Organocatalysis Paradigm Revisited: Are Metal-Free Catalysts Really

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Organocatalysis Paradigm Revisited: Are Metal-free Catalysts really Harmless? Amandine Nachtergael, Olivier Coulembier, Philippe Dubois, Maxime Helvenstein, Pierre Duez, Bertrand Blankert, and Laetitia Mespouille Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm5015443 • Publication Date (Web): 09 Dec 2014 Downloaded from http://pubs.acs.org on December 15, 2014

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Biomacromolecules

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Organocatalysis Paradigm Revisited: Are Metal-

2

free Catalysts really Harmless?

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Amandine Nachtergael1, Olivier Coulembier2, Philippe Dubois2, Maxime Helvenstein3,

4

Pierre Duez1, Bertrand Blankert3*, Laetitia Mespouille2*

5

1

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Pharmacy, University of Mons – UMONS, 23 Place du Parc, 7000 Mons (Belgium).

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2

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Polymeric and Composite Materials, University of Mons - UMONS, 23 Place du Parc, 7000

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Mons (Belgium).

Laboratory of Therapeutic Chemistry and Pharmacognosy, Faculty of Medicine and

Center of Innovation and Research in Materials and Polymers (CIRMAP), Laboratory of

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3

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Mons - UMONS, 23 Place du Parc, 7000 Mons (Belgium).

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All three laboratories are members of the Research Institute for Health Sciences and

13

Technology

Laboratory of Pharmaceutical Analysis, Faculty of Medicine and Pharmacy, University of

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KEYWORDS: amino-based organic catalysts, amidine, metal-free ROP, polyester,

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cytotoxicity.

17 18 19 20

ACS Paragon Plus Environment

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Biomacromolecules

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ABSTRACT

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Catalysts are commonly used in polymer synthesis. Traditionally, catalysts used to be

3

metallic compounds but some studies have pointed out their toxicity for human health and

4

environment and the removal of metal impurities from synthetic polymer is quite expensive.

5

To address these issues, organocatalysts have been intensively synthetized and are now

6

widely used in ring-opening polymerization (ROP) reactions. However, for most of them,

7

there is not any evidence of their safety. The present study attempts to assess if well-

8

established organo-based ROP catalysts used for the preparation of FDA-approved polyesters

9

may present or not a certain level of cytotoxicity. In vitro toxicity is evaluated using a

10

methyl-thiazol-tetrazolium (MTT) cytotoxicity assay on two cell models (FHs74Int and

11

HepaRG). Among the investigated organocatalysts, only functionalized thiourea (fTU) shows

12

an important cytotoxicity on both cell models. 4-Dimethylaminopyridine (DMAP), 1,5,7-

13

triazabicyclo[4.4.0]dec-5-ene (TBD) and meta-(trimethylammonio)phenolate betaine (m-BE)

14

show cytotoxicity against HepaRG cell line only at a high concentration.

15 16 17 18 19 20 21


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INTRODUCTION

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The interest in organocatalysis has spectacularly risen over those last few years as a result of

3

increased environmental awareness. Organocatalysis has many advantages in the context of

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“green chemistry” and, consequently, classical chemical procedures are increasingly replaced

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by metal-free alternatives 1. The world of polymeric materials does not contravene this trend

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and a real effort is concentrated on developing new metal-free synthesis options to replace

7

well-established metal-based routes

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ring-opening polymerization (ROP) reactions has become notably important to the field 3.

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The most obvious advantage of organocatalysis is to dispense from the costly removal of

10

metal impurities from the synthesized polymers, which, beyond economic standpoints, could

11

hamper their application in the medical or microelectronic added-value fields or simply raise

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environmental concerns if used for biodegradable polymer-based packaging production. For

13

that reason, many research groups, including us, have contributed to the development and

14

improvement of ROP metal-free catalysts as being critically important for designing

15

polymer-based materials for targeted biomedical applications

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catalytic systems avoid residual metal trace issues in the polymer material, no real

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investigation has been addressed yet to confirm or disprove their biocompatibility.

