Carbosilane DendronâPeptide Nanoconjugates as Antimicrobial

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CARBOSILANE DENDRONS-PEPTIDES NANOCONJUGATES AS ANTIMICROBIAL AGENTS Jael Fernández, Gerardo Acosta, Daniel Pulido, Marek Maly, José L. Copa-Patiño, Juan Soliveri, Miriam Royo, Rafael Gómez, Fernando Albericio, Paula Ortega, and F. Javier de la Mata Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.9b00222 • Publication Date (Web): 22 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

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Molecular Pharmaceutics

1

Manuscript submitted to Molecular Pharmaceutics

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Article Type: Regular paper

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Corresponding Author: Fco. Javier de la Mata de la Mata

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Departamento de Química Orgánica y Química Inorgánica

5

Universidad de Alcalá

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Campus Universitario. Edificio de Farmacia

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28871 Alcalá de Henares. Spain.

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Tel: (+34) 91 8854679. Fax: (+34) 91 8854683.

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E-mail: [email protected]

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CARBOSILANE DENDRONS-PEPTIDES NANOCONJUGATES AS

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ANTIMICROBIAL AGENTS

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Jael Fernandez, 1,2,3 Gerardo Acosta,2,4 Daniel Pulido,2,5 Marek Malý,7 José L. Copa-Patiño,8 Juan

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Soliveri,8 Miriam Royo,2,5 Rafael Gómez,1,2,3 Fernando Albericio,2,4,6 Paula Ortega,1,2,3* F. Javier

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de la Mata. 1,2,3*

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1Departamento

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de

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[email protected]

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2

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BBN), Spain. E-mail: [email protected].

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3 Instituto

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4

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Spain.

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5

24

6 School

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7 Faculty

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Republic.

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8 Departamento

Química Orgánica y Química Inorganica, Universidad de Alcalá, Spain. Instituto

Investigación

Química

"Andrés

M.

del

Río"

(IQAR),

UAH,

Spain.

Email:

Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-

Ramón y Cajal de Investigación Sanitaria, IRYCIS, Spain.

Deparment of Organic and Inorganic Chemistry, University of Barcelona, 08028 Barcelona,

Institute for Advanced Chemistry of Catalonia-CSIC, Spain. of chemistry and physics, University of KwaZulu-Natal, Durban 4001, South Africa. of Science, J. E. Purkinje University, České mládeže 8, 400 96 Ústí nad Labem, Czech

Biomedicina y Biotecnología, Universidad de Alcalá, Spain.

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1 ACS Paragon Plus Environment

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ABSTRACT

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Over last decades, multidrug resistant bacteria have emerged and spread, increasing the number

3

of bacteria against which, commonly used antibiotics are no longer effective. It has become a

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serious public health problem whose solution requires medical research in order to explore novel

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effective antimicrobial molecules. On one hand, antimicrobial peptides (AMPs) are regarded as a

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good alternative due to their generally broad-spectrum activities, but sometimes they can be easily

7

degraded by the organism or be toxic to animal cells. On the other hand, cationic carbosilane

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dendrons, whose focal point can be functionalized in many different ways, have also shown good

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antimicrobial activity. In this work, we synthetized first and second generation cationic

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carbosilane dendrons with a maleimide molecule on their focal point, enabling their

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functionalization with three different AMPs. After different microbiology studies, we found an

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additive effect between first generation dendron and AMP3 whose study reveals three interesting

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effects: (i) bacteria aggregation due to AMP3, which could facilitate bacteria detection or even

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contribute to antibacterial activity by preventing host cell attack, (ii) bacteria disaggregation

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capability of second generation cationic dendron and (iii) a higher AMP3 aggregation ability

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when dendron was added previously to peptide treatment. These compounds and their different

17

effect observed over bacteria constitute an interesting system for further mechanism studies.

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Keywords: antibacterial peptides, carbosilane dendrons, molecular modelling and molecular

19

dynamics.

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Abbreviations AMPs

antimicrobial peptides

TMS

trimethylsilane

HSQC- NMR

Heteronuclear single quantum coherence spectroscopy

HMBC- NMR

Heteronuclear Multiple Bond Correlation

COSY- NMR

COrrelation SpectroscopY

NOESY- NMR

Nuclear Overhauser Effect

TOCSY- NMR

Total Correlation Spectroscopy

HPLC

High performance liquid chromatography 2 ACS Paragon Plus Environment

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TFA

Trifluoroacetic acid

TOF LC/MS

High Resolution Mass Spectrometry

ESI-TOF

Electrospray ionisation time-of-flight mass spectrometry

DMF

Dimethyformamide

SPPS

Solid Phase Peptide Synthesis

MHB

Muller-Hinton broth

PCA

plate count agar

MIC

Minimal Inhibitory Concentration

MBC

Minimal Biocidal Concentration

NB

Nutrient broth

FICI

Fractional Inhibitory Concentration Index

FIC

fractional inhibitory concentration

HEPES buffer

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

EDTA

Ethylenediaminetetraacetic acid

PBS

Phosphate Buffer Solution

RESP

Restrained Electrostatic Potential

GAFF

Generalized Amber Force Field

LIPID

Lipid Simulation

1 2 3

1.

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In the last years, the emergence of resistant bacteria is exacerbated and accelerated by the

5

inappropriate use of antibiotics and constitutes a risk to the patient and public health.1, 2 Resistance

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to antibiotics is a natural part of the evolution of bacteria and unavoidable given the many types

7

of bacteria and the susceptibility of the human host. For this reason, it is necessary to discover

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new treatments in order to continue fighting illness caused by bacteria infection.3, 4

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In order to find new therapeutic drugs, antimicrobial peptides (AMPs) have attracted interest in

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this context largely due to its lower, although possible, resistant generation5-7. AMPs are key

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components of the innate immune system, also known as host defense antimicrobial peptides, and

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they are characterized by cationic amphipathic properties with broad spectrum antimicrobial

INTRODUCTION



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activity against bacteria, viruses, and fungi.8-10 AMPs interact electrostatically with negatively

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charged bacterial surface and with lipid bilayers, forming transmembrane pores that provoke the

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death of the bacteria. However, they are not devoid of drawbacks because AMPs exhibit

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unfavorable properties, they are labile depending on the surrounding environments (presence of

5

proteases or pH changes), the hemolytic activity, the high cost of production, salt sensitivity, and

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a non-specific broad spectrum of action.11

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Several delivery systems have been initially investigated as AMP carriers. For example,

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surfactants, lipids, polymers or diverse nanoparticles have been evaluated.12 One of these delivery

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systems could be dendritic structures that, in contrast to conventional linear polymers, exhibit

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tridimensional and well-defined highly branched structures with a precise number of terminal

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groups. Dendrimers are defined as synthetic, extremely branched, globular, monodisperse

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macromolecules with spatial arrangement and nanometric size and are used as nanocarriers for

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drug delivery and to solubilize poorly water-soluble drugs.13-15 Dendrimers are synthesized by an

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iterative sequence of reaction steps, leading to a unique branching architecture.16 A monodisperse

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nature, controllable molecular weight, large number of readily available functional groups on the

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surface and an extraordinary ability for encapsulation of guest molecules within the internal

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hydrophobic environment are some of the unique properties of dendrimers that add to their caliber

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as new scaffolds for drug delivery.13, 17 Over recent years, the conjugation between peptides and

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dendrimers has been widely studied. The so-called “peptide dendrimers” are macromolecules in

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which some of their parts, such as branching units, core or surface functional groups are composed

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by peptides.18 The combination of both systems may entail certain advantages such a synergic

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action due to: (i) multivalence that could affect the biological effects of the conjugated dendrimer-

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peptides and offer the possibility to interact with many receptors at the same time (ii) protective

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effect of dendrimers over the peptide avoiding their degradation and increasing bioavailability.

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Carbosilane dendrimers have demonstrated high antibacterial potency against Gram-positive and

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Gram-negative bacteria strains when they are in contact with an aqueous suspension of bacteria.19,

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These dendritic structures do not generate resistances and keep their activity against resistant 4 ACS Paragon Plus Environment

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bacterial strains. The presence of hydrophobic chains in their skeleton favors an increment of the

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activity probably due to their easier penetration into the phospholipid bilayer, leading to the

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disintegration of the bacterial membrane.

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For these reasons, the formation of peptide-carbosilane dendrimer nanoconjugates could present

5

interest since it could lead to the establishment of synergistic or collaborative interactions between

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both types of molecules. In addition, dendrimers could help to protect or transport AMPs inside

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bacterial membranes helping them to exhibit their antibacterial activity. The formation of peptide-

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dendrimer nanoconjugates through different types of interactions would allow to evaluate how

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the nature of the bonding between these two molecules could affect their activity. For this reason,

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in this work we have considered the combination of carbosilane dendrons with different AMPs in

11

order to study their antibacterial activity against two types of bacteria, Staphylococcus aureus

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(Gram +) and Escherichia coli (Gram -). To achieve this goal we have synthesized cationic

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carbosilane dendrons with a maleimide group in the focal point capable of covalently linking

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AMPs containing an additional cysteine residue, through thiol-ene addition reactions rendering

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AMP-carbosilane dendron conjugates. To estimate the effect of the conjugation in the

16

antibacterial activity, we have also evaluated the corresponding dendron-peptide complexes

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formed through non-covalent interactions (electrostatic, hydrophobic, hydrogen bonding…).

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2.

MATERIAL AND METHODS

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2.1.

Chemistry - general description

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All reactions were carried out under inert atmosphere and solvents were purified from appropriate

21

drying agents when necessary. The starting materials used to prepare the peptide dendritic systems

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are carbosilane dendritic wedges with–NH2 group in the focal point,21 NH2Gn(S(CH2)2NMe2)m

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(n=1,m=2(I); n=2,m=4(II); n=3,m=8(II)). NMR spectra were recorded on a Varian Unity VXR-

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300 (300.13 (1H), 75.47 (13C) MHz) or on a Bruker AV400. Chemical shifts (δ) are given in ppm.

