Scalable and cost-effective tosylation-mediated synthesis of antifungal and fungal diagnostic 6″-Modified amphiphilic kanamycins

Article abstract of DOI:10.1016/j.ejmech.2019.111639

Amphiphilic kanamycins bearing hydrophobic modifications at the 6″ position have attracted interest due to remarkable antibacterial-to-antifungal switches in bioactivity. In this report, we investigate a hurdle that hinders practical applications of these amphiphilic kanamycins: a cost-effective synthesis that allows the incorporation of various connecting functionalities to which the hydrophobic moieties are connected to the kanamycin core. A cost-effective tosylation enables various modifications at the 6″ position, which is scalable to a 90-g scale. The connecting functionalities, such as amine and thiol, were not the dominant factor for biological activity. Instead, the linear chain length played the decisive role. Amphiphilic kanamycin attached with tetradecyl (C14) or hexadecyl (C16) showed strong antifungal and modest antibacterial activities than with shorter chains (C6–C10). However, increases in chain length were closely correlated with an increase in HeLa cell toxicity. Thus, a compromise between the antimicrobial activities and cytotoxicities, for optimal efficacy of amphiphilic kanamycins may contain chain lengths between C8 and C12. Finally, the described synthetic protocol also allows the preparation of a fluorescent amphiphilic kanamycin selective toward fungi.

Full text of DOI:10.1016/j.ejmech.2019.111639

European Journal of Medicinal Chemistry 182 (2019) 111639
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Scalable and cost-effective tosylation-mediated synthesis of antifungal
and fungal diagnostic 600-Modified amphiphilic kanamycins
Yagya Prasad Subedi a, Uddav Pandey a, Madher N. Alfindee a, Heath Montgomery a,
Paul Roberts a, Jeffrey Wight a, Gavin Nichols a, Michell Grilley b, Jon Y. Takemoto b,
,
*
Cheng-Wei Tom Chang a
a Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, UT, 84322-0300, USA
b Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT, 84322-5305, USA
a r t i c l e i n f o
a b s t r a c t
Amphiphilic kanamycins bearing hydrophobic modifications at the 600 position have attracted interest
due to remarkable antibacterial-to-antifungal switches in bioactivity. In this report, we investigate a
hurdle that hinders practical applications of these amphiphilic kanamycins: a cost-effective synthesis
that allows the incorporation of various connecting functionalities to which the hydrophobic moieties
are connected to the kanamycin core. A cost-effective tosylation enables various modifications at the 600
position, which is scalable to a 90-g scale. The connecting functionalities, such as amine and thiol, were
not the dominant factor for biological activity. Instead, the linear chain length played the decisive role.
Amphiphilic kanamycin attached with tetradecyl (C14) or hexadecyl (C16) showed strong antifungal and
modest antibacterial activities than with shorter chains (C6eC10). However, increases in chain length
were closely correlated with an increase in HeLa cell toxicity. Thus, a compromise between the anti-
microbial activities and cytotoxicities, for optimal efficacy of amphiphilic kanamycins may contain chain
lengths between C8 and C12. Finally, the described synthetic protocol also allows the preparation of a
fluorescent amphiphilic kanamycin selective toward fungi.
Article history:
Received 24 July 2019
Received in revised form
20 August 2019
Accepted 21 August 2019
Available online 22 August 2019
Keywords:
Amphiphilic kanamycin
Antifungal
Fungal infection
Fungal diagnostic
Fluorescent aminoglycoside
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
or cations. To address these shortcomings, we decided to employ
kanamycin sulfate as the starting material and explore toluene-
Amphiphilic kanamycin (AK) such as those synthesized from
kanamycin A, kanamycin B and tobramycineall with hydrophobic
moieties attached at the 600-positionehas attracted great interest
due to their broad spectrum antibacterial and antifungal activities
[1e5]. The reported synthetic approaches have allowed studies on
structure-activity relationships (SARs), including the effects of
connecting functionalities, such as thiol, sulfone, amide, ether, and
1,2,3-triazole) and the optimal chain length of the linear hydro-
phobic groups. Despite the advances, cost-effective and scalable
production of the lead compounds has not been satisfactorily
resolved. In addition, there is no AK bearing an amine as the con-
necting functionality. Amines can amend the solubility or
bioavailability of the AK (especially for those with longer (>C14)
linear hydrophobic chains) by bringing additional hydrogen bonds
sulfonyl as the connecting functionality in the chemical synthesis of
AK-carrying amino hydrophobic moieties attached at the 600-posi-
tion. Commercially available kanamycin sulfate (containing >95%
kanamycin A) is more cost-effective compared to kanamycin B,
tobramycin, or other kanamycin-class aminoglycosides (Fig. 1).
2. Rationale, design and synthesis
To implement modifications at the hydroxyl groups of kana-
mycin, the amino groups need to be protected or masked as azido
groups. The former is commonly achieved using carbamate-type
protecting groups, e.g. t-butoxycarbonyl (Boc) or carboxybenzyl
(Cbz) groups. The latter can be done via amine/azide trans-
formation. We favor the use of Boc groups for two reasons. First, the
reagent (Boc2O) is inexpensive and much safer to handle as
compared to azide needed for the amine/azide transformation.
Second, Boc groups can be removed without trace using TFA,
whereas removal of Cbz requires hydrogenolysis using a costly and
* Corresponding author.
E-mail address: tom.chang@usu.edu (C.-W.T. Chang).
https://doi.org/10.1016/j.ejmech.2019.111639
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
Y.P. Subedi et al. / European Journal of Medicinal Chemistry 182 (2019) 111639
pathogen), and Fusarium graminearium B4-5A (plant pathogen),
using K20 as the control. The minimum inhibitory concentrations
(MICs) were determined (Table 1). From the MICs, AKs bearing
tetradecyl (C14) and hexadecyl (C16) were most potent regardless
of the connecting functionalities (amine and thiol). This is consis-
tent with those thiol analogs reported by others. Interestingly, K20
compounds carrying octanesulfonyl groups are only slightly less
active than the AK bearing tetradecyl (C14) and hexadecyl (C16)
groups, while those with octyl or decyl chains are much less active,
especially against Candida sp. In addition, strains of the genuses
Cryptococcus, Rhodotorula, and Fusarium become more prone to the
increase in chain length, as compounds 4b,c and 5b,c attached with
octyl or decyl groups started to show increased antifungal activity.
Based on these findings, it is concluded that the connecting func-
tionality does not play a key role in level of antifungal activity.
Rather, the chain length and the strain of fungi are the main factors
that govern the antifungal activity of AK with 600-modification.
