Molecular Umbrellas Modulate the Selective Toxicity of Polyene Macrolide Antifungals

Article abstract of DOI:10.1021/acs.bioconjchem.8b00136

Antifungal polyene macrolide antibiotics Amphotericin B (AmB) and Nystatin (NYS) were conjugated through the ω-amino acid linkers with diwalled "molecular umbrellas" composed of spermidine-linked deoxycholic or cholic acids. The presence of "umbrella" substituents modulated biological properties of the antibiotics, especially their selective toxicity. Some of the AmB-umbrella conjugates demonstrated antifungal in vitro activity comparable to that of the mother antibiotic but diminished mammalian toxicity, especially the hemolytic activity. In contrast, antifungal in vitro activity of NYS-umbrella conjugates was strongly reduced and all these conjugates demonstrated poorer than NYS selective toxicity. No correlation between the aggregation state and hemolytic activity of the novel conjugates was found.

Full text of DOI:10.1021/acs.bioconjchem.8b00136

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Article
Molecular umbrellas modulate the selective
toxicity of polyene macrolide antifungals
Andrzej Skwarecki, Kornelia Skarbek, Dorota Martynow, Marcin
Serocki, Irena Bylinska, Maria Jolanta Milewska, and Slawomir Milewski
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00136 • Publication Date (Web): 27 Feb 2018
Downloaded from http://pubs.acs.org on March 3, 2018
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Molecular umbrellas modulate the selective toxicity of
polyene macrolide antifungals
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Andrzej S. Skwarecki, Kornelia Skarbek, Dorota Martynow, Marcin
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Serocki, Irena Bylińska, Maria J. Milewska, Sławomir Milewski, *
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Department of Organic Chemistry, Gdańsk University of Technology, 11/12 G.
Narutowicza Str., 80ꢀ233 Gdańsk, Poland
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Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of
Technology, 11/12 G. Narutowicza Str., 80ꢀ233 Gdańsk, Poland
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Department of Biomedical Chemistry, University of Gdańsk, 63 Wita Stwosza Str.,
80ꢀ308 Gdańsk, Poland
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The first two coauthors (ASS and KS) participated equally to this work.
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ABSTRACT
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Antifungal polyene macrolide antibiotics Amphotericin B (AmB) and Nystatin (NYS)
were conjugated through the ωꢀamino acid linkers with diwalled “molecular
umbrellas” composed of spermidineꢀlinked deoxycholic or cholic acids. Presence of
“umbrella” substituents modulated biological properties of the antibiotics, especially
their selective toxicity. Some of the AmBꢀumbrella conjugates demonstrated
antifungal in vitro activity comparable to that of the mother antibiotic but diminished
mammalian toxicity, especially the hemolytic activity. In contrast, antifungal in vitro
activity of NYSꢀumbrella conjugates was strongly reduced and all these conjugates
demonstrated poorer than NYS selective toxicity. No correlation between the
aggregation state and haemolytic activity of the novel conjugates was found.
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INTRODUCTION
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Disseminated (invasive) fungal infections remain one of the major problems in
modern chemotherapy. The estimated number of cases is more than 2 million/year
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worldwide, with over 1.5 million deaths. Candida species are the most common
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fungal etiological agents of lifeꢀthreatening invasive infections in
immunocompromised hosts, transplant recipients and patients hospitalized in
intensive care units. These species are also the fourth most common cause of
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nosocomial (hospitalꢀacquired) bloodstream infections. The high mortality rate of
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invasive mycoses is due to the several factors, including shortcomings of diagnosing,
a limited number of effective antifungal chemotherapeutics and increasing fungal
resistance to available drugs. Polyene macrolide antibiotics constitute an important
group of antifungals, of which Amphotericin B, Nystatin and Pimaricin are the
approved drugs but only the first of them is used for the treatment of invasive fungal
infections. Nystatin (NYS) and Pimaricin are used exclusively for the topical
applications. Amphotericin B (AmB) is known as a “golden standard” of antifungal
chemotherapy, since it is fungicidal, demonstrates broad antifungal spectrum and
lack of fungal resistance. The only (but important) drawback is its substantial
mammalian toxicity, especially nephrotoxicity, which is a consequence of a
mechanism of biological action.
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Currently there are two major hypotheses on this mechanism. According to the
“barrelꢀstaveꢀpore” mechanism, AmB molecules form complexes with ergosterol in
the membrane and a few such complexes (4 – 12) selfꢀassemble to barrelꢀstaveꢀlike
transꢀmembrane pores, giving rise to leakage of low molecular weight cell
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components, including ions. In an alternative mechanism, called a “sterol sponge”
model, AmB extracts ergosterol from the hydrophobic interior of a fungal membrane
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and deposits it on the membrane’s outer leaflet as single complexes or a “pile” of
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complexes. In both mechanisms, binding of AmB to ergosterol is the prerequisite
for antifungal action but the antibiotic also binds to cholesterol in mammalian cell
membranes. This is only a slightly higher affinity to ergosterol than to cholesterol that
constitutes a molecular basis for selective toxicity of AmB. In consequence, a minimal
concentration at which AmB is toxic to mammalian cells is only 5 – 10 times higher
than the minimal fungicidal concentration. Mechanism of antifungal action of NYS is
much less known, although there is a little doubt that binding to ergosterol is also
crucial for the biological activity of this antibiotic. AmB, as other polyene macrolides,
is poorly soluble in aqueous solutions. Monomeric form of AmB exists in water at
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concentrations below 10 M. At higher concentrations, AmB undergoes complex
processes of selfꢀassociation, formation of dimers and soluble oligomers. Finally, at
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concentrations higher than 10 M insoluble aggregates are observed. It was shown
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that waterꢀsoluble aggregates of AmB are toxic to erythrocytes and fungal cells, while
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the monomers are toxic only to fungal cells.
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It is believed that selective toxicity of polyene macrolides, especially AmB, can be
improved by a proper chemical modifications of the antibiotic molecule concerning
the carboxyl functionality or the amino sugar substituent. A number of derivatives of
AmB were obtained but only some of them, especially those modified at the
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mycosamine residue, exhibited improved selective toxicity. Truly spectacular
effects, i.e. elimination of mammalian in vitro toxicity, with retention or at most
minimal decrease of antifungal activity, have been recently achieved for 2’ꢀ
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deoxyAmB, AmB urea derivatives and a conjugate of AmB with the diwalled
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molecular umbrella. Previously, it was shown that conjugates of AmB with tetraꢀ
and octawalled molecular umbrellas demonstrated low tendency to aggregate and
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negligible hemolytic activity. Herein, we present the results of our studies on
synthesis and biological activity of several AmB and NYS conjugates with diwalled
molecular umbrellas.
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RESULTS AND DISCUSSION
Synthesis of Conjugates. Twelve conjugates of molecular umbrella and polyene
macrolides were synthesized. Eight of them were derivatives of a deoxycholic acidꢀ
based diwalled molecular umbrella with covalently attached molecule of AmB (13aꢀd)
or NYS (14aꢀd). Four conjugates consisted of a cholic acidꢀbased diwalled molecular
umbrella and AmB (13eꢀf) or NYS (14eꢀf). The choice of diwalled (not tetraꢀ or
octawalled) umbrellas as the optimal components of conjugates was justified by the
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results of previous studies of Janout et al.
Molecular umbrella:polyene macrolide conjugates 13a-f and 14a-f were prepared
according to the multistep procedure developed by Janout and coꢀworkers for the
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synthesis of two umbrella:AmB conjugates, with a slight modification at the product
isolation step. Synthesis of molecular umbrellas with ωꢀamino acid linkers (Scheme
1) was based on an active ester approach and started with formation of nonꢀlabile ωꢀ
amino acid linkers, obtained from commercially available ωꢀamino acids 1aꢀd, amino
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groups of which were protected with Boc, using Boc O as an acylating agent. The Nꢀ
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protected derivatives 2aꢀd as crude products were applied in formation of Nꢀ
hydroxysuccinimide (NHS) active esters 3aꢀd, using the NHS/DCC method, followed
by purification by crystallization.
The starting materials in the synthesis of molecular umbrellas 6a and 6b were
commercially available deoxycholic 4a and cholic 4b acids, which were transformed
to corresponding NHS active esters 5aꢀb, using the NHS/DCC technique. Derivatives
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5a and 5b were subsequently condensed with spermidine by generation of amide
bonds, what resulted in formation of diwalled molecular umbrellas 6aꢀb.