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1H

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TMS = 0 ppm. When necessary, assignment of resonances was done from HSQC, HMBC, COSY,

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TOCSY, and NOESY NMR experiments. Elemental analyses were performed on a LECO CHNS-

and 13C resonances were measured relative to internal deuterated solvents peaks considering



Page 6 of 39

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932 instrument. Mass spectra were obtained an Agilent 6210 TOF LC/MS instrument for ESI-

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TOF in MeOH/H2O with (NH4)(HCO2). Analytical HPLC were performed on a Waters Alliance

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2695 with an automated injector and a photodiode array detector Waters 2998, using a XBridgeTM

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C18 reversed-phase analytical column (4.6 mm×100 mm, 5 μm) and either a linear gradient 5–

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100% CH3CN (0.036% TFA) in H2O (0.045% TFA) over 8 min at a flow rate of 1 mL/min

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(System A), or a linear gradient 0–50% CH3CN (0.036% TFA) in H2O (0.045% TFA) over 8 min

7

at a flow rate of 1 mL/min (System B). HPLC-MS analyses were performed on a Waters Alliance

8

2795 with an automated injector and a photodiode array detector Waters 2996 coupled to an

9

electrospray ion source (ESI-MS) Micromass ZQ mass detector, using either a XSelectTM C18

10

reversed-phase analytical column (4.6 mm×50 mm, 3.5 μm), or a Symmetry 300 C4 reversed-

11

phase analytical column (4.6 mm×50 mm, 3.5 μm) and the MassLynx 4.1 software. The

12

instrument was operated in the positive ESI (+) ion mode. HPLC-MS analyses were carried out

13

with several elution systems. System A (C18 column): a linear gradient 5–100% CH3CN (0.07%

14

HCOOH) in H2O (0.1% HCOOH) over 4.5 min at a flow rate of 2 mL/min; and System B (C4

15

column): a linear gradient 5–100% CH3CN (0.07% HCOOH) in H2O (0.1% HCOOH) over

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30 min at a flow rate of 1 mL/min.

17

2.1.1. Synthesis of C2H2(CO)2N(CH2)4[G1(S(CH2)2NMe2)2]; MalG1(SNMe2)2 (1). In an

18

ampule,

19

NH2(CH2)4[G1(S(CH2)2NMe2)2] (0.49 g, 1.3 mmol) maleic anhydride C2H2(CO)2O was added

20

(0.13 g, 1.3 mmol). The mixture was stirred at room temperature for 2 h, after which a Kaiser test

21

was carried out obtaining negative result. Then, solvent was removed in vacuum and yellowish

22

oil was redissolved in acetic anhydride (5 mL). Sodium acetate CH3CO2Na was added (0.05 g,

23

0.65 mmol) and reaction mixture was heated at 60 °C for 16 h. After that, solvent was evaporated

24

under reduced pressure and CH2Cl2/H2O(NaCl) extraction was performed, obtaining

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C2H2(CO)2N(CH2)4[G1(S(CH2)2NMe2)2] as a brown oil after the organic phase was dried over

26

MgSO4 and the volatiles were evaporated. Yield: 0.53 g, 1.16 mmol, (89%). 1H-RMN (CDCl3):

27

δ -0.04 (3 H, s, SiMe), 0.53 (2 H, t, NCH2CH2CH2CH2Si), 0.84 (4 H, t, SiCH2CH2S), 1.23 (2 H,

to

a

dry

dichloromethane

solution


of

carbosilane

dendron

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1

m, NCH2CH2CH2CH2Si), 1.55 (2 H, m, NCH2CH2CH2CH2Si), 2.22 (12 H, s, SCH2CH2NMe2),

2

2.45-2.52 (8 H, m, CH2SCH2CH2NMe2), 2.59 (4 H, t, SCH2CH2NMe2), 3.45 (2 H, t,

3

NCH2CH2CH2CH2Si), 6.64 (2 H, s, C2H2(CO)2N). 13C{1H}-RMN (CDCl3): δ -5.28 (SiMe), 13.06

4

(NCH2CH2CH2CH2Si), 14.55 (SiCH2CH2S), 20.99 (NCH2CH2CH2CH2Si), 27.67 (SiCH2CH2S),

5

29.90 (SCH2CH2NMe2), 32.26 (NCH2CH2CH2CH2Si), 37.36 (NCH2CH2CH2CH2Si), 45.41

6

(SCH2CH2NMe2), 59.26 (SCH2CH2NMe2), 133.94 (C2H2(CO)2N), 171.02 (C2H2(CO)2N). Anal.

7

Calcd for C21H41N3O2S2Si (459.78 g mol-1): C, 54.86; H, 8.99; N, 9.14; S, 13.95 %. Found: C,

8

54.1; H, 8.42; N, 8.66; S, 11.47 %. ESI-MS: [ M + H ]+ 460.24 uma (Calcd 460.25 uma).

9

2.1.2. Synthesis of C2H2(CO)2N(CH2)4[G2(S(CH2)2NMe2)4]; MalG2(SNMe2)4 (2). Following

10

the procedure described for compound 1, compound 2 was obtained as brown oil from the reaction

11

of NH2(CH2)4[G2(S(CH2)2NMe2)4] (0.36 g, 0.44 mmol), C2H2(CO)2O (0.04 g, 0.44 mmol) and

12

CH3CO2Na (0.02 mg, 0.22 mmol). Yield: 0.33 g, 0.37 mmol, (83%). 1H-RMN (CDCl3): δ -0.12

13

(3 H, s, SiMe), -0.01 (6 H, s, SiMe), 0.51 (6 H, m, NCH2CH2CH2CH2Si and SiCH2CH2CH2Si),

14

0.59 (4 H, t, SiCH2CH2CH2Si), 0.87 (8 H, t, SiCH2CH2S), 1.25 (6 H, m, NCH2CH2CH2CH2Si and

15

SiCH2CH2CH2Si), 1.56 (2 H, m, NCH2CH2CH2CH2Si), 2.23 (24 H, s, SCH2CH2NMe2), 2.47 (8

16

H, t, SCH2CH2NMe2), 2.53 (8 H, t, SiCH2CH2S), 2.6 (8 H, t, SCH2CH2NMe2), 3.48 (2 H, t,

17

NCH2CH2CH2CH2Si), 6.66 (2 H, s, C2H2(CO)2N). 13C{1H}-RMN (CDCl3): δ -5.30 (SiMe), 13.65

18

(NCH2CH2CH2CH2Si),

19

(SiCH2CH2CH2Si), 18.42 (SiCH2CH2CH2Si), 18.64 (SiCH2CH2CH2Si), 27.68 (SiCH2CH2S),

20

29.76 (SCH2CH2NMe2), 32.30 (NCH2CH2CH2CH2Si), 37.49 (NCH2CH2CH2CH2Si), 45.30

21

(SCH2CH2NMe2), 59.23 (SCH2CH2NMe2), 134.00 (C2H2(CO)2N), 170.84 (C2H2(CO)2N). Anal.

22

Calcd for C41H87N5O2S4Si3 (894.68 g mol-1): C, 55.04; H, 9.80; N, 7.83; S, 14.34 %. Found: C,

23

52.71; H, 9.44; N, 7.02; S, 12.58 %.

24

2.1.3. Synthesis of C2H2(CO)2N(CH2)4[G1(SN+Me2H∙Cl-)2]; MalG1(SN+Me2H∙Cl-)2 (3). To a

25

diethyl ether solution of compound 1 (0.07 g, 0.16 mmol), an excess of hydrogen chloride in

26

diethyl ether (0.94 mL, 0.94 mmol) was added under inert atmosphere. The mixture was stirred

27

at room temperature for 1 h. Then, solvent and hydrogen chloride excess were evaporated under

14.65

(SiCH2CH2S),

18.28


(NCH2CH2CH2CH2Si),

18.31


Page 8 of 39

1

reduced pressure and compound 10 was obtained as brown oil. Yield: 0.09 g, 1.16 mmol, (100%).

2

1H-RMN

3

SiCH2CH2S), 1.16 (2 H, m, NCH2CH2CH2CH2Si), 1.46 (2 H, m, NCH2CH2CH2CH2Si), 2.53 (4

4

H, t, SiCH2CH2S), 2.78 (12 H, s, SCH2CH2NMe2), 2.79 (4 H, m, SCH2CH2NMe2), 3.22 (4 H, t,

5

SCH2CH2NMe2), 3.38 (2 H, t, NCH2CH2CH2CH2Si), 6.70 (2 H, s, C2H2(CO)2N). 13C{1H}-RMN

6

(D2O):

7

(NCH2CH2CH2CH2Si),

8

(NCH2CH2CH2CH2Si),

9

(SCH2CH2NMe2),

(D2O): δ -0.11 (3 H, s, SiMe), 0.52 (2 H, t, NCH2CH2CH2CH2Si), 0.80 (4 H, t,

δ

-3.97

(SiMe),

14.35

27.65 39.64

136.77

(NCH2CH2CH2CH2Si), (SCH2CH2NMe2),

29.16

(NCH2CH2CH2CH2Si),

(C2H2(CO)2N),

16.05

175.91

45.11

(SiCH2CH2S),

22.73

(SiCH2CH2S),

33.60

(SCH2CH2NMe2),

58.76

(C2H2(CO)2N).

Anal.

Calcd

for

10

C21H43Cl2N3O2S2Si2+ (532.70 g mol-1): C, 47.35; H, 8.14; N, 7.89; S, 12.04 %. Found: C, 45.79;

11

H, 8.21; N, 7.65; S, 10.43 %.

12

2.1.4. Synthesis of C2H2(CO)2N(CH2)4[G2(SN+Me2H∙Cl-)4]; MalG2(SN+Me2H∙Cl-)4 (4).

13

Following the procedure described for compound 10, compound 11 was obtained as a brown oil

14

from the reaction of (0.12 g, 0.13 mmol) of 2 and hydrogen chloride in diethyl ether (1.34 mL,

15

1.34 mmol). Yield: 0.14 g, 0.13 mmol, (100%). 1H-RMN (D2O): δ -0.17 (3 H, s, SiMe), -0.05 (6

16

H, s, SiMe), 0.44 (2 H, m, NCH2CH2CH2CH2Si), 0.49 (4 H, m, SiCH2CH2CH2Si), 0.59 (4 H, m,

17

SiCH2CH2CH2Si), 0.85 (8 H, t, SiCH2CH2S), 1.19 (2 H, m, NCH2CH2CH2CH2Si), 1.28 (4 H, m,

18

SiCH2CH2CH2Si), 1.49 (2 H, m, NCH2CH2CH2CH2Si), 2.59 (8 H, m, SiCH2CH2S), 2.80 (24 H,

19

s, SCH2CH2NMe2), 2.87 (8 H, m, SCH2CH2NMe2), 3.25 (8 H, m, SCH2CH2NMe2), 3.42 (2 H, t,

20

NCH2CH2CH2CH2Si), 6.76 (2 H, s, CHCH(CO)2N).