Fig. 1. Structure of kanamycin class aminoglycosides.
hazardous metal catalyst. We have previously reported the large
scale synthesis of Boc-protected kanamycin (300 g scale) without
the need of column purification [6]. The next hurdle is the regio-
selective incorporation of a hydrophobic group at the 600-position.
This is conducted by selectively converting the 600-OH into a leaving
3.2. Antibacterial activity of amphiphilic kanamycin
The antibacterial activity of AK was also tested against a panel of
bacteria, including Escherichia coli (ATCC 25922, Gram negative, G-
), Staphylococcus aureus (ATCC 25923, Gram positive, Gþ), S. aureus
(ATCC 33591, MRSA), and S. aureus (ATCC 43300, MRSA) using
kanamycin A and vancomycin as the controls (Table 2). The results
showed that these AK exerted only modest antibacterial activity
even for those with C14 or C16 chains, and again the connecting
functionalities did not have obvious effect. Nevertheless, the anti-
bacterial SAR is similar to the antifungal SAR implying the lack of
selectivity between antibacterial and antifungal activity. Interest-
ingly, compound 3 with its toluenesulfonyl group displayed an
activity profile like kanamycin A; it was active against amino-
glycoside susceptible strains while being inactive against MRSA.
group, followed by nucleophilic substitution.
A bulky 2,4,6-
triisopropylbenzenesulfonyl (TIPBS) chloride is commonly used to
enable selective incorporation at the 600-OH [7,8]. However, TIPBSCl
is not cost-effective and usually needs to be employed in excess
amount (5 equiv.). We believe that toluenesulfonyl (Ts) chloride is a
better option. After several attempts, we achieved comparable
yields as those obtained using TIPBSCl but with only 2.5 equiv. of
TsCl to generate compound 2 (Scheme 1). Furthermore, we
demonstrated that the reaction can be conducted at larger scales
(ca. 90 g scale). In contrast, the reported synthesis using TIPBSCl
was often conducted on a scale less than 5 g. The tosylation site was
confirmed from the 1He1H COSY of compound 3.
3.3. Cytotoxicity of amphiphilic kanamycin against HeLa cells
Following the successful synthesis of tosylated kanamycin de-
rivative, nucleophilic substitution using linear alkylamines (hexyl,
octyl, decyl, dodecyl, tetradecyl and hexadecyl amines) followed by
column fractionation and deprotection of Boc groups provided the
desired products. To compare similar compounds as reported by
others, we also used linear alkyl thiols (hexyl, octyl, decyl, dodecyl,
tetradecyl, and hexadecyl thiols) as the nucleophiles. Without pu-
rification using column chromatography, the Boc groups of the
substitution products were removed using TFA and furnished the
final AK ready for biological assay. In addition, we synthesized a
fluorescent member, 7 using an amide as the connecting func-
tionality. Compound 7 enables fluorescence-based evaluation of
microbe selectivity (fungi vs. bacteria), which will be discussed
later. Finally, it is important to mention that the tosylated kana-
mycin derivative can also lead to the synthesis of AK with ether,
amide, or sulfone connecting functionalities using established
protocols [3,9].
Selected AKs with good antifungal activity were tested for their
cytotoxicity towards HeLa cells (Fig. 2). The IC50 values of the
compounds against HeLa cells are presented in Table 3. AK equip-
ped with 12-carbon chain or less did not show toxicity up to tested
concentrations while the AK with a carbon chain of fourteen or
longer have shown significant toxicity at 100 mg/mL. These results
suggest that it may be necessary to make compromises with less
than optimal antifungal activities and cytotoxicities for selecting
efficacious AK analogs (such as in the case of K20), for clinical and
agricultural applications.
Treatments for fungal diseases can be challenging due to the
emergence of drug-resistant fungi and the unclear distinction of
fungal infections from bacterial infections. The latter can be difficult
for physicians due to similar symptoms from bacteria and fungi.
Nevertheless, in order to offer prompt therapeutic options, it is
pivotal to identify the nature of the microbes that cause the
infection since antifungal agents are, in general, inactive against
bacteria and vice versa. Current practices for detection of fungi,
such as culture method and PCR may take days to yield the result
[10]. Amphiphilic kanamycin has been shown to exert a fast
membrane permeabilization (within a few minutes) and endocy-
tosis selective toward fungi over bacteria [11]. Therefore, we
decided to synthesize the fluorescent AK, 7 as described in Scheme
1 for the potential application of offering prompt differentiation of
fungi from bacteria. Two strains of fungi, C. albicans 64124 (azole-
resistant) and C. albicans MYA2876 (azole susceptible), and two
strains of bacteria, E. coli (G-) and S. aureus (Gþ) were selected for
3. Results and discussion
3.1. Antifungal activity of amphiphilic kanamycin
The antifungal activity of newly synthesized AK was tested
against a panel of fungi, including Candida albicans 64124 (human
pathogen, azole-resistant), C. albicans MYA2876 (human pathogen,
azole susceptible), C. albicans B-311 (human pathogen), C. rugosa
95e967 (human pathogen), C. parapsolis Cas08-0490 (human
pathogen, azole-resistant), C. tropicalis 95-41 (human pathogen),
Cryptococcus neoformans H99 (human pathogen), C. neoformans VR-
54 (human pathogen), Rhodotorula pilimanae (ATCC 26423, plant
the imaging study. Fungi were treated with compound 7 at 32
mg/
mL, which is about ¼ of the MIC. Bacteria were also treated with

Y.P. Subedi et al. / European Journal of Medicinal Chemistry 182 (2019) 111639
3
Scheme 1. Synthesis of amphiphilic kanamycin.
Table 1
Antifungal activity of AK.a.
Compound
Ab
Bb
Cb
Db
Eb
Fb
Gb
Hb
Ib
Jb
3
4a
4b
4c
>256
>256
256
128
16
>256
>256
>256
>256
64
>256
>256
>256
256
32
>256
>256
>256
256
16
>256
>256
>256
256
32
>256
>256
>256
>256
32
>256
128
16
8
>256
128
16
8
128
128
16
8
128
64
32
32
2
4d
2
2
2
4e
4
8
8
4
4
4
2
2
2
2
4f
4
8
4
4
4
4
2
2
2
2
5a
5b
5c
5d
>256
128
64
>256
256
64
>256
>256
128
32
>256
256
64
>256
>256
128
32
>256
>256
128
32
128
128
8
64
64
8
32
32
4
256
128
32
4
16
16
16
2
2
2
5e
4
4
8
8
16
4
2
2
2
2
5f
7
K20
4
128
16
4
256
16
4
4
ND
32
4
ND
16
4
ND
16
2
64
4
2
64
4
2
32
4
2
32
8
NDc
16
Kanamycin A
ꢀ256
ꢀ256
ꢀ256
ND
ND
ND
ND
ꢀ256
ꢀ256
ꢀ256
a
Unit:
m
g/mL.
b
A: C. albicans 64124, B: C. albicans MYA2876, C: C. albicans B-311, D: C. rugosa 95e967, E: C. parapsolis Cas08-0490 (azole resistant), F: C. tropicalis 95-41, G: C. neoformans
H99, H: C. neoformans VR-54, I: R. pilimanae (ATCC 26423), J: F. graminearium B4-5A.
c
ND: Not determined.
compound 7 at 32
MIC for S. aureus. The images were taken after 5 and 60 min of
incubation with compound 7 (Fig. 3).
m
g/mL, which is the MIC for E. coli and ½ of the
the fungal yeast Candida strains even with 5 min of incubation and
at less than MIC concentrations. In contrast, no fluorescent staining
can be observed for both bacteria (E. coli and S. aureus) after 5 min.