Condensation of derivatives 6aꢀb with previously obtained NHS active esters 3aꢀd,
followed by deprotection of amino groups in acidic conditions lead to conjugates 8aꢀf,
i.e. molecular umbrellas carrying a nonꢀlabile ωꢀamino acid linker, necessary for
coupling of a polyene macrolide molecule.
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Scheme 1. Synthesis of molecular umbrellas with ωꢀamino acid linkers. Reagents
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2 3 2 2
and conditions. i) Na CO , H O, 0°C, Boc O, 1,4ꢀdioxane, 1 h, 0°Cꢀ>rt,
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24 h, HCl; ii) NHS, THF, 0°C, DCC, 0°Cꢀ>rt, 24 h; iii) NHS, THF, 0°C,
DCC, 0°Cꢀ>rt, 26 h; iv) spermidine, CHCl /DMF, rt, 48 h; v) CHCl /DMF,
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Et
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N, rt, 60 h; vi) MeOH/HCl, rt, overnight.
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The further steps are shown in Scheme 2. NꢀFmocꢀprotected AmB 9 and Nys 10
were converted into their 3,4ꢀdihydroꢀ4ꢀoxoꢀ1,2,3ꢀbenzotriazinꢀ3ꢀyl active esters 11
and 12, respectively, with Oꢀ(3,4ꢀdihydroꢀ4ꢀoxoꢀ1,2,3ꢀbenzotriazinꢀ3ꢀyl)ꢀN,N,N’,N’ꢀ
tetramethyluronium tetrafluoroborate (TDBTU) as a substrate. The esters obtained
(11 and 12) were used in subsequent reactions without further purification.
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Scheme 2. Conjugation of polyene macrolides with molecular umbrellas. Reagents
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and conditions. i) FmocꢀNHS; ii) DMF, diisopropylethylamine, TDBTU, rt,
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20 min; iii) DMF, Et N, rt, 5 h, piperidine, rt, overnight.
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Formation of molecular umbrellaꢀpolyene conjugates was accomplished by
condensation of active esters 11 and 12 with one of the molecular umbrellaꢀlinker
conjugates 8aꢀf, in the presence of triethylamine. Condensation was directly followed
by deprotection of the amino group of mycosamine with piperidine, affording products
13aꢀf and 14aꢀf which were purified by preparative thin layer chromatography.
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The thus obtained preparations of NYS conjugates 14a-f, contained tiny amounts
(less than 4% by HPLC analysis, exemplary analysis of 14c preparation in
Supporting Information) of respective isomeric forms (additional peak in HPLC, hardly
distinguishable by preparative TLC, identified by HRMS and NMR analysis). These
isomers were the products of the previously described intramolecular
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translactonization of NYS, resulting in formation of isoꢀnystatin (isoꢀNYS). For the
purpose of physicoꢀchemical and biological studies, each preparation was further
purified by semiꢀpreparative HPLC, so that all results presented below were obtained
for pure NYS conjugates, free of the isoꢀNYS derivatives.
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Antifungal and Hemolytic Activity of Conjugates. All the conjugates 13a-f and
14a-f were tested for antifungal in vitro activity against the model C. albicans SC
5314 strain. Assay was performed in RPMIꢀ1640 medium and values of MIC were
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determined. It should be noted, that formation of isoꢀNYS or its derivatives was not
observed (HPLC control) for NYS and its conjugates 14a-f under conditions of this
assay (incubation for 24 h at 37°C in RPMIꢀ1640 medium buffered to 7.0), although
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possibility of NYS isomerisation at pH 6.0 or 7.5 was previously suggested. The
same set of compounds was also tested for hemolytic activity against human
erythrocytes and the EH50 values were determined as a measure of this activity.
Results of these determinations are shown in Table 1.
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Table 1. Fungistatic and hemolytic activities of AmB, NYS and their conjugates with
molecular umbrellas and selective toxicity indexes. MIC values were
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determined in RPMIꢀ1640 medium against C. albicans SC 5314. EH50
values were determined against human erythrocytes. Selective toxicity
index is defined as EH50 [ꢀM]/MIC80 [ꢀM]. ND = not determined
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Compound
MIC80
AmB
13a
13b
13c
13d
13e
13f
NYS
14a
14b
14c
14d
14e
14f
0.3/
0.32
7.1/
3.8
5.5/
2.0
1.8/
0.9/
1.0/
0.6/
0.8/
0.86 9.62
18/
14/
33/
38/
45/
127/
ꢀ1
[ꢀg mL /ꢀM]
0.94 0.46 0.52 0.44
7.42 17.2 19.6 23.5 64.3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EH50
4.0/
4.3
8.5/
4.6
12/
6.4
15/
7.85
31/
16
42/
22
64/
32.5
41/
44.3
6.5/
3.5
5.0/
24/
37/
47/
ꢀ1
ND
ꢀ
[
ꢀg mL /ꢀM]
2.65 12.5 19.1 24.5
Selective
toxicity index
13.4
1.2
3.19
8.35 34.8 42.3 73.9
51.5 0.36
0.36 0.73 0.97 1.04
1
1
1
1
80
81
82
83
All conjugates exhibited lower anticandidal activity that their mother compounds, i.e.
AmB and NYS, respectively. In the case of AmB conjugates, the lowest difference
between their MIC80 values and that of AmB was found for compounds 13d, 13e and
13f. In terms of molar concentrations, MICs of these compounds were almost the
same as that of the mother antibiotic. Interestingly, for conjugates with deoxycholic
acidꢀbased molecular umbrellas (13aꢀ13d), the length of the ωꢀamino acid linker
determined the antifungal activity and the conjugates with short linkers, 13a (n = 1)
and 13 b (n = 2) demonstrated much lower activity that their counterparts with longer
linkers, 13c (n = 4) and 13 d (n = 6). In contrast, both conjugates with the cholic acidꢀ
based molecular umbrellas 13e (n = 2) and 13 f (n = 6) exhibited similar antifungal
activity. On the other hand, all the NYS conjugates were strongly less active that NYS
itself and MIC80 of the most active conjugate 14b (n = 2) was almost 9ꢀfold higher
than that of NYS. Paradoxically, the lowest activity was found for the conjugate 14f,
which is a counterpart of the most active AmB conjugate 13f. A qualitative difference
between the hemolytic activities of AmB and NYS conjugates was found. The former
were less hemolytic than AmB, while the EH50 values of the latter were lower than
that of NYS. Again, the length of the ωꢀamino acid linker determined the hemolytic
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1
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1
1
1
1
91
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activity of AmB conjugates with deoxycholic acidꢀbased molecular umbrellas but the
higher activity was found for the conjugates with shorter linkers. Notably, a similar
trend was observed by Yu and coꢀworkers for the AmB derivatives, conjugated with
“molecular semi umbrellas” composed of cholic acid and diamine linkers of variable
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01
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0
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0
16
length.
The selective toxicity indexes, defined as EH50/MIC80, of three AmB conjugates,
namely 13d, 13e and 13f, were better than that of AmB, while for most of the NYS
derivatives, the EH50 values were lower than MICs80, and a selective toxicity of all
NYS conjugates was poorer than that of the mother antibiotic.
206
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07
08
09
10
11
The anticandidal spectrum of compounds 13d, 13f and 14b was compared to that of
AmB and NYS. The MIC50 and MIC80 values of these compounds determined against
7 Candida spp. and S. cerevisiae are summarized in Table 2.
Table 2. Antifungal in vitro activities of AmB, NYS and conjugates 13d, 13f and 14b.
MIC80 and MIC50 values were determined in RPMIꢀ1640 medium.
