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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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Bioconjugate Chemistry
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Molecular umbrellas modulate the selective toxicity of
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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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†
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Narutowicza Str., 80-233 Gdańsk, Poland
Department of Organic Chemistry, Gdańsk University of Technology, 11/12 G.
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‡
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Technology, 11/12 G. Narutowicza Str., 80-233 Gdańsk, Poland
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§
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80-308 Gdańsk, Poland
Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of
Department of Biomedical Chemistry, University of Gdańsk, 63 Wita Stwosza Str.,
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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)
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were conjugated through the ω-amino acid linkers with diwalled “molecular
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umbrellas” composed of spermidine-linked deoxycholic or cholic acids. Presence of
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“umbrella” substituents modulated biological properties of the antibiotics, especially
31
their selective toxicity. Some of the AmB-umbrella conjugates demonstrated
32
antifungal in vitro activity comparable to that of the mother antibiotic but diminished
33
mammalian toxicity, especially the hemolytic activity. In contrast, antifungal in vitro
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activity of NYS-umbrella conjugates was strongly reduced and all these conjugates
35
demonstrated poorer than NYS selective toxicity. No correlation between the
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aggregation state and haemolytic activity of the novel conjugates was found.
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Bioconjugate Chemistry
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INTRODUCTION
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Disseminated (invasive) fungal infections remain one of the major problems in
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modern chemotherapy. The estimated number of cases is more than 2 million/year
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worldwide, with over 1.5 million deaths.1 Candida species are the most common
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fungal etiological agents of life-threatening invasive infections in
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immunocompromised hosts, transplant recipients and patients hospitalized in
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intensive care units. These species are also the fourth most common cause of
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nosocomial (hospital-acquired) bloodstream infections.2 The high mortality rate of
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invasive mycoses is due to the several factors, including shortcomings of diagnosing,
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a limited number of effective antifungal chemotherapeutics and increasing fungal
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resistance to available drugs. Polyene macrolide antibiotics constitute an important
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group of antifungals, of which Amphotericin B, Nystatin and Pimaricin are the
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approved drugs but only the first of them is used for the treatment of invasive fungal
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infections. Nystatin (NYS) and Pimaricin are used exclusively for the topical
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applications. Amphotericin B (AmB) is known as a “golden standard” of antifungal
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chemotherapy, since it is fungicidal, demonstrates broad antifungal spectrum and
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lack of fungal resistance. The only (but important) drawback is its substantial
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mammalian toxicity, especially nephrotoxicity, which is a consequence of a
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mechanism of biological action.
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Currently there are two major hypotheses on this mechanism. According to the
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“barrel-stave-pore” mechanism, AmB molecules form complexes with ergosterol in
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the membrane and a few such complexes (4 – 12) self-assemble to barrel-stave-like
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trans-membrane pores, giving rise to leakage of low molecular weight cell
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components, including ions.3 In an alternative mechanism, called a “sterol sponge”
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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.4,5 In both mechanisms, binding of AmB to ergosterol is the prerequisite
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for antifungal action but the antibiotic also binds to cholesterol in mammalian cell
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membranes. This is only a slightly higher affinity to ergosterol than to cholesterol that
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constitutes a molecular basis for selective toxicity of AmB. In consequence, a minimal
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concentration at which AmB is toxic to mammalian cells is only 5 – 10 times higher
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than the minimal fungicidal concentration. Mechanism of antifungal action of NYS is
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much less known, although there is a little doubt that binding to ergosterol is also
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crucial for the biological activity of this antibiotic. AmB, as other polyene macrolides,
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is poorly soluble in aqueous solutions. Monomeric form of AmB exists in water at
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concentrations below 10-6 M. At higher concentrations, AmB undergoes complex
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processes of self-association, formation of dimers and soluble oligomers. Finally, at
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concentrations higher than 10-5 M insoluble aggregates are observed. It was shown
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that water-soluble aggregates of AmB are toxic to erythrocytes and fungal cells, while
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the monomers are toxic only to fungal cells.6
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It is believed that selective toxicity of polyene macrolides, especially AmB, can be
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improved by a proper chemical modifications of the antibiotic molecule concerning
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the carboxyl functionality or the amino sugar substituent. A number of derivatives of
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AmB were obtained but only some of them, especially those modified at the
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mycosamine residue, exhibited improved selective toxicity.7-9 Truly spectacular
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effects, i.e. elimination of mammalian in vitro toxicity, with retention or at most
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minimal decrease of antifungal activity, have been recently achieved for 2’-
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deoxyAmB,10 AmB urea derivatives11 and a conjugate of AmB with the diwalled
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molecular umbrella.12 Previously, it was shown that conjugates of AmB with tetra-
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and octawalled molecular umbrellas demonstrated low tendency to aggregate and
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Bioconjugate Chemistry
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negligible hemolytic activity.13 Herein, we present the results of our studies on
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synthesis and biological activity of several AmB and NYS conjugates with diwalled
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molecular umbrellas.
105 106
RESULTS AND DISCUSSION
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Synthesis of Conjugates. Twelve conjugates of molecular umbrella and polyene
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macrolides were synthesized. Eight of them were derivatives of a deoxycholic acid-
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based diwalled molecular umbrella with covalently attached molecule of AmB (13a-d)
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or NYS (14a-d). Four conjugates consisted of a cholic acid-based diwalled molecular
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umbrella and AmB (13e-f) or NYS (14e-f). The choice of diwalled (not tetra- or
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octawalled) umbrellas as the optimal components of conjugates was justified by the
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results of previous studies of Janout et al.12,13
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Molecular umbrella:polyene macrolide conjugates 13a-f and 14a-f were prepared
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according to the multistep procedure developed by Janout and co-workers for the
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synthesis of two umbrella:AmB conjugates,12 with a slight modification at the product
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isolation step. Synthesis of molecular umbrellas with ω-amino acid linkers (Scheme
118
1) was based on an active ester approach and started with formation of non-labile ω-
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amino acid linkers, obtained from commercially available ω-amino acids 1a-d, amino
120
groups of which were protected with Boc, using Boc2O as an acylating agent. The N-
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protected derivatives 2a-d as crude products were applied in formation of N-
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hydroxysuccinimide (NHS) active esters 3a-d, using the NHS/DCC method, followed
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by purification by crystallization.
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The starting materials in the synthesis of molecular umbrellas 6a and 6b were
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commercially available deoxycholic 4a and cholic 4b acids, which were transformed
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to corresponding NHS active esters 5a-b, using the NHS/DCC technique. Derivatives
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5a and 5b were subsequently condensed with spermidine by generation of amide
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bonds, what resulted in formation of diwalled molecular umbrellas 6a-b.
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Condensation of derivatives 6a-b with previously obtained NHS active esters 3a-d,
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followed by deprotection of amino groups in acidic conditions lead to conjugates 8a-f,
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i.e. molecular umbrellas carrying a non-labile ω-amino acid linker, necessary for
132
coupling of a polyene macrolide molecule.
133 134
Scheme 1. Synthesis of molecular umbrellas with ω-amino acid linkers. Reagents
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and conditions. i) Na2CO3, H2O, 0°C, Boc2O, 1,4-dioxane, 1 h, 0°C->rt,
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24 h, HCl; ii) NHS, THF, 0°C, DCC, 0°C->rt, 24 h; iii) NHS, THF, 0°C,
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DCC, 0°C->rt, 26 h; iv) spermidine, CHCl3/DMF, rt, 48 h; v) CHCl3/DMF,
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Et3N, rt, 60 h; vi) MeOH/HCl, rt, overnight. 6 ACS Paragon Plus Environment
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Bioconjugate Chemistry
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The further steps are shown in Scheme 2. N-Fmoc-protected AmB 9 and Nys 10
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were converted into their 3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl active esters 11
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and 12, respectively, with O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N’,N’-
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tetramethyluronium tetrafluoroborate (TDBTU) as a substrate. The esters obtained
143
(11 and 12) were used in subsequent reactions without further purification.
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145 146
Scheme 2. Conjugation of polyene macrolides with molecular umbrellas. Reagents
147
and conditions. i) Fmoc-NHS; ii) DMF, diisopropylethylamine, TDBTU, rt,
148
20 min; iii) DMF, Et3N, rt, 5 h, piperidine, rt, overnight.
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Formation of molecular umbrella-polyene conjugates was accomplished by
150
condensation of active esters 11 and 12 with one of the molecular umbrella-linker
151
conjugates 8a-f, in the presence of triethylamine. Condensation was directly followed
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by deprotection of the amino group of mycosamine with piperidine, affording products
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13a-f and 14a-f which were purified by preparative thin layer chromatography.
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The thus obtained preparations of NYS conjugates 14a-f, contained tiny amounts
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(less than 4% by HPLC analysis, exemplary analysis of 14c preparation in
156
Supporting Information) of respective isomeric forms (additional peak in HPLC, hardly
157
distinguishable by preparative TLC, identified by HRMS and NMR analysis). These
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isomers were the products of the previously described intramolecular
159
translactonization of NYS, resulting in formation of iso-nystatin (iso-NYS).14 For the
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purpose of physico-chemical and biological studies, each preparation was further
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purified by semi-preparative HPLC, so that all results presented below were obtained
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for pure NYS conjugates, free of the iso-NYS derivatives.