Only after incubating for 60 min, did E. coli and S. aureus begin to
From the images, it is clear that compound 7 can quickly stain

4
Y.P. Subedi et al. / European Journal of Medicinal Chemistry 182 (2019) 111639
Table 2
MICs of AK against bacterial strains.a.
Compound
Ab
Bb
Cb
Db
Compound
Ab
Bb
Cb
Db
3
4a
4b
4c
4d
4e
4f
16
256
256
64
16
32
32
4
32
64
128
128
64
16
16
4
>256
>256
>256
256
64
16
32
>256
>256
>256
128
64
32
16
5a
5b
5c
5d
5e
5f
7
128
128
64
16
16
256
128
64
32
16
16
64
ND
>256
256
64
32
16
32
>256
4
>256
256
32
16
16
16
>256
2
32
32
>256
32
Kanamycin A
Vancomycin
NDc
a
Unit: mg/mL.
b
c
A: E. coli (ATCC 25922), B: S. aureus (ATCC 25923), C: S. aureus (ATCC 33591) MRSA, D: S. aureus (ATCC 43300) MRSA.
ND: Not determined.
the cytotoxicity evaluation revealed that as chain length increases
(ꢀC14), cytotoxicity also increases. Combining these results, we
conclude that AK equipped with longer chain length exert superior
bioactivity against fungi and bacteria, but are associated with
increased cytotoxicity, making these AKs behave more like non-
selective antiseptic agents. In contrast, K20 developed previously
in our group represents one of the few examples of AK that best
compromises between antifungal/antibacterial activity and toxicity
to mammalian cells; it is selectively active toward fungi but inactive
against bacteria or mammalian cells. The synthesis of fluorescent
compound 7 and its fungal selective properties may pave the way
for the development of diagnostic tools that can promptly differ-
entiate fungal infection from diseases of bacterial origin.
5. Experimental section
Fig. 2. Cytotoxicity of AK against HeLa cells.
Materials and Methods. Chemicals purchased from the com-
mercial source were used without further purification unless
otherwise mentioned. Pyridine was dried by keeping over calcium
hydride for 72 h and recycled for further use. DMF was dried by
treating with a molecular sieve for 72 h before using for the reac-
tion. 1H NMR, 1He1H COSY, and 13C NMR spectra of the compounds
were obtained using a Brucker AvanceIII HD Ascend-500 at
500 MHz and 125 MHz respectively. Multiplicity of the peaks is
reported as s ¼ singlet, d ¼ doublet, t ¼ triplet, m ¼ multiplet,
b ¼ broad. Coupling constants are presented in the hertz (Hz).
Emission and excitation spectra of 7 were recorded in SHIMADZu
RF-5301PC Spectrofluorophotometer. Emission wavelength was
fixed at 520 nm while scanning excitation wavelength and the
compounds were excited at 480 nm for emission spectra of 7.
General Procedure for the Synthesis of compound 4a - 4f. To a
solution of 2 (0.104 g, 1 equiv, 0.1 mmol) dissolved in anhydrous
dioxane, 3 equiv of alkyl amine was added and the reaction mixture
was refluxed for 24 h. Solvent was removed under reduced pressure
and the solid residue was washed with 3 ꢁ 10 mL of water. During
the washing, 10 mL of water was added to the solid, stirred for
15 min, and then filtered. This process was repeated two more
times before moving to the next step. After washing, the reaction
mixture was loaded onto a silica gel column and eluted with sol-
vents ranging from 100% DCM to 10% MeOH in DCM. The eluted
fractions were analyzed by TLC (5% methanol in DCM), and those
that contained desired products were combined and concentrated
to provide a yellowish white precipitate. The collected precipitate
was re-dissolved in anhydrous DCM (10 mL) and trifluoroacetic acid
(TFA) (1 mL) was added. After being stirred for 6 h, solvents were
removed, and the residual reaction mixture was loaded onto a
column packed with Amberlite CG50 ion exchange resin, and eluted
with a gradient of solvent (100% water to 20% NH4OH in water).
Pure compound obtained after CG50 purification was further sub-
jected to the ion exchange using anion exchange resin IR410 (Clꢂ
Table 3
IC50 value of the AK against HeLa cells.a.
Compound
IC50 value
4d
4e
4f
5d
5e
5f
7
>100
60.79 3.13
41.56 10.23
>100
52.88 5.78
37.73 7.33
>100
a
Unit ¼
mg/mL.
show slight fluorescent staining. Since the concentration of 7
employed was not fungicidal, but closer to bactericidal, the rela-
tively strong fluorescence emitted from fungal cells when treated
with compound 7 indicates that fluorescent AK analogs like com-
pound 7 can be employed for differential diagnostic detection of
fungi vs. bacteria at single-cell imaging resolution.
4. Conclusion
We have developed a practical and scalable route for the syn-
thesis of 600-modified AK that is suitable for the introduction of
various connecting functionalities between the kanamycin core and
hydrophobic moieties. Two libraries of AK bearing amine and thiol
connecting functionalities were synthesized. Similar SARs show
that tetradecyl (C14) and hexadecyl (C16) are the optimal chain
length to exert broad-spectrum antifungal activity and modest
antibacterial activity. Little or no difference in bioactivity displayed
by the two libraries containing amine or thiol connecting func-
tionalities suggests a minor role of these functionalities. However,

Y.P. Subedi et al. / European Journal of Medicinal Chemistry 182 (2019) 111639
5
Fig. 3. Images of fungi and bacteria incubated with 7.
form) to yield the desired amphiphilic kanamycin as a chloride salt.