212
213
ꢀ1
ꢀ1
MIC50 [ꢀg mL ]
MIC80 [ꢀg mL ]
AmB
0.3
0.3
0.4
0.3
0.5
0.6
0.4
0.2
13d
0.7
13f
0.25
0.4
1.3
1.3
1.5
1.7
24
NYS
14b
12
AmB
0.4
0.5
0.5
0.4
0.6
0.7
0.5
0.3
13d
13f
0.5
0.75
1.6
1.7
1.8
0.9
32
NYS
14b
15
C. albicans ATCC 10231
C. albicans B3
C. albicans B4
C. glabrata
0.7
1.0
1.2
0.45
0.9
1.1
0.8
0.7
0.8
0.9
1.0
1.1
0.9
0.8
1.0
1.3
1.4
0.7
0.8
13
15
2.3
10
3.6
15
2.7
16
3.6
22
C. krusei
3.0
17
3.75
1.2
31
C. parapsilosis
C. tropicalis
0.7
15
16
12.5
0.75
3.4
3.0
16
4.0
3.8
S. cerevisiae
0.6
0.95
0.8
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215
In general, the difference between MICs of the conjugates 13d and 13f and that of
AmB was higher for Candida spp. other than C. albicans, especially in the case of C.
tropicalis. Interestingly enough, MICs of these conjugates against the C. albicans B4
strain, multidrug resistant due to the overexpression of the MDR1 gene, were 2ꢀ4
times higher than those against nonꢀresistant C. albicans B3, while in the case of the
NYS conjugate 14b, there was no difference in susceptibilities of B3 and B4 strains.
2
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0
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0
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0
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4
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0
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26
Mammalian cytotoxicity of conjugates. Selected conjugates 13b, 13d, 13f and
14b were evaluated for cytotoxicity against three mammalian cell lines: pig kidney
epithelial cells LLCꢀPK1, human embryonic kidney cells HEKꢀ293T and human
hepatocellular carcinoma cells HepG2. Values of IC , i.e. concentrations at which
50
50% inhibition of control growth was observed are summarized in Table 3. All values
are means of at least two independent experiments, each done in duplicate.
227
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2
2
29
30
Table 3. Cytotoxicity of AmB, NYS and their conjugates 13b, 13d, 13f and 14b
toward three mammalian cell lines in tissue culture.
ꢀ
1
IC50 ±SD [ꢁg mL ]
Compound
HEKꢀ293T cells LLCꢀPK1 cells Hep G2 cells
0.488 ±0.101 2.688 ±0.950 0.565 ±0.111
AmB
1
3b
13d
3f
NYS
4b
16.708 ±3.571 8.471 ±1.204
>50
5.565 ±0.253 6.454 ±0.992 25.209 ±2.313
17.742 ±2.642 15.411 ±1.018 33.071 ±2.907
6.173 ±0.602 11.652 ±2.332 7.864 ±0.540
2.176 ±0.122 2.505 ±0.397 16.102 ±4.089
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All the AmB conjugates were less cytotoxic than AmB. The difference between the
232
IC values of conjugates 13b, 13d and 13f and that of AmB was especially large in
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the case of the Hep G2 cells and the lowest for the LLCꢀPK1 cells. On the other
hand, the NYS conjugate 14b was less cytotoxic than NYS only against the Hep G2
cells.
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Aggregation of Conjugates in Solutions. AmB and NYS undergo aggregation in
aqueous solutions in a concentrationꢀdependent manner. AmB in a monomeric state
exhibits a characteristic absorption band at 409 nm, molar absorptivity of which
decreases upon aggregation. For the solutions containing both monomeric and
238
2
2
2
39
40
41
aggregated forms, ε = ε
p
+ (ε
m
ꢀ ε
P
)m/T, where T and m represent the total and
and ε stand for
2
42
monomeric concentration of the antibiotic or its conjugate and ε, ε
m
p
243
the apparent molar absorptivity, molar absorptivity of the monomeric component and
molar absorptivity of the micellar component, respectively. By measuring the
apparent molar absorptivity as a function of the reciprocal of AmB or its conjugate
concentration, one may estimate cac (critical aggregation concentration) from the
2
2
2
2
44
45
46
47
17
intercept of two straight lines. Such plots obtained by us for compounds 13d and
13f are shown in Figure 1. The cac values for AmB and the conjugates were as
follows: AmB – 1 ꢀM; 13a – 1.5 ꢀM; 13b – 0.2 ꢀM; 13d – 3.2 ꢀM; 13f – 3.8 ꢀM.
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51
Figure 1. Plots of molar absorptivity (λmax = 409 nm) as a function of the reciprocal
concentration of 13d (A) and 13f (B).
2
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2
52
53
54
Application of the same methodology for studies on aggregation of NYS and its
conjugates was not possible, since the concentrationꢀdependent decrease of
molecular absorptivity at 409 nm is not observed. On the other hand, this antibiotic
demonstrates fluorescence at λ = 410 nm upon excitation at λ = 320 and changes in
an emission spectrum may be monitored to follow the aggregation state of NYS and
255
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2
57
58
59
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18
its derivatives in solution. In his work, the fluorescence intensity and steadyꢀstate
anisotropy were measured as a function of antibiotic/conjugate concentration in
buffered solution. As shown in Figure 2, at low concentrations of NYS, the anisotropy
increased with the concentration of antibiotic in solution; however, above ∼ 2 ꢀM
antibiotic, the anisotropy reached a maximum of 0.25 ± 0.03 and became
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63
64
65
66
concentrationꢀindependent. At this antibiotic concentration, there was also a sharp
increase in the fluorescence intensity of NYS. These observations are consistent with
19
an aggregation of the antibiotic in aqueous solution. The cac value of NYS
determined in this way was 2 ꢀM, for the conjugate 14a cac = 5.5 ꢀM and for
conjugates 14b and 14d cac = 4 ꢀM.
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Figure 2. Dependence of the steadyꢀstate fluorescence anisotropy <r> on
concentration of NYS, 14a, 14b and 14d.
2
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71
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73
These results clearly indicate that the cac values of AmB, NYS and their conjugates
with molecular umbrellas were similar, inside the 0.2 – 5.5 ꢁM range, so that they do
not seem to determine the hemolytic activity of these compounds.
274
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76
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79
It should be noted however, that although the conjugate 13f demonstrated in our
hands the most favourable biological properties among all the AmB conjugates
tested, parameters characterizing its mammalian cytotoxicity were markedly different
from that reported by Janout and coꢀworkers, who found for this compound EH50
=
1
2
280
375 ꢀM toward sheep erythrocytes and little if any toxicity toward HEK293 T cells.
To determine whether the inhibition of C. albicans growth by was related to
the amount of binding of NYS and AmB conjugates to yeast cells, a
2
2
2
2
81
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83
84
binding assay was performed. Cell suspensions of different cell density were
incubated with AmB, NYS or any of the conjugates at 30 ꢀM, cells were spun down
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and concentration of an antifungal agent remaining in supernatant was measured by
Uvꢀvis spectrophotometry at 409 nm. Data presented in Figures 3AB clearly show
that all compounds tested were bound by C. albicans cells and percent of compound
bound was cell density dependent. No binding was observed for E. coli cells (less
than 2% binding at cell density corresponding to A660 = 1.0) lacking sterols in the
plasma membrane, thus indicating that binding to C. albicans cells was due to the
interaction with ergosterol. Taking percent of NYS and its conjugates bound at cell
density corresponding to A660 = 1.0 as a semiꢀquantitative measure of their affinity to
the ergosterolꢀcontaining yeast cells, some correlation of these values with antifungal
activity expressed as MIC80 was found. This correlation, shown in Figure 4, although
not very good (r = 0.87), confirms dependence of antifungal in vitro activity of NYS
conjugates on their binding affinity to yeast cells. The respective graph for AmB and
its conjugates is not shown, because both the MIC80 values and binding affinities of
all these compounds were very similar, so that any possible correlation is not
especially informative.
3
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93
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97
298
299
3
3
3
00
01
02
Figure 3. Binding of polyene macrolide antibiotics and their conjugates to C. albicans
cells. Cell suspensions of various cell density were incubated with a given compound
(30 ꢀM) for 1 h and concentration of the unbound compound was determined by UVꢀ
vis in supernatant left after centrifugation. A. AmB and its conjugates. B. NYS and its
conjugates. Data are the means of three independent determinations ±SD.
303
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06
Figure 4. Correlation between cell binding affinity and antifungal activity of NYS and
its conjugates. Percent of compound bound to C. albicans cells at A660 = 1.0 was
plotted against log MIC80 values. Data are the means of three independent
determinations ±SD.
307
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Conclusions. Some previous reports showed that attachment of bulky substituents,
like tetrawalled and octawalled molecular umbrellas or poly(ethylene glycol) chains
to AmB, resulted in a significant increase of the cac values of the conjugates, in
3
3
3
3
3
13
14
15
16
17
comparison to that of AmB alone, and in consequence, in an adequate increase of
13,17
concentrations at which they exhibited a hemolytic activity.