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Antifungal and Hemolytic Activity of Conjugates. All the conjugates 13a-f and
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14a-f were tested for antifungal in vitro activity against the model C. albicans SC
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5314 strain. Assay was performed in RPMI-1640 medium and values of MIC80 were
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determined. It should be noted, that formation of iso-NYS or its derivatives was not
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observed (HPLC control) for NYS and its conjugates 14a-f under conditions of this
168
assay (incubation for 24 h at 37°C in RPMI-1640 medium buffered to 7.0), although
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possibility of NYS isomerisation at pH 6.0 or 7.5 was previously suggested.15 The
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same set of compounds was also tested for hemolytic activity against human
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erythrocytes and the EH50 values were determined as a measure of this activity.
172
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
175
molecular umbrellas and selective toxicity indexes. MIC80 values were
176
determined in RPMI-1640 medium against C. albicans SC 5314. EH50
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values were determined against human erythrocytes. Selective toxicity
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index is defined as EH50 [µM]/MIC80 [µM]. ND = not determined
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Bioconjugate Chemistry
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Compound
AmB
13a
13b
13c
13d
13e
13f
NYS
14a
14b
14c
14d
14e
14f
MIC80 [µg mL-1/µM]
0.3/ 0.32
7.1/ 3.8
5.5/ 2.0
1.8/ 0.94
0.9/ 0.46
1.0/ 0.52
0.6/ 0.44
0.8/ 0.86
18/ 9.62
14/ 7.42
33/ 17.2
38/ 19.6
45/ 23.5
127/ 64.3
EH50 [µg mL-1/µM]
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/ 2.65
24/ 12.5
37/ 19.1
47/ 24.5
ND
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
-
180 181 182
All conjugates exhibited lower anticandidal activity that their mother compounds, i.e.
183
AmB and NYS, respectively. In the case of AmB conjugates, the lowest difference
184
between their MIC80 values and that of AmB was found for compounds 13d, 13e and
185
13f. In terms of molar concentrations, MICs of these compounds were almost the
186
same as that of the mother antibiotic. Interestingly, for conjugates with deoxycholic
187
acid-based molecular umbrellas (13a-13d), the length of the ω-amino acid linker
188
determined the antifungal activity and the conjugates with short linkers, 13a (n = 1)
189
and 13 b (n = 2) demonstrated much lower activity that their counterparts with longer
190
linkers, 13c (n = 4) and 13 d (n = 6). In contrast, both conjugates with the cholic acid-
191
based molecular umbrellas 13e (n = 2) and 13 f (n = 6) exhibited similar antifungal
192
activity. On the other hand, all the NYS conjugates were strongly less active that NYS
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itself and MIC80 of the most active conjugate 14b (n = 2) was almost 9-fold higher
194
than that of NYS. Paradoxically, the lowest activity was found for the conjugate 14f,
195
which is a counterpart of the most active AmB conjugate 13f. A qualitative difference
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between the hemolytic activities of AmB and NYS conjugates was found. The former
197
were less hemolytic than AmB, while the EH50 values of the latter were lower than
198
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activity of AmB conjugates with deoxycholic acid-based molecular umbrellas but the
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higher activity was found for the conjugates with shorter linkers. Notably, a similar
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trend was observed by Yu and co-workers for the AmB derivatives, conjugated with
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“molecular semi umbrellas” composed of cholic acid and diamine linkers of variable
203
length.16
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The selective toxicity indexes, defined as EH50/MIC80, of three AmB conjugates,
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namely 13d, 13e and 13f, were better than that of AmB, while for most of the NYS
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derivatives, the EH50 values were lower than MICs80, and a selective toxicity of all
207
NYS conjugates was poorer than that of the mother antibiotic.
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The anticandidal spectrum of compounds 13d, 13f and 14b was compared to that of
209
AmB and NYS. The MIC50 and MIC80 values of these compounds determined against
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7 Candida spp. and S. cerevisiae are summarized in Table 2.
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Table 2. Antifungal in vitro activities of AmB, NYS and conjugates 13d, 13f and 14b.
212
MIC80 and MIC50 values were determined in RPMI-1640 medium.
213 MIC50 [µg mL-1]
MIC80 [µg mL-1]
AmB
13d
13f
NYS
14b
AmB
13d
13f
NYS
14b
C. albicans ATCC 10231
0.3
0.7
0.25
0.7
12
0.4
0.8
0.5
1.0
15
C. albicans B3
0.3
0.8
0.4
1.0
13
0.5
0.9
0.75
1.1
15
C. albicans B4
0.4
2.3
1.3
1.2
10
0.5
3.6
1.6
0.9
15
C. glabrata
0.3
2.7
1.3
0.45
16
0.4
3.6
1.7
0.8
22
C. krusei
0.5
3.0
1.5
0.9
17
0.6
3.75
1.8
1.0
31
C. parapsilosis
0.6
0.7
1.7
1.1
15
0.7
1.2
0.9
1.3
16
C. tropicalis
0.4
12.5
24
0.8
3.4
0.5
16
32
1.4
4.0
S. cerevisiae
0.2
0.75
0.6
0.7
3.0
0.3
0.95
0.8
0.7
3.8
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Bioconjugate Chemistry
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In general, the difference between MICs of the conjugates 13d and 13f and that of
216
AmB was higher for Candida spp. other than C. albicans, especially in the case of C.
217
tropicalis. Interestingly enough, MICs of these conjugates against the C. albicans B4
218
strain, multidrug resistant due to the overexpression of the MDR1 gene, were 2-4
219
times higher than those against non-resistant C. albicans B3, while in the case of the
220
NYS conjugate 14b, there was no difference in susceptibilities of B3 and B4 strains.
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Mammalian cytotoxicity of conjugates. Selected conjugates 13b, 13d, 13f and
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14b were evaluated for cytotoxicity against three mammalian cell lines: pig kidney
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epithelial cells LLC-PK1, human embryonic kidney cells HEK-293T and human
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hepatocellular carcinoma cells HepG2. Values of IC50, i.e. concentrations at which
226
50% inhibition of control growth was observed are summarized in Table 3. All values
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are means of at least two independent experiments, each done in duplicate.
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Table 3. Cytotoxicity of AmB, NYS and their conjugates 13b, 13d, 13f and 14b toward three mammalian cell lines in tissue culture. IC50 ±SD [µg mL-1] Compound HEK-293T cells LLC-PK1 cells Hep G2 cells AmB
0.488 ±0.101
2.688 ±0.950
0.565 ±0.111
13b
16.708 ±3.571
8.471 ±1.204
>50
13d
5.565 ±0.253
6.454 ±0.992 25.209 ±2.313
13f
17.742 ±2.642 15.411 ±1.018 33.071 ±2.907
NYS
6.173 ±0.602
11.652 ±2.332 7.864 ±0.540
14b
2.176 ±0.122
2.505 ±0.397 16.102 ±4.089 11
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All the AmB conjugates were less cytotoxic than AmB. The difference between the
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IC50 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 LLC-PK1 cells. On the other
234
hand, the NYS conjugate 14b was less cytotoxic than NYS only against the Hep G2
235
cells.
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Aggregation of Conjugates in Solutions. AmB and NYS undergo aggregation in
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aqueous solutions in a concentration-dependent manner. AmB in a monomeric state
239
exhibits a characteristic absorption band at 409 nm, molar absorptivity of which
240
decreases upon aggregation. For the solutions containing both monomeric and
241
aggregated forms, ε = εp + (εm - εP)m/T, where T and m represent the total and
242
monomeric concentration of the antibiotic or its conjugate and ε, εm and εp stand for
243
the apparent molar absorptivity, molar absorptivity of the monomeric component and
244
molar absorptivity of the micellar component, respectively. By measuring the
245
apparent molar absorptivity as a function of the reciprocal of AmB or its conjugate
246
concentration, one may estimate cac (critical aggregation concentration) from the
247
intercept of two straight lines.17 Such plots obtained by us for compounds 13d and
248
13f are shown in Figure 1. The cac values for AmB and the conjugates were as
249
follows: AmB – 1 µM; 13a – 1.5 µM; 13b – 0.2 µM; 13d – 3.2 µM; 13f – 3.8 µM.
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Bioconjugate Chemistry
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Figure 1. Plots of molar absorptivity (λmax = 409 nm) as a function of the reciprocal concentration of 13d (A) and 13f (B).
253 254
Application of the same methodology for studies on aggregation of NYS and its
255
conjugates was not possible, since the concentration-dependent decrease of
256
molecular absorptivity at 409 nm is not observed. On the other hand, this antibiotic
257
demonstrates fluorescence at λ = 410 nm upon excitation at λ = 320 and changes in
258
an emission spectrum may be monitored to follow the aggregation state of NYS and
259
its derivatives in solution.18 In his work, the fluorescence intensity and steady-state
260
anisotropy were measured as a function of antibiotic/conjugate concentration in
261
buffered solution. As shown in Figure 2, at low concentrations of NYS, the anisotropy
262
increased with the concentration of antibiotic in solution; however, above ∼ 2 µM
263
antibiotic, the anisotropy reached a maximum of 0.25 ± 0.03 and became
264
concentration-independent. At this antibiotic concentration, there was also a sharp
265
increase in the fluorescence intensity of NYS. These observations are consistent with
266
an aggregation of the antibiotic in aqueous solution.19 The cac value of NYS
267
determined in this way was 2 µM, for the conjugate 14a cac = 5.5 µM and for
268
conjugates 14b and 14d cac = 4 µM. 13 ACS Paragon Plus Environment
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Figure 2. Dependence of the steady-state fluorescence anisotropy on concentration of NYS, 14a, 14b and 14d.
