Bioinspired Amphiphilic Peptide Dendrimers as Specific and Effective Compounds against Drug Resistant Clinical Isolates of E. coli

Article abstract of DOI:10.1021/acs.bioconjchem.8b00544

Evolution-derived natural compounds have been inspirational for design of numerous pharmaceuticals, e.g., penicillins and tetracyclines. Herein, we present a bioinspired strategy to design peptide dendrimers for the effective therapy of E. coli infections where the selection of appropriate amino acids and the mode of their assembly are based on the information gained from research on membranolytic natural antimicrobial peptides (AMP's). On the molecular level two opposite effects were explored: the effect of multiple positive charges necessary for membrane disintegration was equilibrated by the anchoring role of tryptophanes. Indeed, a series of Trp-terminated dendrimers exhibited high potency against clinical isolates of antibiotic resistant ESBL E. coli strains, stability in human plasma along with very low hemo- and genotoxicity. Investigation of the underlying antimicrobial mechanism indicated that the dendrimers studied at minimal inhibitory concentration showed weak permeability toward membranes. Solid-state 2D NMR studies revealed their presence on and inside the model membranes. Therefore, their biological properties might be explained by targeting of extra- or intracellular receptors. Our results point to a new approach to design novel branched antimicrobials with high therapeutic index.

Full text of DOI:10.1021/acs.bioconjchem.8b00544

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Article
Bioinspired Amphiphilic Peptide Dendrimers as Specific and Effective
Compounds Against Drug Resistant ClinicalIisolates of E. coli
Marta Sowinska, Anna Laskowska, Adam Gu#piel, Jolanta Solecka, Marta Bochy#ska-
Czy#, Andrzej W. Lipkowski, Katarzyna Trzeciak, and Zofia Urbanczyk-Lipkowska
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00544 • Publication Date (Web): 21 Sep 2018
Downloaded from http://pubs.acs.org on September 22, 2018
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Bioinspired Amphiphilic Peptide Dendrimers as Specific and Effective
Compounds Against Drug Resistant Clinical Isolates of E. coli
†
‡
‡
‡
Marta Sowińska, Anna Laskowska, Adam Guśpiel, Jolanta Solecka, Marta Bochynskaꢀ
§
§
†
,&
†
Czyż, Andrzej W. Lipkowski, Katarzyna Trzeciak, Zofia UrbanczykꢀLipkowska*
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†
Institute of Organic Chemistry PAS, Kasprzaka Str. 44/54, Warsaw 01ꢀ224, Poland
National Institute of Public Health–National Institute of Hygiene, Chocimska Str. 24,
‡
Warsaw 00ꢀ791, Poland
§
Mossakowski Medical Research Centre PAS, Pawinskiego Str. 5, 02ꢀ106 Warsaw,
Poland
&
Centre of Molecular and Macromolecular Studies PAS, Sienkiewicza 112, 90ꢀ363 Lodz,
Poland
Corresponding author: Zofia UrbanczykꢀLipkowska
Institute of Organic Chemistry PAS,
Kasprzaka Str. 44/52
01ꢀ224 Warsaw, Poland
Eꢀmail: zofia.lipkowska@icho.edu.pl
Tel. +48 (22) 343 2207
ABSTRACT
Evolutionꢀderived natural compounds have been inspiration for design of numerous
pharmaceuticals, e.g. penicillins, tetracyclines, etc. Herein, we present a bioinspired strategy
to design peptide dendrimers for the effective therapy of E. coli infections where the selection
of appropriate amino acids and the mode of their assembly are based on the information
gained from research on membranolytic natural antimicrobial peptides (AMP’s). On
molecular level two opposite effects were explored: effect of multiple positive charges
necessary for membrane disintegration was equilibrated by anchoring role of tryptophanes.
Indeed, a series of Trpꢀterminated dendrimers exhibited high potency against clinical isolates
of antibiotic resistant ESBL E. coli strains, stability in human plasma along with very low
hemoꢀ and genotoxicity. Investigation of the underlying antimicrobial mechanism indicated
that studied dendrimers at MIC showed weak permeability towards membranes. Solid state
2
D NMR studies revealed their presence on and inside of the model membranes. Therefore,
their biological properties might be explained by targeting of extraꢀ or intracellular receptors.
Our results point to a new approach to design novel branched antimicrobials with high
therapeutic index.
KEYWORDS:
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Bioinspired; antibiotic resistance; dendrimers; Trpꢀrich, E.coli; ESBL, solid state NMR;
membrane
INTRODUCTION
Development of drug resistance and shortage of suitable antibiotics cause that microbial
infections are becoming one of the challenging diseases encountered in clinical practice.
Herein, we present a bioinspired strategy to design peptide dendrimers for the effective
therapy of E. coli infections where both, selection of appropriate amino acids and mode of
their assembly are based on information gained from research on natural antimicrobial
peptides (AMP’s). AMP’s are an essential component of the innate immunity system
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protecting all forms of life against microbial, fungal, viral, etc. infections. Their advantage
over clinical antibiotics is multiple mechanism of action that makes the resistance to these
molecules uncommon. The remarkable feature of AMPs with broad spectrum of activity is a
3D sorting of cationic and hydrophobic amino acid side chains during supramolecular
interactions with negatively charged phospholipids distributed on the microbial surface. As
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revealed by structural studies of AMP’s, performed by NMR, circular dichroism (CD),
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and Xꢀray studies,
in presence of membrane models majority of the studied linear 10ꢀ50ꢀ
peptides undergo conformational conversion to proteinꢀlike secondary structures (helical, βꢀ
sheet, etc.), resulting in perturbation of microbial membrane and at least inhibition of
microbial growth. Several recent clinical trials of synthetic and semiꢀsynthestic analogs of
16
AMPs show a promising new perspective. However, adverse effects after systemic
administration often restrict this class of compounds to topical use only.
Nanometer sized molecules with branched structure and multiple cationic surface
groups called dendrimers represent another promising alternative to fight pathogen infections.
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7ꢀ
Due to their prevailing homofunctional structure, antimicrobial dendrimers like PAMAMs,
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20,21
22ꢀ23
PPIs,
poly(Lꢀlysine), carbosilane,
and viologenꢀphosphorus etc., are very potent but
often not selective antimicrobials. However, dendrimeric scaffolds can be functionalized with
a variety of active biomolecules providing specificity of binding to particular cells, i.e. with
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4ꢀ28
29ꢀ33
MAPs
or glycodendrimers designed as selective inhibitors of the adhesion process.
Another approach that we successfully applied was to design broadly active small
dendrimeric mimics of AMP’s. In these molecules, basic amino acids (Lys, Orn, Arg)
embedded in the dendrimer structure with tailorꢀmade localization of lipophilic protective
groups (or amino acid side chains) yielded molecules with both variableꢀlength branching and
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cationic, amphipatic character. This is a strategy of high potential as such peptide mimics
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cannot be prepared as recombinant products by ribosomal syntheses or by combinatorial
methods, since these methods produce only natural (linear) AMPs.
The analysis of recent data relating structure and function of designed AMP analogs
with low toxicity turned our attention to a group of peptides with two cationic fragments
connected by lipophylic sequence. For example, several studies by Deber et al., indicated that
in a relatively small linear 11ꢀ or 19ꢀpeptides, the positively charged terminal amino acid
sequences (consisting of Lys and/or Arg) separated by a peptide linker prepared from
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hydrophobic amino acids yielded highly active and nonꢀhemolytic derivatives.
In contrast,
peptides with substitution of terminal lysines by arginines and replacement of alanine by
leucine in the central part (enhancement of hydrophobicity) resulted in a significant increase
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of hemolysis. Similarly, Shai et al., detected very high hemolysis for the cationic 15ꢀpeptide
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KKKLLLLLLLLLKKKꢀNH sequence. Moreover, the very short but less charged peptides
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(RWRWRWꢀNH or RRRWWWꢀNH ), designed by Svendsen et al., expressed high
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antimicrobial potency with relatively mild hemolysis. The above data show that the
antibacterial activity and most of all selectivity of the cationic peptides can be modulated to a
large extend through alteration of the peptide's hydrophobic fragments. As it was shown
above, not only cationic character but also selection of appropriate amino acids with
lipophylic side chains significantly adds to their antimicrobial profile. Within a collection of
short AMPs, the interesting properties of indolicidin, its synthetic analog omiganan, and other
40ꢀ42
short Trpꢀrich peptides also pointed to an intriguing biological role of tryptophane.
On the molecular level two opposite effects known from the structureꢀactivity studies
of AMP’s were reproduced using the dendrimer tree. Presence in the molecule of multiple
positive charges that is necessary for microbial membrane disintegration (often nonselective)
was equilibrated by introduction of several copies of tryptophane that is known for its
anchoring properties (membrane healing). Moreover, extended hydrophobic fragments were
introduced into the central part of the dendrimer that according to the above mentioned
studies might reduce toxicity.
On the basis of the above foundations, we hypothesized that small amphiphilic peptide
dendrimers designed as AMP mimics, constructed from hydrophobic cores and flexible,
charged arms would exhibit enhanced and more selective antimicrobial activity. We also
anticipated that presence of tryptophan might help to immobilize dendrimers at the lipid/water
interface and therefore, reduce toxicity. Indeed, in this paper we report the successful creation
of the first bioinspired, targetꢀspecific active per se antimicrobial dendrimers, via rational
design of their molecular structure that is based on molecular characteristics of AMP’s, as
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regards choice of suitable amino acids and 3D structure. In order to meet the above criteria the
resulting series of amphiphilic dendrimers include novel hydrophobic, resistant to protonation
2 1 2
cores, constructed from a new branching units of equal (AB ) or variable (AB B ) arm
lengths. The branches are functionalized either with Zꢀ or (2ꢀClꢀZ)ꢀsubstituted lysine or
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variable net charge, arm lengths, lipophilicity and topology of the surface groups as well as
variability of residues located at Cꢀterminus.