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The present paper attempts to assess and verify if well-established organo-based ROP

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catalysts used for the preparation of FDA-approved polyesters may present or not a certain

20

level of cytotoxicity. For this purpose, two cell models have been selected: epithelial

21

intestinal cells (FHs74Int) and differentiated human hepatocellular carcinoma cells

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(HepaRG). As FHs74Int cells are derived from normal human fetal intestine and show mature

23

epithelial-like characteristics 8, they are widely used to study food or drug effects on

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biological processes

2, 3

; the application of metal-free strategies to mediate

4-7

. If, conceptually, metal-free

9, 10

. HepaRG cells have become very useful to study xenobiotic


3

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metabolism and toxicity because they express major hepatic transporters and drug

2

metabolizing enzymes 11.

3

MATERIALS AND METHODS

4

1. Chemicals

5

The metal-free catalysts were (+)-Sparteine (CAS 90-39-1), a home-prepared functionalized

6

thiourea

7

Dimethylaminopyridine (DMAP) (CAS 1122-58-3), meta-(trimethylammonio)phenolate

8

betaine (m-BE), 1,8-diazabicycloundec-7-ene (DBU) (CAS 6674-22-2) and an imidazolium

9

salt (I-TFA). (+)-Sparteine (Aldrich, 99%), DBU (Acros, 98%) and TBD (Aldrich, 96%)

10

were dried over BaO for 48h, distilled under reduced pressure and stored under N2. fTU was

11

prepared according to a well-established protocol

12

N2. DMAP was dried under vacuum at 60 °C for 12 hours and stored under N2. The m-BE

13

and imidazolium salt were prepared as described in the literature

14

Metal-based catalysts were tin(IV) bromide (CAS 7789-67-5, Aldrich) and dibutyltin bis(2-

15

ethylhexanoate) (CAS 2781-10-4, Aldrich). (+)-Sparteine/fTU, DMAP and dibutyltin bis(2-

16

ethylhexanoate) were dissolved in DMSO (100 mM), whereas TBD, DBU, m-BE,

17

imidazolium salt and tin (IV) bromide were prepared in ultrapure water (100 mM) without

18

any preliminary purification.

19

2. Cell culture

20

The FHs74Int fetal human intestinal epithelial cell line (ATCC number CCL-241) was

21

obtained from the American Type Culture Collection (St Cloud, USA). Cells were cultured in

22

Dulbecco's Modified Eagle Medium high glucose (Lonza, Belgium) supplemented with 10 %

23

fetal bovine serum Gold, 10 mM HEPES, 2 mM L-glutamine, 1 % non-essential amino acids,

24

100 U/mL penicillin, 100 µg/mL streptomycin, 30 ng/µL epidermal growth factor and 10

25

µg/µL insulin, and maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air. The

(fTU),

1,5,7-triazabicyclo[4.4.0]dec-5-ene

(TBD)

(CAS

5807-14-7),

4-

12

, dried under vacuum and stored under

13, 14

and stored under N2.


4

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HepaRG human hepatocellular carcinoma cell line was obtained from Biopredic International

2

(Saint-Grégoire, France). Cells were cultured in William’s E medium supplemented with 10

3

% of fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL

4

streptomycin, 5 µg/mL insulin and 5 x 10-5 M hydrocortisone hemisuccinate. After 2 weeks,

5

the medium was changed to a differentiation medium provided by the manufacturer

6

corresponding to "supplemented" William’s E medium with 2 % DMSO for 2 more weeks.

7

This proprietary medium allows differentiating cells into biliary-like and hepatocyte-like

8

cells.