272 273
These results clearly indicate that the cac values of AmB, NYS and their conjugates
274
with molecular umbrellas were similar, inside the 0.2 – 5.5 µM range, so that they do
275
not seem to determine the hemolytic activity of these compounds.
276
It should be noted however, that although the conjugate 13f demonstrated in our
277
hands the most favourable biological properties among all the AmB conjugates
278
tested, parameters characterizing its mammalian cytotoxicity were markedly different
279
from that reported by Janout and co-workers, who found for this compound EH50 =
280
375 µM toward sheep erythrocytes and little if any toxicity toward HEK293 T cells.12
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To determine whether the inhibition of C. albicans growth by was related to
282
the amount of binding of NYS and AmB conjugates to yeast cells, a
283
binding assay was performed. Cell suspensions of different cell density were
284
incubated with AmB, NYS or any of the conjugates at 30 µM, cells were spun down
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Bioconjugate Chemistry
285
and concentration of an antifungal agent remaining in supernatant was measured by
286
Uv-vis spectrophotometry at 409 nm. Data presented in Figures 3AB clearly show
287
that all compounds tested were bound by C. albicans cells and percent of compound
288
bound was cell density dependent. No binding was observed for E. coli cells (less
289
than 2% binding at cell density corresponding to A660 = 1.0) lacking sterols in the
290
plasma membrane, thus indicating that binding to C. albicans cells was due to the
291
interaction with ergosterol. Taking percent of NYS and its conjugates bound at cell
292
density corresponding to A660 = 1.0 as a semi-quantitative measure of their affinity to
293
the ergosterol-containing yeast cells, some correlation of these values with antifungal
294
activity expressed as MIC80 was found. This correlation, shown in Figure 4, although
295
not very good (r = 0.87), confirms dependence of antifungal in vitro activity of NYS
296
conjugates on their binding affinity to yeast cells. The respective graph for AmB and
297
its conjugates is not shown, because both the MIC80 values and binding affinities of
298
all these compounds were very similar, so that any possible correlation is not
299
especially informative.
300
Figure 3. Binding of polyene macrolide antibiotics and their conjugates to C. albicans
301
cells. Cell suspensions of various cell density were incubated with a given compound
302
(30 µM) for 1 h and concentration of the unbound compound was determined by UV-
303
vis in supernatant left after centrifugation. A. AmB and its conjugates. B. NYS and its
304
conjugates. Data are the means of three independent determinations ±SD.
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Page 16 of 39
306
Figure 4. Correlation between cell binding affinity and antifungal activity of NYS and
307
its conjugates. Percent of compound bound to C. albicans cells at A660 = 1.0 was
308
plotted against log MIC80 values. Data are the means of three independent
309
determinations ±SD.
310 311
Conclusions. Some previous reports showed that attachment of bulky substituents,
312
like tetrawalled and octawalled molecular umbrellas or poly(ethylene glycol) chains
313
to AmB, resulted in a significant increase of the cac values of the conjugates, in
314
comparison to that of AmB alone, and in consequence, in an adequate increase of
315
concentrations at which they exhibited a hemolytic activity.13,17 In our hands, although
316
the cac values of AmB conjugates demonstrating much lower than AmB hemolytic
317
activity were slightly higher than that of the antibiotic, they were still well below the
318
EH50 values, thus confirming a previous observation of Janout and co-workers, who
319
found that the AmB conjugate with the cholic acid-based molecular umbrella
320
containing an 8-aminooctanoic acid linker (compound 13f in our study) was much
321
less haemolytic than AmB, despite the fact that its cac value was almost the same as
322
that of AmB.12 A similar phenomenon was recently noted by Yu et al. for AmB
323
conjugates with cholic acid.16 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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Bioconjugate Chemistry
325
valid for all kinds of AmB derivatives. Nevertheless, there is no doubt that conjugation
326
of AmB with diwalled molecular umbrellas affords conjugates demonstrating better
327
selective toxicity that the mother antibiotic. On the other hand, application of the
328
same strategy for the construction of NYS conjugates did not result in a similar effect,
329
since the obtained conjugates demonstrated much lower antifungal activity and
330
higher mammalian cytotoxicity than NYS. This observation is another evidence that
331
strategies of AmB chemical modification aimed at improvement of selective toxicity of
332
this antibiotic cannot be directly transferred to NYS, despite the apparent structural
333
analogy between these antibiotics. This was previously demonstrated for NYS
334
derivatives containing bulky substituents at the amino group of mycosamine, the
335
selective toxicity of which were much poorer than those of their AmB-derived
336
counterparts.20
337
Conjugation of AmB with a diwalled molecular umbrella or a “semi umbrella”, i.e. a
338
facially amphiphilic sterol, resulting in increasing the cellular selectivity of this
339
antibiotic, due to the dramatic reduction in hemolytic activity and significant retention
340
of antifungal activity, is known as a “taming strategy”.12,16 It was postulated that this
341
strategy could be applied also for other classes of membrane-disrupting agents.
342
Results described in this work provide evidence that application of the taming
343
strategy for NYS, structurally similar to AmB, results in substantial decreasing,
344
instead of increasing, of cellular selectivity, due to the enhancement of hemolytic
345
activity and substantial diminishment of antifungal activity of conjugates in
346
comparison with those of the mother antibiotics. It seems that the latter might be due
347
to the decreased affinity of NYS conjugates to ergosterol, since some correlation
348
between their antifungal activity and binding affinity to yeast cells containing
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Page 18 of 39
349
ergosterol in plasma membranes was found. One may thus conclude that the general
350
applicability of the taming strategy may be somewhat limited.
351 352
MATERIALS AND METHODS
353
Chemistry. 1H NMR spectra were recorded on Unity 500 plus Varian spectrometer at
354
500 MHz and on Bruker Advance III HD 400 MHz at 400 MHz. HRMS-ESI spectra
355
were recorded on Aqilent Technologies 6540 UHD Accurate – Mass Q-TOF LC/MS
356
apparatus. Purification of reaction products was carried out by liquid preparative
357
column chromatography on silica gel Geduran® Si 60 (40-63 µm) and preparative
358
TLC silica gel 60 F250, 1 mm. Separation of NYS and iso-NYS conjugates was
359
performed by semi-preparative HPLC on Agilent 1200 Series apparatus, Zorbax SB-
360
C18 (5 µm, 9.4 × 250 mm) column, isocratic elution with acetonitrile/H20, 50:50 v/v
361
solvent system. HPLC analysis was performed with Agilent 1290 Infinity system,
362
Poroshell 120 EC-C18 (2.7 µm, 4.6 ×150mm) column, isocratic elution with
363
acetonitrile/ H20, 10:90 v/v solvent system, containing 0.1% formic acid. Mass
364
detection with 6540 UHD Q-TOF spectrometer using positive ESI ionization mode.
365
The melting points are uncorrected. Commercial grade reagents and solvents were
366
used without further purification.
367 368
NHS Esters of Boc-ω ω-amino acids – General Procedure. ω-Amino acid (34 mmol)
369
and Na2CO3 (51 mmol) were dissolved in water (20 mL) and the resulting solution
370
was cooled to 0°C in an ice bath. di-tert-Butyl dicarbonate (34 mmol) dissolved in 50
371
mL of 1,4-dioxane was added dropwise. The reaction mixture was stirred at 0°C for 1
372
hour and next allowed to warm to room temperature. After 24 hours, dioxane was
373
evaporated under reduced pressure, the aqueous solution was washed with AcOEt
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Bioconjugate Chemistry
374
and subsequently acidified with 1M HCl to pH 1-2. The mixture was then extracted
375
with AcOEt and an organic layer was dried over anhydrous MgSO4. After removal of
376
the desiccant and concentration under reduced pressure, the crude Boc-ω-amino
377
acid (oil) was used in the next step without further purification. The equimolar
378
amounts of Boc-ω-amino acid N-hydroxysuccinimide (NHS) were dissolved in dry
379
THF. The solution was cooled in an ice bath and a dicyclohexylcarbodiimide (DCC)
380
solution in dry THF (1.2 × molar excess in respect to Boc-ω-amino acid) was added
381
dropwise, while the mixture was stirred vigorously. The mixture was allowed to warm
382
to room temperature and then stirred for additional 24 hours. The dicyclohexylurea
383
(DCU) formed was filtered off and the filtrate was concentrated under reduced
384
pressure. The residue was dissolved in CHCl3 and washed consecutively with water,
385
a saturated solution of NaHCO3, and water. The organic layer was then dried over
386
anhydrous MgSO4 and concentrated under reduced pressure. The residue was
387
crystallized from the AcOEt/hexane mixture.
388
N-succinimidyl N-Boc-3-aminopropanoate 3a. Starting from 3.05 g (34 mmol) of 3-
389
aminopropanoic acid 1a, 6.30 g (22 mmol, 66%) of the 3a product was obtained as a
390
white solid (Rf 0.46, hexane/AcOEt, 1/1, v/v); m.p. 95-97°C (lit.21 100°C). 1H NMR
391
(500 MHz, CDCl3) δ: 5.14 (s, 1H), 3.53 (m, 2H), 2.86 (m, 6H), 1.45 (s, 9H).