To assess the effect of such structural features on the microbicidal activity, in vitro
screening against broad spectrum of the reference bacterial strains was performed. Their
toxicity against red blood cells, human melanoma and fibroblast cell lines was assayed. For
several representative compounds, the minimal inhibitory concentration (MIC) values were
assayed in human serum. For the compound 46, which expressed the highest therapeutic
index, the MIC was measured against 30 antibiotic susceptible and 38 strains producing
extended spectrum betaꢀlactamase (ESBL) clinical E. coli isolates as well as in the presence
ꢀ
+
of two genetically modified strains of Bacillus subtilis ꢀ M45 rec and H17 rec .
Details of the antimicrobial mechanism was studied by several methods using low to
high dendrimer concentrations. Bacterial viability and membrane integrity analysis using the
LIVE/DEAD assay revealed that relatively mild membrane disruption is one of the possible
sources of their microbicidal activity. Permeability of fluorophore filled large unilamelar
vesicles mimicking various cellular membranes (prepared from POPC, POPG or
POPC/POPG (3:1) phospholipids) in presence of dendrimers was studied by fluorescence
spectroscopy. In this paper we describe also application of solidꢀstate NMR spectroscopy
techniques i.e. combination of the magic angle spinning (MAS) and the nuclear Overhauser
enhancement spectroscopy (NOESY) to determine at the atomic level interactions of the
dendrimer 46 immobilized in phospholipid membranes. Solidꢀstate NMR spectroscopy used
for the above purpose is emerging as a new approach in the structural investigations of
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peptides and proteins in membrane bilayers but is scarce in investigation of dendrimers.
RESULTS AND DISCUSSION
General strategy of dendrimer synthesis. The general strategy for the synthesis of a
series of amphipatic dendrimers containing amino acids on the surface involves synthesis of a
1 2 1 2
Bocꢀprotected new hydrophobic branching units of the AB B type, where B and B denote
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alkylamino chains of equal or different length, followed by coupling them with the N or N
atoms of lysine functionalized at Cꢀterminus with benzylethylamine, tryptamine,
dodecylamine or WOMe fragment. After deprotection of amino groups such core molecules
are then coupled with suitably protected lysines or LysꢀTrp residue.
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Preparation of new branching units. Preparation of new branching monomer with
variable arm length of AB
2 1 2
or AB B structure from 3,5ꢀdihydroxybenzoic acid is shown in
Scheme 1. The monomer of AB type 10 with equal arm length was synthesized via
2
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modification of a procedure published by Liskamp et al., that yielded disubstituted as well
as monosubstituted product 9. Finally, the monomer of AB type 12 was obtained from 9
by reaction with 5ꢀ(Bocꢀamino)pentyl mesylate 7. A larger quantity of compound 9 was
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obtained according to the Brouwer and Liskamp method (42.7%).
Preparation of new hydrophobic cores and final dendrimers. New hydrophobic
cores 22 – 28 were obtained by coupling the respective amides of lysine 18ꢀ21 (Scheme 2)
and various amines or KWꢀOMe dipeptide with symmetric AB or nonsymmetric AB B
2
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monomers obtained from 3,5ꢀdihydroxybenzoic acid that after deprotection yielded
compounds 29 – 35, used in the next coupling reaction with suitably protected lysine.
(
Scheme 3). Classic solution peptide chemistry using DCC/HOBt coupling was employed for
preparation of lysineꢀbased core molecules and the final dendrimers (described before in ref.
34). Core molecules 29 – 35 were synthesized by coupling reaction between symmetric AB
10) or unsymmetric AB B (12) branching monomer based on 3,5ꢀdihydroxybenzoic acid
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(
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with unprotected Lꢀlysine amides 18 – 21. The synthesis of dendrimers 36 and 37 terminated
ε
ε
with polar N ꢀ2ꢀchlorocarbobenzoxy lysines and N ꢀtryptophan lysines 42 – 45 are shown in
Scheme 4 and 5, respectively. Scheme 6 presents the protocol for preparation of dendrimer
46.
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Scheme 1. Synthesis of branching monomers: bis[2ꢀ(Bocꢀamino)ethoxy]benzoic acid (21) and
3ꢀ[5ꢀ(Bocꢀamino)penthoxy]ꢀ5ꢀ[2ꢀ(Bocꢀamino)ethoxy]benzoic acid (23). (i) MeOH, cat.
H SO , reflux., 12 h; (ii) K CO , DMF, 60 °C, 48 h; (iii) 1M NaOH, 50 °C, 4 h; (iv) Boc O,
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Scheme 2. Preparation of Lysꢀamide dihydrochlorides 18–21: (i) DCC/HOBt, PEAꢁHCl/Et N
(TA, DDA, dDDA, WꢀOMe), DMF/DCM (1:1), 24 ꢀ 48 h.; (ii) sat. HCl/EtOAc.
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Scheme 3. Preparation of core molecules from Cꢀterminus modified Lys (18–21): (i)
DCC/HOBt, PEAꢁHCl/Et N (TA, DDA, dDDA, WꢀOMe), DMF/DCM (1:1), 24 ꢀ 48 h.; (ii)
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nas. HCl/EtOAc.
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Scheme 4. Synthesis of peptide dendrimers 36 and 37 terminated with Lys (2ꢀClꢀZ) residue.
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Scheme 5. Synthesis of peptide dendrimers terminated with N −tryptophan (42 – 45): (i) Bocꢀ
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TrpꢀOH, EDC/HOBt, Et N, DMF, 48 – 72 h; (ii) HCl/EtOAc.
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Scheme 6. Synthesis of dendrimer 46.
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All dendrimers were purified by size exclusion chromatography using a Sephadex LH20
column. The molecular mass of the dendrimers was established on the basis of ESI MS
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spectra and their purity was confirmed by H and C NMR spectroscopy. The synthetic
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procedures, analytical data (ESIMS, H and C NMR chemical shifts assignment, melting
points and molar ellipticity, etc.) for key compounds are given in Supplementary Data.
In vitro testing of dendrimers
Antimicrobial potency. Primary screening of the antibacterial activity of the
dendrimers involved four Gramꢀpositive and eight Gramꢀnegative reference bacteria strains,
including MRSA for Gramꢀpositive as well as ESBL and VISA strains for Gramꢀnegative
antibioticꢀresistant pathogens. The species and results of the antimicrobial testing against
these bacterial strains are summarized in Table 1. Two groups of dendrimers were obtained ꢀ
either terminated with orthogonally substituted lysine (36ꢀ37) or tryptophane (42ꢀ46) with the
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respective four or eight cationic centers located beneath the hydrophobic surface of the
molecules. The first group of dendrimers carrying four positive charges is broadly active
against Gramꢀpositive and Gramꢀnegative bacteria with MIC values in the range of 15.5 – 31
ε
ꢀ
M. However, further coupling of N ꢀatoms of Lys with Trp (42 – 45 and 46) yielded
compounds with 4ꢀ8 times higher potency and preference towards Gramꢀnegative bacteria.
Compound 46, that exhibited the lowest MIC against ESBL reference strains (1.7 ꢀM), was
selected for testing against 30 antibiotic susceptible and 38 antibiotic resistant clinical isolates
of E. coli. The respective results are summarized in Fig. 1, whereas detailed information about
the species, source, and MIC of the dendrimers are given in Table S2 of the Supporting
information. According to these data, dendrimer 46 exhibits particularly high potency against
extended spectrum βꢀlactamase producing clinical isolates ꢀ ESBL(+). For the selected
dendrimers 44 and 46, the equal value of the MIC measured against E. coli ATCC 25922 in
human serum and in buffered media demonstrated that these dendrimers were not susceptible
to enzymatic degradation (Table 1).
0
1
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0
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1
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8
9
0
1
2
3
4
5
6
7
8
9
0
Fig. 1. Potency of dendrimer 46 against 68 clinical isolates of E. coli: 38 isolates producing extended
spectrum betaꢀlatamase [ESBL(+)] and 30 isolates susceptible to antibiotics [ESBL(ꢀ)].
Genotoxicity. A negligible ability of the dendrimers (42, 44, 46) to produce microbial
mutations was confirmed by measuring MICs against vulnerable, genetically modified strains
of Bacillus subtilis: M45 recꢀ and H17 rec+ strains performed according to a procedure
4
5ꢀ47
ꢀ
published by Kada et. al.
The B. subtilis M45 (rec ) is devoid of the recombinantꢀbased
DNA repair mechanism and, therefore, is much more susceptible to genotoxic damage than its
+
rec counterpart, which is evidenced by a wider zone of growth inhibition for this strain
around the reference genotoxin (4ꢀnitroquinoline Nꢀoxide)ꢀcarrying discs. The diameter of the
ꢀ
+
zone of growth inhibition reached 24 mm for the rec strain and 12 mm for the rec strain.
Since the examined dendrimers did not inhibit the growth of the tested B. subtilis strains they
were determined as not genotoxic.
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Table 1. Minimal inhibitory concentration (MIC, ꢀM) for dendrimers.
Strain*
Minimal inhibitory concentration MIC (µM)
3
6
37
42
43
28
28
56.1
3.5
7
44
45
46
S. aureus
ATCC 29213
S. aureus
ATCC 43300
S. aureus
ATCC 700787
S. epidermidis
ATCC 12228
E. coli
62
62
62
31
> 119
> 119
59.5
29.8
29.8
59.5
> 119
29.8
29.8
44.6
nt
41.2
27.5
27.5
3.4
1.7
6.9
6.9
3.4
1.7
3.4
55
27
6.7
5.0
6.7
1.7
3.3
13.4
3.3
3.3
1.7
3.3
13.4
26.8
53.2
53.2
106.5
3.3
1
1
1
1
1
1
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1
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2
2
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2
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4
4
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4
4
4
4
4
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5
5
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6
0
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9
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9
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9
0
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8
9
0
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8
9
0
27
13.5
3.4
3.4
6.8
6.8
3.4
1.7
3.4
ATCC BAAꢀ198 15.5
1.7
E. coli
ATCC 25922
A. baumanii
ATCC 19606
S. enterica
31
62
31
31
15.5
nt
7
3.4
28
3.5
3.5
3.5
53.2
6.7
ATCC 10708
B.bronchiseptica
ATCC 4617
S. maltophilia
ATCC 13637
P. aeruginosa
ATCC 27853
K. pneumoniae
ATCC 13882
E. coli
1.7
3.3
> 112.2 27
> 106.5
> 106.5
nt
nt
55
112.2
nt
54.1
ATCC 25922
(
Human serum) nt
nt
nt
6.8
nt
2.7
*
red ꢀ Gramꢀnegative bacteria , black ꢀ Gramꢀpositive bacteria; nt ꢀ not tested
Recently, Kalenbach and coꢀworkers explored the significance of Trp presence in
various natural antimicrobial peptides and their linear and branched analogs. It has been found
that TrpꢀArg sequence (Argꢀcovered surface) is superior to a combination of LysꢀTrp (Trpꢀ
covered surface), yielding extension of the microbicidal power into Gramꢀnegative bacteria,
2
6,27
salt tolerancy and activity against E. coli biofilms.