In our hands, although
the cac values of AmB conjugates demonstrating much lower than AmB hemolytic
activity were slightly higher than that of the antibiotic, they were still well below the
EH50 values, thus confirming a previous observation of Janout and coꢀworkers, who
found that the AmB conjugate with the cholic acidꢀbased molecular umbrella
containing an 8ꢀaminooctanoic acid linker (compound 13f in our study) was much
less haemolytic than AmB, despite the fact that its cac value was almost the same as
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23
12
that of AmB. A similar phenomenon was recently noted by Yu et al. for AmB
16
conjugates with cholic acid. It seems therefore, that the well confirmed correlation
324
between the aggregation state and haemolytic activity of AmB does not have to be
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valid for all kinds of AmB derivatives. Nevertheless, there is no doubt that conjugation
of AmB with diwalled molecular umbrellas affords conjugates demonstrating better
selective toxicity that the mother antibiotic. On the other hand, application of the
same strategy for the construction of NYS conjugates did not result in a similar effect,
since the obtained conjugates demonstrated much lower antifungal activity and
higher mammalian cytotoxicity than NYS. This observation is another evidence that
strategies of AmB chemical modification aimed at improvement of selective toxicity of
this antibiotic cannot be directly transferred to NYS, despite the apparent structural
analogy between these antibiotics. This was previously demonstrated for NYS
derivatives containing bulky substituents at the amino group of mycosamine, the
selective toxicity of which were much poorer than those of their AmBꢀderived
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20
counterparts.
Conjugation of AmB with a diwalled molecular umbrella or a “semi umbrella”, i.e. a
facially amphiphilic sterol, resulting in increasing the cellular selectivity of this
antibiotic, due to the dramatic reduction in hemolytic activity and significant retention
338
339
12,16
3
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3
40
41
42
43
of antifungal activity, is known as a “taming strategy”.
It was postulated that this
strategy could be applied also for other classes of membraneꢀdisrupting agents.
Results described in this work provide evidence that application of the taming
strategy for NYS, structurally similar to AmB, results in substantial decreasing,
instead of increasing, of cellular selectivity, due to the enhancement of hemolytic
activity and substantial diminishment of antifungal activity of conjugates in
comparison with those of the mother antibiotics. It seems that the latter might be due
to the decreased affinity of NYS conjugates to ergosterol, since some correlation
between their antifungal activity and binding affinity to yeast cells containing
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ergosterol in plasma membranes was found. One may thus conclude that the general
applicability of the taming strategy may be somewhat limited.
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55
MATERIALS AND METHODS
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Chemistry. H NMR spectra were recorded on Unity 500 plus Varian spectrometer at
500 MHz and on Bruker Advance III HD 400 MHz at 400 MHz. HRMSꢀESI spectra
were recorded on Aqilent Technologies 6540 UHD Accurate – Mass QꢀTOF LC/MS
apparatus. Purification of reaction products was carried out by liquid preparative
3
56
®
357
column chromatography on silica gel Geduran Si 60 (40ꢀ63 ꢀm) and preparative
3
3
3
58
59
60
TLC silica gel 60 F250, 1 mm. Separation of NYS and isoꢀNYS conjugates was
performed by semiꢀpreparative HPLC on Agilent 1200 Series apparatus, Zorbax SBꢀ
2
C18 (5 ꢀm, 9.4 × 250 mm) column, isocratic elution with acetonitrile/H 0, 50:50 v/v
361
solvent system. HPLC analysis was performed with Agilent 1290 Infinity system,
3
3
3
3
3
62
63
64
65
66
Poroshell 120 ECꢀC18 (2.7 ꢂm, 4.6 ×150mm) column, isocratic elution with
acetonitrile/ H 0, 10:90 v/v solvent system, containing 0.1% formic acid. Mass
2
detection with 6540 UHD QꢀTOF spectrometer using positive ESI ionization mode.
The melting points are uncorrected. Commercial grade reagents and solvents were
used without further purification.
367
3
3
3
3
3
68
69
70
71
72
NHS Esters of Boc-ω-amino acids – General Procedure. ωꢀAmino acid (34 mmol)
and Na CO (51 mmol) were dissolved in water (20 mL) and the resulting solution
2
3
was cooled to 0°C in an ice bath. diꢀtertꢀButyl dicarbonate (34 mmol) dissolved in 50
mL of 1,4ꢀdioxane was added dropwise. The reaction mixture was stirred at 0°C for 1
hour and next allowed to warm to room temperature. After 24 hours, dioxane was
evaporated under reduced pressure, the aqueous solution was washed with AcOEt
373
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and subsequently acidified with 1M HCl to pH 1ꢀ2. The mixture was then extracted
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
375
with AcOEt and an organic layer was dried over anhydrous MgSO . After removal of
4
3
3
3
76
77
78
the desiccant and concentration under reduced pressure, the crude Bocꢀωꢀamino
acid (oil) was used in the next step without further purification. The equimolar
amounts of Bocꢀωꢀamino acid Nꢀhydroxysuccinimide (NHS) were dissolved in dry
THF. The solution was cooled in an ice bath and a dicyclohexylcarbodiimide (DCC)
solution in dry THF (1.2 × molar excess in respect to Bocꢀωꢀamino acid) was added
dropwise, while the mixture was stirred vigorously. The mixture was allowed to warm
to room temperature and then stirred for additional 24 hours. The dicyclohexylurea
(DCU) formed was filtered off and the filtrate was concentrated under reduced
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
379
3
3
3
3
80
81
82
83
384
385
pressure. The residue was dissolved in CHCl and washed consecutively with water,
3
a saturated solution of NaHCO
3
, and water. The organic layer was then dried over
3
3
3
3
3
86
87
88
89
90
anhydrous MgSO and concentrated under reduced pressure. The residue was
4
crystallized from the AcOEt/hexane mixture.
N-succinimidyl N-Boc-3-aminopropanoate 3a. Starting from 3.05 g (34 mmol) of 3ꢀ
aminopropanoic acid 1a, 6.30 g (22 mmol, 66%) of the 3a product was obtained as a
21
1
f
white solid (R 0.46, hexane/AcOEt, 1/1, v/v); m.p. 95ꢀ97°C (lit. 100°C). H NMR
391
(500 MHz, CDCl ) δ: 5.14 (s, 1H), 3.53 (m, 2H), 2.86 (m, 6H), 1.45 (s, 9H).
3
3
3
3
92
93
94
N-succinimidyl N-Boc-4-aminobutanoate 3b. Starting from 2.89 g (28 mmol) of 4ꢀ
aminobutanoic acid 1b, 5.40 g (18 mmol, 64%) of the 3b product was obtained as a
22
1
white solid (R
f
0.60, hexane/AcOEt, 1/1, v/v); m.p. 112ꢀ115°C (lit. 110°C). H NMR
395
396
(500 MHz, CDCl
3
) δ: 4.77 (s, 1H), 3.24 (m, 2H), 2.85 (s, 4H), 2.67 (t, J=7.2 Hz, 2H),
1.95 (m, 2H), 1.45 (s, 9H).
3
3
97
98
N-succinimidyl N-Boc-6-aminohexanoate 3c. Starting from 3.02 g (23 mmol) of 6ꢀ
aminohexanoic acid 1c, 4.93 g (15 mmol, 65%) of the 3c product was obtained as a
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2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
99
white solid (R
f
0.40, hexane/AcOEt, 1/1, v/v); m.p. 82ꢀ86°C. HRMSꢀESI found m/z
+.
1
400
24 2 6 3
329.1602 [M+1] calcd for C15H N O 328.1634. H NMR (500 MHz, CDCl ) δ: 4.60
4
4
4
4
01
02
03
04
(s, 1H), 3.12 (m, 2H), 2.84 (m, 4H), 2.60 (t, J=7.4 Hz, 2H), 1.77 (qv, J=7.5 Hz, 2H),
1.51 (m, 2H), 1.45 (m, 1H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
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3
4
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6
7
8
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0
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2
3
4
5
6
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8
9
0
N-succinimidyl N-Boc-8-aminooctanoate 3d. Starting from 716 mg (4.5 mmol) of 8ꢀ
aminooctanoic acid 1d, 1.03 g (2.88 mmol, 64%) of the 3d product was obtained as a
405
white solid (R 0.58, hexane/AcOEt, 1/1, v/v); m.p. 96ꢀ99°C. HRMSꢀESI found m/z
f
+.