21

(SiMeCH2CH2S),

22

(SiCH2CH2S), 19.62 (SiCH2CH2CH2Si), 20.21 (SiCH2CH2CH2Si), 20.54 (SiCH2CH2CH2Si),

23

23.31

24

(NCH2CH2CH2CH2Si),

25

(SCH2CH2NMe2),

26

C41H91Cl4N5O2S4Si34+ (1040.51 g mol-1): C, 47.33; H, 8.82; N, 6.73; S, 12.32 %. Found: C, 45.67;

27

H, 8.35; N, 6.05; S, 9.47 %.

-3.09

(NCH2CH2CH2CH2SiMe),

(NCH2CH2CH2CH2Si), 39.90

137.12

27.85

13C{1H}-RMN

15.20

(SCH2CH2NMe2),

(NCH2CH2CH2CH2Si),

(C2H2(CO)2N),

175.72

(D2O): δ

-3.67

(NCH2CH2CH2CH2Si),

16.41

29.56

45.25

(SiCH2CH2S),

33.80

(SCH2CH2NMe2),

58.70

(C2H2(CO)2N).


Anal.

Calcd

for

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1

2.1.5. Synthesis of L-CysHClSucG1(S(CH2)2NMe2)2 (5). In a schlenk under inert atmosphere,

2

0.10 g (0.21 mmol) of compound 1 dissolved in DMF (2 mL) were mixed with 0.03 g (0.21 mmol)

3

of L-Cysteine hydrochloride anhydrous and stirred at room temperature for 5 h. After that, solvent

4

was evaporated under reduced pressure and compound 5 was obtained as a brown oil. 1H-RMN

5

(D2O): δ -0.07 (3 H, s, SiMe), 0.51 (2 H, t, NCH2CH2CH2CH2Si), 0.81 (4 H, t, SiCH2CH2S), 1.18

6

(2 H, m, NCH2CH2CH2CH2Si), 1.47 (2 H, m, NCH2CH2CH2CH2Si), 2.54 (4 H, t, SiCH2CH2S),

7

2.79 (12 H, s, SCH2CH2NMe2), 2.81 (4 H, m, SCH2CH2NMe2), 3.04-3.09 (2 H, dd, Cl-

8

(COOH)(N+H3)CHCH2S), 3.19-3.27 (6 H, m, SCHCH2(CO)2N and SCH2CH2NMe2), 3.39 (4 H,

9

m,

NCH2CH2CH2CH2Si

and

Cl-(COOH)(N+H3)CHCH2S),

3.87-3.90

(1

H,

m,

Cl-

10

(COOH)(N+H3)CHCH2S), 3.91-3.94 (1 H, m, SCHCH2(CO)2N). 13C{1H}-RMN (D2O): δ -3.51

11

(SiMe), 14.75 (NCH2CH2CH2CH2Si), 16.34 (SiCH2CH2S), 23.04 (NCH2CH2CH2CH2Si), 27.90

12

(SCH2CH2NMe2),

13

(COOH)(N+H3)CHCH2S) 35.65 (SCHCH2(CO)2N), 41.35 (NCH2CH2CH2CH2Si), 45.34

14

(SCH2CH2NMe2), 56.11 (Cl-(COOH)(N+H3)CHCH2), 56.59 (SCHCH2(CO)2N), 180.73, 181.84,

15

182.43 (COOH, SCHCH2CON and SCHCON). Anal. Calcd for C24H50ClN4O4S3Si+ OH- (635.41

16

g mol-1): C, 45.37; H, 8.09; N, 8.82; S, 15.14 %. Found: C, 43.94; H, 9.42; N, 8.21; S, 13.64 %.

17

ESI-MS: [ M + H ]+ 581.27 uma (Calcd 581.27 uma), [ M + 2H ]2+ 291.14 uma (Calcd 291.14

18

uma).

19

2.1.6. Synthesis of L-CysHClSucG2(S(CH2)2NMe2)4 (6). Following the procedure described for

20

compound 5, compound 6 was obtained as a brown oil from the reaction of 0.07 g (0.07 mmol)

21

of compound 2 and 0.01 g (0.07 mmol) of L-Cysteine hydrochloride anhydrous. 1H-RMN (D2O):

22

δ -0.13 (3 H, s, SiMe), -0.00 (6 H, s, SiMe), 0.47 (2 H, m, NCH2CH2CH2CH2Si), 0.51 (4 H, m,

23

SiCH2CH2CH2Si), 0.59 (4 H, m, SiCH2CH2CH2Si), 0.87 (8 H, m, SiCH2CH2S), 1.21-1.33 (6 H,

24

m, NCH2CH2CH2CH2Si and SiCH2CH2CH2Si), 1.49 (2 H, m, NCH2CH2CH2CH2Si), 2.61 (8 H,

25

m, SiCH2CH2S), 2.84 (24 H, s, SCH2CH2NMe2), 2.87 (8 H, m, SCH2CH2NMe2), 2.93-2.99 (2 H,

26

dd, Cl-(COOH)(N+H3)CHCH2S), 3.25-3.31 (10 H, , SCHCH2(CO)2N and SCH2CH2NMe2), 3.40

27

(4 H, m, NCH2CH2CH2CH2Si and Cl-(COOH)(N+H3)CHCH2S), 3.88-3.90 (1 H, m, Cl-

29.40

(SiCH2CH2S),

32.91

(NCH2CH2CH2CH2Si),


34.66

(Cl-


Page 10 of 39

1

(COOH)(N+H3)CHCH2S), 3.94-3.98 (1 H, m, SCHCH2(CO)2N), 7.82 (4 H, m, N+HNMe2), 8.33

2

(3 H, m, Cl-(COOH)(N+H3)).

3


4

(SiCH2CH2CH2Si),

5

(NCH2CH2CH2CH2Si),

6

(NCH2CH2CH2CH2Si), 34.72 (Cl-(COOH)(N+H3)CHCH2S), 35.74 (SCHCH2(CO)2N), 41.24

7

(NCH2CH2CH2CH2Si), 45.31 (SCH2CH2NMe2), 55.71 (Cl-(COOH)(N+H3)CHCH2S), 55.71 (Cl-

8

(COOH)(N+H3)CHCH2), 56.23 (SCHCH2(CO)2N), 59.02 (SCH2CH2NMe2), 174.81, 174.45,

9

167.40 (COOH and SCHCH2CON and SCHCON). Anal. Calcd for C44H98ClN6O4S5Si33+3OH-

10

(1106.33 g mol-1): C, 47.77; H, 9.20; N, 7.60; S, 14.49 %. Found: C, 43.64; H, 9.55; N, 7.32; S,

11

12.52 %. [ M + 2H ]2+ 508.27 uma (Calcd 508.27 uma).

12

2.1.7. Synthesis of N-Acetyl-L-CysSucG1(S(CH2)2NMe2)2 (7). In a schlenk under inert

13

atmosphere, 0.08 g (0.17 mmol) of compound 1 dissolved in DMF (2 mL) were mixed with 0.03

14

g (0.17 mmol) of N-Acetyl-L-Cysteine and stirred at room temperature for 5 h. After that, solvent

15

was evaporated under reduced pressure and compound 7 was obtained as a brown oil. 1H-RMN

16

(D2O): δ -0.06 (3 H, s, SiMe), 0.51 (2 H, t, NCH2CH2CH2CH2Si), 0.83 (4 H, t, SiCH2CH2S), 1.17

17

(2 H, m, NCH2CH2CH2CH2Si), 1.49 (2 H, m, NCH2CH2CH2CH2Si), 1.95 (3 H, t, CH3OCNH),

18

2.54-2.60 (6 H, m, SiCH2CH2S and SCHCH2(CO)2N), 2.80 (12 H, s, SCH2CH2NMe2), 2.81 (4 H,

19

m, SCH2CH2NMe2), 2.87-2.91 and 3.20-3.23 (2 H, 2dd, (CH3OCNH)CHCH2S), 3.16-3.23 (2 H,

20

m, SCHCH2(CO)2N), 3.25 (4 H, t, SCH2CH2NMe2), 3.41 (2 H, t, NCH2CH2CH2CH2Si), 3.96 (1

21

H, m, SCHCH2(CO)2N), 4.35 (1 H, m, (CH3OCNH)CHCH2S), 7.85 (1 H, m, CH3OCNH).

22

13C{1H}-RMN

23

(NCH2CH2CH2CH2Si), 24.71 (CH3OCNH), 27.97 (SCH2CH2NMe2), 29.48 (SiCH2CH2S), 33.02

24

(NCH2CH2CH2CH2Si), 36.25-36.63 ((CH3OCNH)CHCH2S), 38.62-39.03 (SCHCH2(CO)2N),

25

41.15 (NCH2CH2CH2CH2Si), 42.35 and 43.07 (SCHCH2(CO)2N), 45.31 (SCH2CH2NMe2), 56.85

26

((CH3OCNH)CHCH2S), 58.98 (SCH2CH2NMe2), 175.96 (CH3OCNH), 178.65 (COOH), 180.78

27

(SCHCH2CON), 181.80 (SCHCON). Anal. Calcd for C26H51N4O5S3Si+OH- (640.99 g mol-1): C,

13C{1H}-RMN

15.88

20.91

(D2O): δ

(NCH2CH2CH2CH2Si),

(SiCH2CH2CH2Si),

28.02

(SCH2CH2NMe2),

-2.77 (SiMeCH2CH2S), -1.81 16.74

21.26 29.61

(SiCH2CH2S),

20.71

(SiCH2CH2CH2Si),

23.75

(SiCH2CH2S),

33.44

(D2O): δ -3.35 (SiMe), 14.89 (NCH2CH2CH2CH2Si), 16.39 (SiCH2CH2S), 23.04


Page 11 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


1

48.72; H, 8.18; N, 8.74; S, 15.00 %. Found: C, 45.91; H, 7.73; N, 8.57; S, 14.51 %. ESI-MS: [ M

2

+ H ]+ 623.22 uma (Calcd 623.28 uma), [ M + 2H ]2+ 312.24 uma (Calcd 312.15 uma).