The presently studied group of the very
potent dendrimeric peptides confirms again that small structural modifications may induce
significant changes in the biological profile. In particular, the design of hydrophobic, aromatic
cores that separate positively charged arms and the proper choice of terminal amino acids
(
Trp) facilitating location of molecules at the target, yielded compounds that possess
selectivity against E. coli.
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Hemolytic activity. The majority of the Trpꢀrich antimicrobial peptides, e.g.
indolicidin, as well as Trpꢀfree dendrimers previously designed in our laboratory were
34
characterized by the lack of specificity regarding the microbial vs human target cells.
Therefore, in the current studies, the concentration dependent hemotoxicity against human
erythrocytes was assayed. As Fig. 2 shows the hemotoxicity of most of the newly designed
dendrimers is below 6 %, up to dendrimer concentrations of 200 ꢀM, i.e., concentrations
significantly exceeding their MIC values. An exception is dendrimer 44 with the Cꢀterminus
substituted with a hydrocarbon chain.
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3
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5
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8
9
0
1
2
3
4
5
6
7
8
9
0
Fig. 2. Concentration dependent hemotoxicity of dendrimers (%); inset shows in detail low
hemolysis range.
Cytotoxicity against mammalian cells. Human melanoma MEW 155 and human
fibroblast AW/4 cell lines were kindly provided by the Institute of Oncology, Warsaw.
Antiproliferative properties of the selected dendrimers carrying tryptophan residue at terminal
positions were tested in concentrations of 5, 10, 20 and 100 ꢀM (Table 2). Results are
expressed as % of cell survival with respect to control and are the mean ± SD from three
independent experiments (P ≤ 0.05) versus vehicle control. For 20 ꢀM all but dendrimer 46
reduce the number of survived cells significantly in relation to a control (100 %). The
dendrimers 44 and 45, which are most active against melanoma cells, are also more
hydrophobic due to substitution of the Cꢀterminus by tryptamine and dodecylamine residues,
respectively. Interestingly, dendrimer 46, which is very active against bacteria, does not have
negative impact on the proliferation of melanoma cells below 20 ꢀM concentration.
Treatment of human fibroblast cell line AW/4, with dendrimers 42 ꢀ 44 at 20 ꢀM
concentration reduced number of the survived cells to 90 ± 5, 96 ± 2 and 85 ± 5 %,
respectively.
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Table 2. In vitro toxicity of 42 ꢀ 46 on human melanoma cell line Mew 155, % of living cells
in relation to control with standard deviations; nt – not tested
Dendrimer
Dendrimer concentration
100 µM
20 µM
10 µM
5 µM
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
0
1
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0
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1
2
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8
9
0
42
43
44
45
46
0
0
0
0
0
18 ± 4
27 ± 6
11 ± 3
0
155 ± 9
111 ± 10
87 ± 8
66 ± 5
nt
155 ± 11
nt
nt
100 ± 9
nt
117 ± 12
LIVE/DEAD staining. The bacterial viability and membrane integrity was analyzed
using the LIVE/DEAD Bac Light Bacterial Viability Assay TM Kit according to the protocol
suggested by the manufacturer. The kit contains two dyes – Syto9 that enters into intact cells
and accumulates in the DNA. It gives a bright green colour after irradiation with a 488 nm
laser. Propidium iodide (PI) is a membrane nonꢀpermeable dye. When the bacterial cell
membrane is rendered permeabilized, PI enters the cell and interacts with cellular DNA. The
uptake of propidium iodide by bacteria can be detected as bright red colour with a
spectropolarimeter.
Fig. 3. The representative fluorescence images of E. coli ATCC 25922 colony after 24ꢀhour
incubation in the absence (left, control) and presence (right) of dendrimer 46 at 13.2 ꢀM.
Fig. 3 shows that after 24 h of incubation with dendrimer 46 at relatively high concentration
(4 times MIC) ca 50 % of the remaining bacterial colonies turned red, confirming that
eradication of bacteria is not very fast. It should be noted also, that at this concentration the
effect is bacteriostatic not bactericidal.
The results of the LIVE/DEAD test performed for compound 46 at 2 times MIC for 24 h
showed that instead of fast killing process typical for the a “carpet pathway”, a much slower
inhibitory action is observed.
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Permeability studies. To examine the relationship between in vitro membrane
perturbation and inhibitory activity of the selected dendrimers, permeability studies involving
fluorescent dyeꢀloaded large unilamellar vesicles were performed (LUV’s). It is wellꢀknown
that cytoplasmic membranes of Gramꢀnegative bacteria contain both anionic and zwitterionic
phospholipids, while membranes of many Gramꢀpositive bacteria contain predominantly
0
1
2
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0
48
anionic lipids. Therefore, the negatively charged POPG and the mixed POPC/POPG (3:1)
LUVs were used to mimic Gramꢀpositive and Gramꢀnegative bacterial membranes,
respectively. The electrically neutral LUVs prepared from POPC were used to mimic the
reference erythrocyte membranes. From the group of Trpꢀrich dendrimers 42 – 44 bearing
(+)8 charge, 42 (phenylethylamine at Cꢀterminus) and 44 (tryptamine at Cꢀterminus) were
selected for testing the concentrationꢀdependent dye release from these phospholipids.
Fig. 4. Leakage of carboxyfluorescein (CF) from LUV’s mimicking various cell systems
induced by Trpꢀrich dendrimers 42 and 44. The percentage of carboxyfluorescein (CF)
released after 15 min of incubation is plotted as a function of peptide concentration.
As Fig. 4 shows the degree of vesicle permeability against CF, when stressed by the
dendrimers, was dependent on the vesicle composition and therefore, the membrane charge
and fluidity. In particular, dendrimer 42 at 1.7 ꢀM concentration (i.e. MIC against E. coli
ATCC BAAꢀ198) released ca. 30 % of the dye from the CF ꢀ entrapped POPC/POPG
vesicles, whereas only 10 % release was observed in case of vesicles made from POPG or
POPC alone. Similarly, dendrimer 44 at the MIC concentration released ca 40 % of the dye
from CF ꢀ loaded POPC/POPG vesicles. Moreover, at 2 times MIC both dendrimers are not
harmful for mimics of RBC (POPC LUV’s). Above this concentration dendrimers become
more destructive for mimics of Gramꢀpositive membranes (POPG). As exemplified in Fig. 5,
further increase of dendrimer 44 concentration becomes harmful also for vesicles prepared
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from zwitterionic phospholipids. In this model, abundant dendrimer molecules not only
significantly quenched calcein fluorescence but also induced significant red shift – (from 516
to 537 nm) evidencing differences between excimer emission of the dye molecule embedded
in vesicle container and released into solution with cationic dendrimer.
Excimer emission of the dye molecules released from vesicles due to dye–solvent or
dye/dendrimer interactions is redꢀshifted in comparison to emission of the dye molecules
embedded in vesicles where dye/dye interactions prevail
1
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9
0
Fig. 5. Emission spectra of 2.5 mmol calcein entrapped in DMPC LUV’s in presence of
dendrimer 44 at 100 and 200 ꢀm concentrations and upon solubilization of the vesicles with
Triton Xꢀ100, 10 %).
As evidenced by the permeability studies, good selectivity of 42 and 44 towards
mimics of Gramꢀnegative microbial membranes composed of POPC:POPG lipids was
accompanied by only moderate lytic properties.
Solid state 2D NMR studies of dendrimer 42 embedded in microbial membrane
mimics. A variety of physicochemical methods are used to study the interactions of
49,50
biologically active compounds with model phospholipid membranes.
However, only the
nuclear magnetic resonance spectroscopy (NMR), especially in the solid state provides
information about such interactions at the atomic level. NMR is well suited for examination of
the organization and dynamics of the membrane after incorporation of the active substance, as
well as the orientation and conformation of the studied substance embedded in the
5
1
membrane. The literature shows numerous examples of applications of solidꢀstate NMR
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spectroscopy (SS NMR) in this type of research, mainly involving peptides, membrane
52
proteins and AMPs.
One of the important types of information concerning interactions with the membrane
is the location of the active compound in the lipid bilayer. Therefore, we used a combination
of the magic angle spinning (MAS) technique and the nuclear Overhauser enhancement
spectroscopy (NOESY) to determine the location of the 42 dendrimer in phospholipid
membranes. It is well documented that bacterial membranes are rich in the negatively charged
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53
lipids, whereas mammalian membranes are mainly composed of the neutral lipids. Fig. 6
1
1
presents Hꢀ H NOESY MAS NMR spectra of dendrimer 42 confined in the multilamellar
vesicles (MLVs) prepared from zwitterionic DMPC (Fig. 6a) or from anionic DMPG (Fig. 6b)
registered with a mixing time of 400 ms at 310 K. It is very difficult to analyze the
interactions of the 42 with DMPC and DMPG membranes in the aliphatic region of the
spectrum (0ꢀ5.5 ppm) because the intense crossꢀpeaks corresponding to the phospholipidꢀ
phospholipid interactions obscure the peptide correlations. This is also result of a small
content (about 6%) of the dendrimer 42 in the membranes. The spectral range from 6 to 9
ppm, showing the interactions of aromatic protons of tryptophan, tryptamine and phenyl
residues with model membrane phospholipids, is more appropriate for diagnostic purposes.