9

3. MTT cytotoxicity assay

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Both FHs74Int and HepaRG cells were seeded in 96-well plates (2 x 104 cells per well) and

11

grown at 37 °C for 22-26 h before a 50 µL aliquot of a base-2 logarithmic dilution of tested

12

compound was added (concentrations 0.8 to 200 µM). After 24 h, the medium was replaced

13

by 200 µL of MTT [3(-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] solution

14

in PBS (0.5 mg/mL) and the plate was incubated at 37 °C. After 4 h, the supernatant was

15

replaced by 100 µL of DMSO to dissolve reduced formazan crystals and the absorbance was

16

measured with a Thermo Labsystems Multiskan Ascent (Thermo Fisher Scientific Inc.,

17

Waltham, USA) at 570 nm versus a 690 nm reference. Percentages of cell viability were

18

expressed compared to control. The fitted curves “% of cell viability” versus “log (catalyst

19

concentration)” allowed the determination of IC10, IC50 and IC90, the catalyst concentrations

20

that inactivate cellular growth by 10, 50 and 90 percent, respectively.

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RESULTS AND DISCUSSION

22

The field of organic catalysis for ROP of cyclic esters and cyclic carbonates has rapidly

23

expanded and a wide range of catalysts are now available for standardized synthesis protocols

24

15

25

ROP activator

. Following the report of Nederberg et al. who used 4-dimethylaminopyridine (DMAP) as a 16

, other organic molecules have demonstrated their high activity for the


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preparation of aliphatic polyesters and polycarbonates with highly competitive kinetic

2

parameters and control levels as compared with their metallic counterparts. Among them are

3

currently

4

triazabicyclo[4.4.0]dec-5-ene (TBD) 17, 21, (+)-Spartein/functionalized thiourea (fTU) 12, 22, 23,

5

an imidazolium salt (I-TFA)

6

(Figure 1). Beyond favorable kinetic and control feature aspects, this widespread

7

development is motivated by the possibility to lift restrictions on the use of metal-free

8

polymeric materials in fields where high levels of quality are a prerequisite, e.g. for in vivo

9

implants, drug formulation, tissue engineering or biodegradable plastics. Therefore, the

10

reported

:

1,8-diazabicyclo[5.4.0]undec-7-ene

14

(DBU)

17-20

,

1,5,7-

and meta-(trimethylammonio)phenolate betaine (m-BE)

24

cytotoxicity of the above listed catalysts has to be evaluated.

Functionalized thiourea

11 12

Figure 1. Typical organocatalysts used in ring-opening polymerizations of lactones and

13

cyclic carbonates.

14

The MTT cytotoxicity assay is based on the measurement of cells metabolic activity. A

15

tetrazolium salt is reduced by metabolically active cells in purple formazan crystals that are

16

dissolved in DMSO for colorimetric measurement. Assuming that the amount of generated

17

formazan is directly proportional to the amount of metabolically active cells, the proportions


6

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1

of viable cells can be inferred from spectrophotometric measurements

. This rapid,

2

sensitive, reproducible and inexpensive MTT assay is considered as a good preliminary

3

indicator for the toxicity and biocompatibility of polymers and their degradation products

4

28

5

concentrations (from 0.8 to 200 µM), presumably extending way over their likely residual

6

levels in polymers. Table 1 presents the IC10, IC50 and IC90 values determined from viability

7

curves fitted from the HepaRG and FHs74Int data (Figures 2 and 3, respectively). As positive

8

control, 5-fluorouracil, a well-known cytotoxic compound used in treatment of cancer, caused

9

40 % of cells death at 193.0 µM on FHs74Int normal cells (full data not shown).

26-

. Catalysts were tested on HepaRG and FHs74Int cell lines over a wide range of

10

Table 1. IC10, IC50 and IC90 determined for organic and metallic catalysts from fitted curves

11

“% of cell viability” versus “log (catalysts concentration)”. Organocatalysts IC10

IC50

IC90

HepaRG

FHs74Int

HepaRG

FHs74Int

HepaRG

FHs74Int

DMAP

138.7 µM

n.a.

n.a.

n.a.

n.a.

n.a.