1
4
4
4
4
06
07
08
09
28 2 6 3
357.1972 [M+1] calcd for C17H N O 356.1947. H NMR (500 MHz, CDCl ) δ: 4.52
(s, 1H), 3.12 (t, J=6.5 Hz, 2H), 2.85 (s, 4H), 2.61 (t, J=7.1 Hz, 2H), 1.75 (m, 2H),
1.55ꢀ1.25 (m, 17H).
23
Diwalled Molecular Umbrellas – Modified General Procedure. The solution
made of a bile acid (13.49 mmol) and NHS (13.49 mmol) in dry THF was cooled to
0°C in an ice bath and then a DCC solution in dry THF (16.60 mmol) was added
dropwise. Subsequently, the reaction mixture was allowed to warm to room
temperature and stirred for additional 36 h. The DCU formed was filtered off and the
filtrate was concentrated under reduced pressure. The residue was dissolved in
410
411
4
4
4
4
12
13
14
15
CHCl
3
and washed consecutively with water, a saturated solution of NaHCO
3
, and
416
417
water. The organic layer was then dried over anhydrous MgSO
4
and concentrated
under reduced pressure. After removal of the desiccant and concentration under
reduced pressure, the residue was crystallized from AcOEt. The product obtained
was used without characterization in the next step. The NHS active ester (8.1 mmol)
4
4
4
4
4
18
19
20
21
22
and spermidine (4.05 mmol) were dissolved in CHCl
DMF. The reaction mixture was stirred at room temperature for 48 hrs and then
washed consecutively with water, a saturated solution of NaHCO , and water. The
organic layer was then dried over anhydrous MgSO , the desiccant was filtered off
3
with addition of small amount of
3
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and the filtrate was concentrated under reduced pressure. The residue was purified
by column chromatography on silica gel eluted with the CHCl /MeOH/NH OH
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
425
3
4
4
4
4
4
26
27
28
29
mixture.
1
3
N ,N - bis-deoxycholylspermidine 6a. Starting from 5.30 g (13.49 mmol) of
deoxycholic acid 4a, 5.17 g (5.8 mmol, 43%) of the 6a product was obtained as a
0
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2
3
4
5
6
7
8
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2
3
4
5
6
7
8
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5
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8
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2
3
4
5
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7
8
9
0
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2
3
4
5
6
7
8
9
0
white solid (R
f
0.31, CHCl
3
/MeOH/NH
4
OH, 4/1.5/0.1, v/v/v). HRMSꢀESI found m/z
+
.
1
430
894.7290 [M+H] calcd. for C55
H
95
N
3
O
6
893.7221. H NMR (500 MHz, CD
3
OD) δ:
4
4
4
4
4
31
32
33
34
35
3.95 (s, 2H), 3.52 (m, 2H), 3.23 (t, J=6.5 Hz, 2H), 3.18 (t, J=6.4 Hz, 2H), 2.70 (t,
J=7,1 Hz, 4H), 2.24 (m, 2H), 2.11 (m, 2H), 1.96ꢀ1.68 (m, 16H), 1.68ꢀ1.22 (m, 32H),
1.22ꢀ1.07 (m, 4H), 1.07ꢀ0.87 (m, 14H), 0.71 (s, 6H).
1
3
N ,N - bis-cholylspermidine 6b. Starting from 7.21 g (17.65 mmol) of cholic acid 4b,
5.48 g (6.0 mmol, 34%) of the 6b product was obtained as a white solid (R 0.50,
f
+
.
436
CHCl
3
/MeOH/NH
4
OH, 6/2/0.5, v/v/v). HRMSꢀESI found m/z 926.7199 [M+H] calcd.
1
4
4
4
4
37
38
39
40
for C H N O 925.7119. H NMR (500 MHz, CD OD) δ: 3.96 (s, 2H), 3.79 (s, 2H),
55
95
3
8
3
3.37 (m, 2H), 3.25 (t, J=6.6 Hz, 2H), 3.19 (t, J=6.6 Hz, 2H), 2.74 (t, J=7.1 Hz, 4H),
2.11 (m, 2H), 2.05ꢀ1.69 (m, 16H), 1.69ꢀ1.48 (m, 16H), 1.48ꢀ1.20 (m, 10H), 1.11 (m,
2H), 1.05ꢀ0.95 (m, 8H), 0.92 (s, 6H), 0.71 (s, 6H).
4
4
41
42
Diwalled Molecular Umbrella/ω-amino acid Conjugates – General Procedure. A
diwalled molecular umbrella (1.11 mmol) and a Bocꢀωꢀamino acid NHS ester (1.33
4
4
4
43
44
45
mmol) were dissolved in CHCl
Et N (1.11 mmol) was added. The reaction mixture was stirred for 60 hrs at room
temperature. Subsequently, the solvents were evaporated and the residue was
purified by column chromatography on silica gel with the CHCl /MeOH/H O mixture.
3
containing a small amount of dry DMF and then dry
3
446
4
4
47
48
3
2
The conjugates thus obtained were deꢀprotected by dissolving in anhydrous MeOH
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1
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1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
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5
5
5
5
5
5
5
6
4
49
saturated with gaseous HCl, stirring overnight and subsequent evaporation of
solvents. The crude products were used in the next steps.
450
1
3
2
4
4
4
4
51
52
53
54
N ,N -bis-deoxycholyl-N -(Boc-3-aminopropanoyl)spermidine 7a. Starting from 1 g
(1.11 mmol) of molecular umbrella 6a and 381 mg (1.33 mmol) of NHS ester of Bocꢀ
3ꢀaminopropionic acid 3a, 1.05 g (1 mmol, 90%) of product 7a was obtained as white
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
solid (R
f
0.44, CHCl
3
/MeOH/H
2
O, 65/10/1, v/v/v); mp 70ꢀ72°C; HRMSꢀESI found m/z
+
.
1
455
1065.8187 [M+H] calcd. for C63
H
108
N
4
O
9
1064.8116. H NMR (500 MHz, CD
3
OD) δ:
4
4
4
4
4
56
57
58
59
60
8.00 (m, 2H), 3.96 (s, 2H), 3.52 (m, 2H), 3.34 (m, 6H), 3.18 (m, 4H), 2.56 (m, 2H),
2.26 (m, 2H), 2.12 (m, 2H), 1.96ꢀ1.69 (m, 16H), 1.67ꢀ1.24 (m, 41H), 1.23ꢀ0.86 (m,
18H), 0.70 (s, 6H).
1
3
2
N ,N -bis-deoxycholyl-N -(Boc-4-aminobutanoyl)spermidine 7b. Starting from 1 g
(1.12 mmol) of molecular umbrella 6a and 402 mg (1.34 mmol) of NHS ester of Bocꢀ
4ꢀaminobutanoic acid 3b, 1.00 g (0.93 mmol, 83%) of product 7b was obtained as
461
462
white solid (R
f
0.55, CHCl
3
/MeOH/H
2
O, 60/10/1, v/v/v); mp 105ꢀ110°C; HRMSꢀESI
+
.
1
4
4
4
63
64
65
found m/z 1079.8343 [M+H] calcd. for C64
H
110
N
4
O
9
1078.8273. H NMR (500 MHz,
CD
3
OD) δ: 3.97 (s, 2H), 3.54 (m, 2H), 3.35 (m, 4H), 3.19 (m, 4H), 3.09 (m, 2H), 2.39
(m, 2H), 2.25 (m, 2H), 2.13 (m, 2H), 1.96ꢀ1.69 (m, 18H), 1.67ꢀ1.24 (m, 41H), 1.22ꢀ
0.85 (m, 18H), 0.7 (s, 6H).
466
467
1
3
2
N ,N -bis-deoxycholyl-N -(Boc-6-aminohexanoyl)spermidine 7c. Starting from 1g
(1.10 mmol) of molecular umbrella 6a and 433 mg (1.32 mmol) of NHS ester of Bocꢀ
6ꢀaminohexanoic acid 3c, 0.73 g (0.66 mmol, 60%) of product 7c was obtained as
4
4
4
4
68
69
70
71
white solid (R
f
0.40, CHCl
3
/MeOH/H
2
O, 65/10/1, v/v/v); mp 125°C; HRMSꢀESI found
+
.