General Procedure for the Synthesis of 5a - 5f. To a solution of
2 (0.104 gm, 1 equiv, 0.1 mmol) dissolved in dimethylformade
(DMF), 5 equiv of alkylthiol and 5 equiv of cesium carbonate was
added and the reaction mixture was stirred for 48 h at room tem-
perature. Solvent was removed, and the precipitate was washed
with water 4 ꢁ 20 mL of water using a Buchner funnel. During the
washing, 20 mL of water was added to the precipitate, stirred for
15 min, and then filtered. This process was repeated three more
times. The precipitate was further washed with 3 ꢁ 10 mL of hex-
ane. The residual solid was dissolved in 10 mL of anhydrous DCM
and 1 mL of TFA was added, and stirred at room temperature for 6 h.
Solvents were removed, and the residual reaction mixture was
loaded onto a column packed with Amberlite CG50 ion exchange
resin, and eluted with a gradient of solvent (100% water to 20%
NH4OH in water). Pure compound obtained after CG50 purification
was further subjected to the ion exchange using anion exchange
resin IR410 (Clꢂ form) to yield the desired amphiphilic kanamycin
as a chloride salt.
butoxycarbonyl)kanamycin A (86.9 gm, 1 equiv, 100 mmol) was
dissolved in 1200 mL of anhydrous pyridine by magnetic stirring at
room temperature. The reaction flask was then transferred to the
ice bath and stirred for 15 min. A solution of toluenesulfonyl chlo-
ride (TsCl) (47.67 gm, 2.5 equiv, 250 mmol) dissolved in 300 mL of
anhydrous pyridine was slowly added to the stirring solution of
1,3,60,300-tetra-N-(tert-butoxycarbonyl)kanamycin A at 0 ꢃC over the
period of an hour. After completion of the addition of TsCl, the re-
action mixture was stirred for additional 30 min. The ice bath was
removed and the reaction mixture was stirred for 3 days allowing
the temperature to rise slowly to room temperature. The reaction
was quenched by addition of methanol (6 mL, 1.5 equiv, 0.15 mol).
After being stirred for 30 min, the solvents were removed using a
rotary evaporator under reduced pressure while keeping the tem-
perature of the water bath below 30 ꢃC. The viscous residue ob-
tained after the removal of the solvent solidified into powder and
was washed with 5 ꢁ 500mL water using a Buchner funnel. The
resulting pale yellowish powder was loaded in to the silica gel
column and purified by eluting with a gradient of solvent (100%
DCM to 5% MeOH in DCM). The compound 2 was obtained as a
white solid (56.4 g, 6.4 mmol, 54%). 1H NMR (500 MHz, CD3OD)
600-O-Toluenesulfonyl-1,3,6′,300-tetra-N-(tert-butoxycarbonyl)
kanamycin
A
(2).
A
solution of 1,3,60,300-tetra-N-(tert-

6
Y.P. Subedi et al. / European Journal of Medicinal Chemistry 182 (2019) 111639
d
7.79 (d, J ¼ 8.0 Hz, 2H), 7.45 (d, J ¼ 8.0 Hz, 2H), 5.08 (b, 1H), 5.00 (b,
d
100.25, 98.78, 84.23, 81.56, 74.38, 72.00, 71.13, 70.84, 68.64, 68.23,
1H), 4.1e4.4 (m, 3H), 3.3e3.8 (m, 12H), 3.03 (t, J ¼ 9.5 Hz, 1H), 2.48
68.14, 67.44, 54.68, 50.40, 48.58, 48.33, 47.75, 40.38, 31.18, 28.77
(3C), 28.69, 28.52, 28.50, 28.42, 25.68, 25.38, 22.03, 13.42. ESI/APCI
calcd for C30H62N5Oþ10 [MH]þ: 652.4497; measured m/e: 652.4487.
(s, 3H), 2.0e2.1 (m, 1H), 1.4e1.5 (m, 37H); 13C NMR (125 MHz,
CD3OD)
d 157.97, 157.82, 156.54, 156.28, 132.99, 129.63 (2C), 127.72
(2C), 101.41, 98.38, 84.34, 79.91, 79.22, 79.01 (2C), 78.78, 75.33,
73.10, 72.52, 70.95, 70.45, 70.33, 70.21, 69.05, 67.71, 55.93, 50.60,
49.37, 40.49, 34.63, 27.53 (3C), 27.49 (3C), 27.45 (6C), 20.31. ESI/APCI
calcd for C45H74N4NaO21Sþ [MNa]þ: 1061.4464; measured m/e:
1061.4474.
6”-(1-Tetradecylamino)-600-deoxykanamycin
A
(4e). The
compound was synthesized according to the general procedure and
obtained as a white solid (40.5 mg, 0.047 mmol, 47%). 1H NMR
(500 MHz, D2O)
d
5.56 (d, J ¼ 3.5 Hz, 1H), 5.06 (d, J ¼ 3.5 Hz, 1H),
4.0e4.2 (m, 1H), 3.9e4.0 (m, 2H), 3.6e3.8 (m, 4H), 3.2e3.6 (m, 8H),
3.1e3.2 (m, 2H), 3.0e3.1 (m, 2H), 2.2e2.3 (m, 1H), 1.7e1.8 (m, 1H),
1.6e1.7 (m, 2H), 1.1e1.3 (m, 22H), 0.78 (t, J ¼ 7.0 Hz, 3H); 13C NMR
600-O-Toluenesulfonylkanamycin A (3). To a solution of 2
(0.104 gm, 0.1 mmol) dissolved in anhydrous DCM (5 mL), tri-
fluoroacetic acid was added (0.5 mL). After being stirred at room
temperature for 6 h, the solvent was removed. The crude product
was subjected to ion exchange using anion exchange resin IR410
(Clꢂ form) to yield the desired amphiphilic kanamycin as a chloride
salt. The compound was synthesized as white solid (74.5 mg,
(125 MHz, D2O)
d 100.43, 98.70, 83.74, 79.93, 74.20, 71.87, 70.99,
70.70, 68.75, 68.29, 68.04, 67.19, 54.64, 50.12, 48.39, 48.34, 47.69,
40.30, 31.19, 28.78 (5C), 28.69, 28.50þ(2C), 28.25, 25.68, 25.37, 22.03,
13.41. ESI/APCI calcd for C32H66N5O10 [MH]þ: 680.4810; measured
m/e: 680.4810.