3

2.1.8. Synthesis of N-Acetyl-L-CysSucG2(S(CH2)2NMe2)4 (8). Following the procedure

4

described for compound 7, compound 8 was obtained as a brown oil from the reaction of 0.02 g

5

(0.02 mmol) of compound 2 and 3.26 mg (0.02 mmol) of N-Acetyl-L-Cysteine. 1H-RMN (D2O):

6

δ -0.15 (3 H, s, SiMe), -0.02 (6 H, s, SiMe), 0.43 (2 H, m, NCH2CH2CH2CH2Si), 0.49 (4 H, m,

7

SiCH2CH2CH2Si), 0.56 (4 H, m, SiCH2CH2CH2Si), 0.85 (8 H, m, SiCH2CH2S), 1.17-1.26 (6 H,

8

m, NCH2CH2CH2CH2Si and SiCH2CH2CH2Si), 1.48 (2 H, m, NCH2CH2CH2CH2Si), 1.93 (3 H,

9

s, CH3OCNH), 2.54-2.61 (10 H, m, SiCH2CH2S and SCHCH2(CO)2N), 2.80 (24 H, s,

10

SCH2CH2NMe2), 2.81 (8 H, m, SCH2CH2NMe2), 2.85-2.90 and 3.12-3.17 (2 H, 2dd,

11

(CH3OCNH)CHCH2S), 3.14-3.21 (2 H, m, SCHCH2(CO)2N), 3.25 (10 H, m, SCH2CH2NMe2),

12

3.38 (2 H, t, NCH2CH2CH2CH2Si), 3.97 (1 H, m, SCHCH2(CO)2N), 4.35 (1 H, m,

13

(CH3OCNH)CHCH2S).

14


15

(SiCH2CH2CH2Si), 20.95 (SiCH2CH2CH2Si), 21.01 (SiCH2CH2CH2Si), 24.75 (CH3OCNH),

16

25.23

17

(NCH2CH2CH2CH2Si), 36.24-36.63 ((CH3OCNH)CHCH2S), 38.64-39.07 (SCHCH2(CO)2N),

18

41.04 (NCH2CH2CH2CH2Si), 42.38 ((CH3OCNH)CHCH2S), 42.33 and 43.04 (SCHCH2(CO)2N),

19

45.30 (SCH2CH2NMe2), 56.75 ((CH3OCNH)CHCH2S), 59.07 (SCH2CH2NMe2), 175.79-178.94

20

(CH3OCNH,

21

C46H99N6O5S5Si33+3OH- (1111.91 g mol-1): C, 49.69; H, 9.25; N, 7.56; S, 14.42 %. Found: C,

22

44.70; H, 8.26; N, 7.06; S, 14.47 %. ESI-MS: [ M + 2H ]2+ 529.28 uma (Calcd 529.28 uma), [ M

23

+ 3H ]3+ 353.20 uma (Calcd 353.19 uma).

24

2.2.

25

The solid-phase peptide synthesis,22, 23 was carried out manually in disposable polypropylene

26

syringes fitted with a polyethylene porous disc using the H-Rink amide ChemMatrix resin (0.5

27

mmol·g-1 loading) and a Fmoc/tBu-based strategy. In this strategy, the α-amino group of the

13C{1H}-RMN

15.97

(NCH2CH2CH2CH2Si),

(COOH),

(D2O):

δ

(NCH2CH2CH2CH2Si),

28.13

(SCH2CH2NMe2),

(SCHCH2CON)

and

-2.74

(SiMeCH2CH2S),

16.78

29.73

(SCHCON)).

(SiCH2CH2S),

(SiCH2CH2S),

Anal.

Calcd

-1.85 20.73

33.55

for

General procedures for Solid Phase Peptide Synthesis (SPPS)



1

amino acids is protected with the Fmoc group, and the side chains of the amino acids by: tert-

2

butyl (tBu) for Asp, Glu, Thr, Tyr and Ser; trityl (Trt) for Asn, Gln, His and Cys; tert-

3

butoxycarbonyl (Boc) for Lys and Trp; 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf)

4

for Arg. Solvents and soluble reagents were removed by vacuum suction. The Fmoc group was

5

eliminated by treatment with piperidine:DMF (20:80 v/v; 2 × 5 min) and coupling reactions were

6

performed using the corresponding standard Fmoc-amino acids (3 equiv), DIC (3 equiv) and

7

OxymaPure (3 equiv) in DMF for 1 h at room temperature. The completion of the coupling was

8

monitored with the Kaiser test. After every step of deprotection and coupling the resin was

9

thoroughly washed with DMF (3 × 1 min), CH2Cl2 (3 × 1 min) and again with DMF (3 × 1 min).

10

The Rhodamine B was incorporated to AMP3 peptide on the side chain of an extra Lys residue

11

coupled to the C-terminus. The Lys residue used was Fmoc-Lys(Alloc)-OH. After peptide chain

12

elongation, the Alloc group was removed by treatment with Pd(PPh3)4 (0.1 eq) and phenylsilane

13

(5 eq) in CH2Cl2 (3 × 15 min). Then, Rhodamine B was coupled in the above mentioned

14

conditions. The cleavage of peptides from the resin and the removal of protecting groups was

15

carried out by treatment with a TFA:H2O:TIS (95:2.5:2.5 v/v/v) mixture (10 mL/g of resin) for

16

180 min at room temperature. Then, TFA was removed by evaporation and the peptides were

17

precipitated by addition of cold diethyl ether, centrifuged and the supernatant was discarded. The

18

resulting crudes were purified by semipreparative reversed-phase HPLC (0 to 50% CH3CN (0.1%

19

HCOOH) in H2O (0.1% HCOOH) in 10 min at a flow rate of 16 mL/min; XBridge BEH 130 C18

20

19 mm × 150 mm 5µm) affording the desired peptides.

21

2.2.1. Synthesis of AMP1 peptide (H-Cys-Ile-Ile-Gly-Gly-Arg-NH2). 74.5 mg (45%). HPLC:

22

System A, tR 3.4 min, >99% (220 nm). ESI-MS: [M + H]+ 617.4 uma (Calcd 616. 8 uma), [M +

23

2H]2+ 309.2 uma (Calcd 309. 4 uma).

24

2.2.2. Synthesis of AMP2 peptide (H-Cys-His-Pro-Gln-Tyr-Asn-Gln-Arg-NH2). 79.6 mg

25

(45%). HPLC: System B, tR 3.2 min, 96% (220 nm). ESI-MS: [M + H]+ 1044.4 uma (Calcd 1044.

26

2 uma), [M + 2H]2+ 522.8 uma (Calcd 523. 1 uma), [M + 3H]3+ 348.9 uma (Calcd 349. 1 uma).


Page 12 of 39

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1

2.2.3.

2

98.0 mg (45%). HPLC: System A, tR 4.1 min, >99% (220 nm). ESI-MS: [M + H]+ 1489.7 uma

3

(Calcd 1488. 8 uma), [M + 2H]2+ 745.0 uma (Calcd 745. 4 uma), [M + 3H]3+ 497.0 uma (Calcd

4

497. 3 uma), [M + 4H]4+ 373.1 uma (Calcd 373. 2 uma).

5

2.2.4. Synthesis of Rhodamine-AMP3 peptide (H-Cys-Arg-Lys-Trp-Val-Trp-Trp-Arg-Asn-

6

Arg-Lys(Rho)-NH2). 21.4 mg (35%). HPLC: System A, tR 5.3 min, 99% (220 nm). ESI-MS: [M

7

+ 2H]2+ 1021.7 uma (Calcd 1022. 2 uma), [M + 3H]3+ 681.5 uma (Calcd 681. 8 uma), [M + 4H]4+

8

511.3 uma (Calcd 511. 6 uma), [M + 5H]5+ 409.3 uma (Calcd 409. 5 uma).

9

2.3.

Synthesis of AMP3 peptide (H-Cys-Arg-Lys-Trp-Val-Trp-Trp-Arg-Asn-Arg-NH2).

General procedures for the synthesis of carbosilane dendrons-peptides conjugates.

10

In a schlenk under inert atmosphere, a solution of the desired AMP peptide (1 equiv) in 1 mL

11

distilled water was added drop by drop over a solution of the corresponding cationic dendron (3

12

or 4) (1 equiv) in 1 mL distilled water. Solution was deoxigenated and stirred at room temperature

13

and the progression of the reactions was followed by mass spectrometry due to the low UV

14

absorption of carbosilane dendrons. In case of non-completed reactions, small portions of dendron

15

(0.1 equiv) dissolved in distilled water were added until no detection of the corresponding AMP.

16

Once the conjugations were completed, solvent was evaporated under reduced pressure and the

17

compounds were used for their evaluation without further purification.

18

2.3.1. Synthesis of AMP1MalG1(S(CH2)2N+Me2H∙Cl-)2 (9). Conjugation carried out between

19

AMP 1 (6.00·10-3 g, 9.73·10-3 mmol) and dendron 3. Compound obtained as a brown oil. ESI-

20

MS: [M + 2H]2+ 538.9 uma (Calcd (amine) 539.3 uma), [M + 3H]3+ 359.8 uma (Calcd (amine)

21

359.9 uma), [M + 4H]4+ 270.2 uma (Calcd (amine) 270.1 uma).

22

2.3.2.

23


24


25

502.3 uma), [M + 4H]4+ 377.0 uma (Calcd (amine) 377.0 uma), [M + 5H]5+ 301.8 uma (Calcd

26

(amine) 301.8 uma).

Synthesis of AMP2MalG1(S(CH2)2N+Me2H∙Cl-)2 (10). Conjugation carried out between



1

2.3.3.

2


3


4

390.7 uma), [M + 6H]6+ 325.7 uma (Calcd (amine) 325.8 uma).

5

2.3.4.

6


7


8


9

(amine) 303.3 uma).



10

2.3.5.

11


12


13


14

(amine) 324.1 uma).

15

2.3.6.

16


17


18


19

(amine) 341.5 uma).

20

2.4.

21

Bacterial strains, culture media and growth conditions. Staphylococcus aureus CECT 240 strain

22

(S. aureus) and Escherichia coli CECT 515 strain (E. coli) were cultured at 37 °C either in Muller-

23

Hinton broth (MHB) under shaking (120 rpm) or on plate count agar (PCA) plates for 24 h.