1
m/z 1107.8666 [M+H] calcd. for C66
H
114
N
4
O
9
1106.8586. H NMR (500 MHz,
472
CD OD) δ: 8.00 (m, 2H), 3.95 (s, 2H), 3.52 (m, 2H), 3.35 (m, 4H), 3.20 (m, 4H), 3.05
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(q, J=6.6 Hz, 2H), 2.37 (m, 2H), 2.26 (m, 2H), 2.11 (m, 2H), 1.96ꢀ1.69 (m, 18H), 1.67ꢀ
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
474
1.23 (m, 45H), 1.23ꢀ1.04 (m, 4H), 1.02 (m, 6H), 0.94 (s, 8H), 0.71 (s, 6H).
4
4
4
4
4
75
76
77
78
79
1
3
2
N ,N -bis-deoxycholyl-N -(Boc-8-aminooctanoyl)spermidine 7d. Starting from 1.25 g
(1.40 mmol) of molecular umbrella 6a and 599 mg (1.68 mmol) of NHS Bocꢀ8ꢀ
aminooctanoic acid 3d, 1.40 g (1.25 mmol, 89%) of product 7d was obtained as oil
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
.
(R 0.62, CHCl /MeOH/H O, 65/10/1, v/v/v); HRMSꢀESI found m/z 1135.8901 [M+H]
f
3
2
1
480
118 4 9 3
calcd. for C68H N O 1134.8899. H NMR (500 MHz, CD OD) δ: 3.96 (s, 2H), 3.52
4
4
4
4
81
82
83
84
(m, 2H), 3.35 (m, 4H), 3.17 (m, 4H), 3.01 (m, 2H), 2.35 (m, 2H), 2.25 (m, 2H), 2.11
(m, 2H), 1.96ꢀ1.69 (m, 18H), 1.67ꢀ1.23 (m, 49H), 1.23ꢀ0.89 (m, 18H), 0.7 (s, 6H).
1
3
2
N ,N -bis-cholyl-N -(Boc-4-aminobutanoyl)spermidine 7e. Starting from 750 mg (0.81
mmol) of molecular umbrella 6b and 291 mg (0.97 mmol) of NHS ester of Bocꢀ4ꢀ
aminobutanoic acid 3b, 604 mg (0.54 mmol, 67%) of product 7e was obtained as oil
485
486
+
.
(R
f
0.49, CHCl
3
/MeOH/H
2
O, 65/10/1, v/v/v); HRMSꢀESI found m/z 1111.8180 [M+H]
1
4
4
4
87
88
89
calcd. for C64
H
110
N
4
O
11 1110.8171. H NMR (500 MHz, CD
3
OD) δ: 3.96 (s, 2H), 3.80
(s, 2H), 3.35 (m, 6H), 3.20 (m, 4H), 3.09 (m, 2H), 2.40 (m, 2H), 2.27 (m, 6H), 2.12 (m,
2H), 2.06ꢀ1.70 (m, 18H), 1.70ꢀ1.22 (m, 35H), 1.21ꢀ0.87 (m, 16H), 0.71 (s, 6H).
1
3
2
490
491
N ,N -bis-cholyl-N -(Boc-8-aminooctanoyl)spermidine 7f. Starting from 2.49 g (2.69
mmol) of molecular umbrella 6b and 1.15 g (3.23 mmol) of NHS ester of Bocꢀ8ꢀ
4
4
4
4
92
93
94
95
aminooctanoic acid 3d, 1.82 g (1.56 mmol, 58%) of product 7f was obtained as oil (R
f
+.
0.40, CHCl /MeOH/H O, 65/10/1, v/v/v); HRMSꢀESI found m/z 1167.8879 [M+H]
3
2
1
calcd. for C68
H
118
N
4
O
11 1166.8797. H NMR (500 MHz, CD
3
OD) δ: 3.96 (s, 2H), 3.80
(s, 2H), 3.35 (m, 6H), 3.17 (m, 4H), 3.03 (m, 2H), 2.35 (m, 2H), 2.27 (m, 6H), 2.13 (m,
2H), 2.06ꢀ1.69 (m, 18H), 1.69ꢀ1.21 (m, 43H), 1.21ꢀ0.86 (m, 16H), 0.72 (s, 6H).
496
97
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1
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2
2
2
2
2
2
2
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3
3
3
3
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3
3
3
3
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4
4
4
4
4
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5
5
5
5
5
5
5
6
4
98
N
-Fmoc-polyene macrolide – General Procedure. A polyene macrolide (0.08
499
mmol) and FmocꢀNHS (0.2 mmol) were dissolved in 6 mL of DMF/MeOH (2:1)
mixture and subsequently 56 ꢂL of pyridine was added. The reaction mixture was the
stirred for 12 hours at room temperature. In the next step, the reaction mixture was
cooled to 0°C and subsequently, 140 mL of diethyl ether was added dropwise. The
precipitated yellow solid was filtered off under reduced pressure and dried under
vacuum for 1 hour.
5
5
5
5
5
00
01
02
03
04
0
1
2
3
4
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505
5
5
5
5
5
06
07
08
09
10
N-Fmoc-AmB 9. Starting from 100 mg (0.09 mmol) of amphotericin B and 56 mg
(0.16 mmol) of FmocꢀNHS, 118 mg (0.1 mmol, 95%) of the 9 product was obtained
as a yellow solid (R
f
0.45, CHCl
3
/MeOH/H
2
O, 4/1/0.1, v/v/v). HRMSꢀESI found m/z
+
.
1
1146.5641 [M+H] calcd. for C H NO 1145.5559. H NMR (500 MHz, pyridineꢀd )
62
83
19
5
δ: 7.9 (d, J=7.6Hz, 2H), 7.7 (m, 2H), 7.4 (m, 2H), 7.2 (m, 2H), 6.8 (m, 2H), 6.7ꢀ6.3 (m,
12H), 5.9 (m, 1H), 5.7 (m,1H) 5.5 (m, 1H), 5.0ꢀ5.4 (m, 4H), 4.9 (t, 1H), 4.8 (m,2H),
4.5ꢀ4.0 (m, 4H), 3.6ꢀ3.7 (m, 2H), 3.0 (m, 1H),2.5ꢀ2.7 (m,4H), 1.8ꢀ2.3 (m, 9H), 1.6 (m,
3H), 1.5 (m, 3H), 1.3 (m, 3H), 1.2 (m, 2H).
511
512
5
5
5
5
13
14
15
16
N-Fmoc-Nys 10. Starting from 100 mg (0.08 mmol) of nystatin and 70 mg (0.2 mmol)
of FmocꢀNHS, 112 mg (0.1 mmol, 90%) of the 10 product was obtained as a yellow
solid (R
f
0.45, CHCl
3
/MeOH/H
2
O, 4/1/0.1, v/v/v). HRMSꢀESI found m/z 1148.5803
+
.
1
517
518
[M+H] calcd. for C62
H
85NO19 1147.5716. H NMR (500 MHz, pyridineꢀd
5
) δ: 8.0 (s,
1H), 7.8 (d, J=7.6Hz 2H), 7.6 (m, 2H), 7.4 (m, 2H), 7.2 (m, 2H), 6.6 (m, 2H), 6.0ꢀ6.4
(m, 12H), 5.6ꢀ5.9 (m, 4H), 5.3 (m, 2H), 5.1 (m, 3H), 5.0 (s, 1H), 4.9 (bd, 1H), 4.8
(m,1H), 4.5ꢀ4.6 (m, 4H), 4.4 (m, 1H), 4.3 (m, 1H), 4,2 (m, 1H), 3.8 (m, 1H), 3.7 (m,
1H), 3.6 (m, 1H), 3.5 (m, 1H),3.4 (s, 1H), 1.8ꢀ2.8 (m, 26H), 1.6 (m, 3H), 1.4 (m, 3H),
1.3 (m, 3H), 1.1 (m, 4H).
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524
TDBTU ester of N-Fmoc-polyene macrolide – general procedure. To the solution of
Fmocꢀpolyene macrolide (0.18 mmol) in dry DMF (5 mL), 0.29 mmol of
diisopropylethylamine was added, followed by 0.18 mmol of TDBTU. The reaction
mixture was stirred for 20 minutes at room temperature. Subsequently, the crude
5
5
5
5
5
25
26
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28
29
0
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3
4
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2
3
4
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6
7
8
9
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2
3
4
5
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7
8
9
0
product was precipitated with 200 mL of Et
purification in the next step.