(+)-Sparteine

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

fTU

4.0 µM

n.a.

11.4 µM

27.5 µM

31.6 µM

119. 5 µM

(+)-Sparteine/fTU

2.6 µM

2.5 µM

6.1 µM

32.4 µM

16.6 µM

114.1 µM

DBU

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

TBD

130.6 µM

n.a.

n.a.

n.a.

n.a.

n.a.

m-BE

75.4 µM

n.a.

n.a.

n.a.

n.a.

n.a.

I-TFA

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

Metallic catalysts IC10

Tin(IV)bromide

IC50

IC90

HepaRG

FHs74Int

HepaRG

FHs74Int

HepaRG

FHs74Int

n.t.

n.a.

n.t.

n.a.

n.t.

n.a.

12

2.5 µM n.t. 3.1 µM n.t. 4.2 µM Dibutyltinbis(2-ethylhexanoate) n.t. Legend : n.a.: not applicable (inhibitory concentration not reached in the tested concentration range). n.t.: not

13

tested in present work.


7

Biomacromolecules

(+)-Sparteine

200 150 100 50 0 0.1

1

10

100

1000

% Cell viability versus control


DMAP 150

100

50

0 0.1

Catalysts concentration [µM]

50

10

100

1000



100

1

100

50

0 0.1

1

100

50

100

1000

100

50

0 0.1

1

10

100

1000

I-TFA

100

50

100

1000


m-BE

10

1000


150

1

100

150


0 0.1

10

TBD % Cell viability versus control


DBU

10

1000


150

1

100

150


0 0.1

10

(+)-Sparteine/fTU

150

0 0.1

1


fTU


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

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200 150 100 50 0 0.1


1

10

100

1000


1 2

Figure 2. Cell viability determined by the MTT assay for organocatalysts on HepaRG human

3

hepatocellular carcinoma cells. Results are expressed as average +/- standard deviation (SD) ;

4

data from 2 separate experiments in triplicate.


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(+)-Sparteine

150

100

50

0 0.1

1

10

100

1000



DMAP 150

100

50

0 0.1


50

10

100

1000



100

1

100

50

0 0.1

1

100

50

100

1000

100

50

0 0.1

1

10

100

1000

I-TFA

100

50

100

1000

Concentration (µM)


m-BE

10

1000


150

1

100

150


0 0.1

10

TBD % Cell viability versus control


DBU

10

1000


150

1

100

150


0 0.1

10

(+)-Sparteine/fTU

150

0 0.1

1


fTU


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

Biomacromolecules

150

100

50

0 0.1

1

10

100

1000


1 2

Figure 3. Cell viability curves determined by the MTT assay for organocatalysts on FHs74Int

3

fetal human intestinal epithelial cells. Results are expressed as average +/- SD ; data from 3

4

separate experiments in triplicate.

5

The association (+)-Sparteine/fTU displays an important cytotoxicity on both cell models. As

6

sparteine alone doesn’t show any toxicity against both cell models, the functionalized


9

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thiourea appears entirely responsible for the observed cytotoxicities. A comparative study of

2

the cytotoxicity of a series of mono- and di-substituted thiourea has shown that substituted

3

thiourea cytotoxicity is structure dependent

4

compound. The first step in the bioactivation of thiourea is the oxidation of the thionosulfur

5

by a flavin-monooxygenase (FMO) which results in the formation of sulfenic acid. The

6

oxidation of glutathione by sulfenic acid leads to oxidative stress that may result in cell death

7

or oxidation of biomolecules, proteins, lipids and nucleic acids. According to Onderwater et

8

al. 29 , the toxicity of N-phenylthiourea would not be the result of a FMO-based aromatic ring

9

bioactivation. However, a possible metabolic oxidation of another part of the molecule by