0.095 mmol, 95%). 1H NMR (500 MHz, D2O)
d
7.72 (d, J ¼ 8.5 Hz, 2H),
6”-(1-Hexadecylamino)-600-deoxykanamycin A (4f). The com-
pound was synthesized according to the general procedure and
obtained as a white solid (17.8 mg, 0.020 mmol, 20%). 1H NMR
7.37 (d, J ¼ 8.5 Hz, 2H), 5.49 (d, J ¼ 4.0 Hz, 1H), 4.94 (d, J ¼ 3.5 Hz,
1H), 4.2e4.3 (m, 2H), 4.0e4.1 (m, 1H), 3.9e4.0 (m, 1H), 3.2e3.9 (m,
12H), 3.1e3.2 (m, 1H), 2.4e2.5 (m, 1H), 2.34 (s, 3H), 1.8e1.9 (m, 1H);
(500 MHz, CD3OD)
d
5.58 (d, J ¼ 3.5 Hz, 1H), 5.22 (d, J ¼ 3.5 Hz, 1H),
13C NMR (125 MHz, D2O)
d
146.75, 130.37, 130.24 (2C), 127.93 (2C),
4.2e4.3 (m, 1H), 4.0e4.1 (m, 1H), 3.7e3.9 (m, 4H), 3.4e3.7 (m, 7H),
3.0e3.3 (m, 6H), 2.4e2.5 (m, 1H), 1.7e1.9 (m, 3H), 1.3e1.4 (m, 26H),
100.55, 96.25, 83.82, 78.20, 72.53, 72.08, 70.82, 70.81, 70.33, 69.06,
68.69, 67.90, 65.19, 54.72, 49.93, 47.89, 40.43, 27.58, 20.97. ESI/APCI
calcd for
639.2543.
0.92 (t, J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz, CD3OD)
d 100.29, 97.75,
C
25H43N4O13Sþ [MH]þ: 639.2547; measured m/e:
84.59, 81.29, 73.42, 72.65, 71.87, 71.66, 69.00, 68.76, 68.56, 67.80,
55.12, 50.67, 48.65, 48.39, one carbon peak underneath the solvent
peak, 40.72, 31.66, 29.40 (6C), 29.36, 29.32, 29.21, 29.07, 28.89,
26.28, 25.64, 22.32, 13.03. ESI/APCI calcd for C34H70N5Oþ10 [MH]þ:
708.5123, measured m/e: 708.5121.
600-(1-Hexylamino)-600-deoxykanamycin
A (4a). The com-
pound was synthesized according to the general procedure and
obtained as a white solid (41.9 mg, 0.056 mmol, 56%). 1H NMR
(500 MHz, D2O)
d
5.75 (d, J ¼ 4.0 Hz, 1H), 5.08 (d, J ¼ 3.5 Hz, 1H),
600-(1-Hexylmercapto)-600-deoxykanamycin A (5a). The com-
pound was synthesized according to the general procedure and
obtained as a white solid (51.8 mg, 0.071 mmol, 71%). 1H NMR
4.1e4.2 (m, 1H), 3.3e4.0 (m, 15H), 3.1e3.2 (m, 1H), 3.03 (t,
J ¼ 9.0 Hz, 2H), 2.4e2.5 (m, 1H), 1.8e1.9 (m, 1H), 1.6e1.7 (m, 2H),
1.2e1.4 (m, 6H), 0.79 (t, J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz, D2O)
(500 MHz, D2O)
d
5.52 (d, J ¼ 4.0 Hz, 1H), 5.01 (d, J ¼ 4.0 Hz, 1H),
d
100.4, 98.5, 83.7, 79.7, 74.1, 71.8, 70.9, 70.6, 68.7, 68.2, 68.0, 67.2,
3.8e4.0 (m, 5H), 3.6e3.8 (m, 2H), 3.2e3.6 (m, 7H), 3.1e3.2 (m, 1H),
2.9e3.0 (m, 1H), 2.6e2.7 (m, 1H), 2.53 (t, J ¼ 7.5 Hz, 2H) 2.3e2.4 (m,
1H), 1.7e1.8 (m, 1H), 1.4e1.5 (m, 2H), 1.1e1.3 (m, 6H), 0.76 (t,
54.6, 50.1, 48.4, 48.3, 47.7, 4þ0.2, 30.4, 28.3, 25.3 (2C), 21.7, 13.2. ESI/
APCI calcd for C24H50N5O10 [MH]þ: 568.3558; Measured m/e:
568.3576.
J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz, D2O)
d 100.33, 96.70, 84.34,
6”-(1-Octylamino)-600-deoxykanamycin A (4b). The compound
was synthesized according to the general procedure and obtained
as a white solid (35.0 mg, 0.045 mmol, 45%). 1H NMR (500 MHz,
78.68, 73.19, 72.19 (2C), 70.88, 70.78, 68.59, 68.44, 68.21, 54.82,
49.78, 47.98, 40.38, 32.41, 32.39, 30.63, 28.79, 28.31, 27.64, 21.89,
13.38. ESI/APCI calcd for
measured m/e: 585.3171.
C
24H49N4O10Sþ [MH]þ: 585.3164;
D2O)
d
5.44 (d, J ¼ 4.0 Hz, 1H), 5.03 (d, J ¼ 3.5 Hz, 1H), 4.1e4.2 (m,
1H), 3.1e4.0 (m, 16H), 3.02 (t, J ¼ 8.0 Hz, 2H), 2.2e2.3 (m, 1H),
6”-(1-Octylmercapto)-600-deoxykanamycin A (5b). The com-
pound was synthesized according to the general procedure and
obtained as a white solid (40.9 mg, 0.054 mmol, 54%). 1H NMR
1.5e1.7 (m, 3H), 1.1e1.3 (m, 10H), 0.78 (t, J ¼ 6.5 Hz, 3H); 13C NMR
(125 MHz, D2O)
d 100.37, 98.44, 83.72, 79.69, 74.18, 71.84, 70.00,
70.75, 68.71, 68.25, 68.04, 67.21, 54.64, 50.13, 48.42, 48.36, 47.71,
40.33, 31.01, 28.35, 28.20, 28.18, 25.66, 25.37, 21.79, 13.40. ESI/APCI
calcd for C26H54N5O10: 596.3871; measured m/e: 596.3864.
600-(1-Decylamino)-600-deoxykanamycin A (4c). The compound
was synthesized according to the general procedure and obtained
as a white solid (47.6 mg, 0.059 mmol, 59%). 1H NMR (500 MHz,
(500 MHz, D2O)
d
5.54 (d, J ¼ 4.0 Hz, 1H), 5.03 (d, J ¼ 3.5 Hz, 1H),
3.8e4.0 (m, 5H), 3.7e3.8 (m, 2H), 3.5e3.6 (m, 4H), 3.2e3.5 (m, 3H),
3.1e3.2 (m, 1H), 2.9e3.0 (m, 1H), 2.6e2.7 (m, 1H), 2.56 (t, J ¼ 7.5 Hz,
2H), 2.4e2.5 (m, 1H), 1.8e1.9 (m, 1H), 1.5e1.6 (m, 2H), 1.1e1.4 (m,
10H), 0.78 (t, J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz, D2O)
d 100.46,
96.48, 84.11, 78.03, 72.95, 72.20, 72.11, 70.80, 70.72, 68.65, 68.38,
68.17, 54.80, 49.69, 47.83, 40.35, 32.41, 32.37, 31.10, 28.81, 28.34,
28.28, 27.95, 27.59, 22.01, 13.42. ESI/APCI Calcd for C26H53N4O10Sþ
[MH]þ: 613.3482; measured m/e: 613.3486.