1
Unfortunately, due to the overlapping of the aromatic H signals, they have not been assigned
1
in the H MAS NMR spectra shown on the F1 and F2 projections. As can be seen in the
NOESY spectra, the aromatic fragments of the dendrimer 42 interact with both the
hydrophilic and hydrophobic components dendrimer of DMPC and DMPG membranes. This
is confirmed by crossꢀpeaks between the aromatic protons of the dendrimer 42 and all protons
of the acyl chains, glycerol as well as choline in case of the DMPC membrane. Interestingly,
the interactions of the aromatic dendrimer fragments with the methylene protons of acyl
chains and choline are stronger than interactions with the glycerol protons (Fig. 6a). These
two types of interactions have the comparable strength in the case of the DMPG membrane
(Fig. 6b). This suggests that the mechanism of interaction of the dendrimer 42 branches with
the zwitterionic DMPC membrane is mainly based on the hydrophobic interactions, whereas
for the anionic DMPG membrane on the hydrophobic and electrostatic interactions resulting
from the amphiphilic and cationic character of the 42 dendrimer are most important.
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Fig. 6. Hꢀ H NOESY MAS NMR spectra of MLVs of a) DMPC b) DMPG containing
dendrimer 42 recorded with a mixing time of 400 ms and spinning rate of 10 kHz at 310 K, c)
molecular structures of DMPC and DMPG with the numbering of atoms. The F1 and F2
1
projections present H MAS NMR spectra with signal assignments to the appropriate protons
of DMPC and DMPG molecules. The interactions between dendrimer and phospholipids are
marked in red.
Moreover, the spectra show the peptideꢀwater correlations at 4.7 ppm. Consequently, it can be
concluded that molecules of the dendrimer 42 are located within the lipidꢀwater interface
defined by the polar head groups of glycerol or choline. This is due to nature of tryptophan for
which location at the waterꢀlipid interface has been repeatedly confirmed, for example by
54,55
molecular modeling or NMR studies.
In this position, tryptophan can interact with lipids
through hydrogen bonds formed between the indole NH and the lipid carbonyl Oꢀatoms, the
phosphate groups and water, while the sixꢀmembered ring can interact with the upper region
56
of the acyl chains of the membrane. Furthermore, tryptophan can also participate in cationꢀπ
57
interactions with the choline group. The ability to determine the position of these residues in
the peptide dendrimers by solidꢀstate NMR is a novel approach and extremely important due
to the fact that within the phospholipid membrane of bacterial cells, tryptophan acts as an
49
anchor for the peptide.
In contrast to natural, linear AMPs,
58,59
the mechanism of action of dendrimeric
peptides is not well understood. Our research will certainly expand knowledge about the
interactions of dendrimeric peptides with membranes, as well as bring new insights in
understanding the interfacial processes in context of biological systems.
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DISCUSSION
This study demonstrates that amphiphilic, highly charged dendrimers designed
according to rules established by Nature, exhibit superior antimicrobial functions against
antibiotic resistant E. coli clinical isolates. Key modification of their molecular structure
involves design of hydrophobic cores that separate cationic domains. In particular, a series of
the Trpꢀterminated antimicrobial dendrimers despite having the same net positive charge and
branched scaffold construction as Lysꢀterminated molecules showed a significantly higher
selectivity and potency against clinical isolates of antibiotic resistant pathogenic E. coli.
0
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9
0
Pharmacological potential of these novel antimicrobial dendrimers defined as the
therapeutic index, TI, i.e. the ratio of the dose expressing 50 % hemotoxicity to the MIC dose
was also evaluated. Table 3 presents a comparison of the potency of the studied dendrimers
and two natural antimicrobial peptides potent against Gramꢀnegative bacteria ꢀ polymyxin B
and indolicidin, along with their TI’s. As evidenced from the literature data, polymyxin B is
four times more potent than 43 and 46. However, an appreciable property of this series of
dendrimers is that they are practically nonhemolytic at 200ꢀ400 ꢀM concentrations, whereas
polymyxin B expresses 50 % hemotoxicity at ca 160 ꢀM.
Table 3. Therapeutic index (TI) of indolicidin, polymyxin B and Trpꢀrich dendrimers 42– 46
against selected Gramꢀpositive and Gramꢀnegative microbial strains.
MIC
MIC
MIC
(µM)
(µM)
(µM)
TI
TI
TI
AMP or
HC *
5
0
S.
aureus
E. coli
E. coli
(
HC50
/
(HC50
/
(HC50/
dendrimer
(µM)
ATCC
ATCC
BAAꢀ198
MIC)
MIC)
MIC)
ATCC
25922
29213
6
0
Indolicidin
68.2
4.2
16.2
3.3
8.4
8.1
ꢀ
ꢀ
6
1
Polymyxin B
42
161.9
49.2
0.38
426
ꢀ
ꢀ
400** 27.5/55
>>10.9 3.4/6.9
>>87.8
>>28.6
>>29.4
14.9
1.7
7
>>235.3
>>28.6
>>58.8
60.6
4
3
200**
200**
200
28
27
>>7.1
>>7.4
29.9
7
44
6.8
13.4
3.4
3.3
1.7
4
4
5
6
6.7
53.2
400**
>>7.5
1.7/3.3 >>178,3
>>235.3
*
*
%
HC50 – concentration that induces 50 % hemolysis;
* not relevant to HC50, since at the given concentration the dendrimer induces only 1.1 – 2.8
hemolysis; nt – not tested; ,,ꢀ” – no data available
1
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Development of understanding the mechanism of interaction of peptide dendrimers
with the lipid bilayer, which is the main component of the biological membrane, is
undoubtedly crucial for pharmacological applications and further directions of synthetic
modification of these compounds. The results of the LIVE/DEAD test performed for
compound 46 showed that instead of the fast killing process anticipated for the “carpet
mechanism”, a much slower and milder inhibitory action is observed. As evidenced by the
permeability studies, the good selectivity of 42 and 44 towards mimics of Gramꢀnegative
microbial membranes was accompanied by only a moderate lytic properties.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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0
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9
0
Application of solid state 2D H NMR for studying interactions of dendrimer 42 with
model membranes supports insertion of its Trpꢀterminated branches in the membrane as well
as location at water/lipid interface. This suggests that 42 and probably also other dendrimers
of this group not only exhibit antimicrobial activity but also can permeate cell membranes and
therefore, may serve as carrier molecule. Since the impact of the Trpꢀterminated dendrimers
on the integrity of microbial and model membranes is mild, their high potency and selectivity
might be explained by their ability to target intraꢀ or extracellular receptors. Several other
studies on Trpꢀrich peptides are known where key role of Trp for development of efficient,
multiple action antimicrobials was emphasized. Close analogs of indolicidin such as
62
puroindolidins among others inhibit the transcription and replication processes in E. coli.
Other analogs of indolicidin obtained by Pro→Lys substitution as reported by Shin et al.,
6
3
expressed high selectivity towards S. aureus and antiꢀinflammatory activity. Our recent
studies on Trpꢀrich group of dendrimers designed for the control of C. albicans infections
64
appear to support this hypothesis.
The high potency against clinical isolates ESBL E. coli strains and lack of hemoꢀ and
genotoxicity expressed by dendrimer 46 and other analogs makes this group of compounds
promising candidates for further studies. However, as Gill et al., pointed out, currently the
isolation of new natural AMPs and designing of their analogs is much more advanced than
65
our current understanding of origins of the variability of their modes of action. In view of
the reported preliminary results, further biophysical and biochemical assays are needed to
thoroughly elucidate the mechanism of action of these new dendrimers. Nevertheless, the
present work lays a foundation for generating targetꢀspecific “smart” antimicrobials to
complement currently available conventional antibiotics.
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6
EXPERIMENTAL
General procedures
All solvents and reagents were of analytical grade and were used without further purification.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
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9
0
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9
0
1ꢀpalmitoylꢀ2ꢀoleoylꢀsnꢀglyceroꢀ3ꢀphosphocholine (POPC), 1ꢀpalmitoylꢀ2ꢀoleoylꢀsnꢀglyceroꢀ
3ꢀphosphoꢀ(1'ꢀracꢀglycerol) (POPG) and 1,2ꢀdimyristoylꢀsnꢀglyceroꢀ3ꢀphosphocholine
(DMPC) and 1,2ꢀdimyristoylꢀsnꢀglyceroꢀ3ꢀphosphoꢀ(1'ꢀracꢀglycerol) (DMPG) were
purchased from Avanti Polar Lipids and used without further purification. Calcein, 5(6)ꢀ
Carboxyfluorescein (CF) and all solvents were obtained from SigmaꢀAldrich. Mass spectra
were recorded with a Mariner ESI timeꢀofꢀflight mass spectrometer (PerSeptive Biosystems)
1
13
for the samples prepared in MeOH. The HꢀNMR and CꢀNMR spectra were recorded using
a Bruker Avance spectrometer at 500/125 or 400/100 MHz, respectively, using deuterated
solvents and TMS as an internal standard. Chemical shifts are reported as δ values in parts per
million, and coupling constants are given in hertz. The optical rotations were measured with
JASCO Jꢀ1020 digital polarimeter. Melting points were recorded on a Köfler hotꢀstage
apparatus and are uncorrected. Thin layer chromatography (TLC) was performed on
aluminum sheets with silica gel 60 F254 from Merck. Column chromatography (CC) was
carried out using silica gel (230–400 mesh) from Merck or Sephadex LH20. The TLC spots
were visualized by treatment with 1 % alcoholic solution of ninhydrin and heating.
For minimal inhibitory concentration (MIC) and hemolysis tests dendrimers were freshly
lyophilized, dissolved in minimal amount of DMSO and then diluted with buffer. Upon
prolong standing buffer solutions became opaque but no dendrimer precipitation was
observed.
2
Synthesis of symmetric AB building block.