10

cytochrome P450 isoenzymes could explain a structure dependency of substituted thiourea

11

cytotoxicity

12

affirm whether the cytotoxicity is due to the thiourea or the fluorinated aromatic ring

13

moieties. This issue needs further investigation notably on the in vitro and in vivo metabolism

14

of the compound. The IC values of functionalized thiourea and (+)-Sparteine/fTU association

15

were compared across the two cell lines using a two-way ANOVA with post-hoc test

16

(Bonferroni correction). Statistically significant difference (p 20 µM). Interestingly the metallic catalyst tin(IV)bromide

23

does not show any cytotoxicity against FHs74Int cells contrary to dibutyltinbis(2-

24

ethylhexanoate) that proved to be very toxic to the intestinal cell model (Figure 4 and Table

25

1).

30

29

but not correlated with the lipophilicity of the

As this is the first time that fTU is assessed for cytotoxicity, we are unable to

31, 32

. This


10

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dibutyltinbis(2-ethylhexanoate)

200 150 100 50 0 0

1

10

100

1000



Tin(IV)bromide % Cell viability versus control

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

Biomacromolecules

150

100

50

0 0

1

10

100

1000


1 2

Figure 4. Cell viability curves determined by the MTT assay for metallic catalysts on

3

FHs74Int fetal human intestinal epithelial cells. Results are expressed as average +/- SD; data

4

from 3 separate experiments in triplicate.


11

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Table 2. In vitro and in vivo toxicity assay performed for catalysts studied in the present paper. Catalyst

In vitro toxicity assay

In vivo toxicity assay

Reference

Tin(IV) bromide CAS : 7789-67-5

n.a.

n.a.

Dibutyltinbis(2-ethyl hexanoate) CAS : 2781-10-4

n.a.

Deer mice : approximate lethal dose (acute oral toxicity) : 1075 mg/kg LDfr1 : 62 mg/kg/day

33

(+)-Sparteine CAS : 90-39-1

n.a.

Wistar rats : LD50 (acute toxicity) of 30 mg/kg

34

Functionalized Thiourea (fTU)

n.a.

n.a. 3

1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) CAS : 5807-14-7

The cytotoxicity of the P(EEP-co-EMEP) copolymers synthetized by copolymerization with TBD. The product was purified by repeated precipitation into cold diethyl ether. Hela cell line: no cytotoxicity for concentrations from 1 to 600 mg/mL(resazurin assay)

n.a.

35

4-dimethylaminopyridine (DMAP) CAS : 1122-58-3

Vibrio fischeri : EC504 of 0.315 mg/mL

LD50 - Deer mice : oral, 450 mg/kg - Mice : oral, 350 mg/kg/day - Rat : oral, 250 mg/mL - Fly : oral, 0.15 mg/mL

33, 36, 37

meta-(trimethylammonio)phenolate betaine (m-BE)

n.a.

n.a.

1,8-diazabicycloundec-7-ene (DBU) CAS : 6674-22-2

Lymphocytes : no significant effect at 0.5 mg/mL Chondrocytes : no effect on cell proliferation up to 1 mg/mL and significant effect (28 % viability) with 2 mg/mL (membrane integrity assay)

n.a.

38

Imidazolium salt n.a. n.a. (I-TFA) Legend : 1LD50, dose that causes death of 50 % animals of the tested group; 2IC50-IC25, concentration which inhibits 50 % and 25 % of cell proliferation, respectively; 3EEP, 2ethoxy-2-oxo-1,3,2-dioxaphospholane and EMEP, 2-ethoxy-4-methyl-2-oxo-1,3,2-dioxaphospholane; 4EC50, effective DMAP concentration at which V. fischeri bioluminescence was reduced by 50 %; 5NR50, concentration of surfactant required to induce a 50 % inhibition in neutral red uptake; n.a., not applicable


12

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Biomacromolecules

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These data indicate that cytotoxicity is certainly compound-dependent and does not correlate

2

with the metallic or non-metallic nature of the catalyst. As polymer usages used to be mostly

3

industrial, there is only a few in vitro and in vivo toxicity assays published for the catalysts

4

studied herein (Table 2).