D2O)
d
5.57 (d, J ¼ 4.0 Hz, 1H), 5.07 (d, J ¼ 4.0 Hz, 1H), 4.1e4.2 (m,
1H), 3.9e4.0 (m, 2H), 3.7e3.9 (m, 4H), 3.3e3.6 (m, 7H) 3.1e3.2 (2H),
3.0e3.1 (t, J ¼ 8.0 Hz 2H), 2.4e2.5 (m, 1H), 1.7e1.8 (m, 1H), 1.6e1.7
(m, 2H), 1.2e1.3 (m, 14H), 0.78 (t, J ¼ 6.5 Hz, 3H); 13C NMR
6”-(1-Decylmercapto)-600-deoxykanamycin A (5c). The com-
pound was synthesized according to the general procedure and
obtained as a white solid (35.4 mg, 0.045 mmol, 45%). 1H NMR
(125 MHz, D2O)
d 100.39, 98.52, 83.73, 79.78, 74.19, 71.86, 71.01,
70.75, 68.73, 68.26, 68.05, 67.21, 54.65, 50.14, 48.42, 48.36, 47.72,
40.34, 31.16, 28.65, 28.51, 28.45, 28þ.35, 28.24, 25.67, 25.38, 22.03,
13.42. ESI/APCI calcd for C28H58N5O10 [MH]þ: 624.4184; measured
m/e: 624.4181.
(500 MHz, D2O)
d
5.52 (d, J ¼ 4.0 Hz, 1H), 5.02 (d, J ¼ 3.5 Hz, 1H),
3.8e4.0 (m, 5H), 3.6e3.8 (m, 3H), 3.2e3.6 (m, 7H), 3.1e3.2 (m, 1H),
2.9e3.0 (m, 1H), 2.6e2.7 (m, 1H), 2.55 (t, J ¼ 7.5 Hz, 2H) 2.3e2.4 (m,
1H), 1.7e1.8 (m, 1H), 1.5e1.6 (m, 2H), 1.1e1.3 (m, 14H), 0.78 (t,
6”-(1-Dodecylamino)-600-deoxykanamycin A (4d). The com-
pound was synthesized according to the general procedure and
obtained as a white solid (54.2 mg, 0.065 mmol, 65%). 1H NMR
J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz, D2O)
d 100.33, 96.88, 84.49,
79.10, 73.30, 72.16, 72.13, 70.91, 70.78, 68.59, 68.46, 68.23, 54.82,
49.83, 48.02, 40.37, 32.42, 32.39, 31.18, 28.80, 28.70, 28.65 (2C),
28.48, 28.30, 27.93, 22.04, 13.43. ESI/APCI calcd for C28H57N4O10Sþ
[MH]þ: 641.3795; measured m/e: 641.3789.
(500 MHz, D2O)
d
5.52 (d, J ¼ 3.5 Hz, 1H), 5.07 (d, J ¼ 3.5 Hz, 1H),
4.1e4.2 (m, 1H), 3.8e4.0 (m, 2H), 3.6e3.8 (m, 4H), 3.2e3.6 (m, 8H),
3.1e3.2 (m, 2H), 3.0e3.1 (m, 2H), 2.2e2.3 (m, 1H), 1.5e1.7 (m, 3H),
1.2e1.3 (m, 18H), 0.77 (t, J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz, D2O)
6”-(1-Dodecylmercapto)-600-deoxykanamycin
A (5d). The

Y.P. Subedi et al. / European Journal of Medicinal Chemistry 182 (2019) 111639
7
compound was synthesized according to the general procedure and
obtained as a white solid (49.7 mg, 0.061 mmol, 61%). 1H NMR
6.6e6.7 (m, 2H), 6.55 (d, J ¼ 9.0 Hz, 1H), 6.4e6.5 (m, 2H), 6.40 (d,
J ¼ 8.5 Hz, 1H), 5.45 (d, J ¼ 3.5 Hz, 1H), 4.60 (d, J ¼ 3.5 Hz, 1H),
3.7e3.9 (m, 3H), 3.6e3.7 (m, 2H), 3.56 (t, J ¼ 10.0 Hz, 1H), 3.3e3.5
(m, 7H), 3.1e3.3 (m, 3H), 2.8e2.9 (m, 1H), 2.4e2.5 (m, 1H), 1.8e1.9
(500 MHz, D2O)
d
5.49 (d, J ¼ 3.5 Hz, 1H), 4.99 (d, J ¼ 3.5 Hz, 1H),
3.8e4.0 (m, 4H), 3.2e3.7 (m, 10H), 3.0e3.1 (m, 1H), 2.9e3.0 (m, 1H),
2.6e2.7 (m, 1H), 2.52 (t, J ¼ 7.5 Hz, 2H), 2.3e2.4 (m, 1H), 1.6e1.7 (m,
1H), 1.4e1.6 (m, 2H), 1.1e1.3 (m, 18H), 0.75 (t, J ¼ 7.0 Hz, 3H); 13C
(m, 1H); 13C NMR (125 MHz, D2O)
d 171.74, 157.40, 156.99, 153.04,
152.92, 152.77, 134.31, 130.09, 129.61, 129.35, 129.32, 124.24, 122.83,
112.56, 112.08, 111.36, 109.21, 102.65, 102.53, 100.24, 96.26, 84.29,
76.28, 73.96, 72.15, 71.97, 70.67, 70.63, 68.62, 67.30, 66.37, 66.27,
53.68, 49.12, 48.26, 41.05, 40.05, 27.47. ESI/APCI calcd for
NMR (125 MHz, D2O)
d 100.27, 97.08, 84.65, 79.61, 73.45, 72.17,
72.09, 70.96, 70.80, 68.56, 68.49, 68.27, 54.82, 49.87, 48.10, 40.36,
32.44 (2C), 31.22, 28.82 (4C), 28.76, 28.68, 28.54, 28.34, 27.97, 22.06,
13.44. ESI/APCI calcd for
measured m/e: 669.4107.
C
30H61N4O10Sþ [MH]þ: 669.4108;
C
38H48N5Oþ14 [MH]þ: 798.3198; measured m/e: 798.3165.