Methyl 3,5ꢀbis[2ꢀ(Bocꢀamino)ethoxy]benzoate(19) and (20)
5.71 g (33.98 mmol, 1 eq.) methyl 3,5ꢀdihydroxybenzoate (15) was dissolved in 80
mL DMF, and 21.13 g (152.91 mmol, 4.5 eq.) of K
2 3
CO was added. Suspension was stirred at
60 ˚C for 1 h and 2ꢀ(Bocꢀamino)ethyl bromide (16, 22.82 g, 101.94 mol, 3 eq.) dissolved in
20 mL DMF was added. After 48 h stirring at temp. 60 ˚C suspension was cooled to r.t.
filtered off (Celite) and evaporated. Crude product was dissolved in 150 mL EtOAc, washed
with H O (3 × 30 mL) and brine (30 mL). Organic fraction was dried over Na SO , filtered
2
2
4
and solvent was evaporated. Crude product was purified twice on silica gel column with
CHCl /MeOH 25:1 as mobile phase: 10.71 g of pure 19 was obtained as white crystalline
3
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powder, 69.5 % yield. From the same reaction a monoalkylated product, methyl 3ꢀhydroxyꢀ5ꢀ
[
2ꢀ(Bocꢀamino)ethoxy]benzoate (20) was isolated, 3.64 g , 23.6 % yield. Both products were
dried over P
2
5
O .
C
22
H
34
O
8
2
N , M = 454.51 g/mol (monoisotopic mas 454.2).
+
LRMS (ESI, MeOH): 477.2 [M + Na ].
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H NMR (400 MHz, MeOD): δ = 1.44 [s, 18H, 2×C(CH ) ], 3.43 (m, 4H, 2×CH2NH), 3.88
3
3
4
(
3 2
s, 3H, OCH ), 4.01 (t, J = 5.5 Hz, 4H, 2×OCH ), 6.75 (m, 1H, C ꢀH Ph), 7.14 (d, J = 2.15
2
,6
Hz, 2H, C ꢀH Ph).
1
3
C NMR (400 MHz, MeOD): δ = 28.7 [C(
C
H ) ], 40.9 (CH NH), 52.7 (OCH ), 68.3
3 3
2
3
4
2,6
1
(OCH
2
), 80.2 [
C
(CH
3
)
3
], 107.4 (C Ph), 109.0 (C Ph), 133.2 (C Ph), 158.5 [C=O (Boc)],
3
,5
161.4 (C Ph), 168.1 (
2
CO Me).
R = 0.70 (CHCl /MeOH 8:1).
f
3
3,5ꢀbis[2ꢀ(Bocꢀamino)ethoxy]benzoic acid (21)
To a 5 g (11 mmol, 1 eq.) of ester 19 dissolved in mixture of 100 mL MeOH and
[
what is the second component of the mixture?] 44 mL (44 mmol, 4 eq.) of 1 M NaOH was
added and stirred for 4 h. Reaction mixture was concentrated, then H O was added, solution
2
was acidified with 1 M HCl to pH = 1.5 and extracted with EtOAc. Combined organic
extracts were dried over MgSO , filtered and evaporated. Product was dried over P O .
4
2
5
Compound 21 was obtained as white semicrystalline solid 4.61g, 95.1 % yield.
C
21
H
32
O
8
2
N , M = 440.49 g/mol (monoisotopic mass 440.2).
+
+
+
+
LRMS (ESI, MeOH): 463.4 [M + Na ] – main signal, 485.4 [M ꢀ H + 2Na ], 439.3 [M ꢀ H ,
ꢀ
negative ions mode] – main signal, 485.3 [M + HCOO , negative ions mode].
f 3
R = 0.43 (CHCl /MeOH 8:1).
1 2
Synthesis of nonsymmetric building block AB B
5ꢀ(Bocꢀamino)ꢀ1ꢀpentanol (17)
To 10 g (96.9 mmol) of 5ꢀaminoꢀ1ꢀpentanol (14) dissolved in 50 mL DCM and
cooled to 0 ˚C, a solution of Boc O (21.15 g, 96.9 mmol) in 20 mL DCM was added
dropwise. After stirring for 12 h at r.t. 50 mL DCM was added. Reaction mixture was washed
with H O, brine and dried over Na SO . Solution was filtered and evaporated. Product was
obtained as colorless semisolid; 19.2 g, 97.5 % yield.
2
2
2
4
C
10
H
21
O
3
N, M = 203.28 g/mol (monoisotopic mass 203.2).
+
LRMS (ESI, MeOH): 226.4 [M + Na ].
2
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f
R = 0.37 (EtOAc/hexane 1:1).
5ꢀ(Bocꢀamino)pentyl mesylate (18)
To a cold solution (ice/H
2
O) of compound 17 (3 g, 14.76 mmol, 1 eq.) and
N (2.25 mL,
16.24 mmol, 1.1 eq.) was added slowly. After stirring for 1 h at r.t. the mixture was
concentrated and redissolved in 80 mL EtOAc, washed with 1M KHSO , H O and brine, then
dried over Na SO , filtered and evaporated. Mesylate 18 was obtained as colorless oil that
methanesulfonyl chloride (1.26 mL, 16.24 mmol, 1.1 eq.) in 15 mL DCM, Et
3
0
1
2
3
4
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9
0
1
2
3
4
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9
0
1
2
3
4
5
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7
8
9
0
1
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3
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7
8
9
0
1
2
3
4
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7
8
9
0
4
2
2
4
solidified to form white solid 97.6 % (4.05 g) and was used directly in the next reaction
without purification.
C
11
H
23
O
5
NS, M = 281.37 g/mol (monoisotopic mass 281.1).
R = 0.49 (EtOAc/hexane 1:1).
f
3ꢀHydroxyꢀ5ꢀ[2ꢀ(Bocꢀamino)ethoxy]benzoic acid methyl ester (20)
Mixture of a 14.25 g (84.8 mmol, 1 eq.) of methyl 3,5ꢀdihydroxybenzoate (15)
dissolved in 460 mL DMF, and 11.72 g (84.8 mmol, 1 eq.) K CO was stirred for 1 h. Then a
2
3
solution of 2ꢀ(Bocꢀamino)ethyl bromide (16, 9.5 g, 42.4 mmol, 0.5 eq.) in 40 mL DMF in
was
four portions was added. After addition of each portion reaction
stirred for 30 min. Then
temperature was raised to 40 ˚C and stirring was continued for 48 h. Finally, the reaction
mixture was filtered (Celite) and solvent was evaporated. Crude product was dissolved in
2 2 4
EtOAc and washed twice with H O and brine. Organic layer was dried over Na SO , filtered
and solvent was evaporated. Crude product was purified on silica gel column using 4 %
acetone/DCM mixture as mobile phase (5ꢀtimes). Product was obtained as white powder; 5.64
g, 42.7 % yield.
1
5
C
H
21
O
6
N, M = 311.33 g/mol (monoisotopic mass 311.1).
+
+
LRMS (ESI, MeOH): 645.4 [2M + Na ], 334.3 [M + Na ] – main signal.
R = 0.51 (CHCl /MeOH 8:1), m.p.: 106ꢀ107 ˚C .
f
3
3ꢀ[5ꢀ(Bocꢀamino)pentoxy]ꢀ5ꢀ[2ꢀ(Bocꢀamino)ethoxy]benzoic acid methyl ester (22)
To a solution of compound 20 (1.72 g, 5.52 mmol, 1 eq.) dissolved in 20 mL DMF,
(3.43 g, 24.84 mmol, 4.5 eq.) K CO was added. A suspension was stirred for 1 h at 60 ˚C.
2 3
Then 3.2 g (11.04 mmol, 2 eq.) of mesylate 18 was added and suspension was stirred for 48 h
at 60 ˚C. Finally, the reaction was cooled to r.t., filtered through Celite and evaporated.
Residue was dissolved in 100 mL EtOAc, washed with H O (2 × 30 mL) and brine (30 mL),
2
2 4
dried over Na SO , filtered and evaporated. Crude product was purified on silica gel using
2
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EtOAc/hexane 3:7. Product 22, obtained as colorless oil was dried over P
2
O
5, 2.3 g, 84 %
yield.
C
25
H
40
O
8
2
N , M = 496.61 g/mol (monoisotopic mass 496.6).
+
+
LRMS (ESI, MeOH): 519.4 [M + Na ] – main signal, 497.4 [M + H ].
1
2 2 2 2 2
H NMR (500 MHz, MeOD): δ = 1.53 [br m, 4H, Oꢀ(CH ) ꢀCH ꢀCH ꢀCH
ꢀNH], partially
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
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7
8
9
0
overlapped with 1.43, 1.44 [2s, 18H, 2×C(CH ) ], 1.79 [m, 2H, OꢀCH ꢀCH2ꢀ(CH ) ꢀNH],
3
3
2
2 3
3
.06 [t, J = 6.7 Hz, 2H, Oꢀ(CH
2
)
4
ꢀC
H
2
ꢀNH], 3.43 (t, J = 5.6 Hz, 2H, OꢀCH
2
ꢀC
H
2
ꢀNH), 3.87
(
s, 3H, OCH
3
), 3.98 [t, J = 6.35 Hz, 2H, OꢀC
H
2
ꢀ(CH
2
)
4
2
ꢀNH], 4.01 (t, J = 5.6 Hz, 2H, OꢀCH ꢀ
4
2,6
CH ꢀNH), 6.71 (t, J = 2.25 Hz, 1H, C ꢀH Ph), 7.12 (m, 2H, C ꢀH Ph).
2
13
C NMR (500 MHz, MeOD): δ = 24.3 [Oꢀ(CH
2
)
2
ꢀ
C
H
2
ꢀ(CH
ꢀNH], 40.9 (OꢀCH
H ꢀ(CH ) ꢀNH], 79.8, 80.2
2
)
2
ꢀN], 28.8 [C(
3 3
CH ) ], 29.9 [Oꢀ
CH
2
ꢀC
H
2
ꢀ(CH
2
)
3
ꢀNH], 30.7 [Oꢀ(CH
)
2 3
ꢀC
H
2
ꢀCH
2
2 2
ꢀCH
ꢀNH), 41.2 [Oꢀ
(CH ) ꢀ
C
H ꢀNH], 52.7 (OCH ), 68.3 (Oꢀ
C
H ꢀCH ꢀNH), 69.2 [Oꢀ
2
C
2
4
2
3
2
2
2 4
4
2,6
1
[
C
3 3
(CH ) ], 107.4 (C Ph), 108.7, 109.0 (C Ph), 133.1 (C Ph), 158.4, 158.5 [C=O (Boc)],
3
,5
161.3, 161.6 (C Ph), 168.2 (
2
CO Me).