392
N-succinimidyl N-Boc-4-aminobutanoate 3b. Starting from 2.89 g (28 mmol) of 4-
393
aminobutanoic acid 1b, 5.40 g (18 mmol, 64%) of the 3b product was obtained as a
394
white solid (Rf 0.60, hexane/AcOEt, 1/1, v/v); m.p. 112-115°C (lit.22 110°C). 1H NMR
395
(500 MHz, CDCl3) δ: 4.77 (s, 1H), 3.24 (m, 2H), 2.85 (s, 4H), 2.67 (t, J=7.2 Hz, 2H),
396
1.95 (m, 2H), 1.45 (s, 9H).
397
N-succinimidyl N-Boc-6-aminohexanoate 3c. Starting from 3.02 g (23 mmol) of 6-
398
aminohexanoic acid 1c, 4.93 g (15 mmol, 65%) of the 3c product was obtained as a 19 ACS Paragon Plus Environment
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Page 20 of 39
399
white solid (Rf 0.40, hexane/AcOEt, 1/1, v/v); m.p. 82-86°C. HRMS-ESI found m/z
400
329.1602 [M+1]+. calcd for C15H24N2O6 328.1634. 1H NMR (500 MHz, CDCl3) δ: 4.60
401
(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),
402
1.51 (m, 2H), 1.45 (m, 1H).
403
N-succinimidyl N-Boc-8-aminooctanoate 3d. Starting from 716 mg (4.5 mmol) of 8-
404
aminooctanoic acid 1d, 1.03 g (2.88 mmol, 64%) of the 3d product was obtained as a
405
white solid (Rf 0.58, hexane/AcOEt, 1/1, v/v); m.p. 96-99°C. HRMS-ESI found m/z
406
357.1972 [M+1]+. calcd for C17H28N2O6 356.1947. 1H NMR (500 MHz, CDCl3) δ: 4.52
407
(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),
408
1.55-1.25 (m, 17H).
409
Diwalled Molecular Umbrellas – Modified General Procedure.23 The solution
410
made of a bile acid (13.49 mmol) and NHS (13.49 mmol) in dry THF was cooled to
411
0°C in an ice bath and then a DCC solution in dry THF (16.60 mmol) was added
412
dropwise. Subsequently, the reaction mixture was allowed to warm to room
413
temperature and stirred for additional 36 h. The DCU formed was filtered off and the
414
filtrate was concentrated under reduced pressure. The residue was dissolved in
415
CHCl3 and washed consecutively with water, a saturated solution of NaHCO3, and
416
water. The organic layer was then dried over anhydrous MgSO4 and concentrated
417
under reduced pressure. After removal of the desiccant and concentration under
418
reduced pressure, the residue was crystallized from AcOEt. The product obtained
419
was used without characterization in the next step. The NHS active ester (8.1 mmol)
420
and spermidine (4.05 mmol) were dissolved in CHCl3 with addition of small amount of
421
DMF. The reaction mixture was stirred at room temperature for 48 hrs and then
422
washed consecutively with water, a saturated solution of NaHCO3, and water. The
423
organic layer was then dried over anhydrous MgSO4, the desiccant was filtered off
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Bioconjugate Chemistry
424
and the filtrate was concentrated under reduced pressure. The residue was purified
425
by column chromatography on silica gel eluted with the CHCl3/MeOH/NH4OH
426
mixture.
427
N1,N3- bis-deoxycholylspermidine 6a. Starting from 5.30 g (13.49 mmol) of
428
deoxycholic acid 4a, 5.17 g (5.8 mmol, 43%) of the 6a product was obtained as a
429
white solid (Rf 0.31, CHCl3/MeOH/NH4OH, 4/1.5/0.1, v/v/v). HRMS-ESI found m/z
430
894.7290 [M+H]+. calcd. for C55H95N3O6 893.7221. 1H NMR (500 MHz, CD3OD) δ:
431
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,
432
J=7,1 Hz, 4H), 2.24 (m, 2H), 2.11 (m, 2H), 1.96-1.68 (m, 16H), 1.68-1.22 (m, 32H),
433
1.22-1.07 (m, 4H), 1.07-0.87 (m, 14H), 0.71 (s, 6H).
434
N1,N3- bis-cholylspermidine 6b. Starting from 7.21 g (17.65 mmol) of cholic acid 4b,
435
5.48 g (6.0 mmol, 34%) of the 6b product was obtained as a white solid (Rf 0.50,
436
CHCl3/MeOH/NH4OH, 6/2/0.5, v/v/v). HRMS-ESI found m/z 926.7199 [M+H]+. calcd.
437
for C55H95N3O8 925.7119. 1H NMR (500 MHz, CD3OD) δ: 3.96 (s, 2H), 3.79 (s, 2H),
438
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),
439
2.11 (m, 2H), 2.05-1.69 (m, 16H), 1.69-1.48 (m, 16H), 1.48-1.20 (m, 10H), 1.11 (m,
440
2H), 1.05-0.95 (m, 8H), 0.92 (s, 6H), 0.71 (s, 6H).
441 442
Diwalled Molecular Umbrella/ω ω-amino acid Conjugates – General Procedure. A
443
diwalled molecular umbrella (1.11 mmol) and a Boc-ω-amino acid NHS ester (1.33
444
mmol) were dissolved in CHCl3 containing a small amount of dry DMF and then dry
445
Et3N (1.11 mmol) was added. The reaction mixture was stirred for 60 hrs at room
446
temperature. Subsequently, the solvents were evaporated and the residue was
447
purified by column chromatography on silica gel with the CHCl3/MeOH/H2O mixture.
448
The conjugates thus obtained were de-protected by dissolving in anhydrous MeOH
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Page 22 of 39
449
saturated with gaseous HCl, stirring overnight and subsequent evaporation of
450
solvents. The crude products were used in the next steps.
451
N1,N3-bis-deoxycholyl-N2-(Boc-3-aminopropanoyl)spermidine 7a. Starting from 1 g
452
(1.11 mmol) of molecular umbrella 6a and 381 mg (1.33 mmol) of NHS ester of Boc-
453
3-aminopropionic acid 3a, 1.05 g (1 mmol, 90%) of product 7a was obtained as white
454
solid (Rf 0.44, CHCl3/MeOH/H2O, 65/10/1, v/v/v); mp 70-72°C; HRMS-ESI found m/z
455
1065.8187 [M+H]+. calcd. for C63H108N4O9 1064.8116. 1H NMR (500 MHz, CD3OD) δ:
456
8.00 (m, 2H), 3.96 (s, 2H), 3.52 (m, 2H), 3.34 (m, 6H), 3.18 (m, 4H), 2.56 (m, 2H),
457
2.26 (m, 2H), 2.12 (m, 2H), 1.96-1.69 (m, 16H), 1.67-1.24 (m, 41H), 1.23-0.86 (m,
458
18H), 0.70 (s, 6H).
459
N1,N3-bis-deoxycholyl-N2-(Boc-4-aminobutanoyl)spermidine 7b. Starting from 1 g
460
(1.12 mmol) of molecular umbrella 6a and 402 mg (1.34 mmol) of NHS ester of Boc-
461
4-aminobutanoic acid 3b, 1.00 g (0.93 mmol, 83%) of product 7b was obtained as
462
white solid (Rf 0.55, CHCl3/MeOH/H2O, 60/10/1, v/v/v); mp 105-110°C; HRMS-ESI
463
found m/z 1079.8343 [M+H]+. calcd. for C64H110N4O9 1078.8273. 1H NMR (500 MHz,
464
CD3OD) δ: 3.97 (s, 2H), 3.54 (m, 2H), 3.35 (m, 4H), 3.19 (m, 4H), 3.09 (m, 2H), 2.39
465
(m, 2H), 2.25 (m, 2H), 2.13 (m, 2H), 1.96-1.69 (m, 18H), 1.67-1.24 (m, 41H), 1.22-
466
0.85 (m, 18H), 0.7 (s, 6H).
467
N1,N3-bis-deoxycholyl-N2-(Boc-6-aminohexanoyl)spermidine 7c. Starting from 1g
468
(1.10 mmol) of molecular umbrella 6a and 433 mg (1.32 mmol) of NHS ester of Boc-
469
6-aminohexanoic acid 3c, 0.73 g (0.66 mmol, 60%) of product 7c was obtained as
470
white solid (Rf 0.40, CHCl3/MeOH/H2O, 65/10/1, v/v/v); mp 125°C; HRMS-ESI found
471
m/z 1107.8666 [M+H]+. calcd. for C66H114N4O9 1106.8586. 1H NMR (500 MHz,
472
CD3OD) 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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Bioconjugate Chemistry
473
(q, J=6.6 Hz, 2H), 2.37 (m, 2H), 2.26 (m, 2H), 2.11 (m, 2H), 1.96-1.69 (m, 18H), 1.67-
474
1.23 (m, 45H), 1.23-1.04 (m, 4H), 1.02 (m, 6H), 0.94 (s, 8H), 0.71 (s, 6H).
475 476
N1,N3-bis-deoxycholyl-N2-(Boc-8-aminooctanoyl)spermidine 7d. Starting from 1.25 g
477
(1.40 mmol) of molecular umbrella 6a and 599 mg (1.68 mmol) of NHS Boc-8-
478
aminooctanoic acid 3d, 1.40 g (1.25 mmol, 89%) of product 7d was obtained as oil
479
(Rf 0.62, CHCl3/MeOH/H2O, 65/10/1, v/v/v); HRMS-ESI found m/z 1135.8901 [M+H]+.