5

With the development of recyclable, biodegradable and biocompatible polymer, the toxicity

6

(both environmental and human) has become an important parameter that has to be taken into

7

account. Catalysts are the most susceptible elements promoting reactions with toxicological

8

impact and have to be carefully tested for toxicity before they can be used in polymer in such

9

application fields. The assumption that organocatalysts are less toxic than metallic catalysts is

10

not supported by objective data in the state-of-the art and we can even conclude from our results

11

that it is not necessarily the case. By using normal intestinal and metabolically active hepatic cell

12

lines we increase the range of toxicity that can be observed because the results reflect also the

13

metabolization aspect but it will be very interesting to further testing the influence of such a

14

metabolization on catalysts cytotoxicity. To link our results with ROP-process, we started from

15

the hypothesis that 100 % of the catalyst is released in vivo from the biodegradable polymer and

16

extrapolated the cell viability for estimated catalyst concentration in 100 µg of poly(lactide)

17

polymer of 20 000 g/mol (Figure 5). By using 100 µg of 20 000 g/mol polymer, we are

18

consistent with in vivo conditions on the one hand, and on the other hand we fit in the range of

19

catalysts concentrations tested for all catalysts whatever their quantity in polymer after ROP-

20

process (Figure 5 A). Except for fTU, our results demonstrate the relatively low toxicity of

21

organocatalysts against normal intestinal and metabolically active hepatic cell models.

22 23


13

Biomacromolecules

A. 1

Catalyst

Equivalent(a)

Quantity per kg of polymer(b)

DMAP

1 to 5 eq.

6 to 30 g

3

(+)-Sparteine

5 eq.

60 g

4

fTU

5 eq.

92.5 g

DBU

1 eq.

8g

TBD

0.01 to 0.1 eq.

0.07 to 0.7 g

m-BE

3 to 5 eq.

24 to 40 g

7

I-TFA

5 eq.

45 g

8

Tin(IV)bromide

1 eq.

22 g

9

Dibutyltin bis(2-ethylhexanoate)

1 eq.

26 g

2

5 6

10

Legend: (a) Equivalent of catalyst relative to the initiator alcohol. (b) Required amount of catalyst to produce 1 kg of poly(lactide) polymer of 20 000 g/mol.

11 B.

50

0

M

D

150

100

50

0

M

100

D

150

A D P( M 1 A e (+ P ( q.) )-S 5 e pa q. rt ) ei ne f TU TB D (0 DB TB .01 U D eq ( m 0.1 .) -B eq m E(3 .) -B e E q. (5 ) eq . I-T ) FA


HepaRG

A P M (1 A (+ P eq )-S (5 .) p a eq rt .) ei ne TB fT U D D TB (0.0 BU D 1 m (0. eq. D -B 1 ) ib ut m E ( eq. yl -B 3 ) Ti E eq nb (5 .) is T in eq (2 ( - e iv I- .) th )b TF yl ro A he m x a id e no at )


FHs74Int

D

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Figure 5. A. Required amount of catalyst to produce 1 kg of poly(lactide) polymer of 20 000

14

g/mol. B. Percentage of cell viability for estimated catalyst concentration in 100 µg of

15

poly(lactide) polymer of 20 000 g/mol using FHs74Int and HepaRG cell models.


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1

Although the MTT assay is a very good preliminary indicator of a compound cellular toxicity,

2

the results should be interpreted with caution. Indeed, although a correlation can often be derived

3

between viable cell number and MTT-reducing activity

4

number of viable cells but also on their metabolic activities and on the capacity of tested

5

compounds to eventually chemically reduce tetrazolium salts

6

compounds could affect cell integrity by targeting specific cell compartments or functions and be

7

missed by MTT measurements, a complete assessment of toxicity should be obtained only by

8

combining different quantitative methods 25, 27.