24

2.4.1. Determination of antibacterial activity



Biological evaluation


Page 14 of 39

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1

The bactericidal effect of peptides, dendrons, covalent nanoconjugates and non-covalent

2

complexes against S. aureus and E. coli in exponential growth phase was evaluated according to

3

ISO 20776-1:2006. Briefly, bacterial cells were grown to an OD625 between 0.08 and 0.11, which

4

means around 108 UFC/mL and then diluted with sterile water until final concentration of

5

approximately 2·107 UFC/mL. Serial twofold dilutions of each antibacterial compound were

6

achieved in sterile water and added (100μL) to MHB 2x (100 μL) at a starting concentration of

7

512 mg/L. Finally, 5 μL of bacterial suspension were inoculated in each well to measure bacterial

8

growth over 20 h by reading absorbance at 630 nm. Each concentration was assayed by triplicate

9

in a 96 well plate. Minimal Inhibitory Concentration (MIC) was determined as the lowest drug

10

concentration which avoids bacterial growth. Then, Minimal Biocidal Concentration (MBC) was

11

determined as the lowest drug concentration at which there was no visible growth after 24 h of

12

incubation at 37 °C of PCA plates inoculated with 5 μL of each well from MIC determination 96

13

well plate.

14

AMP1 and AMP2 antibacterial activities were also evaluated using Nutrient broth (NB).

15

2.4.2. Checkerboard microdilution

16

The MICs of single drugs A and B (MICA and MICB) and in combination (MICAB and MICBA)

17

were determined after 20 h of incubation at 37 °C. MICAB was defined as the MIC of drug A in

18

the presence of drug B and MICBA was defined as the MIC of drug B in the presence of drug A.

19

Then, the Fractional Inhibitory Concentration Index (FICI) was calculated using fractional

20

inhibitory concentration (FIC) for each drug and the following formula: FICI=FICA+FICB,

21

where FICA equals the MIC of drug A in combination divided by the MIC of drug A alone and

22

FICB equals the MIC of drug B in combination divided by the MIC of drug B alone. The FICIs

23

were interpreted as: synergy for FICI ≤ 0.5; additivity for 0.5 > FICI ≤ 1; no interaction for 1
4.

25

2.4.3. Cytoplasmic depolarization assay



1

The depolarization of the cytoplasmic membrane of S. aureus ATCC 25923 and E. coli CECT

2

515 by cationic dendrons, AMP3 and nanoconjugates was determined using the membrane

3

potential-sensitive dye 3,3'-Dipropylthiadicarbocyanine Iodide (DiSC3(5)) from Thermo Fisher.

4

S. aureus in exponential growth phase were washed in 5 mM HEPES buffer (pH 7.2) containing

5

20 mM glucose and resuspended in the same buffer at an OD625 of 0.09. The cell suspension was

6

placed in a white polystyrene 96 well plate and incubated with 0.6 μM final concentration

7

DiSC3(5) while fluorescence was monitored in a Perkin Elmer Wallac 1420 Victor2 microplate

8

reader at an excitation wavelength of 595 nm and an emission wavelength of 642 nm. After 15

9

mins, the reduction in fluorescence was stable due to DiSC3(5) maximal uptake and quenching in

10

the cell because of a normal membrane potential. Then, cell suspension well collected and 100

11

mM KCl was added to equilibrate internal and external K+ concentrations. Cell suspension was

12

washed with 5 mM HEPES buffer (pH 7.2) containing 20 mM glucose and 100 mM KCl by

13

centrifuging 3 min at 8.000 rpm in order to remove the external DiSC3(5) that was not internalized

14

by bacteria. Bacteria were then resuspended in the same buffer and placed again in a new white

15

polystyrene 96 well plate. Cationic dendrons, AMP3, nanoconjugates, negative control (water)

16

and positive control (gramicidine 250 mg/L) were then added by triplicate and fluorescence was

17

again continuously monitored.

18

For E. coli membrane depolarization assay, previous treatment was required in order to allow the

19

DiSC3(5) uptake. Same than before, E. coli suspension was washed and adjusted at an optical

20

density at 625 nm of 0.09. Cells were then treated during 30 mins with 0.2 mM EDTA (pH 8.0)

21

for outer membrane permeabilization. Then, protocol was carried out as previously described for

22

S. aureus, using polimixin B (48 mg/mL) as a positive control. It was checked that 0.2 mM EDTA

23

treatment of E. coli did not interfere with bacteria viability (Figure S10).

24

Additionally, same experiments were carried out with cell suspensions and also replacing cells

25

by 5 mM HEPES buffer (pH 7.2) containing 20 mM glucose but omitting washing step after

26

DiSC3(5) uptake.

27

2.4.4. Confocal Microscopy 16 ACS Paragon Plus Environment

Page 16 of 39

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1

The bacterial internalization of assayed compounds has been performed by ICTS “NANBIOSIS”

2

more specifically by the Confocal Microscopy Service: Ciber in Bioengineering, Biomaterials &

3

Nanomedicine (CIBER-BNN) at the Alcala University (CAI Medicine Biology). For this purpose,

4

S. aureus and E. coli were cultured as described above and diluted until 0.09 OD625 with Phosphate

5

Buffer Solution (PBS). These bacterial solutions (1 mL) were incubated in eppendorfs at 37 °C

6

during desired time (15 or 60 min) with Rhodamine B isothiocyanate labeled AMP3; Rho-AMP3,

7

fluorescein isothiocyanate labeled cationic dendron FITC-G2(SNMe3I)4 or 14 at MIC

8

concentration of its analogous unlabeled compounds. After desired incubation time, all samples

9

were washed once with PBS by centrifuging 3 min at 8.000 rpm. Sequential treatments required

10

washing steps after 30 min treatment of first compound and again after 30 min treatment of second

11

compound. After centrifuging, samples were vigorously resuspended with 20 μL of PBS and 5

12

μL were placed on a glass slide for further microscopy visualization. Confocal images were

13

collected using a Leica SP5 inverted laser scanning confocal microscope (Leica microsystems

14

CMS. GmbH). Bright field microscopy images were taken using a 488 nm argon laser. A 561 nm

15

He-Ne excitation laser and BP 570-620 nm filter was used to obtain fluorescence images of the

16

Rho-AMP-3 while a 488 nm argon laser and BP 500-560nm filter was used to obtain fluorescence

17

images of the FITC-G2(SNMe3)+4. All images were collected using a N.A 1.25 HCX PL APO

18

CS 40X objetive OIL UV lens.

19

2.4.5. Scanning Electron Microscopy

20

For SEM studies, bacteria treated with corresponding dendrons, peptide and peptide-dendrons

21

conjugates at MIC were fixed in Milloning’s solution containing 2% glutaraldehyde, washed in

22

Milloning’s solution with 0.5% glucose and dehydrated first through an ethanol series and finally

23

with anhydrous acetone. Samples were critical-point dried using a Polaron CPD7501 critical-

24

point drying system and sputter coated with 200 Å gold–palladium using a Polaron E5400. SEM

25

was performed at 5–15 kV on a Zeiss DSM950 SEM.

26

2.5.

Computer Modeling - methods



1

3D computer models of dendrons were created using dendrimer builder, as implemented

2

in the Materials Studio software package from BIOVIA (formerly Accelrys). Model of

3

solvated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) bilayer (160 lipid

4

molecules at each side (leaflet) ) was created using CHARMM-GUI Membrane Builder.24

5

The RESP technique25 was used for calculation of dendron atoms partial charges. GAFF

6

(Generalized Amber Force Field)26 and LIPID force field were used for parameterization

7

of dendrimers and lipids, respectively. The AMP1, AMP2 and AMP3 peptide computer

8

models were assembled using program leap belonging to AMBER package. The final

9

tertiary structures of these peptides at the given conditions (T = 295.15 K, P = 0.1 MPa)

10

were obtained by molecular dynamics simulations in implicit (500 ns) and consequently

11

in explicit (100 ns) water using ff14SB force field. The pmemd.cuda module27 from

12

Amber16 package28 was used for Molecular Dynamics simulations of dendron/peptid-

13

POPG systems (explicit water, length 1000 ns, NPT, P = 1bar, T = 295.15 K,

14

neutralization of dendron/peptid-lipid systems using Na+ ions) and also peptid clusters

15

(200 ns). Please see Supporting Information (SI) and eventually references for more

16

details.

17

3.

RESULTS AND DISCUSION

18

3.1.

Synthesis and Characterization


Page 18 of 39

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1

To carry out the formation of dendron-peptide nanoconjugates through a covalent bond by means

2

of thiol-ene addition "click" reactions, it was necessary to initially derivatize the selected

3

antimicrobial peptides (AMPs) with a cysteine residue and functionalize the focal point of the

4

carbosilane dendrons used in this work with a maleimide group. Cationic AMPs used in this work

5

were selected considering the antimicrobial capacity described in the literature.29, 30 Amino acid

6

sequences and characteristics of these peptides are listed in Table 1 and Scheme 2, together with

7

some of the new nanoconjugates formed. For simplicity, the following nomenclature will be used

8

to name dendrons described in this work: Mal-Gn-(Y)m, and R-Suc-Gn-(Y)m, where ‘Gn’

9

represents carbosilane dendron generation,‘Y’ denotes the nature of peripheral groups, “m” is the

10

number of these groups, ‘Mal’, is the maleimide group of the focal point of the dendrons that are

11

precursors of peptide-dendron conjugates, ‘Suc’ is the succinimide moiety resulting from the

12

conjugation and ‘R’ the thiol derivative conjugated to maleimide dendrons.

13

Maleimide dendrons have been prepared by condensation reaction of NH2Gn(S(CH2)2NMe2)m

14

(n=1, m=2 (I); n=2, m=4 (II)), previously reported by our research group21 and maleic anhydride,

15

followed by the cyclization of the resulting maleamic acid with sodium acetate, to obtain

16

MalGn(S(CH2)2NMe2)m (n=1, m=2 (1); n=2, m=4 (2)) as brown oils. NMR spectroscopy

17

confirmed modification of the focal point of these dendrons, as the 1H NMR signal of the initial

18

-CH2NH2 group were not observed whereas two new signals at 3.45 and 6.65 ppm were

19

respectively attributed to the methylene group bonded to maleimide moiety and the two alkene

20

protons of this group.