2
O, filtered off and used without
530
5
5
5
5
5
31
32
33
34
35
O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N-Fmoc-amphotericin B 11. Starting
from 206 mg (0.18 mmol) of FmocꢀAmB 9 and 63 mg (0.18 mmol) of TDBTU, 220 mg
f
(0.17 mmol, 93%) of TDBTU active ester 11 was obtained as a solid (R 0.48,
ꢀ
.
CHCl /MeOH/H O, 65/10/1, v/v/v). HRMSꢀESI found m/z 1289.5742 [Mꢀ1] calcd. for
3
2
1
69 86 4 3
C H N O20 1290.5835. H NMR (500 MHz, CDCl ) δ: 8.40 (d, J=7.5Hz, 1H), 8.00
536
(d, J=6.8Hz, 1H), 7.80 (t, J=7.9Hz 1H), 7.60 (m, 3H), 7.50 (m, 1H), 7.40 (bt, 2H),
7.00ꢀ7.30 (m, 3H), 5.90ꢀ6.40 (m, 14H), 5.30 (bd, 1H) 5.20 (m, 1H), 4.10ꢀ4.40 (m, 4H),
3.00ꢀ3.90 (m, 10H), 1.40ꢀ2.60 (m, 18H), 1.20ꢀ2.40 (m, 17H), 1.10 (d, J=6.4Hz, 3H),
1.00 (d, J=6.4Hz, 3H), 0.90 (d, J=8.3Hz 3H).
5
5
5
5
37
38
39
40
O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N-Fmoc-nystatin A 12. Starting from
199 mg (0.173 mmol) of FmocꢀNys 10 and 60 mg (0.173 mmol) of TDBTU, 146 mg
541
542
f
(0.113 mmol, 65%) of TDBTU active ester 12 was obtained as a solid (R 0.46,
ꢀ
.
5
5
5
5
43
44
45
46
CHCl
3
/MeOH/H
2
O, 65/10/1, v/v/v). HRMSꢀESI found m/z 1291.6053 [MꢀH] calcd. for
1
C
69
H
88
N
4
O
20 1292.5992. H NMR (500 MHz, CDCl
3
) δ: 8.38 (d, J=7.6 Hz, 1H), 8.15
(d, J= 8.2 Hz, 1H), 7.89 (dd, J=9.8, 4.9 Hz, 1H), 7.84ꢀ7.68 (m, 3H), 7.59ꢀ7.45 (m, 1H),
7.45ꢀ7.20 (m, 4H), 6.57ꢀ6.01 (m, 12H), 5.51ꢀ5.26 (m, 2H), 5.34 (dd, J=14.5 and 10.3
Hz, 1H), 4.44 (d, J=9.5 Hz, 1H), 4.23ꢀ4.09 (m, 2H), 4.07 (d, J= 6.8 Hz, 1H), 3.79ꢀ3.60
547
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5
48
(m, 3H), 3.53ꢀ3.40 (m, 1H), 3.19 (dt, J= 14.9 and 7.5 Hz, 2H), 3.01 (s, 2H), 2.88 (d,
J= 0.6 Hz, 2H), 2.77ꢀ2.56 (m, 2H), 2.51ꢀ2.11 (m, 4H), 1.93 (dd, J= 13.4 and 9.4 Hz,
1H), 1.90ꢀ1.45 (m, 6H), 1.46ꢀ1.24 (m, 10H), 1.20 (d, J= 6.4 Hz, 3H), 1.12 (d, J= 6.4
Hz, 3H), 1.03 (t, J= 8.3 Hz, 3H).
549
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50
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52
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Diwalled molecular umbrella/ω-amino acid/polyene conjugates – General
Procedure. To a solution of TDBTU ester of Fmocꢀpolyene (40.8 ꢁmol) in dry DMF,
39.7 ꢁmol of a molecular umbrella/ωꢀamino acid conjugate in dry 2 ml DMF
554
5
5
5
5
55
56
57
58
3
containing 2.3 mmol of dry Et N was added dropwise. The reaction mixture was
stirred for 5 hours at room temperature. Subsequently, 1.5 ꢁmol of piperidine was
added and the mixture was stirred at room temperature overnight. Next, the crude
product was precipitated with Et O, filtered off and purified by preparative PLC
2
559
560
chromatography.
5
5
5
5
61
62
63
64
Diwalled deoxycholic molecular umbrella/3-aminopropanoic acid/AmB conjugate 13a.
Starting from 38 mg of molecular umbrella 8a and 53 mg of TDBTU active ester of
FmocꢀAmB 11, 16 mg (0.008 mmol, 20%) of conjugate 13a was obtained (R 0.13,
f
+
.
CHCl
3
/MeOH/H
2
O, 65/10/1, /v/v). HRMSꢀESI found m/z 1871.2412 [M+H] calcd. for
1
565
C
105
H
171
N
5
O
23 1870.2365. H NMR (500 MHz, CDCl
3
/CD
3
OD, 1/3) δ: 6.0ꢀ6.5 (m,
5
5
5
5
5
66
67
68
69
70
14H), 5.3ꢀ5.4 (m, 2H), 3.0ꢀ4.6 (m, 27H), 3.9 (bs, 4H), 1.4ꢀ2.6 (m, 104H), 0.9ꢀ1.4 (m,
20H), 0.7 (s, 6H).
Diwalled deoxycholic molecular umbrella/4-aminobutanoic acid/AmB conjugate 13b.
Starting from 39 mg of molecular umbrella 8b and 53 mg of TDBTU active ester of
f
FmocꢀAmB 11, 63 mg (0.03 mmol, 80%) of conjugate 13b was obtained (R 0.13,
+.
571
72
CHCl
3
/MeOH/H
2
O, 65/10/1, v/v/v). HRMSꢀESI found m/z 1885.2586 [M+H] calcd.
1
5
for C106
H
173
N
5
O
23 1884.2521. H NMR (500 MHz, CDCl
3
/CD
3
OD, 1/3) δ: 6.0ꢀ6.5 (m,
2
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Bioconjugate Chemistry
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14H), 5.3ꢀ5.4 (m, 2H), 3.0ꢀ4.6 (m, 27H), 3.9 (bs, 4H), 2.9 (m, 2H) 1.4ꢀ2.6 (m, 104H),
0.9ꢀ1.4 (m, 20H), 0.7 (s, 6H).
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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2
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3
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5
5
6
574
5
5
5
5
75
76
77
78
Diwalled deoxycholic molecular umbrella/6-aminohexanoic acid/AmB conjugate 13c.
Starting from 40 mg of molecular umbrella 8c and 53 mg of TDBTU active ester of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
f
FmocꢀAmB 11, 27 mg (0.01 mmol, 34%) of conjugate 13c was obtained (R 0.13,
+.
CHCl
3
/MeOH/H
2
O, 65/10/1, v/v/v). HRMSꢀESI found m/z 1913.2872 [M+H] calcd.
1
579
for C108
H
177
N
5
O
23 1912.2834. H NMR (500 MHz, CDCl
3
/CD
3
OD, 1/3) δ: 6.0ꢀ6.5 (m,
5
5
5
5
5
80
81
82
83
84
14H), 5.3ꢀ5.4 (m, 2H), 3.0ꢀ4.6 (m, 25H), 3.9 (bs, 4H), 1.4ꢀ2.6 (m, 105H), 1.0ꢀ1.4 (m,
20H), 0.9 (s, 6H), 0.7 (s, 6H).
Diwalled deoxycholic molecular umbrella/8-aminooctanoic acid/AmB conjugate 13d.
Starting from 41 mg of molecular umbrella 8d and 53 mg of TDBTU active ester of
FmocꢀAmB 11, 43 mg (0.02 mmol, 54%) of conjugate 13d was obtained (R 0.13,
f
+.
585
CHCl
3
/MeOH/H
2
O, 65/10/1, v/v/v). HRMSꢀESI found m/z 1941.3156 [M+H] calcd.
1
5
5
5
5
86
87
88
89
for C110H181N O 1940.3147. H NMR (500 MHz, CDCl /CD OD, 1/3) δ: 6.0ꢀ6.5 (m,
5
23
3
3
14H), 5.3ꢀ5.4 (m, 2H), 3.0ꢀ4.6 (m, 32H), 3.9 (s, 2H), 3.8 (s, 2H), 0.9ꢀ2.4 (m, 123H),
0.7 (s, 6H).