480
calcd. for C68H118N4O9 1134.8899. 1H NMR (500 MHz, CD3OD) δ: 3.96 (s, 2H), 3.52
481
(m, 2H), 3.35 (m, 4H), 3.17 (m, 4H), 3.01 (m, 2H), 2.35 (m, 2H), 2.25 (m, 2H), 2.11
482
(m, 2H), 1.96-1.69 (m, 18H), 1.67-1.23 (m, 49H), 1.23-0.89 (m, 18H), 0.7 (s, 6H).
483
N1,N3-bis-cholyl-N2-(Boc-4-aminobutanoyl)spermidine 7e. Starting from 750 mg (0.81
484
mmol) of molecular umbrella 6b and 291 mg (0.97 mmol) of NHS ester of Boc-4-
485
aminobutanoic acid 3b, 604 mg (0.54 mmol, 67%) of product 7e was obtained as oil
486
(Rf 0.49, CHCl3/MeOH/H2O, 65/10/1, v/v/v); HRMS-ESI found m/z 1111.8180 [M+H]+.
487
calcd. for C64H110N4O11 1110.8171. 1H NMR (500 MHz, CD3OD) δ: 3.96 (s, 2H), 3.80
488
(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,
489
2H), 2.06-1.70 (m, 18H), 1.70-1.22 (m, 35H), 1.21-0.87 (m, 16H), 0.71 (s, 6H).
490
N1,N3-bis-cholyl-N2-(Boc-8-aminooctanoyl)spermidine 7f. Starting from 2.49 g (2.69
491
mmol) of molecular umbrella 6b and 1.15 g (3.23 mmol) of NHS ester of Boc-8-
492
aminooctanoic acid 3d, 1.82 g (1.56 mmol, 58%) of product 7f was obtained as oil (Rf
493
0.40, CHCl3/MeOH/H2O, 65/10/1, v/v/v); HRMS-ESI found m/z 1167.8879 [M+H]+.
494
calcd. for C68H118N4O11 1166.8797. 1H NMR (500 MHz, CD3OD) δ: 3.96 (s, 2H), 3.80
495
(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,
496
2H), 2.06-1.69 (m, 18H), 1.69-1.21 (m, 43H), 1.21-0.86 (m, 16H), 0.72 (s, 6H).
497
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Page 24 of 39
498
N-Fmoc-polyene macrolide – General Procedure. A polyene macrolide (0.08
499
mmol) and Fmoc-NHS (0.2 mmol) were dissolved in 6 mL of DMF/MeOH (2:1)
500
mixture and subsequently 56 µL of pyridine was added. The reaction mixture was the
501
stirred for 12 hours at room temperature. In the next step, the reaction mixture was
502
cooled to 0°C and subsequently, 140 mL of diethyl ether was added dropwise. The
503
precipitated yellow solid was filtered off under reduced pressure and dried under
504
vacuum for 1 hour.
505 506
N-Fmoc-AmB 9. Starting from 100 mg (0.09 mmol) of amphotericin B and 56 mg
507
(0.16 mmol) of Fmoc-NHS, 118 mg (0.1 mmol, 95%) of the 9 product was obtained
508
as a yellow solid (Rf 0.45, CHCl3/MeOH/H2O, 4/1/0.1, v/v/v). HRMS-ESI found m/z
509
1146.5641 [M+H]+. calcd. for C62H83NO19 1145.5559. 1H NMR (500 MHz, pyridine-d5)
510
δ: 7.9 (d, J=7.6Hz, 2H), 7.7 (m, 2H), 7.4 (m, 2H), 7.2 (m, 2H), 6.8 (m, 2H), 6.7-6.3 (m,
511
12H), 5.9 (m, 1H), 5.7 (m,1H) 5.5 (m, 1H), 5.0-5.4 (m, 4H), 4.9 (t, 1H), 4.8 (m,2H),
512
4.5-4.0 (m, 4H), 3.6-3.7 (m, 2H), 3.0 (m, 1H),2.5-2.7 (m,4H), 1.8-2.3 (m, 9H), 1.6 (m,
513
3H), 1.5 (m, 3H), 1.3 (m, 3H), 1.2 (m, 2H).
514
N-Fmoc-Nys 10. Starting from 100 mg (0.08 mmol) of nystatin and 70 mg (0.2 mmol)
515
of Fmoc-NHS, 112 mg (0.1 mmol, 90%) of the 10 product was obtained as a yellow
516
solid (Rf 0.45, CHCl3/MeOH/H2O, 4/1/0.1, v/v/v). HRMS-ESI found m/z 1148.5803
517
[M+H]+. calcd. for C62H85NO19 1147.5716. 1H NMR (500 MHz, pyridine-d5) δ: 8.0 (s,
518
1H), 7.8 (d, J=7.6Hz 2H), 7.6 (m, 2H), 7.4 (m, 2H), 7.2 (m, 2H), 6.6 (m, 2H), 6.0-6.4
519
(m, 12H), 5.6-5.9 (m, 4H), 5.3 (m, 2H), 5.1 (m, 3H), 5.0 (s, 1H), 4.9 (bd, 1H), 4.8
520
(m,1H), 4.5-4.6 (m, 4H), 4.4 (m, 1H), 4.3 (m, 1H), 4,2 (m, 1H), 3.8 (m, 1H), 3.7 (m,
521
1H), 3.6 (m, 1H), 3.5 (m, 1H),3.4 (s, 1H), 1.8-2.8 (m, 26H), 1.6 (m, 3H), 1.4 (m, 3H),
522
1.3 (m, 3H), 1.1 (m, 4H).
24 ACS Paragon Plus Environment
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Bioconjugate Chemistry
523 524
TDBTU ester of N-Fmoc-polyene macrolide – general procedure.12 To the solution of
525
Fmoc-polyene macrolide (0.18 mmol) in dry DMF (5 mL), 0.29 mmol of
526
diisopropylethylamine was added, followed by 0.18 mmol of TDBTU. The reaction
527
mixture was stirred for 20 minutes at room temperature. Subsequently, the crude
528
product was precipitated with 200 mL of Et2O, filtered off and used without
529
purification in the next step.
530 531
O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N-Fmoc-amphotericin B 11. Starting
532
from 206 mg (0.18 mmol) of Fmoc-AmB 9 and 63 mg (0.18 mmol) of TDBTU, 220 mg
533
(0.17 mmol, 93%) of TDBTU active ester 11 was obtained as a solid (Rf 0.48,
534
CHCl3/MeOH/H2O, 65/10/1, v/v/v). HRMS-ESI found m/z 1289.5742 [M-1]-. calcd. for
535
C69H86N4O20 1290.5835. 1H NMR (500 MHz, CDCl3) 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),
537
7.00-7.30 (m, 3H), 5.90-6.40 (m, 14H), 5.30 (bd, 1H) 5.20 (m, 1H), 4.10-4.40 (m, 4H),
538
3.00-3.90 (m, 10H), 1.40-2.60 (m, 18H), 1.20-2.40 (m, 17H), 1.10 (d, J=6.4Hz, 3H),
539
1.00 (d, J=6.4Hz, 3H), 0.90 (d, J=8.3Hz 3H).
540
O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N-Fmoc-nystatin A 12. Starting from
541
199 mg (0.173 mmol) of Fmoc-Nys 10 and 60 mg (0.173 mmol) of TDBTU, 146 mg
542
(0.113 mmol, 65%) of TDBTU active ester 12 was obtained as a solid (Rf 0.46,
543
CHCl3/MeOH/H2O, 65/10/1, v/v/v). HRMS-ESI found m/z 1291.6053 [M-H]-. calcd. for
544
C69H88N4O20 1292.5992. 1H NMR (500 MHz, CDCl3) δ: 8.38 (d, J=7.6 Hz, 1H), 8.15
545
(d, J= 8.2 Hz, 1H), 7.89 (dd, J=9.8, 4.9 Hz, 1H), 7.84-7.68 (m, 3H), 7.59-7.45 (m, 1H),
546
7.45-7.20 (m, 4H), 6.57-6.01 (m, 12H), 5.51-5.26 (m, 2H), 5.34 (dd, J=14.5 and 10.3
547
Hz, 1H), 4.44 (d, J=9.5 Hz, 1H), 4.23-4.09 (m, 2H), 4.07 (d, J= 6.8 Hz, 1H), 3.79-3.60
25 ACS Paragon Plus Environment
Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 26 of 39
548
(m, 3H), 3.53-3.40 (m, 1H), 3.19 (dt, J= 14.9 and 7.5 Hz, 2H), 3.01 (s, 2H), 2.88 (d,
549
J= 0.6 Hz, 2H), 2.77-2.56 (m, 2H), 2.51-2.11 (m, 4H), 1.93 (dd, J= 13.4 and 9.4 Hz,
550
1H), 1.90-1.45 (m, 6H), 1.46-1.24 (m, 10H), 1.20 (d, J= 6.4 Hz, 3H), 1.12 (d, J= 6.4
551
Hz, 3H), 1.03 (t, J= 8.3 Hz, 3H).
552
Diwalled molecular umbrella/ω ω-amino acid/polyene conjugates – General
553
Procedure. To a solution of TDBTU ester of Fmoc-polyene (40.8 µmol) in dry DMF,
554
39.7 µmol of a molecular umbrella/ω-amino acid conjugate in dry 2 ml DMF
555
containing 2.3 mmol of dry Et3N was added dropwise. The reaction mixture was
556
stirred for 5 hours at room temperature. Subsequently, 1.5 µmol of piperidine was
557
added and the mixture was stirred at room temperature overnight. Next, the crude
558
product was precipitated with Et2O, filtered off and purified by preparative PLC
559
chromatography.