21

Treatment of dendrons 1-2 with a solution of HCl in diethyl ether during 1 hour causes the

22

peripheral amine quaternization to give the cationic dendrons MalGn(S(CH2)2N+Me2H∙Cl-)m (n=1,

23

m=2 (3); n=2, m=4 (4)) as brown oils, soluble and stable in water, alcohols (like methanol or

24

ethanol) and dimethylsulfoxide (Scheme 1). Compounds 3-4 can be stored without decomposition

25

for long time periods. Their 1H NMR spectra confirmed the quaternization of the amine groups

26

resulting in a deshielding of the chemical shift of the -CH2NMe2H groups consistent with the

27

presence of positive charges on the nitrogen atoms. 19 ACS Paragon Plus Environment


Page 20 of 39

1

With the purpose of optimizing the reaction conditions that will be subsequently extrapolated to

2

the introduction of AMPs, the thiol-maleimide addition reaction was initially studied for the

3

conjugation of small molecules containing a thiol group, such as L-cysteine hydrochloride and N-

4

acetyl-L-cysteine. The reaction of these two cysteine derivatives with dendrons 1-2 at room

5

temperature

6

CysHClSucGn(S(CH2)2NMe2)m

7

CysSucGn(S(CH2)2NMe2)m (n=1, m=2 (7) n=2, m=4 (8)). Dendrons 5-8 were isolated as brown

8

oils in moderate yields, and they are soluble in polar solvents as alcohols or water (Scheme 1).

in

DMF

during

5

h

(n=1,

led

m=2

to

(5);

the n=2,

corresponding m=4

(6))

derivatives

and

L-

N-Acetyl-L-

9 O Gn

H2N

(S(CH2)2NMe2)m

O

O

O

O N

O

Gn

O

ONa

(S(CH2)2NMe2)m

R-SH

n=1, m=2 (1) n=2, m=4 (2)

HCl 2M in Et2O O n=1, m=2 (3) n=2, m=4 (4)

N

Gn

N

RS

Gn

(S(CH2)2NMe2)m

O

R = L- Cysteine

n=1, m=2 (5) n=2, m=4 (6)

R = N-Acetyl-L-cysteine

n=1, m=2 (7) n=2, m=4 (8)

(S(CH2)2NHMe2 Cl)m

O

L-CysSucGn(S(CH2)2NMe2)m

N-Acetyl-L-CysSucGn(S(CH2)2NMe2)m n=2 O O

n=1

N

HO

S NH

O

S Si

Si

Si

S S S

N n=2 O

N N

N

O HO

N

S NH3 Cl

Si Si

Si

S S S

O

O

10

n=1

S

N N N N

n=1, m=2 (5) n=2, m=4 (6)

n=1, m=2 (7) n=2, m=4 (8)

11

Scheme 1. Synthesis of cationic carbosilane dendrons with a maleimide group at the focal point and

12

coupling of thiol containing small molecules to this focal point through thiol-ene addition reactions.

13

NMR spectra of compounds 5-8 are complex (Fig. S4-S9) due to the presence of diastereoisomer

14

mixtures of thioether succinimide derivatives. 2D 1H,13C-HSQC; 1H,13C-HMBC and 1H,1H-

15

COSY NMR experiments were used to characterize compounds 5-8 and to confirm the

16

introduction of cysteine derivatives at the focal point due to the disappearance of maleimide

17

alkene protons at 6.65 ppm (Fig. S1(A)) and the observation of new signals corresponding to

18

protons bonded to chiral centers (Fig. S4-S9). In the case of N-Acetyl-L-cysteine derivatives, 20 ACS Paragon Plus Environment

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1

protons bonded to chiral centers, localized in the succinimide ring and in N-Acetyl-L-cysteine

2

fragment, appear in the 1H NMR as a doublet of doublets at 3.96 and 4.35 ppm respectively (Fig.

3

S7-S8). In 13C-NMR two resonances were found for each chiral carbon due to the presence of two

4

diastereoisomers. Resonances at δ=43.07 and 42.35 ppm are attributed to succinimide chiral

5

carbon while N-Acetyl-L-cysteine chiral carbon appears at 56.70 and 56.85 ppm (Fig. S7 and

6

S9). In addition, the ESI spectra corroborated the proposed structures since a single signal

7

corresponding to the expected ions [M + H]+ at 581.27 and at 623.22 were observed in the mass

8

spectrums of first generation compounds 5 and 7 respectively. Similar results were observed in

9

the case of dendritic wedges of second generation.

10

Once the reaction conditions to introduce thiol derivatives to the maleimide group of the focal

11

point of our selected dendrons had been established, different dendron-peptides nanoconjugates

12

were formed using cationic carbosilane dendrons of first and second generation containing either

13

2 or 4 ammonium groups on the surface respectively, and three different AMPs: AMP1, AMP2

14

and AMP3, previously derivatized with a cysteine residue in the N-terminus that provides the

15

required thiol group for maleimide conjugation. In these cases, reactions were carried out in water

16

due to the solubility of the cationic dendrons and peptides in this media (Scheme 2). The integrity

17

of peptide-dendron conjugates was assessed by MS.

18



Page 22 of 39

HN HN

H 2N

NH2 NH

O HS

N H

NH2

H N

O N H

O

H N

N H O

NH2

O

N

O

AMP1

H N

N H

O

O

NH2

N H

O

O OH

N H

HN

NH2 O H N

O

NH2 NH

O

O

O N H

O

O

NH2

SH

H 2N N

AMP2

NH2 O

NH NH2 HS

O

H N

N H

O

O

H N

H N

N H

O

O NH

O

O

H N

N H

N H

O

O

+

NH2

Si

N

S

O NH

NH

NHMe2 Cl

S

O

NH

HN H 2N

NH2 O H N

HN

NHMe2 Cl

NH

NH2

HN

(3)

NH2

AMP3 H2O, 72h, T.a

Cl Me2HN

NH2

O

S S

Si

N

S

O

Cl Me2HN

O

NH NH2

H N

O

O N H

H N

O

O

H N

N H

O

O

H N

N H

NH

O

O

NH2

O

NH

HN H 2N

NH2 O H N

N H

NH NH

HN

NH2

NH HN

NH2

1

AMP3Suc(3)

2

Scheme 2. Structure of AMPs selected in this work and synthetic protocol for dendron-peptide

3

nanoconjugates formation.

(11)

4 5

Table 1. Dendrons, antimicrobial peptides and nanoconjugates included in this study.

Compound

Amino acid sequence/Chemical formula

Charge

Theorical molar mass

AMP1

H-CIIGGR-NH2

+2

616.78

AMP2

H-CHPQYNQR-NH2

+2

1044.16

AMP3

H-CRKWVWWRNR-NH2

+5

1488.79

(3)

MalG1(S(CH2)2N+Me2H∙Cl-)2

+2

532.71

(4)

MalG2(S(CH2)2N+Me2H∙Cl-)4

+4

1040.51

[AMP1Suc(3)] (9)

AMP1SucG1(S(CH2)2N+Me2H∙Cl-)2

+4

1148.49

[AMP2Suc(3)] (10)


+4

1575.87

[AMP3Suc(3)] (11)


+7

2020.50

[AMP1Suc(4)] (12)


+6

1656.29


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


[AMP2Suc(4)] (13)


+6

2083.67

[AMP3Suc(4)] (14)


+9

2528.3

1 2

3.2.

3

The MIC (minimum inhibitory concentration) and the MBC (minimum bactericidal

4

concentration) of first and second generation dendrons, peptides and dendron-peptide

5

nanoconjugates formed either by covalent thioether bonds ([AMP1Suc(3)] (9), [AMP2Suc(3)]

6

(10), [AMP3Suc(3)] (11), [AMP1Suc(4)] (12), [AMP2Suc(4)] (13), [AMP3Suc(4)] (14)) or by

7

non-covalent interactions in the case of AMP3 (AMP3 + (3) (15) and AMP3 + (4) (16)) were

8

determined against two strains of bacteria S. aureus and E. coli, and their values are shown in

9

Table 2. Although the peptides AMP1 and AMP2 had shown some antibacterial activity in

10

previous works described in the literature,29 in our hands both peptides were not active against

11

any of the two types of bacteria, whereas AMP3 showed a moderate antibacterial activity, with

12

MICs values of 32 mg/L and 64 mg/L against E. coli and S. aureus respectively. For this reason,

13

peptides AMP1 and AMP2 were not used for nanoconjugates preparation via non-covalent

14

interactions and only AMP3 was used to compare nanoconjugates activity depending on the bond

15

type between dendrons and peptides.

16

Antibacterial activity of the AMP3-first generation nanoconjugate (11) showed the same values

17

as those of the dendron (3) alone against E. coli, 16 mg/L, nevertheless it is necessary to remark

18

that the dendron concentration in this nanoconjugate is just 4,2 mg/L, pointing to certain

19

cooperative effect between dendron and peptide inside the nanoconjugate. This effect is more

20

accused against S. aureus where the reduction goes from 128 mg/L in the free dendron (3) to 16.9

21

mg/L in the nanoconjugate 11. In the case of the AMP3 nanoconjugate with second generation

22

dendron (14) this cooperative effect is barely appreciable and activity of this nanoconjugate is

23

pretty similar to those of the free dendron (4) (MIC (4): 8 mg/L; MIC (14): 16 mg/L; where

24

dendron concentration was just 6,6 mg/L) . We think that in this case, dendron 4 activity is so

25

strong that it is not possible to appreciate collaborative effects between dendron and peptide.

Antimicrobial activity



Page 24 of 39

1

Similar results were observed in the case of nanoconjugates formed by non-covalent interactions

2

(15 and 16). Again, the cooperative effect is more evident in the case of the complex formed by

3

a mixture of AMP3 and dendron 3 against S. aureus (MIC (3): 128 mg/L; MIC (15): 32 mg/L)

4

in respect to activity against E. coli, showing no significant reduction of MICs values respect to

5

the dendron alone for second generation derivatives.

6

Table 2. MICs and MBCs of maleimide carbosilane dendrons, cationic peptides, covalent nanoconjugates

7

and non-covalent conjugates against gram-negative bacteria E. coli and gram-positive bacteria S. aureus.

8

For nanoconjugates, number in brackets corresponds to dendron concentration present on nanoconjugates.

9

()* Indicate the concentration mg/L of dendron in the conjugate. E. coli

S. aureus

COMPOUND

MIC (mg/L)

MBC (mg/L)

MIC (mg/L)

MBC (mg/L)

MalG1(S(CH2)2N+Me2H∙Cl-)2 (3)

16

16

128

128

MalG2(S(CH2)2N+Me2H∙Cl-)4 (4)

4

8

8

4-8

AMP3

32

32

64

64

[AMP3-(3)] (11)

16 (4,2)*

16 (4,2) *

64 (16,9) *

64 (16,9) *

[AMP3-(4)] (14)

16 (6,6) *

32 (13) *

16 (6,6) *

16-32 (6,6-13) *

[AMP3 + (3)] (15)

16

16

32

64

[AMP3 + (4)] (16)

4

4

4-8

8

10 11

3.3.