R = 0.26 (EtOAc/hexane 3:7).
f
[5ꢀ(Bocꢀamino)pentoxy]ꢀ5ꢀ[2ꢀ(Bocꢀamino)ethoxy]benzoic acid (23)
Product 23 was obtained by hydrolysis of ester 22. Namely, to 2.22 g (4.47 mmol, 1
eq.) of ester 22 dissolved in 40 mL MeOH and 18 mL (17.9 mmol, 4 eq.) of 1 M NaOH was
added and mixture was stirred for 2 hrs. Isolated colorless, oily product after drying over
2 5
P O , turned into white solid (2.1 g, 97.7 % yield).
C
24
H
38
O
8
2
N , M = 482.57 g/mol (monoisotopic mass 482.3).
+
LRMS (ESI, MeOH): 505.4 [M + Na ].
R
f
= 0.54 (CHCl
3
/MeOH 8:1).
Synthesis of dendrimers 36 – 37, 42 – 46
ε
Synthetic method for obtaining dendrimers 36 and 37 with N atoms of four terminal lysines
substituted with 2ꢀClꢀZ generally follows procedures described in ref. 34 . Details of synthesis
ε
of dendrimers 42 – 46, containing N atoms of the terminal lysines substituted with tryptophan
are included in Supplementary Data S1.
Analytical data for the final dendrimers 42 – 46
Dendrimer 42
2
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Yellow powder, obtained from 0.34 g (0.12 mmol) of dendrimer 70; 0.27 g, yield 96.4%.
C
110 151 15
H O N23×8HCl, M = 2327.21 g/mol (monoisotopic mass of nonprotonated dendrimer ꢀ
2034.2).
LRMS (ESI, MeOH): 1044.9 [M + MeOH + H + Na ] , 1017.9 [M + 2H ] , 679.0 [M +
+
+ 2+
+ 2+
+ 4+
+ 3+
+
+ 3+
3
H ] , 696.9 [M + MeOH + 2H + Na ] ꢀ main signal, 536.4 [M + 4Na ] , 523.0 [M +
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+ 4+
MeOH + 3H + Na ] .
1
2
H NMR (500 MHz, MeOD): δ = 1.25ꢀ1.87 [br m, 42H, 5×β, γ, δCH L-Lys and core, 2×Oꢀ
2 2 3 2 2 2
CH ꢀ(CH ) ꢀCH ꢀNH], 2.75 (t, J = 7.2 Hz, 2H, CH ꢀAr PEA), 2.98ꢀ3.40 [br m, 23H, 5×εCH
L-Lys and core, βCH L-Trp, CH2ꢀNH PEA, 2×Oꢀ(CH ) ꢀCH2ꢀNH], 3.44 (m, 1H, CH2ꢀNH
2
2 4
PEA), 3.54, 3.68 (2m, 4H, 2×OꢀCH
2
ꢀC
H
2
2 2 4
ꢀNH), 3.81ꢀ3.96 [br m, 8H, 2×OꢀCH ꢀ(CH ) ꢀNH,
2 2
4×αCH L-Lys], 4.08 (br m, 8H, 2×OꢀCH ꢀCH ꢀNH, 4×αCH L-Trp), 4.46 (m, 1H, αCH core),
4
2,6
2,6
5
6
7
.60, 6.65 (2m, 2H, C ꢀH Ph), 6.96 (m, 2H, C ꢀH Ph), 7.03 (m, 6H, C ꢀH Ph, C ꢀH L-Trp),
6
2,3,4,5,6
2
7
.12 (m, 9H, C ꢀH L-Trp, C
ꢀH PEA), 7.21 (m, 4H, C ꢀH L-Trp), 7.37 (m, 4H, C ꢀH L-
4
Trp), 7.55 (m, 4H, C ꢀH L-Trp).
13
C NMR (500 MHz, MeOD): δ = 23.1, 23.2 (γC), 24.6 [2×Oꢀ(CH ) ꢀ
C
H ꢀ(CH ) ꢀNH], 28.9,
2
2
2
2 2
2
9.0 (βC L-Trp), 29.5, 29.9, 30.0, 30.1 [δC, 2×OꢀCH
2.8 (βC), 36.5 (
ꢀNH], 40.7 (εC core), 42.0 (CH
2
ꢀ
C
H
2
ꢀCH
2
ꢀ
C
H
2
ꢀCH
2
ꢀNH], 32.2, 32.3,
3
C
H
2
ꢀAr PEA), 40.1 (εC L-Lys), 40.2 (2×OꢀCH
2 2
ꢀCH ꢀNH), 40.6 [2×Oꢀ
(CH
2
)
4
ꢀC
H
2
2
ꢀNH PEA), 54.4 (4×αC L-Lys), 55.2 (4×αC L-
Trp), 55.8 (αC core), 67.6, 67.7 (2×Oꢀ
C
H
2
ꢀCH
2
ꢀNH), 69.2, 69.3 [2×Oꢀ
C
H
2
ꢀ(CH
2
)
4
ꢀNH],
4
2,6
3
7
1
05.7, 106.0 (C Ph), 106.8, 107.1, 107.2, 107.6 (C Ph), 108.1 (C L-Trp), 112.6 (C L-
4
5
6
2
4
Trp), 119.4 (C L-Trp), 120.2 (C L-Trp), 122.9 (C L-Trp), 125.6 (C L-Trp), 127.4 (C PEA),
3
a
2,3,5,6
1
7a
128.4 (C L-Trp), 129.5, 129.9 (C
PEA), 137.2, 137.8 (C Ph), 138.2 (C L-Trp), 140.4
1
3,5
(
C PEA), 161.2, 161.7 (C Ph), 169.8 (CONH Ph), 170.0, 170.1, 170.4, 174.5 (CONH) .
2
D
5
[α]
= +27.5 (c 1, MeOH), m. p.: 195ꢀ207˚C.
Dendrimer 43
Pale yellow powder, obtained from 0.38 g (0.136 mmol) of dendrimer 71; 0.3 g, yield 96.8%.
C
106 140 15
H O N24×8HCl, M = 2282.08 g/mol (monoisotopic mass of nonprotonated dendrimer ꢀ
1989.1).
+
2+
+ 3+
LRMS (ESI, MeOH): 995.6 [M + 2H ] , 664.5 [M + 3H ] ꢀ main signal, 498.6 [M +
+
4+
4
H ] .
1
2 2
H NMR (500 MHz, MeOD): δ = 1.30 (br m, 14H, 4×γCH L-Lys, 3×δCH L-Lys), 1.35ꢀ1.50
(br m, 4H, δCH L-Lys, γCH core), 1.62 (br m, 2H, δCH core), 1.67ꢀ1.86 (br m, 10H,
2
2
2
5×βCH
2
L-Lys i rdzenia), 2.91 (t, J = 7.2 Hz, 2H, CH
ꢀAr TA), 2.95ꢀ3.09 (br m, 8H, 4×εCH
2 2
L-Lys), 3.20 (m, 4H, 2×βCH
2
2 2
L-Trp), 3.34 (m, 6H, 2×βCH L-Trp, εCH rdzenia), 3.42 (m,
2
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1
H, C
H
2
ꢀNH TA), 3.52 (m, 5H, 2×OꢀCH
2
ꢀC
H
2
ꢀNH, C
H
2
ꢀNH TA), 3.65 (m, 4H, 2×OꢀCH
, 4×αCH L-Trp), 4.47
(m, 1H, αCH core), 6.63, 6.69 (2m, 2H, C ꢀH Ph), 6.94 (m, 1H, C ꢀH TA), 6.97ꢀ7.06 (br m,
2
ꢀ
C
H
2
ꢀNH), 3.78 (m, 4H, 4×αCH L-Lys), 3.99ꢀ4.10 (br m, 12H, 4×OꢀCH
2
4
5
2
,6
5
2,6
6
2
10H, C ꢀH Ph, C ꢀH L-Trp, C ꢀH TA), 7.09 (m, 4H, C ꢀH L-Trp), 7.19 (m, 4H, C ꢀH L-
7
7
Trp), 7.31 (d, J = 8.04 Hz, 1H, C ꢀH TA), 7.36 (d, J = 8.11 Hz, 4H, C ꢀH L-Trp), 7.51 (d, J =
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
4
7.75 Hz, 1H, C ꢀH TA), 7.63 (m, 4H, C ꢀH L-Trp).
1
3
C NMR (500 MHz, MeOD): δ = 23.2, 24.5 (γC), 26.2 (
L-Trp), 30.1 (δC), 32.7, 32.9 (βC), 40.1 (εC L-Lys), 40.2 (4×OꢀCH
rdzenia), 41.4 (CH ꢀNH TA), 54.6 (4×αC L-Lys), 55.5 (4×αC L-Trp), 55.8 (αC core), 67.7,
C
H
2
ꢀAr TA), 29.3 (δC), 29.6 (δC, βC
2 2
ꢀCH
ꢀNH), 40.7 (εC
2
4
2,6
3
2 2
67.8 (4×OꢀCH ꢀCH ꢀNH), 105.8, 106.2 (C Ph), 107.3, 107.6 (C Ph), 108.5 (C L-Trp),
7
7
3
4
4
5
112.3 (C TA), 112.6 (C L-Trp), 113.1 (C TA), 119.3 (C TA), 119.4 (C L-Trp), 119.6 (C
5
6
6
2
2
TA), 120.2 (C L-Trp), 122.3 (C TA), 122.8 (C L-Trp), 123.6 (C TA), 125.5 (C L-Trp),
3a
3a
1
7a
7a
128.5 (C L-Trp), 128.7 (C TA), 137.4 (C Ph), 137.9 (C L-Trp), 138.2 (C TA), 161.3
3
,5
(C Ph), 169.6 (CONH Ph), 170.8, 170.9, 171.4, 174.3 (CONH).