560 561
Diwalled deoxycholic molecular umbrella/3-aminopropanoic acid/AmB conjugate 13a.
562
Starting from 38 mg of molecular umbrella 8a and 53 mg of TDBTU active ester of
563
Fmoc-AmB 11, 16 mg (0.008 mmol, 20%) of conjugate 13a was obtained (Rf 0.13,
564
CHCl3/MeOH/H2O, 65/10/1, /v/v). HRMS-ESI found m/z 1871.2412 [M+H]+. calcd. for
565
C105H171N5O23 1870.2365. 1H NMR (500 MHz, CDCl3/CD3OD, 1/3) δ: 6.0-6.5 (m,
566
14H), 5.3-5.4 (m, 2H), 3.0-4.6 (m, 27H), 3.9 (bs, 4H), 1.4-2.6 (m, 104H), 0.9-1.4 (m,
567
20H), 0.7 (s, 6H).
568
Diwalled deoxycholic molecular umbrella/4-aminobutanoic acid/AmB conjugate 13b.
569
Starting from 39 mg of molecular umbrella 8b and 53 mg of TDBTU active ester of
570
Fmoc-AmB 11, 63 mg (0.03 mmol, 80%) of conjugate 13b was obtained (Rf 0.13,
571
CHCl3/MeOH/H2O, 65/10/1, v/v/v). HRMS-ESI found m/z 1885.2586 [M+H]+. calcd.
572
for C106H173N5O23 1884.2521. 1H NMR (500 MHz, CDCl3/CD3OD, 1/3) δ: 6.0-6.5 (m, 26 ACS Paragon Plus Environment
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Bioconjugate Chemistry
573
14H), 5.3-5.4 (m, 2H), 3.0-4.6 (m, 27H), 3.9 (bs, 4H), 2.9 (m, 2H) 1.4-2.6 (m, 104H),
574
0.9-1.4 (m, 20H), 0.7 (s, 6H).
575
Diwalled deoxycholic molecular umbrella/6-aminohexanoic acid/AmB conjugate 13c.
576
Starting from 40 mg of molecular umbrella 8c and 53 mg of TDBTU active ester of
577
Fmoc-AmB 11, 27 mg (0.01 mmol, 34%) of conjugate 13c was obtained (Rf 0.13,
578
CHCl3/MeOH/H2O, 65/10/1, v/v/v). HRMS-ESI found m/z 1913.2872 [M+H]+. calcd.
579
for C108H177N5O23 1912.2834. 1H NMR (500 MHz, CDCl3/CD3OD, 1/3) δ: 6.0-6.5 (m,
580
14H), 5.3-5.4 (m, 2H), 3.0-4.6 (m, 25H), 3.9 (bs, 4H), 1.4-2.6 (m, 105H), 1.0-1.4 (m,
581
20H), 0.9 (s, 6H), 0.7 (s, 6H).
582
Diwalled deoxycholic molecular umbrella/8-aminooctanoic acid/AmB conjugate 13d.
583
Starting from 41 mg of molecular umbrella 8d and 53 mg of TDBTU active ester of
584
Fmoc-AmB 11, 43 mg (0.02 mmol, 54%) of conjugate 13d was obtained (Rf 0.13,
585
CHCl3/MeOH/H2O, 65/10/1, v/v/v). HRMS-ESI found m/z 1941.3156 [M+H]+. calcd.
586
for C110H181N5O23 1940.3147. 1H NMR (500 MHz, CDCl3/CD3OD, 1/3) δ: 6.0-6.5 (m,
587
14H), 5.3-5.4 (m, 2H), 3.0-4.6 (m, 32H), 3.9 (s, 2H), 3.8 (s, 2H), 0.9-2.4 (m, 123H),
588
0.7 (s, 6H).
589
Diwalled cholic molecular umbrella/4-aminobutanoic acid/AmB conjugate 13e.
590
Starting from 40 mg of molecular umbrella 8e and 53 mg of TDBTU active ester of
591
Fmoc-AmB 11, 18 mg (0.009 mmol, 22%) of conjugate 13e was obtained (Rf 0.13,
592
CHCl3/MeOH/H2O, 65/10/1, v/v/v). HRMS-ESI found m/z 1917.2433 [M+H]+. calcd.
593
for C106H173N5O25 1916.2420. 1H NMR (500 MHz, CDCl3/CD3OD, 1/3) δ: 6.0-6.5 (m,
594
14H), 5.3-5.4 (m, 2H), 3.0-4.6 (m, 27H), 3.9 (bs, 4H), 2.9 (m, 2H) 1.4-2.6 (m, 102H),
595
0.9-1.4 (m, 20H), 0.7 (s, 6H).
596
Diwalled cholic molecular umbrella/8-aminooctanoic acid/AmB conjugate 13f.
597
Starting from 42 mg of molecular umbrella 8f and 53 mg of TDBTU active ester of
27 ACS Paragon Plus Environment
Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 28 of 39
598
Fmoc-AmB 11, 32 mg (0,016 mmol, 40%) of conjugate 13f was obtained (Rf 0.13,
599
CHCl3/MeOH/H2O, 65/10/1, v/v/v). HRMS-ESI found m/z 1973.3061 [M+H]+. calcd.
600
for C110H181N5O25 1972.3046. 1H NMR (500 MHz, CDCl3/CD3OD, 1/3) δ: 6.0-6.5 (m,
601
14H), 5.3-5.4 (m, 2H), 3.0-4.6 (m, 25H), 3.9 (s, 2H), 3.8 (s, 2H), 1.4-2.6 (m, 90H),
602
1.2-1.4 (m, 25H), 1.21-0.9 (m, 16H), 0.7 (s, 6H).
603
Diwalled deoxycholic molecular umbrella/3-aminopropanoic acid/nystatin conjugate
604
14a. Starting from 38 mg of molecular umbrella 8a and 53 mg of TDBTU active ester
605
of Fmoc-Nys 12, 10 mg (0.005 mmol, 13%) of conjugate 14a was obtained (Rf 0.13,
606
CHCl3/MeOH/H2O, 65/10/1, v/v/v). HRMS-ESI found m/z 1873.2529 [M+H]+. calcd.
607
for C105H173N5O23 1872.2521. 1H NMR (500 MHz, CDCl3/CD3OD, 1/3) δ: 5.2-6.2 (m,
608
12H), 3.0-4.6 (m, 29H), 2.0-2.8 (m, 18H), 0.9-2.0 (m, 114H), 0.7 (s, 6H).
609
Diwalled deoxycholic molecular umbrella/4-aminobutanoic acid/nystatin conjugate
610
14b. Starting from 39 mg of molecular umbrella 8b and 53 mg of TDBTU active ester
611
of Fmoc-Nys 12, 58 mg (0.03 mmol, 73%) of conjugate 14b was obtained (Rf 0.13,
612
CHCl3/MeOH/H2O, 65/10/1, v/v/v). HRMS-ESI found m/z 1887.2696 [M+H]+. calcd.
613
for C106H175N5O23 1886.2678. 1H NMR (500 MHz, CDCl3/CD3OD, 1/3) δ: 5.2-6.2 (m,
614
12H), 3.0-4.6 (m, 29H), 2.0-2.8 (m, 20H), 0.9-2.0 (m, 114H), 0.7 (s, 6H).
615
Diwalled deoxycholic molecular umbrella/6-aminohexanoic acid/nystatin conjugate
616
14c. Starting form 40 mg of molecular umbrella 8c and 53 mg of TDBTU active ester
617
of Fmoc-Nys 12, 21 mg (0.01 mmol, 27%) of conjugate 14c was obtained (Rf 0.13,
618
CHCl3/MeOH/H2O, 65/10/1, v/v/v). HRMS-ESI found m/z 1915.3124 [M+H]+. calcd.
619
for C108H179N5O23 1914.2991. 1H NMR (500 MHz, CDCl3/CD3OD, 1/3) δ: 5.2-6.2 (m,
620
12H), 3.0-4.6 (m, 25H), 3.9 (bs, 4H), 1.3-2.4 (m, 103H), 0.9-1.3 (m, 35H), 0.7 (s, 6H).
621
Diwalled deoxycholic molecular umbrella/8-aminooctanoic acid/nystatin conjugate
622
14d. Starting from 41 mg of molecular umbrella 8d and 53 mg of TDBTU active ester 28 ACS Paragon Plus Environment
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Bioconjugate Chemistry
623
of Fmoc-Nys 12, 36 mg (0.02 mmol, 45%) of conjugate 14d was obtained (Rf 0.13,
624
CHCl3/MeOH/H2O, 65/10/1, v/v/v). HRMS-ESI found m/z 1943.3346 [M+H]+. calcd.
625
for C110H183N5O23 1942.3304. 1H NMR (500 MHz, CDCl3/CD3OD, 1/3) δ: 5.2-6.2 (m,
626
12H), 2.9-4.6 (m, 37H), 1.2-2.6 (m, 96H), 1.2-0.8 (m, 34H) 0.7 (s, 6H).
627
Diwalled cholic molecular umbrella/4-aminobutanoic acid/nystatin conjugate 14e.
