4.32 (d, J = 9.6 Hz,
1H), 4.15 (d, J = 10.0 Hz, 1H), 4.04 (dd, J = 10.3, 8.9 Hz, 1H), 3.9 – 4.0 (m, 1H), 3.3 – 3.8 (m, 15H),
2.4 (m, 1H), 1.5 – 1.7 (m, 3H), 1.0 ꢀ 1.4 (m, 10H), 0.86 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 75 MHz)
δ 138.8, 138.7, 138.1 (2 carbons), 137.83, 137.77, 128.64 (3 carbons), 128.57 (3 carbons), 128.49 (4
carbons), 128.25 (3 carbons), 128.20 (4 carbons), 128.1 (3 carbons), 128. 0 (3 carbons), 127.9 (2
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