25
[α]D = +14.1 (c 1, MeOH), m. p.: 203ꢀ212˚C.
Dendrimer 44
24×8HCl, M = 2366.24 g/mol (monoisotopic mass of nonprotonated dendrimer ꢀ
073.2), pale grey powder.
C
112 152 15
H O N
2
+
2+
+ 3+
+ 4+
LRMS (ESI, MeOH): 1037.6 [M + 2H ] , 692.1 [M + 3H ] , 519.3 [M + 4H ] ꢀ main
signal.
1
H NMR (500 MHz, MeOD): δ = 1.25ꢀ1.50 [br m, 20H, 5×γCH
ꢀC ꢀC ꢀCH ꢀNH], 1.53ꢀ1.65 (br m, 8H, 4×δCH L-Lys), 1.66ꢀ1.89 [br m, 14H,
L-Lys and core, 2×OꢀCH ꢀC ꢀ(CH ꢀNH], 2.92 (t, J = 7.2 Hz, 2H, CH ꢀAr TA),
.04 [m, 4H, 2×Oꢀ(CH ꢀC ꢀNH], 3.14 (m, 4H, 2×εCH L-Lys), 3.23 (m, 8H, 2×βCH L-
L-Lys), 3.34 (m, 6H, 2×βCH L-Trp, εCH ꢀNH TA),
2 2
L-Lys and core, δCH core,
2×Oꢀ(CH
2
)
2
H
2
H
2
2
2
5
3
×βCH
2
2
H
2
2
)
3
2
2
)
4
H
2
2
2
Trp, 2×εCH
2
2
2
core), 3.43 (m, 1H, CH
2
3.53 (m, 3H, OꢀCH ꢀCH2ꢀNH, CH2ꢀNH TA), 3.67 (br m, 2H, OꢀCH ꢀCH2ꢀNH), 3.81ꢀ3.96 [br
2
2
m, 8H, 2×OꢀC
H
2
ꢀ(CH
2
4
) ꢀNH, 4×αCH L-Lys], 4.08 (br m, 8H, 2×OꢀC
H
2
ꢀCH
2
ꢀNH, 4×αCH L-
4
2,6
Trp), 4.47 (m, 1H, αCH rdzenia), 6.59, 6.64 (2m, 2H, C ꢀH Ph), 6.96 (m, 4H, C ꢀH Ph),
5
2,5,6
6
2
7
7
.02 (m, 7H, C ꢀH L-Trp, C ꢀH TA), 7.10 (m, 4H, C ꢀH L-Trp), 7.21 (m, 4H, C ꢀH L-Trp),
7
7
4
.30 (m, 1H, C ꢀH TA), 7.37 (m, 4H, C ꢀH L-Trp), 7.51 (d, J = 7.86 Hz, H, C ꢀH TA), 7.64
4
(m, 4H, C ꢀH L-Trp).
1
3
C NMR (500 MHz, MeOD): δ = 23.2 (γC), 24.5, 24.6 [2×Oꢀ(CH ) ꢀ
C
H ꢀ(CH ) ꢀNH], 26.2
2
2
2
2 2
(C
H
2
ꢀAr TA), 28.9, 29.0 (βC L-Trp), 29.5, 29.9, 30.0 [δC, 2×OꢀCH
2
ꢀC
H
2
2 2 2
ꢀCH ꢀCH ꢀCH ꢀNH],
32.2, 32.3, 32.9 (βC), 40.1 (εC L-Lys), 40.2 (2×OꢀCH ꢀCH ꢀNH), 40.6 [2×Oꢀ(CH ) ꢀCH ꢀ
2 2 2 4 2
2
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NH], 40.7 (εC core), 41.4 (CH
2
ꢀNH TA), 54.4 (4×αC L-Lys), 54.5 (4×αC L-Trp), 55.3 (αC
4
core), 67.6, 67.7 (2×Oꢀ ꢀCH
C
H
2
2
ꢀNH), 69.2 [2×Oꢀ
C
H
2
ꢀ(CH
2
)
4
ꢀNH], 105.7, 106.0 (C Ph),
2,6
3
7
7
3
106.9, 107.2, 107.5 (C Ph), 108.1 (C L-Trp), 112.3 (C TA), 112.6 (C L-Trp), 113.1 (C
4
4
5
5
6
6
TA), 119.3 (C L-Trp, C TA), 119.6 (C TA), 120.2 (C L-Trp), 122.3 (C TA), 122.9 (C L-
2
2
3a
3a
1
Trp), 123.6 (C TA), 125.6 (C L-Trp), 128.4 (C L-Trp), 128.7 (C TA), 137.8 (C Ph),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
a
7a
3,5
138.1 (C L-Trp), 138.2 (C TA), 161.2, 161.8 (C Ph), 169.8 (CONH Ph), 170.0, 170.1,
170.4 (CONH).
2
D
5
[α]
= +28.1 (c 1, MeOH), m. p.: 197ꢀ206˚C.
Dendrimer 45
Pale yellow powder, obtained from 0.3 g (0.1 mmol) of dendrimer 73; 0.24 g, yield 96%.
C
114 167 15
H O N23×8HCl, M = 2391.37 g/mol (monoisotopic mass of nonprotonated dendrimer ꢀ
2098.3).
+
3+
+ 4+
LRMS (ESI, MeOH): 700.9 [M + 3H ] ꢀ main signal, 526.1 [M + 4H ] .
1
3
H NMR (500 MHz, MeOD): δ = 0.87 (br t, 3H, CH DDA), 1.20ꢀ1.94 [br m, 62H, 5×β, γ,
δCH L-Lys i rdzenia, 2×OꢀCH ꢀ(C
H
) ꢀCH ꢀNH, CH ꢀ2ꢀ11 DDA], 2.95ꢀ3.26 [br m, 18H,
2
2
2 3
2
2
4
×εCH
2
L-Lys, 2×βCH
2
L-Trp, 2×Oꢀ(CH
2
)
4
ꢀC
H
2
2 2
ꢀNH, CH ꢀ1 DDA], 3.35 (m, 6H, 2×βCH L-
2 2 2
Trp, εCH rdzenia), 3.55, 3.67 (2m, 4H, 2×OꢀCH ꢀCH ꢀNH), 3.85 (m, 4H, 4×αCH L-Lys),
3.93 [m, 4H, 2×OꢀC
H
2
ꢀ(CH
2
)
4
ꢀNH], 4.09 (m, 8H, 2×OꢀC
H
2
ꢀCH
2
ꢀNH, 4×αCH L-Trp), 4.49
4
2,6
2,6
(m, 1H, αCH rdzenia), 6.63 (m, 2H, C ꢀH Ph), 6.96 (m, 2H, C ꢀH Ph), 7.03 (m, 6H, C ꢀH
5
6
2
7
Ph, C ꢀH L-Trp), 7.10 (m, 4H, C ꢀH L-Trp), 7.21 (m, 4H, C ꢀH L-Trp), 7.37 (m, 4H, C ꢀH L-
4
Trp), 7.65 (m, 4H, C ꢀH L-Trp).
1
3
12
11
C NMR (500 MHz, MeOD): δ = 14.5 (C DDA), 23.1, 23.2, 23.7, 24.6 [γC, C DDA,
3
2×Oꢀ(CH
2
)
2
ꢀC
H
2
ꢀ(CH
2
2
) ꢀNH], 28.0 (C DDA), 28.9, 29.0 (βC L-Trp), 29.5, 29.9, 30.0, 30.1,
2
4
10
3
0.4ꢀ30.8 [δC, C , C ꢀC DDA, 2×OꢀCH
2
ꢀC
H
2
ꢀCH
2
ꢀC
H
2
ꢀCH
2
ꢀNH], 32.2, 32.3, 32.9, 33.1
1
(βC), 40.1 (εC L-Lys), 40.2 (2×OꢀCH
2
ꢀC
H
2
ꢀNH), 40.6 [2×Oꢀ(CH
2
)
4
ꢀC
H
2
ꢀNH, C DDA], 40.8
(εC rdzenia), 54.4 (4×αC L-Lys), 55.2 (4×αC L-Trp), 55.8 (αC rdzenia), 67.6 (2×OꢀCH ꢀCH ꢀ
2 2
4
2,6
2 2 4
NH), 69.2 [2×OꢀCH ꢀ(CH ) ꢀNH], 105.7, 106.0 (C Ph), 106.8, 107.1, 107.2 (C Ph), 108.1
3
7
4
5
6
2
(
C L-Trp), 112.6 (C L-Trp), 119.4 (C L-Trp), 120.2 (C L-Trp), 122.9 (C L-Trp), 125.6 (C
3
a
1
7a
3,5
L-Trp), 128.4 (C L-Trp), 137.2, 137.8 (C Ph), 138.2 (C L-Trp), 161.2, 161.8 (C Ph),
169.7 (CONH Ph), 170.0, 170.1, 170.5, 174.4 (CONH).
25
D
[α]
= +25.5 (c 1, MeOH), m. p.: 187ꢀ195˚C.
Dendrimer 46
2
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Compound 46 was obtained as pale yellow solid with yield 95.2% (0.5 g) z 0.63 g
(
0.205 mmol) of dendrimer 41a.
C
126 191 15
H O N23×8HCl, M = 2559.69 g/mol (monoisotopic mass of nonprotonated dendrimer ꢀ
2
266.5).