628
Starting from 40 mg of molecular umbrella 8e and 53 mg of TDBTU active ester of
629
Fmoc-Nys 12, 12 mg (0.006 mmol, 15%) of conjugate 14e was obtained (Rf 0.13,
630
CHCl3/MeOH/H2O, 65/10/1, v/v/v). HRMS-ESI found m/z 1919.2657 [M+H]+. calcd.
631
for C106H175N5O25 1918.2576. 1H NMR (500 MHz, CDCl3/CD3OD, 1/3) δ: 5.0-6.2 (m,
632
12H), 3.0-4.6 (m, 25H), 3.9 (s, 2H), 3.8 (s, 2H), 1.2-2.6 (m, 115H), 1.2-0.9 (m, 16H),
633
0.7 (s, 6H).
634
Diwalled cholic molecular umbrella/8-aminooctanoic acid/nystatin conjugate 14f.
635
Starting from 42 mg of molecular umbrella 8f and 53 mg of TDBTU active ester of
636
Fmoc-NysA 12, 20 mg (0.01 mmol, 25%) of conjugate 14f was obtained (Rf 0.13,
637
CHCl3/MeOH/H2O, 65/10/1, v/v/v). HRMS-ESI found m/z 1975.3222 [M+1]+. calcd. for
638
C110H183N5O25 1974.3202. 1H NMR (500 MHz, CDCl3/CD3OD, 1/3) δ: 5.0-6.2 (m,
639
12H), 3.0-4.6 (m, 25H), 3.9 (s, 2H), 3.8 (s, 2H), 1.4-2.6 (m, 90H), 1.2-1.4 (m, 28H),
640
1.21-0.9 (m, 16H), 0.7 (s, 6H).
641 642
Determination of Critical Aggregation Concentration (cac) of Amphotericin B
643
and its Conjugates. Stock solutions of AmB and its conjugates (1 mM) were
644
prepared in DMSO. Aliquots of 75 µL were serially diluted with DMSO and then
645
introduced into test tubes containing 4.925 mL of phosphate-buffered saline (PBS),
646
pH 7.4 at 37°C, to give concentrations ranging from 0.2 to 15 µM. After vortex mixing
647
for 10 s, the solutions were transferred to a 1.60 mL UV cuvette that was maintained
29 ACS Paragon Plus Environment
Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 30 of 39
648
at 37oC. The UV spectrum was then recorded in the range of 250-500 nm. The
649
absorbance at 409 nm was plotted as a function of the reciprocal value of the
650
concentration of AmB or a conjugate and the critical aggregation concentration of
651
compound tested was determined graphically.
652
Determination of Aggregation Level of Nystatin and its Conjugates by Steady-
653
State Fluorescence Anisotropy. Stock solutions of NYS and its conjugates (1 mM)
654
were prepared in DMSO. Aliquots of 75 µL were serially diluted with DMSO and then
655
introduced into test tubes containing 4.925 mL of phosphate-buffered saline (PBS),
656
pH 7.4 at 37°C, to give concentrations ranging from 0.2 to 15 µM. After vortex mixing
657
for 10 s, the solutions were transferred to 0.5 cm path length quartz cuvettes.
658
Fluorescence intensities were measured with a FluoroMax-4 Horiba
659
spectrofluorimeter with a double emission monochromator and a thermostated
660
cuvette holder (37 ± 1°C). Fluorescence intensities were measured at 315 nm
661
excitation and 415 nm emission wavelengths, with spectral bandwidths of 0.9 nm and
662
9.0 nm, respectively.
663
The steady-state anisotropy, , defined by the relationship24 < > =
−
+ 2
664
was obtained by measuring the vertical and horizontal components of the
665
fluorescence emission with excitation vertical (IVV and IVH, respectively) and
666
horizontal (IHV and IHH, respectively) to the emission axis. The G factor (G = IHV/IHH)
667
corrects for the transmissivity bias introduced by the detection system.
668 669
Microbial Strains and Culture Conditions. The reference strains used in this study
670
were: Candida albicans ATCC 10231, Candida albicans SC 5314, Candida glabrata
671
DSM 11226, Candida krusei DSM 6128, Candida parapsilosis DSM 578, 30 ACS Paragon Plus Environment
Page 31 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Bioconjugate Chemistry
672
Saccharomyces cerevisiae ATCC 9763 and Escherichia coli ATCC 9637. C. albicans
673
B3, B4, Gu4 and Gu5 clinical isolates were kindly provided by Joachim
674
Morschhäuser, Würzburg, Germany. Gu4 and B3 are fluconazole-sensitive isolates
675
obtained from early infection episodes, while Gu5 and B4 are the corresponding
676
fluconazole-resistant isolates obtained from later episodes in the same patients
677
treated with fluconazole.25 Yeast strains were grown at 30°C in YPD medium (2%
678
glucose, 1% Yeast Extract and 1% Bacto Peptone) and stored on YPD agar plates
679
containing 2% agar. E. coli cells were grown at 37°C in LBB medium (0.5% Yeast
680
Extract, 1% Tryptone, 1% NaCl).
681 682
Antifungal In vitro Activity Determination. Susceptibility testing was performed by
683
the serial two-fold dilution method, using the 96-well microtiter plates, in RPMI-1640
684
medium w/o sodium bicarbonate, with L-glutamine + 2% glucose + 3.45% MOPS, pH
685
adjusted to 7.0, under conditions outlined in the CLSI recommendations26 except for
686
the end-point readout that was done by spectrophotometric determination of cell
687
density at 660 nm. Turbidity in individual wells was measured with a microplate
688
reader (Victor3; Perkin Elmer). The in vitro growth inhibitory activity of antifungals was
689
quantified by determination of MIC80 values that were defined as the lowest drug
690
concentration that gave at least an 80% decrease in turbidity, relative to that of the
691
drug-free growth control.
692 693
Hemolysis Assay. Whole human blood (sodium heparin) was obtained from the
694
Gdańsk Regional Blood Donation Centre, stored at 4 °C and used within two days of
695
receipt. To a 50 mL Falcon tube, 25 mL of whole human blood was added and
696
centrifuged at 10,000 g for 2 minutes. The supernatant was removed and the
31 ACS Paragon Plus Environment
Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 32 of 39
697
erythrocyte pellet was washed with 20 mL of sterile saline and centrifuged at 10,000
698
g for 2 minutes. The saline wash was repeated for a total of three washes. The
699
erythrocyte pellet was suspended in 25 mL of saline to form the erythrocyte stock
700
suspension. The stock solution was diluted with saline, to give suspension of 2×107
701
cells mL-1 (hemocytometer count).
702
The stock 1 mg mL-1 solutions of AmB, NYS and the conjugates were prepared in
703
DMSO and 50 µL aliquots of serial two-fold dilutions were placed in Eppendorf tubes.
704
Tubes containing 50 µL of DMSO and 50 µL of 2% aqueous Triton X-100 solution
705
were included as a negative and positive control, respectively. To each tube, 950 µL
706
of the erythrocyte suspension was added and mixed by inversion to give the final
707
concentrations of compounds tested in the 100 – 0.78 µg mL-1 range. The samples
708
were incubated at 37 °C for 30 min, then mixed by inversion and centrifuged at
709
10,000 g for 5 minutes. Samples of the supernatants (100 µL) were carefully
710
collected and added to individual wells in 96-well microplates. Absorbances were
711
read at 540 nm using a microplate reader. Experiments were performed in triplicate
712
and the reported EH50 values represent an average of three experiments.
713
The results obtained for each compound were plotted as A540 = f(c) and the
714
interpolated values of concentrations at which A540 reached 50% of the value noted
715
for the positive control (sample containing 0.1% Triton X-100) were taken as EH50.
716 717
Cytotoxicity Assay. Cell culture media, antibiotics and serum were from Corning.
718
Stock solutions of AmB, nystatin and conjugates were freshly prepared by dissolving
719
compounds in DMSO. All cells were from ATCC and showed no mycoplasma
720
contamination as revealed by Roche ELISA-based test. Cytotoxicity parameters were
721
determined against three mammalian cell lines: Hep G2, human liver hepatocellular 32 ACS Paragon Plus Environment
Page 33 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Bioconjugate Chemistry
722
carcinoma; LLC-PK1, normal, epithelial adherent kidney proximal tubule cells isolated
723
from kidney of 3-4 weeks old male pig; HEK-293T, human embryonic kidney cells.
724
Multiwell (24–well) plates were seeded at 2.5 × 104 LLC-PK1 cells/well in Medium
725
199 supplemented with 5% FBS or 4×104 Hep G2 cells/well in MEM Eagle medium
726
supplemented with 10% FBS or 1.25×104 HEK-293T cells/well in DMEM medium
727
supplemented with 10% FBS. All media were supplemented with L-glutamate and
728
antibiotics (penicillin/streptomycin, 100 µg/mL). Cells were allowed to attach
729
overnight. Compounds tested were added to wells in 10 µL aliquots of 200-times
730
concentrated solutions in DMSO. To control wells, 10 µL of DMSO was added. Cells
731
were incubated with studied compounds for 72 h at 37oC and 95%/5% CO2
732
atmosphere. Subsequently, 200 µL aliquots of MTT solution in PBS (4 mg/ml) were
733
added to each well and plates were further incubated for 1–4 h at 37oC. Absorbance
734
was measured after solubilization of formazan crystals in 1 mL DMSO, using a
735
multiwell plate reader at λ = 540 nm. Cytotoxicity was determined compared to non-
736
treated cells (% control). All experiments were performed in biological duplicates.