12

In order to quantify the cooperative effect between cationic dendron 3 and AMP3, we carried out

13

the study of the fractional inhibitory concentration index (FICI) that is commonly used to evaluate

14

cooperative effects in most combination studies of antifungal and antibacterial agents. The effect

15

is defined as, synergic for FICI ≤ 0.5; additive for 0.5 > FICI ≤ 1; no effect for 1 < FICI ≤ 4 or

16

antagonism for FICI > 4.31 FICI index analysis between dendron 3 and AMP3 revealed a slight

17

additive effect in both types of bacteria (FICI = 1 for E. coli and FICI = 0.75 for S. aureus).

18

3.4.

Checkerboard microdilution

Cytoplasmic membrane depolarization assay 24 ACS Paragon Plus Environment

Page 25 of 39

1

To further study the action mechanism of these compounds, firstly we carried out tests of

2

cytoplasmic bacterial membrane depolarization using the membrane potential-sensitive dye

3

DiSC3(5), which is distributed between cells and brothum depending on the magnitude of the

4

cytoplasmic membrane electrical potential gradient, resulting in a self-quenching of fluorescence

5

when it is accumulated in the cytoplasmic membrane. In this way, after adding DiSC3(5) to

6

bacteria suspension, fluorescence stabilized after 2-3 min for S. aureus and 25-30 min for E. coli.

7

Then, if the cytoplasmic membrane is disrupted or forms channels, DiSC3(5) will be released into

8

the medium, causing an increase in fluorescence.32 In our case, when we carried out the

9

experiments we observed a fluorescence interference probably due to an interaction between our

10

compounds and DiSC3(5), for this reason it was necessary to make an additional experiment in

11

order to determine the amount of DiSC3(5) fluorescence in buffer before and after compounds

12

addition and so corroborate the total internalization of DiSC3(5) by bacteria. The results for both

13

type

of

bacteria,

E.

coli

(

12000 H20

10000

Polimixin 48mg/L (3) 16mg/L

Fluorescence (Ua)

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


8000

(4) 4mg/L AMP-3 32mg/L

6000

(11) 16mg/L (14) 16mg/L

4000 2000 0 0

5

10

15

20

25

30

35

40

45

50

55

60

65

time (min)

14 15

Figure 1) and S. aureus (data not shown), indicated that peptide AMP3, dendrons of first (3) and

16

second (4) generation and the corresponding nanoconjugates (11 and 14), caused a fast and

17

complete membrane depolarization at their MIC concentrations.

18 25 ACS Paragon Plus Environment


12000 H20

10000

Polimixin 48mg/L (3) 16mg/L

Fluorescence (Ua)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 39

8000

(4) 4mg/L AMP-3 32mg/L

6000

(11) 16mg/L (14) 16mg/L

4000 2000 0 0

5

10

15

20

25

30

35

40

45

50

55

60

65

time (min)

1 2

Figure 1. Cytoplasmic membrane permeabilization of E. coli showed by DiSC3(5) fluorescence intensity

3

changes after Polimixin B (48 mg/L, positive control), AMP3 (32 mg/L), 11 (16 mg/L), 14 (16 mg/L), 3

4

(16 mg/L), 4 (4 mg/L) and water (negative control) treatment of bacteria preincubated with 0.6 M

5

DiSC3(5).

6

3.5.

7

To investigate how AMP3 peptide and dendritic derivatives are localized within the bacteria,

8

confocal microscopy was used on E. coli and S. aureus after incubation for 15 and 60 min with a

9

fluorescein labeled second generation dendron (FITC-G2(SNMe3I)4) and a Rhodamine labeled

10

peptide 3 (Rho-AMP3) at MIC of respective unlabeled compounds (for FITC-G2(SNMe3I)4 MIC

11

E. coli 4mg/L and S. aureus 8mg/L; for Rho-AMP3 MIC E. coli 32mg/L and S. aureus 64 mg/L

12

). Confocal images showed that Rho-AMP3 and FITC-G2(SNMe3I)4 were inside both types of

13

bacteria after just 15 min and remained there after 60 min. Also, it could be observed that Rho-

14

AMP3 gave rise to the formation of bacteria aggregates while FITC-G2(SNMe3I)4 did not produce

15

aggregation. This effect was more evident after 15 min incubation in S. aureus than in E. coli,

16

where longer exposure times were necessary to observe aggregates formation (Figure 2Error!

17

Reference source not found.). In order to evaluate the effect of non-covalent conjugates formed

18

by dye-labeled systems on bacteria, E. coli and S. aureus were treated with different combinations

Confocal Microscopy


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


1

of FITC-G2(SNMe3I)4 and Rho-AMP3 in two different ways (i) by simultaneous addition, (ii) by

2

sequential addition with 30 min treatment of each compound. The results showed that in a

3

simultaneous addition the bacteria aggregation does not occur, while by sequential treatment the

4

aggregates formation depends on the addition order. First, if the dendron is added in the first place

5

followed by peptide addition, then it is possible to observe aggregates formation, whose size is

6

quite bigger than the ones formed after 60 min treatment with only Rho-AMP3. To quantify the

7

size difference, the area of ten aggregates per sample was measured from confocal images

8

showing that, the average of aggregates’ areas changed from 958 m2 to 10230 m2 for S. aureus

9

and from 820 m2 to 3482 m2 for E. coli when we compare 60 min treatment of Rho-AMP3 with

10

sequential treatment of 30 min FITC-G2(SNMe3I)4 followed by 30 min treatment of Rho-AMP3

11

(Figures 2 and 3). Secondly, if the peptide is added first, it is only possible to observe a few small

12

aggregates, showing that the dendron seems to disaggregate the previously bacteria aggregates

13

formed by AMP3 (Figure 3).

14

These results may be partly explained as follows. Dendrons can penetrate relatively rapidly into

15

the membrane, probably mainly due to their hydrophobic skeleton, while the peptide interaction

16

dynamics with the membrane is probably slower (as also indicated by the results of computer

17

modeling - see supporting information), so it is possible that they could remain attached to the

18

bacterial surface for sufficiently long time periods to mediate, together with another peptide

19

molecules attached to this primary ones, bacterial aggregation. One possible mechanism could be

20

creation of peptide oligomer "bridges" between adjacent bacteria, acting as some "glue", which

21

keeps the bacteria together.33 Connections between peptides ability to aggregate bacteria and

22

oligomerization ability of these peptides themselves was already described.34, 35 From this point

23

of view the disaggregation property of studied dendrons at the bacterial level might be justified

24

by the same property at the peptide level. This ability of some dendrimers with respect to peptides

25

was already described e.g. in some "dendrimer-amyloid" studies.36, 37 Computer modeling of small

26

AMP3 cluster indicates the ability of this positively charged peptide to create oligomers (Figure

27

S17) and this ability is likely to be enhanced near the negatively charged surface of the bacterium. 27 ACS Paragon Plus Environment


1

This explanation would correlate with increase size of aggregates observed after sequential

2

treatment, first dendron and second peptide, where dendron would penetrate first into bacteria and

3

could act as an additive barrier for peptide penetration, and so more peptide would remain for

4

longer time on bacteria surface, supporting aggregation effect.

5

Finally, let us note that the ability to aggregate bacteria per se can also be in some cases

6

understood as certain antibacterial activity preventing or complicating host cell attack34 or it could

7

be useful for bacterial detection and diagnostic purpose.38 From this point of view, synergic effect

8

found after sequential application of first, dendrons and second AMP3 might be of particular

9

interest due to the increase of AMP3 aggregation ability achieved.

10

11 12

Figure 2. Overlay of S. aureus and E. coli confocal fluorescence and bright-field images of Rho-AMP3 or

13

FITC-G2(SNMe3I)4 at MIC of respective unlabeled compounds after 15 and 60 min of treatment.


Page 28 of 39

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


1 2

Figure 3. Overlay of S. aureus and E. coli confocal fluorescence and bright-field images of Rho-AMP3 (P)

3

and FITC-G2(SNMe3I)4 (D) at MIC of respective unlabeled compounds after 60 min of simultaneous

4

treatment or 30 min treatment with Rho-AMP3 (P) followed by 30 min treatment with FITC-G2(SNMe3I)4

5

(D) and vice versa. Images confirm the AMP3 aggregation effect but also show the described

6

disaggregation effect of cationic dendron.

7

3.6.

8

The impact of AMP3, dendrons 3-4, and covalent conjugates 11 and 14 on the bacteria surface

9

morphology was studied by scanning electron microscopy (SEM). It allowed us to observe cell

10

elongation, rupture of cell membranes and the release of cellular contents of E. coli and S. aureus.

11

As presented in Figure 4, untreated E. coli and S. aureus cells showed a normal, smooth, intact

12

surface and after treatment with the tested systems, both bacteria exhibited significant

13

morphological alterations with damaged cellular integrity and signiﬁcant deformation in cell

14

morphology, regardless of the type of compound evaluated. In addition, it is possible to observe

15

how, after AMP3 treatment, E. coli media length increase from 1.2 m to 2.5 m some of the E.

16

coli bacteria have not been able to produce cell division, resulting in extremely long bacteria

17

which reach 13.8 m in length (Figure S11).

Scanning Electron Microscopy



1

2 3

Figure 4. Scanning electron microscopy of E. coli and S. aureus untreated or treated with respectively

4

MIC value of AMP3, MalG2(S(CH2)2N+Me2H∙Cl-)4 (4) and AMP3-(4) (14).

5

3.7.

6

In order to better understand the different behavior of peptides and dendrons, computer

7

simulations were performed to deeply investigate the nature of these molecules interaction with

8

negatively charged phospholipid bilayer (POPG) as a simplified model of bacterial membrane.

9

Simulations results show that all studied molecules seem to well interact with the POPG

10

membrane as a result of which they are stabilized in surface region of membrane. However,

11

simulations also show differences between the interaction of dendrons and peptides with this

12

membrane.

13

Computer simulations at the given time scale of 1000 ns revealed faster and deeper internalization

14

of dendrons in surface region of POPG membrane comparing to peptides (see Figure 5 and

15

supporting information for more details). This is clearly attributed to the optimal amphiphilic

16

nature of dendrons which allows them to optimally interact with also amphiphilic phospholipids.

