Molecular Umbrellas Modulate the Selective Toxicity of Polyene

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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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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

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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

29

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

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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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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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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

112

octawalled) umbrellas as the optimal components of conjugates was justified by the

113

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

116

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

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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-

121

protected derivatives 2a-d as crude products were applied in formation of N-

122

hydroxysuccinimide (NHS) active esters 3a-d, using the NHS/DCC method, followed

123

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

126

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

128

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,

131

i.e. molecular umbrellas carrying a non-labile ω-amino acid linker, necessary for

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coupling of a polyene macrolide molecule.

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Scheme 1. Synthesis of molecular umbrellas with ω-amino acid linkers. Reagents

135

and conditions. i) Na2CO3, H2O, 0°C, Boc2O, 1,4-dioxane, 1 h, 0°C->rt,

136

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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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

141

and 12, respectively, with O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N’,N’-

142

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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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

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condensation of active esters 11 and 12 with one of the molecular umbrella-linker

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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

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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

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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

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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.

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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

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molecular umbrellas and selective toxicity indexes. MIC80 values were

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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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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

-

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All conjugates exhibited lower anticandidal activity that their mother compounds, i.e.

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AmB and NYS, respectively. In the case of AmB conjugates, the lowest difference

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between their MIC80 values and that of AmB was found for compounds 13d, 13e and

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13f. In terms of molar concentrations, MICs of these compounds were almost the

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same as that of the mother antibiotic. Interestingly, for conjugates with deoxycholic

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acid-based molecular umbrellas (13a-13d), the length of the ω-amino acid linker

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determined the antifungal activity and the conjugates with short linkers, 13a (n = 1)

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and 13 b (n = 2) demonstrated much lower activity that their counterparts with longer

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linkers, 13c (n = 4) and 13 d (n = 6). In contrast, both conjugates with the cholic acid-

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based molecular umbrellas 13e (n = 2) and 13 f (n = 6) exhibited similar antifungal

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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

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than that of NYS. Paradoxically, the lowest activity was found for the conjugate 14f,

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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

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were less hemolytic than AmB, while the EH50 values of the latter were lower than

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that of NYS. Again, the length of the ω-amino acid linker determined the hemolytic 9 ACS Paragon Plus Environment


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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

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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

206

derivatives, the EH50 values were lower than MICs80, and a selective toxicity of all

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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

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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.

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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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In general, the difference between MICs of the conjugates 13d and 13f and that of

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AmB was higher for Candida spp. other than C. albicans, especially in the case of C.

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tropicalis. Interestingly enough, MICs of these conjugates against the C. albicans B4

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strain, multidrug resistant due to the overexpression of the MDR1 gene, were 2-4

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times higher than those against non-resistant C. albicans B3, while in the case of the

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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

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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

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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

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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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250 251 252

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

281

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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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.

305 15 ACS Paragon Plus Environment


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


Page 17 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


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



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


Page 19 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


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


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


Page 21 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


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



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


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


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


Page 23 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


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


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


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



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).


Page 25 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


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



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

Page 27 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


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


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


586


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


593


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



Page 28 of 39

598

Fmoc-AmB 11, 32 mg (0,016 mmol, 40%) of conjugate 13f was obtained (Rf 0.13,

599


600


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


607


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


613


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


619


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

Page 29 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


623

of Fmoc-Nys 12, 36 mg (0.02 mmol, 45%) of conjugate 14d was obtained (Rf 0.13,

624


625


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


631


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



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


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



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


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


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

764

Notes

765

The authors declare no competing financial interest.

766 767

ACKNOWLEDGEMENTS

768

This work was supported by the UMO-2014/13/B/NZ7/02305 grant from the Polish

769

National Science Centre to MJM. The authors are grateful to Dr. Katarzyna

770

Kozłowska-Tylingo and Dr. Paweł Kubica for their help in analysis and preparative

771

separation by HPLC.


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772 773 774

REFERENCES

775

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776

White, T.C. (2012) Hidden killers: Human fungal infections. Sci. Transl. Med. 4,

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1-9.

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779

Edmond, M. B. (2004) Nosocomial bloodstream infections in US hospitals:

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Analysis of 24,179 cases from a prospective nationwide surveillance study. Clin.

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(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.

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(4) Palacios, D. S., Dailey, I., Endo, M. M., Uno, B. E., Wilcock, B. C. and Burke, M.

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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

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forms an extramembranous and fungicidal sterol sponge. Nat. Chem. Biol. 10,

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400−406.

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(6) Legrand, P,. Romero, E. A., Cohen, B. E. and Bolard, B. Effects of aggregation

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794

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(1997) N-methyl-N-D-fructopyranosylamphotericin B methyl ester, new

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amphotericin B derivative of low toxicity. J. Antibiot. 50, 709–711.

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(9) Volmer, A. A., Szpilman, A. M. and Carreira, E. M. (1997) Synthesis and

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biological evaluation of amphotericin B derivatives. Nat. Prod. Rep. 27,

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1329−1349.

804

(10) Wilcock, B. C., Endo, M. M., Uno, B. E. and Burke, M. D. (2013) C2'-OH of

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amphotericin B plays an important role in binding the primary sterol of human

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cells but not yeast cells. J. Am. Chem. Soc. 135, 8488–8491.

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(11) Davis, S. A., Vincent, B. M., Endo, M. M., Whitesell, L., Marchillo, K., Andes, D.

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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.,

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Kubica, P., Sowiński, P., Pawlak, J. and Borowski, E. (2016) Intramolecular

817

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818

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Wallace, T., Cossum, P. and Rex, T.H. (2001) Differential antifungal activity of

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824

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827

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amphiphilic antibiotic, J. Am. Chem. Soc. 117, 6249–6253.

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(18) Castanho, M.A.R.B., Coutinho, A. and Prieto, M.J.E. (1992) Absorption and

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fluorescence spectra of polyene antibiotics in the presence of cholesterol, J. Biol.

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(19) Coutinho, A. and Prieto, M. (1995) Self-association of the polyene antibiotic

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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.,

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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.

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(21) Zou, X., Zhao, H., Fu, Y., Zhang, X. and Xu, P. (2003) Synthesis of Boc-

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Asp(OBzl)-β-Ala-Asp(OBzl)-N(OMe)Me as a useful precursor of aspartyl peptide

841

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Molecular Umbrellas Modulate the Selective Toxicity of Polyene

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