737 738
Determination of Binding to Intact Cells. C. albicans SC 5314 cells were grown to
739
the mid-logarithmic phase in YPD medium at 30°C. As a negative control, the E. coli
740
cells were grown to the logarithmic phase in 100 mL of LBB medium at 37°C. The
741
cells were harvested by centrifugation (rt, 3 500 × g, 10 min.), washed twice with PBS
742
and re-suspended in a small volume of PBS. A series of 2 mL cell suspensions were
743
prepared ranging from an A660 of 0 to 2.0. The suspensions were centrifuged (rt,
744
3 500 × g, 10 min.) and re-suspended in the same volume of PBS containing 30 µM
745
AmB, NYS or any of the conjugates. Cells re-suspended in PBS served as the zero
746
control and solutions of AmB, NYS or any of the conjugates in PBS as the positive 33 ACS Paragon Plus Environment
Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
747
controls. The cell suspensions were incubated for 1 h at rt with shaking (500 rpm)
748
and then spun down (rt, 3 500 × g, 10 min.). The amount of AmB, NYS or any
749
conjugate remaining in the supernatants was determined by UV-vis absorption
750
measurements at 409 nm.
Page 34 of 39
751 752 753 754
ASSOCIATED CONTENT
755
Supporting Information
756
Supplementary materials providing NMR spectra and results of HPLC analysis
757
(PDF).
758 759
AUTHOR INFORMATION
760
Corresponding Author
761
*E-mail address:
[email protected] 762
ORCID
763
Sławomir Milewski: 0000-0003-2616-4495
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Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGEMENTS
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This work was supported by the UMO-2014/13/B/NZ7/02305 grant from the Polish
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National Science Centre to MJM. The authors are grateful to Dr. Katarzyna
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Kozłowska-Tylingo and Dr. Paweł Kubica for their help in analysis and preparative
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separation by HPLC.
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REFERENCES
775
(1) Brown, G. D., Denning, D. W., Gow, N. A. R., Levitz, S. M., Netea, M. G. and
776
White, T.C. (2012) Hidden killers: Human fungal infections. Sci. Transl. Med. 4,
777
1-9.
778
(2) Wisplinghoff, H., Bischoff, T., Tallent, S. M., Seifert, H., Wenzel, R. P. and
779
Edmond, M. B. (2004) Nosocomial bloodstream infections in US hospitals:
780
Analysis of 24,179 cases from a prospective nationwide surveillance study. Clin.
781
Infect. Dis. 39, 309–317.
782
(3) Gray, K. C., Brajtburg, J., Powderly, W. G., Kobayashi, G.S. and Medoff, G.
783
(1990) Amphotericin B: current understanding of mechanisms of action.
784
Antimicrob. Agents Chemother. 34 183–188.
785
(4) Palacios, D. S., Dailey, I., Endo, M. M., Uno, B. E., Wilcock, B. C. and Burke, M.
786
D. (2012) Amphotericin primarily kills yeast by simply binding ergosterol. Proc.
787
Natl. Acad. Sci. U.S.A. 109, 2234-2239.
788
(5) Anderson, T. M., Clay, M. C., Cioffi, A. G., Diaz, K. A., Hisao, G. S., Tuttle, M.D.,
789
Nieuwkoop, A. J, Comellas, G., Maryum, N,. Wang, S. et al. (2014) Amphotericin
790
forms an extramembranous and fungicidal sterol sponge. Nat. Chem. Biol. 10,
791
400−406.
792
(6) Legrand, P,. Romero, E. A., Cohen, B. E. and Bolard, B. Effects of aggregation
793
and solvent on the toxicity of amphotericin B to human erythrocytes. (1992)
794
Antimicrob. Agents Chemother. 36, 2518–2522.
35 ACS Paragon Plus Environment
Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
795
(7) Wright, J. J. K., Albarella, J. A., Krepski, L. R. and Loebenberg, D. (1982) N-
796
aminoacyl derivatives of polyene macrolide antibiotics and their esters. J.
797
Antibiot. 35, 911–914.
798
(8) Grzybowska, J., Sowiński, P., Gumieniak, J., Zieniawa, T. and Borowski, E.
799
(1997) N-methyl-N-D-fructopyranosylamphotericin B methyl ester, new
800
amphotericin B derivative of low toxicity. J. Antibiot. 50, 709–711.
801
(9) Volmer, A. A., Szpilman, A. M. and Carreira, E. M. (1997) Synthesis and
802
biological evaluation of amphotericin B derivatives. Nat. Prod. Rep. 27,
803
1329−1349.
804
(10) Wilcock, B. C., Endo, M. M., Uno, B. E. and Burke, M. D. (2013) C2'-OH of
805
amphotericin B plays an important role in binding the primary sterol of human
806
cells but not yeast cells. J. Am. Chem. Soc. 135, 8488–8491.
807
Page 36 of 39
(11) Davis, S. A., Vincent, B. M., Endo, M. M., Whitesell, L., Marchillo, K., Andes, D.
808
R., Lindquist, S. and Burke, M. D. (2015) Nontoxic antimicrobials that evade drug
809
resistance, Nat. Chem. Biol. 11, 481−487.
810 811
(12) Janout, V. Schell, W., Thévenin, D., Yu, Y., Perfect, J. R. and Regen, S. L. (2015) Taming Amphotericin B. Bioconjugate Chem. 26, 2012−2024.
812
(13) Janout, V., Bienvenu, C., Schell, W., Perfect, J. R. and Regen, S. L. (2014)
813
Molecular umbrella−Amphotericin B conjugates. Bioconjugate Chem. 25,
814
1408−1411.
815
(14) Szwarc, K., Płosiński, M., Czerniejewska, K., Laskowski, T., Leniak, A., Czub, J.,
816
Kubica, P., Sowiński, P., Pawlak, J. and Borowski, E. (2016) Intramolecular
817
transformation of an antifungal antibiotic nystatin A1 into its isomer, iso-nystatin
818
A1 – structural and molecular modeling studies. Magn. Reson. Chem. 54, 953–
819
961.
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820
Bioconjugate Chemistry
(15) Ostrosky-Zeichner, L., Bazemore, S., Paetznik, V.L., Rodriguez, J.R., Chen, E.,
821
Wallace, T., Cossum, P. and Rex, T.H. (2001) Differential antifungal activity of
822
isomeric forms of Nystatin. Antimicrob. Agents Chemother. 45, 2781–2786.
823
(16) Yu, Y., Sabulski, M. J., Schell, W. A., Pires, M. M., Perfect, J. R. and Regen,
824
S.L. (2016) Simple strategy for taming membrane-disrupting antibiotics.
825
Bioconjugate Chem. 27, 2850-2853.
826
(17) Yamashita, K., Janout, V., Bernard, E.,M., Armstrong, D. and Regen, S. L.
827
(1995) Micelle/monomer control over the membrane-disrupting properties of an
828
amphiphilic antibiotic, J. Am. Chem. Soc. 117, 6249–6253.
829
(18) Castanho, M.A.R.B., Coutinho, A. and Prieto, M.J.E. (1992) Absorption and
830
fluorescence spectra of polyene antibiotics in the presence of cholesterol, J. Biol.
831
Chem. 267, 204–209.
832
(19) Coutinho, A. and Prieto, M. (1995) Self-association of the polyene antibiotic
833
nystatin in dipalmitoylphosphatidylcholine vesicles: a time-resolved fluorescence
834
study. Biophys. J. 69, 2541–2557.
835
(20) Boros‑Majewska, J., Salewska, N., Borowski, E., Milewski, S,. Malic, S., Wei, X.,
836
Hayes, A.J., Wilson, M.J. and Williams, D. W. (2014) Novel Nystatin A1
837
derivatives exhibiting low host cell toxicity and antifungal activity in an in vitro
838
model of oral candidosis. Med. Microbiol. Immunol. 203, 341–355.
839
(21) Zou, X., Zhao, H., Fu, Y., Zhang, X. and Xu, P. (2003) Synthesis of Boc-
840
Asp(OBzl)-β-Ala-Asp(OBzl)-N(OMe)Me as a useful precursor of aspartyl peptide
841
aldehyde derivative. J. Chin. Pharm. Sci. 12, 123–126.
842
(22) Guenin, E., Manteil, M., Bouchemal, N., Prange, T. and Lecouvey, M. (2007)
843
Syntheses of phosphonic esters of alendronate, pamidronate and neridronate.
844
Eur. J. Org. Chem. 72, 3380–3391.
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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
845 846 847 848
849
Page 38 of 39
(23) Janout, V., Lanier, M. and Regen, S. L. (1997) Design and synthesis of molecular umbrellas. J. Am. Chem. Soc. 119, 640–647. (24) Lakowicz, J. R. (2006) Principles of Fluorescence Spectroscopy, 3rd Edition, Springer-Verlag, New York. (25) Franz, R., Kelly, S. L., Lamb, D. C., Kelly, D. E., Ruhnke, M. and Morschhäuser,
850
J. (1998) Multiple molecular mechanisms contribute to a stepwise development
851
of fluconazole resistance in clinical Candida albicans strains. Antimicrob. Agents
852
Chemother. 42, 3065–3072.
853
(26) Clinical Laboratory Standards Institute (2008). Reference method for broth
854
dilution antifungal susceptibility testing of yeasts. Second Edition. Approved
855
Standard M27-A3.Wayne, PA
856
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Graphic for TOC 82x84mm (120 x 120 DPI)
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