17

Positively charged peptides seem to interact mainly with POPG hydrophilic heads through the

18

hydrogen bonds and electrostatic interaction. For stabilizing interaction with the hydrophobic

Computational Simulations


Page 30 of 39

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


1

interior of the lipid membrane, peptides have worse assumptions which depend on the amount

2

and location of eventual hydrophobic amino acids.

3

Simulations suggest that the AMP3 is the best interacting/stabilized peptide since, apart from

4

electrostatic interaction (thanks to its positive charge +5), it also interacts with hydrophilic POPG

5

heads via 17 hydrogen bonds and is also able to turn towards the hydrophobic interior of

6

membrane of its 3 tryptophans (two ideally, one partly) (Figure 5).

7

Conversely, in the case of AMP2 (charge +2, 6 hydrogen bonds), after a certain phase of the

8

stabilization process (200 ns long), the peptide escaped from the membrane surface and after

9

another 500 ns of movement near the membrane surface this peptide was again successfully

10

captured by the membrane (Figure S13), which could indicate the weakest favorable interaction

11

with the given membrane within the peptides used. Confirmation of this hypothesis would require

12

longer simulations. In this context is good to mention, that on contrary to AMP1 and AMP3, the

13

AMP2 peptide do not contain any clearly hydrophobic amino acid except one “semi-

14

hydrophobic” proline (which is moreover in the final configuration located too far from the

15

hydrophobic POPG interior) so the contact with hydrocarbon tails is here minimal (Figure S14)

16

and hence hydrophobic stabilization contribution as well.

17

Peptide AMP1 (charge +2, 9 hydrogen bonds) was stabilized very fast with a good contact with

18

hydrophobic interior of the membrane thanks to the ability to optimally turn both of its Isoleucines

19

(Figure 5 and supporting information for details). The highest positive charge (+5) and strongest

20

interaction of AMP3 with POPG membrane could explain why its antibacterial activity is better

21

than in AMP1 and AMP2 case.

22

In separate, just 200 ns long, simulations of small peptide systems (15 peptide molecules in water)

23

we also confirmed the ability of all studied peptides to create oligomers (Figure S17). The biggest

24

clusters which were created in this water/peptide solution during the given simulations (i.e.

25

composed of 8, 7 and 3 AMP1, AMP2 and AMP3 molecules, respectively) had a similar total



1

charge around +15. Near the negatively charged membrane, the ability of these cationic peptides

2

to create oligomers might be considerably higher.

3

These computational studies allow us to propose a reasonable explanation of the AMP3 capability

4

to aggregate bacteria. In this scenario, the key moment is, that AMP3 molecules or rather their

5

oligomers are stabilized at the bacterial surface for sufficient time (mainly thanks to the many

6

hydrogen bonds) to mediate bacterial aggregation where one role of peptides might be direct

7

participation on "gluing" of bacteria via interconnecting sufficiently close bacterial surfaces by

8

peptide oligomer chains.

9

Moreover sufficiently long interaction between peptide oligomers with bacterial surface may

10

destabilize relevant parts of the surface membrane (e.g. as the result of displacement of stabilizing

11

ions), as a result of which, some local reorganization of the membrane surface molecules may

12

occur up to local destruction of the membrane surface, which may promote bacterial aggregation.

13

This secondary effect of peptides interaction with bacterial surface is relevant especially in the

14

case of Gram-negative E. coli which has a lipid-based outer membrane.

15

Disaggregation effect of dendrons at bacterial level (after previously induced bacterial

16

aggregation using AMP3) may be in this context explained by their ability to destabilize peptide

17

oligomers. Let’s note that this ability of some dendritic molecules was already described e.g. in

18

several "dendrimer-amyloid" studies.36 A further supporting aspect here could eventually be the

19

more effective interaction of these ideally amphiphilic molecules with the lipid membrane and

20

hence the advantage in competition with peptides allowing the preferential occupancy of bacterial

21

surface by these dendritic molecules (eventual destabilization of already bound peptides) and its

22

charge neutralization, promoting destabilization of the bacterial clusters. So, dendrons play 3

23

active roles in bacterial aggregates destruction: i) to destabilize eventual peptide chains ii) to

24

destabilize "peptidic anchors" bound to the bacterial surface iii) they may eventually neutralize

25

negative charge of bacterial surface (instead of peptides). Promising future aspects are to study of

26

the disaggregation ability of dendrons with respect to bacterial biofilms.


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1

Also the opposite effect of dendrons (i.e., bacterial aggregation support when bacteria were first

2

treated with dendron 4 and subsequently with AMP3) that was also observed in this study could

3

be interesting because bacterial clustering can in some cases complicate attack on target cells by

4

these bacteria, and may eventually somehow reduce the spread of bacteria in a given organism.34,

5

35

6

purposes.38

In addition, this phenomenon can also be used for the bacterial detection and diagnostic

7

8 9

Figure 5. Computer models of dendron/POPG and peptide/POPG systems after 1000 ns of molecular

10

dynamics simulation. Ligands atoms (peptides, dendrons) are highlighted using “ball and stick”

11

representation, wire representation is used for POPG molecules. Water and ions are omitted for better

12

clarity. Colors: C – gray (lipid carbons) or black (dendron and peptide carbons), H – white, O – red, P –

13

orange, S – yellow, Si – beige. Hydrogen bonds are visualized as the cyan lines. More visualizations are

14

available in supporting information.

15

CONCLUSIONS

16

We have synthetized first and second generation cationic carbosilane dendrons functionalized

17

with a maleimide molecule on their focal point in order to allow thiol molecules conjugation. 33 ACS Paragon Plus Environment


1

After having demonstrated the solidity of synthetized dendron for thiol molecules conjugation,

2

three different AMPs were covalenting linked to cationic dendrons. Then, after having carried out

3

antibacterial activity, synergy and preliminary mechanism studies of dendrons, AMPs and

4

covalent and no covalent conjugates against S. aureus and E. coli it can be said that cationic

5

dendrons show good antibacterial activity against both assayed bacteria while from peptides, only

6

AMP3 reveals moderate activity. In addition, it was checked that both dendrons and AMP3 are

7

internalized by bacteria after 15 min and remain inside after 60 min. Synergy studies show an

8

additive effect between dendron 4 and AMP3. Additional studies demonstrate that dendrons,

9

AMP3 and also their covalent conjugates are able to permeabilize bacterial membrane, causing

10

significant morphological alterations and cellular integrity damages.

11

Furthermore, all these studies revealed a bacteria aggregation ability of AMP3 and a

12

disaggregation capability of cationic dendrons. Interestingly, it was found that this disaggregation

13

ability of dendrons can increase aggregates size when acting before, instead of after, AMP3. For

14

deeper comprehension of this observation, computational modelling was used to simulate the

15

interaction profiles of molecules against bacteria surface. Results agree with the hypothesis that

16

AMP3, and the oligomers it can form, interact with bacterial surface in a slower manner than

17

dendrons do it. This “slow” interaction contribute to aggregation process in two different ways:

18

(i) directly by joining close bacterial surfaces when peptide oligomer chains, due to this slower

19

interaction, are able to interact with more than one bacterial surface at the same time and (ii)

20

generating local membrane destruction due to membrane destabilization. In agreement with this,

21

computational modelling also provides an explanation for dendron disaggregation ability, which

22

is probably due to its higher effective interaction with membrane. Thus, aggregates formed after

23

AMP3 treatment are disrupted through dendron treatment because of dendron higher affinity for

24

the membrane, which provides it with the advantage in competition over AMP3 for membrane

25

interaction.

26

In this way, after sequential treatment with dendron followed by AMP3, aggregates size was

27

bigger than after AMP3 alone because in the sequential treatment, dendron interact first with 34 ACS Paragon Plus Environment

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1

bacteria and once dendron has penetrated into bacteria, it acts as an additive barrier for peptide

2

penetration, allowing it to remain longer time on bacteria surface, stimulating aggregation effect

3

described.

4

Future challenges are directed to better understand the mechanism and its difference in Gram-

5

negative and Gram-positive bacteria in order to better design AMPs, dendrons and conjugate them

6

to improve antibacterial activity, facilitate bacteria detection, contribute to antibacterial activity

7

by preventing host cell attack or even obtain peptides-dendron conjugates capable of breaking

8

biofilms and kill bacteria from them.

9

ACKNOWLEDGEMENTS

10

This work has been supported by grants from CTQ2017-86224-P (MINECO), Consortiums

11

NANODENDMED II-CM ref B2017/BMD-3703 and IMMUNOTHERCAN-CM B2017/BMD-

12

3733 (CAM) to UAH. This study was funded by grants from the Spanish Ministerio de Economía,

13

Industria y Competitividad (SAF2014-60138-R to MR those grants may include FEDER funds)

14

and Generalitat de Catalunya (2017SGR1439). This work was also supported from ERDF/ESF

15

project "UniQSurf - Centre of biointerfaces and hybrid functional materials" (No.

16

CZ.02.1.01/0.0/0.0/17_048/0007411) and from the Research Infrastructure NanoEnviCz,

17

supported by the Ministry of Education, Youth and Sports of the Czech Republic under project

18

No. LM2015073.

19

CIBER-BBN as an initiative funded by VI National R-D-i Plan 2008-2011, Iniciativa Ingenio

20

2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with

21

assistance from the European Regional Development Fund. A.B. Acknown Ministerio de

22

Economía y Competitividad for a predoctoral fellowship.

23

ASSOCIATED CONTENT

24

*Supporting Information. The Supporting Information is available free of charge on the ACS

25

Publications websiteat DOI: 35 ACS Paragon Plus Environment


1

The following files are available free of charge (file type; PDF).

2

Supporting Figures: (Fig. S1 – S9) NMR spectra of compound 1- 9 Figure S10. E. coli survival

3

after 0.2 mM EDTA treatment., Figure S11. Scanning electron microscopy of E. coli and S.

4

aureus untreated or treated with respectively MIC value of AMP3, MalG1(S(CH2)2N+Me2H∙Cl-

5

)2 (3) and AMP3-(3) (11). Figure S18-S28 HPLC-MASS of compounds.

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

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Nanoconjugates dendron/AMP as antibacterials 338x190mm (96 x 96 DPI)

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Carbosilane DendronâPeptide Nanoconjugates as Antimicrobial

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