Diwalled cholic molecular umbrella/4-aminobutanoic acid/AmB conjugate 13e.
Starting from 40 mg of molecular umbrella 8e and 53 mg of TDBTU active ester of
590
591
f
FmocꢀAmB 11, 18 mg (0.009 mmol, 22%) of conjugate 13e was obtained (R 0.13,
+.
5
5
5
5
92
93
94
95
CHCl
3
/MeOH/H
2
O, 65/10/1, v/v/v). HRMSꢀESI found m/z 1917.2433 [M+H] calcd.
1
for C106
H
173
N
5
O
25 1916.2420. H NMR (500 MHz, CDCl
3
/CD
3
OD, 1/3) δ: 6.0ꢀ6.5 (m,
14H), 5.3ꢀ5.4 (m, 2H), 3.0ꢀ4.6 (m, 27H), 3.9 (bs, 4H), 2.9 (m, 2H) 1.4ꢀ2.6 (m, 102H),
0.9ꢀ1.4 (m, 20H), 0.7 (s, 6H).
596
97
Diwalled cholic molecular umbrella/8-aminooctanoic acid/AmB conjugate 13f.
Starting from 42 mg of molecular umbrella 8f and 53 mg of TDBTU active ester of
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
5
5
5
6
5
98
f
FmocꢀAmB 11, 32 mg (0,016 mmol, 40%) of conjugate 13f was obtained (R 0.13,
+.
599
CHCl /MeOH/H O, 65/10/1, v/v/v). HRMSꢀESI found m/z 1973.3061 [M+H] calcd.
3
2
1
6
6
6
6
00
01
02
03
181 5 3 3
for C110H N O25 1972.3046. H NMR (500 MHz, CDCl /CD OD, 1/3) δ: 6.0ꢀ6.5 (m,
14H), 5.3ꢀ5.4 (m, 2H), 3.0ꢀ4.6 (m, 25H), 3.9 (s, 2H), 3.8 (s, 2H), 1.4ꢀ2.6 (m, 90H),
1.2ꢀ1.4 (m, 25H), 1.21ꢀ0.9 (m, 16H), 0.7 (s, 6H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Diwalled deoxycholic molecular umbrella/3-aminopropanoic acid/nystatin conjugate
14a. Starting from 38 mg of molecular umbrella 8a and 53 mg of TDBTU active ester
604
6
6
6
6
05
06
07
08
f
of FmocꢀNys 12, 10 mg (0.005 mmol, 13%) of conjugate 14a was obtained (R 0.13,
+.
CHCl
3
/MeOH/H
2
O, 65/10/1, v/v/v). HRMSꢀESI found m/z 1873.2529 [M+H] calcd.
1
for C105
H
173
N
5
O
23 1872.2521. H NMR (500 MHz, CDCl
3
/CD
3
OD, 1/3) δ: 5.2ꢀ6.2 (m,
12H), 3.0ꢀ4.6 (m, 29H), 2.0ꢀ2.8 (m, 18H), 0.9ꢀ2.0 (m, 114H), 0.7 (s, 6H).
609
610
Diwalled deoxycholic molecular umbrella/4-aminobutanoic acid/nystatin conjugate
14b. Starting from 39 mg of molecular umbrella 8b and 53 mg of TDBTU active ester
6
6
6
6
11
12
13
14
f
of FmocꢀNys 12, 58 mg (0.03 mmol, 73%) of conjugate 14b was obtained (R 0.13,
+.
CHCl
3
/MeOH/H
2
O, 65/10/1, v/v/v). HRMSꢀESI found m/z 1887.2696 [M+H] calcd.
1
175 5 3 3
for C106H N O23 1886.2678. H NMR (500 MHz, CDCl /CD OD, 1/3) δ: 5.2ꢀ6.2 (m,
12H), 3.0ꢀ4.6 (m, 29H), 2.0ꢀ2.8 (m, 20H), 0.9ꢀ2.0 (m, 114H), 0.7 (s, 6H).
615
Diwalled deoxycholic molecular umbrella/6-aminohexanoic acid/nystatin conjugate
14c. Starting form 40 mg of molecular umbrella 8c and 53 mg of TDBTU active ester
6
6
6
6
16
17
18
19
f
of FmocꢀNys 12, 21 mg (0.01 mmol, 27%) of conjugate 14c was obtained (R 0.13,
+.
CHCl /MeOH/H O, 65/10/1, v/v/v). HRMSꢀESI found m/z 1915.3124 [M+H] calcd.
3
2
1
179 5 3 3
for C108H N O23 1914.2991. H NMR (500 MHz, CDCl /CD OD, 1/3) δ: 5.2ꢀ6.2 (m,
620
12H), 3.0ꢀ4.6 (m, 25H), 3.9 (bs, 4H), 1.3ꢀ2.4 (m, 103H), 0.9ꢀ1.3 (m, 35H), 0.7 (s, 6H).
Diwalled deoxycholic molecular umbrella/8-aminooctanoic acid/nystatin conjugate
14d. Starting from 41 mg of molecular umbrella 8d and 53 mg of TDBTU active ester
6
6
21
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f
of FmocꢀNys 12, 36 mg (0.02 mmol, 45%) of conjugate 14d was obtained (R 0.13,
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
.
624
CHCl /MeOH/H O, 65/10/1, v/v/v). HRMSꢀESI found m/z 1943.3346 [M+H] calcd.
3
2
1
6
6
6
6
25
26
27
28
183 5 3 3
for C110H N O23 1942.3304. H NMR (500 MHz, CDCl /CD OD, 1/3) δ: 5.2ꢀ6.2 (m,
12H), 2.9ꢀ4.6 (m, 37H), 1.2ꢀ2.6 (m, 96H), 1.2ꢀ0.8 (m, 34H) 0.7 (s, 6H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Diwalled cholic molecular umbrella/4-aminobutanoic acid/nystatin conjugate 14e.
Starting from 40 mg of molecular umbrella 8e and 53 mg of TDBTU active ester of
629
FmocꢀNys 12, 12 mg (0.006 mmol, 15%) of conjugate 14e was obtained (R 0.13,
f
+.
6
6
6
6
30
31
32
33
CHCl
3
/MeOH/H
2
O, 65/10/1, v/v/v). HRMSꢀESI found m/z 1919.2657 [M+H] calcd.
1
for C106H175N O 1918.2576. H NMR (500 MHz, CDCl /CD OD, 1/3) δ: 5.0ꢀ6.2 (m,
5
25
3
3
12H), 3.0ꢀ4.6 (m, 25H), 3.9 (s, 2H), 3.8 (s, 2H), 1.2ꢀ2.6 (m, 115H), 1.2ꢀ0.9 (m, 16H),
0.7 (s, 6H).
634
635
Diwalled cholic molecular umbrella/8-aminooctanoic acid/nystatin conjugate 14f.
Starting from 42 mg of molecular umbrella 8f and 53 mg of TDBTU active ester of
6
6
6
6
36
37
38
39
f
FmocꢀNysA 12, 20 mg (0.01 mmol, 25%) of conjugate 14f was obtained (R 0.13,
+.
CHCl
3
/MeOH/H
2
O, 65/10/1, v/v/v). HRMSꢀESI found m/z 1975.3222 [M+1] calcd. for
1
C
110
H
183
N
5
O
25 1974.3202. H NMR (500 MHz, CDCl
3
/CD
3
OD, 1/3) δ: 5.0ꢀ6.2 (m,
12H), 3.0ꢀ4.6 (m, 25H), 3.9 (s, 2H), 3.8 (s, 2H), 1.4ꢀ2.6 (m, 90H), 1.2ꢀ1.4 (m, 28H),
1.21ꢀ0.9 (m, 16H), 0.7 (s, 6H).
640
6
6
6
6
6
41
42
43
44
45
Determination of Critical Aggregation Concentration (cac) of Amphotericin B
and its Conjugates. Stock solutions of AmB and its conjugates (1 mM) were
prepared in DMSO. Aliquots of 75 ꢂL were serially diluted with DMSO and then
introduced into test tubes containing 4.925 mL of phosphateꢀbuffered saline (PBS),
pH 7.4 at 37°C, to give concentrations ranging from 0.2 to 15 ꢂM. After vortex mixing
for 10 s, the solutions were transferred to a 1.60 mL UV cuvette that was maintained
646
47
6
2
9
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