Procedure for the antifungal activity. Minimum inhibitory
6”-(1-Tetradecylmercapto)-600-deoxykanamycin A (5e). The
compound was synthesized according to the general procedure and
obtained as a white solid (36.2 mg, 0.043 mmol, 43%). 1H NMR
concentrations of the compounds were evaluated by two-fold
dilution method following standard protocol [14,15]. All the
fungal strains were grown in RPMI medium supplement with
0.165 M MOPS buffer (pH ¼ 7) at 28 ꢃC with gentle shaking for 48 h.
Compounds were dissolved in water to make stock solution of
10 mg/mL. The compounds from stock were diluted in 96 well plate
using growth medium to the final concentration ranging from 512
(500 MHz, CD3OD)
d
5.61 (d, J ¼ 4.0 Hz, 1H), 5.13 (d, J ¼ 3.5 Hz, 1H),
4.0e4.1 (m, 2H), 3.2e4.0 (m, 12H), 3.0e3.1 (m, 2H), 2.6e2.8 (m, 3H),
2.4e2.5 (m, 1H), 1.8e2.0 (m, 1H), 1.5e1.7 (m, 2H), 1.2e1.5 (m, 22H),
0.92 (t, J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz, CD3OD)
d 100.41, 95.59,
85.34, 79.29, 73.07 (2C), 72.57, 71.68, 71.64, 68.90, 68.85, 68.63,
55.27, 50.14, one peak underneath the solvent peak, 40.81, 32.77,
32.67, 31.67, 29.49, 29.40 (3C), 29.37 (3C), 29.23, 29.07 (2C), 28.59,
22.33, 13.03. ESI/APCI Calcd for C32H65N4O10Sþ[MH]þ: 697.4421;
measured m/e: 697.4425..
to 0.25
mg/mL 50
mL of fungi with recommended confluence from
the standard protocol was added to each well containing 50
mL of
diluted compounds and incubated for 24e36 h. The minimum
concentration of compound that inhibits fungal growth to visible
clearance is reported as the MIC value.
Procedure for the antibacterial activity. Bacterial strains were
grown in Lysogeny broth (LB) at 35 ꢃC. MIC of compounds against
was also determined by two-fold dilution method. The compounds
were diluted in a 96-well plate using water to the final concen-
6”-(1-Hexadecylmercapto)-600-deoxykanamycin A (5f). The
compound was synthesized according to the general procedure and
obtained as a white solid (27.0 mg, 0.031 mmol, 31%). 1H NMR
(500 MHz, CD3OD)
d
5.50 (d, J ¼ 3.5 Hz, 1H), 5.02 (d, J ¼ 3.5 Hz, 1H),
3.9e4.0 (m, 2H), 3.1e4.0 (m, 12H), 2.9e3.0 (m, 2H), 2.5e2.7 (m, 3H),
2.3e2.4 (m, 1H), 1.7e1.8 (m, 1H), 1.4e1.6 (m, 2H), 1.1e1.4 (m, 26H),
tration from 512 to 0.25
mg/mL 50 mL of bacteria growth with
adjusted confluence (OD600 ¼ 0.08e0.1) was added to each well of
0.80 (t, J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz, CD3OD)
d
100.42, 95.56,
the 96-well plate with 50 mL of diluted compounds. The plate was
85.26, 79.17, 73.06 (2C), 72.57, 71.69, 71.65, 68.88, 68.85, 68.63,
55.28, 50.13, one peak underneath the solvent peak, 40.82, 32.77,
32.67, 31.67, 29.49, 29.39 (6C), 29.35 (2C), 29.07 (3C), 28.59, 22.33,
incubated for 18 h at 35 ꢃC and read visually. The minimum con-
centration of compound that inhibited the bacterial growth to
visible clearance is reported as the MIC value.
13.04. ESI/APCI calcd for
measured m/e: 725.4730.
C
34H69N4O10Sþ[MH]þ: 725.4734;
Procedure for the evaluation of cytotoxicity towards human
cell. Cytotoxicity of the compounds was evaluated by colorimetric
assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) dye. The compounds’ cytotoxicity towards human
cells was evaluated against the HeLa cell, a cervical human cell.
Cells were grown in DMEM medium (Corning) supplemented with
10% fetal bovine serum and 100 U/mL each of penicillin and
600-(2-(6-hydroxy-3-oxo-3H-xanthen-9-yl))benzamido)-600-
deoxykanamycin A (7). To a solution of 2 (1.04 gm, 1 equiv,
1.00 mmol) dissolved in 20 mL of anhydrous DMF, sodium azide
(0.214, 3.3 equiv, 3.3 mmol) was added, and the reaction was stirred
at 80 ꢃC under nitrogen atmosphere. After being stirred for 48 h,
solvent was removed under reduced pressure and the solid residue
was washed with water 3 ꢁ 10 mL. The washed precipitate was
dried under vacuum for 24 h. The dried crude compound was then
dissolved in 10 mL of 1:1 solution of degassed MeOH and AcOH.
Catalytic amount of 10% Pd/C was added and the reaction mixture
was stirred at room temperature for 12 h under hydrogen gas
environment. The reaction mixture was filtered through Celite and
the solvents were removed. The solid residue was washed with
2 ꢁ 5 mL of 10% sodium carbonate aqueous solution. The residue
was further washed with 2 ꢁ 5 mL of DCM to obtain compound 6
[12]. For a solution of compound 6 (0.100 gm, 1 equiv, 0.11 mmol)
dissolved in 10 mL of DMF, NHS activated fluorescein (0.095 gm, 2
equiv, 0.22 mmol) [13], was added. The reaction mixture was stir-
red for 24 h at room temperature. Solvent was removed, and the
viscous residue was washed with 2 ꢁ 5 mL water and dried under
vacuum. The dried precipitate was treated with a solution of 5 mL
of anhydrous DCM and 1 mL of trifluoro acetic acid, and the reac-
tion mixture was stirred for 6 h at room temperature. After removal
of solvents, the viscous residue was loaded to a column packed with
by Amberlite CG50 and eluted with a gradient of solvent (100%
water to 20% NH4OH in water). Pure compound obtained after CG50
purification was further subjected to the ion exchange using anion
exchange resin IR410 (Clꢂ form) to yield the desired amphiphilic
kanamycin as a chloride salt (24.0 mg, 0.025 mmol, 23%). 1H NMR
streptomycin under 5% CO2 in humid environment at 37 ꢃC. 200
mL
of cell suspension was added to each well of a 96-well plate from
25,000 cells/mL stock and incubated for 24 h. The medium was
removed, and the compounds were? diluted in growth medium
from 100
mg/mL - 0.1
mg/mL with ten-fold dilution along with just
medium was added to the cells. After 24 h of incubation, 20
mL of
MTT dye (5 mg/mL in PBS) was added and incubated for 5 more hrs.