9

CONCLUSION

26

, MTT data depend not only on the

39

. Furthermore, as some

10

This study demonstrated the relatively low toxicity of organocatalysts over concentrations

11

presumably extending way over their likely residual levels in polymers, which will contribute to

12

promote their interest in the field of green chemistry for all applications where toxicity and

13

environmental concerns are a key issue. While many papers have hypothesized the absence of

14

toxicity of metal-free catalysts, herein we clearly stated that most of them are indeed

15

biocompatible for the range of concentration investigated at the exception of functionalized

16

thiourea that was found to be very toxic against both cell models. This one, at the equivalency of

17

Tin IV-based structures, should be subjected of thorough toxicity studies before validating its

18

usage in medical device or in green chemistry applications. Given the probable long-term use of

19

polymeric implantable devices, further research is warranted on the biological consequences of

20

residual catalysts and initiators possibly slowly leeching from the polymer.

21 22 23


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Biomacromolecules

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1

AUTHOR INFORMATION

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Corresponding Author

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*Professor Bertrand Blankert. Laboratory of Pharmaceutical Analysis, Faculty of Medicine and

4

Pharmacy, University of Mons (UMONS) 23, Place du Parc, 7000 Mons, Belgium.

5

Tel : 0032 65 37 34 92 ; Fax : 0032 65 37 34 26 ; email : [email protected]

6

*Dr Laetitia Mespouille. Center of Innovation and Research in Materials and Polymers

7

(CIRMAP). Laboratory of Polymeric and Composite Materials (LPCM). University of Mons

8

(UMONS) 23, Place du Parc, 7000 Mons, Belgium

9

Tel : 0032 65 37 34 82 ; Fax : 0032 65 37 34 84 ; email : [email protected]

10

Author Contributions

11

The manuscript was written through contributions of all authors. All authors have given approval

12

to the final version of the manuscript.

13

ACKNOWLEDGMENT

14

Amandine Nachtergael gratefully acknowledges the fellowship granted to her by UMONS.

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HepaRG cells (patented by Inserm Transfert) are supplied by Drs. Christiane Guguen-Guillouzo,

16

Philippe Gripon & Christian Trepo (INSERM’s laboratories U522 & U271) and used under a

17

Material Transfer Agreement between INSERM U522 & U271 and the University of Mons,

18

Belgium. CIRMAP is grateful to the “Belgian Federal Government Office Policy of Science

19

(SSTC)” for general support in the frame of the PAI-7/05, the European Commission and the

20

Wallonia Region (FEDER Program) and OPTI2MAT program of excellence. The laboratory of

21

Pharmaceutical Analysis expresses its gratitude to FNRS (Fonds National de la Recherche) for

22

support via the FRSM Grant 3.4614.11. O.C. is Research Fellow of the F.R.S.-FNRS.


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1

Biomacromolecules

ABBREVIATIONS

2 3

ROP, ring-opening polymerization; FDA, food and drug administration; MTT, methyl-thiazol-

4

tetrazolium; fTU, home-prepared functionalized thiourea; TBD 1,5,7-triazabicyclo[4.4.0]dec-5-

5

ene; DMAP, 4-dimethylaminopyridine; m-BE, meta-(trimethylammonio)phenolate betaine;

6

DBU, 1,8-diazabicycloundec-7-ene; I-TFA, imidazolium salt; DMSO, dimethysulfoxide; PBS,

7

phosphate buffered saline; IC, inhibitory concentration; SD, standard deviation; ANOVA,

8

analysis of variance.

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Insert Table of Contents Graphic and Synopsis Here

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REFERENCES

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229x128mm (96 x 96 DPI)


Organocatalysis Paradigm Revisited: Are Metal-Free Catalysts Really

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