+ 2+
+ 3+
+
+ 2+
+ 4+
LRMS (ESI, MeOH): 1134.2 [M + 2H ] , 1161.2 [M + MeOH + H + Na ] , 774.5 [M +
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+ 3+
+
MeOH + 2H + Na ] , 756.5 [M + 3H ] , 581.1 [M + MeOH + 3H + Na ] , 567.6 [M +
+
4+
4
1
H ] ꢀ main signal
3
H NMR (500 MHz, MeOD): δ = 0.87 (m, 6H, 2×CH dDDA), 1.15ꢀ1.87 [br m, 82H, 5×β, γ,
δCH L-Lys i rdzenia, 2×OꢀCH ꢀ(C
H
) ꢀCH ꢀNH, 2×CH ꢀ2ꢀ11 dDDA], 3.0ꢀ3.41 [br m, 26H,
2
2
2 3
2
2
5
×εCH
2
L-Lys i rdzenia, 4×βCH
2
L-Trp, 2×Oꢀ(CH
2
)
4
ꢀC
H
2
ꢀNH, 2×CH
ꢀ(CH
.09 (m, 8H, 4×αCH L-Trp, 2×OꢀCH2ꢀCH ꢀNH), 4.96 (m, 1H, αCH rdzenia), 6.63 (m, 2H,
2
ꢀ1 dDDA], 3.50ꢀ3.73
(2m, 4H, 2×OꢀCH
2
ꢀC
H
2
ꢀNH), 3.80ꢀ3.96 [br m, 8H, 2×OꢀC
H
2
2 4
) ꢀNH, 4×αCH L-Lys],
4
2
4
2,6
2,6
5
1
6
C ꢀH Ph), 6.97 (m, 2H, C ꢀH Ph), 7.02 (m, 6H, C ꢀH Ph, C ꢀH L-Trp), 7. 1 (m, 4H, C ꢀH
2
7
4
L-Trp), 7.22 (m, 4H, C ꢀH L-Trp), 7.37 (m, 4H, C ꢀH L-Trp), 7.65 (m, 4H, C ꢀH L-Trp).
13
12
C NMR (500 MHz, MeOD): δ = 14.5 (2×C dDDA), 23.1, 23.2, 23.7, 23.8, 24.6, 27.9,
3
,11
2
2
3
8.0, 28.6 [γC, 2×C
2 2 2 2 2
dDDA, 2×Oꢀ(CH ) ꢀCH ꢀ(CH ) ꢀNH], 28.9, 29.0 (βC L-Trp), 29.5ꢀ
2
4
10
9.9, 30.0, 30.2, 30.4ꢀ30.8 [δC, 2×C , 2×C ꢀC dDDA, 2×OꢀCH
2
ꢀ
C
H
2
ꢀCH
2
ꢀ
C
H
2
ꢀCH
2
ꢀNH],
ꢀNH],
2.3, 33.1 (βC), 40.1 (εC L-Lys), 40.2 (2×OꢀCH
2
ꢀC
H
2
ꢀNH), 40.6 [2×Oꢀ(CH ) ꢀCH
2 4
2
1
40.7 (εC core), 47.7 (2×C dDDA), 51.5 (αC core), 54.4 (4×αC L-Lys), 55.2 (4×αC L-Trp),
4
6
1
7.6 (2×Oꢀ
C
H
2
ꢀCH
2
ꢀNH), 69.2 [2×Oꢀ
C
H
2
ꢀ(CH
2
)
4
ꢀNH], 105.7, 106.0 (C Ph), 106.8, 106.9,
2
,6
3
7
4
5
07.2, 107.5 (C Ph), 108.1 (C L-Trp), 112.6 (C L-Trp), 119.4 (C L-Trp), 120.2 (C L-
6
2
3a
1
7a
Trp), 122.9 (C L-Trp), 125.6 (C L-Trp), 128.4 (C L-Trp), 137.2, 137.7 (C Ph), 138.2 (C
3,5
L-Trp), 161.2, 161.8 (C Ph), 169.6, 169.7 (CONH Ph), 170.0, 170.1, 170.5, 173.7 (CONH).
2
D
5
[α]
= +25.2 (c 1, MeOH), m.p.: 195ꢀ198˚C.
Antimicrobial testing
concentrations
The antimicrobial spectrum of dendrimers was evaluated by the minimal inhibitory
(
MIC) method using the serial twoꢀfold dilution method under standard conditions as
described in the Clinical and Laboratory Standards Institute (CLSI) reference method M07ꢀ
®
A8. A panel of Gramꢀpositive bacterial strains, Staphylococcus aureus ATCC 29213
®
(sensitive to methicillin, (MSSA)), S. aureus ATCC 43300 (resistant to methicillin,
®
TM
(
MRSA)), S. aureus ATCC 700787 (intermediate to vancomycin, (VISA)), S. epidermidis
®
TM
®
TM
ATCC 12228 , and Gramꢀnegative, Escherichia coli ATCC 25922 , E. coli ATCC
2
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®
BAAꢀ198 (ESBL), Acinetobacter baumanii ATCC 19606, Salmonella enterica ATCC
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0708, Bordetella bronchiseptica ATCC 4617, Stenotrophomonas maltophilia ATCC 13637,
Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 13882 were used. 68
clinical isolates of Escherichia coli (ESBL+, ꢀ) obtained from Medical University of Warsaw,
Poland were also used. The clinical isolates were collected from various patients hospitalized
in several clinics. Complete list of hospital acquired E. coli strains used in these studies along
with source and antibiotic susceptibility is shown in Supplementary Data Table S2. Bacterial
strains were cultivated on tryptic soy agar (TSA) according to ATCC recommendation. All
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strains were incubated for 24 hours at 37 C.
Reference method (broth microdilution susceptibility test) was as follows: The dendrimers
were dissolved in DMSO. A series of the twoꢀfold compounds dilutions were diluted with
cation–adjusted Mueller–Hinton broth (CAMHB). 95 µl aliquots were dispensed into
4
microdilution sterile plates (MarꢀFour). Then, 5 µl of bacteria inoculum, containing 5 x 10
ꢀ1
ꢀ1
CFU ml , was added. The final concentration of compounds ranged from 256 to 2 µg ml all
in twoꢀfold dilution steps. The experiments for each sample were conducted in triplicate.
ꢀ1
o
Penicillin G were used as controls (from 8 – 0.15 µg ml ). The plates were incubated at 37 C
o
(
35 C) for 18 to 24 hours depending on bacterial strain. Results were obtained with the use of
Spectrostar Omega (BMG Labtech, Ortenberg, Germany), absorbance was measured at λ=
40 nm and λ= 595 nm. MIC was defined as the lowest drug concentration that reduced
growth by 100 %. MIC value of the reference compound, penicillin G against S. aureus
5
®
ꢀ1
ATCC 29213 – 2.0 µg ml .
Genotoxicity
Genotoxicity of the selected dendrimers (42, 44, 46) was performed using genetically
modified Bacillus subtilis M45 recꢀ i H17 rec+ obtained from dr. Yoshito Sadaie (Department
of Induced Mutation, National Institute of Genetics, Mishima, Shizuokaꢀken 411, Japan) by
45ꢀ47
the discꢀdiffusion method according to Kada’s procedure.
Sterile filter paper discs (9 mm
diameter, Whatman No3) were dripped with tested dendrimers solution to load 256 µg of the
compound per disc. As a standard 4ꢀnitroquinoline Nꢀoxide (2 µg per disc) was used. Results
of the genotoxicity test were read after 18 hours of incubation of the discs at 35 °C.
Cell Proliferation Assays
The established human melanoma cell line MeW155 and human fibroblast AW/4, were
obtained from the institutional repository at the Maria SkłodowskaꢀCurie Memorial Institute
and Oncology Centre in Warsaw. The melanoma and the fibroblast cell lines were cultured in
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Page 29 of 39
Bioconjugate Chemistry
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Eagle’s 1959 MEM (BiomedꢀLublin) supplemented with 10 % fetal calf serum (Invitrogen),
50 mg/mL penicillin G, 50 mg/ml streptomycin, and 0.1 % glutamine in a humidified
atmosphere with 5 % CO . Adherent cell cultures were plated in 24ꢀwell plates. The initial
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cell number in culture and timing of medium supplementation was adjusted for each cell line
to achieve the optimal cell growth. The culture timing was adjusted to harvest cells at the state
of subꢀconfluency. Adherent cells were harvested using 0.25 % trypsin with EDTA (200
mg/L). Following trypsinization, the harvested cells were counted directly in a
haemacytometer under a microscope, and cell viability was evaluated according to trypan blue
staining.
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Live/dead staining
LIVE/DEAD staining was performed with the LIVE/DEAD BacLight bacterial viability kit
(Invitrogen GmbH, Darmstadt, Germany). Briefly, cultures were grown to midꢀexponential
growth phase under anaerobic conditions, kit reagents were added according to the
manufacturer’s protocol, and 1ꢀmL aliquots of cultures supplemented with reagents were
dispensed to anaerobic Hungate tubes. AMPs were supplemented to different final
concentrations, as given in the Results section. After 15 min incubation in the dark with
dendrimers, cells were microscopically examined at a 1,000 magnification using an Optaꢀ
Tech MN800FL (Poland, Warsaw) supplied with a digital camera.
Preparation of Large Unilamellar Vesicles (LUV’s)
Dyeꢀencapsulated LUV’s composed of POPC, POPG, POPC:POPG (3:1, w/w) and DMPC
were prepared by dissolving 10 mg of lipids in chloroform with a few drops of trifluorethanol
(if necessary), drying then under a stream of nitrogen gas and placing the lipid films under
vacuum overnight to remove any traces of organic solvent. The dried lipids were resuspended
in 1 mL of a dye buffer solution (100 mM CF, 10 mM TRIS, 100 mM NaCl, pH 7.4) or water
containing 2.5 mM calcein by vortexing. The lipid suspension was repeatedly freezeꢀthawed in
liquid nitrogen, and then extruded twenty times through a 0.1 ꢀM pore polycarbonate membrane.
The dyeꢀencapsulated vesicles were separated from free dye by gel filtration chromatography,
using Sephadex Gꢀ50.
Fluorescence spectroscopy measurements
Fluorescence measurements were performed using a Hitachi Fꢀ7000 spectrofluorimeter at an
excitation wavelength of 485 nm and an emission wavelength of 520 nm. All experiments
were conducted at room temperature in 1ꢀcm path length cuvettes. Samples were prepared by
adding 20 µL of solutions containing various concentrations of dendrimer 42 or 44 and 20 µL
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