The medium was removed, and the reduced formazan dye was
dissolved in 200 mL of DMSO. Absorption of formazan solution was
measured at 570 nm with 650 nm as the background absorption.
The experiment was done in triplicate of triplicate. During the
analysis, absorption from the control was converted to 100% cell
viability and the cell viability at different concentration of the
compounds were calculated relative to the control.
Procedure for the imaging of fungi incubated with com-
pound 7. Fungi were grown in the PDB medium with gentle shaking
at 28 ꢃC for 48 h. 1 mL of fungal growth was transferred to the
centrifuge tube and spun down at 10,000 rpm for 2 min in a Fisher
Scientific accuSpin MicroCentrifuge at room temperature. Cell
pallets were washed one more time using the same volume of the
water. The fungal cells were then incubated with 1 mL of 32 mg/mL
of compound 7 for 5 or 60 min. The final confluence of fungal cells
was maintained to 1 ꢁ 107 cells/mL during the incubation with
compound 7. After the incubation with compound 7, fungal cells
were washed with the same volume of water by centrifugation and
(500 MHz, D2O)
d 7.8e7.9 (m, 1H), 7.5e7.6 (m, 2H), 6.9e7.0 (m, 1H),

8
Y.P. Subedi et al. / European Journal of Medicinal Chemistry 182 (2019) 111639
resuspended in 1 mL of water. Images of fungi were taken using an
Olympus IX71 microscope using 100X oil immersion objective
under phase contrast and green channel.
References
[1] Y.P. Subedi, M.N. AlFindee, J.Y. Takemoto, C.-W.T. Chang, Antifungal amphi-
philic kanamycins: new life for an old drug, MedChemComm
9 (2018)
Procedure for the imaging of bacteria incubated with com-
pound 7. Bacteria were grown in LB medium at 37 ꢃC for 18 h 1 mL
of bacteria in each centrifuge tube was spun down at 10,000 rpm
for 2 min in a Fisher Scientific accuSpin Micro at room temperature.
Bacterial cells were washed with water twice using same volume of
water by centrifugation. Bacterial cells were then treated with 1 mL
909e919.
[2] S.K. Shrestha, M.Y. Fosso, K.D. Green, S. Garneau-Tsodikova, Amphiphilic
tobramycin analogues as antibacterial and antifungal agents, Antimicrob.
Agents Chemother. 59 (2015) 4861e4869.
[3] M.Y. Fosso, S.K. Shrestha, K.D. Green, S. Garneau-Tsodikova, Synthesis and
bioactivities of kanamycin B-derived cationic amphiphiles, J. Med. Chem. 58
(2015) 9124e9132.
[4] R. Dhondikubeer, S. Bera, G.G. Zhanel, F. Schweizer, Antibacterial activity of
amphiphilic tobramycin, J. Antibiot. 65 (2012) 495.
[5] I.M. Herzog, K.D. Green, Y. Berkov-Zrihen, M. Feldman, R.R. Vidavski, A. Eldar-
Boock, R. Satchi-Fainaro, A. Eldar, S. Garneau-Tsodikova, M. Fridman, 600-Thi-
oether tobramycin analogues: towards selective targeting of bacterial mem-
branes, Angew. Chem. 51 (2012) 5652e5656.
of 32
were washed with water twice. After washing, cells were resus-
pended in 100 L of water. Images were taken in Olympus IX71
mg/mL of compound 7. After 5 or 60 min of incubation, cells
m
microscope using 100X oil immersion objective under phase
contrast and green channel.
[6] C,-W. T. Chang, J. Takemoto, US Patent 8865665, 2014.
[7] A. Van Schepdael, J. Delcourt, M. Mulier, R. Busson, L. Verbist,
H. Vanderhaeghe, M.-P. Mingeot-Leclercq, P.M. Tulkens, P. Claes, New de-
rivatives of kanamycin B obtained by modifications and substitutions in po-
sition 6''. 1 synthesis and microbiological evaluation, J. Med. Chem. 34 (1991)
1468e1475.
[8] M.D. Disney, O.J. Barrett, An aminoglycoside microarray platform for directly
monitoring and studying antibiotic resistance, Biochemistry 46 (2007)
11223e11230.
[9] K.B. Steinbuch, R.I. Benhamou, L. Levin, R. Stein, M. Fridman, Increased degree
of unsaturation in the lipid of antifungal cationic amphiphiles facilitates se-
lective fungal cell disruption, ACS Infect. Dis. 4 (2018) 825e836.
[10] S.F. Yeo, B. Wong, Current status of nonculture methods for diagnosis of
invasive fungal infections, Clin. Microbiol. Rev. 15 (2002) 465e484.
[11] Y.P. Subedi, P. Roberts, M. Grilley, J.Y. Takemoto, C.-W.T. Chang, Development
of fungal selective amphiphilic kanamycin: cost-effective synthesis and use of
fluorescent analogs for mode of action investigation, ACS Infect. Dis. 5 (2019)
473e483.
Author contributions
Alkyl AKs were synthesized and characterized by Y.P.S., U.P.,
H.M., P. R., J.W., G. N. and antifungal assays were performed by Y.P.S.,
M.N.A., M.G. Synthesis and imaging with the fluorescent analog,
antibacterial activity, cytotoxicity were performed and analyzed by
Y.P.S. The manuscript was written and edited by C.-W. T. C., Y.P.S,
J.Y.T. and M.G.
Acknowledgements
We acknowledge support from the Department of Chemistry
and Biochemistry, Utah State University and the Utah Science,-
Technology and Research (USTAR) initiative, State of Utah. This
work was supported in part by NSF Award CHE-1429195 for a
500 MHz Bruker NMR.
[12] R.J. Fair, M.E. Hensler, W. Thienphrapa, Q.N. Dam, V. Nizet, Y. Tor, Selectively
guanidinylated aminoglycosides as antibiotics, ChemMedChem
7 (2012)
1237e1244.
[13] J. Gao, P. Wang, R.W. Giese, Xanthamide fluorescent dyes, Anal. Chem. 74
(2002) 6397e6401.
[14] CLSI, Reference Method for Broth Dilution Antifungal Susceptibility Testing of
Yeasts; Approved StandarddSecond EditiondDocument M27-A2, CLSI,
Wayne, PA, USA, 2002.
Appendix A. Supplementary data
[15] CLSI, Reference Method for Broth Dilution Antifungal Susceptibility Testing of
Filamentous Fungi; Approved StandarddSecond EditiondDocument M38-A2,
CLSI, Wayne, PA, USA, 2008.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111639.