Studies on antimicrobial activity of arginine-based surfactants - CSIC

249 Pages·2007·4.19 MB·English

STUDIES ON ANTIMICROBIAL ACTIVITY OF
ARGININE-BASED SURFACTANTS AND CHEMO-
ENZYMATIC SYNTHESIS OF NOVEL AMPHIPHILES
BASED ON L-ARGININE AND D-FAGOMINE

José Antonio Castillo Expósito
Thesis, 2006
UNIVERSIDAD AUTÓNOMA DE BARCELONA
FACULTAD DE QUÍMICA

INSTITUTO DE INVESTIGACIONES QUÍMICAS
Y AMBIENTALES DE BARCELONA
CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS
(IIQAB-CSIC)

Studies on antimicrobial activity
of arginine-based surfactants and chemo-
enzymatic synthesis of novel amphiphiles
based on L-arginine and D-fagomine

José Antonio Castillo Expósito
Barcelona, September 2006

Thesis submitted to Universitat Autónoma de Barcelona
in partial fulfilment of the requirements for the degree of PhD
(field of Chemistry)

Dr. Pere Clapés Dr. Àngels Manresa Dr. José Peral
(director) (director) (tutor)
Dejar de aprender es como ir sentado en un vagón de espaldas a lo que venga,
se te va escapando el paisaje y siempre es tarde para ver lo que venía.

Antoine Martán
ACKNOWLEDGEMENTS

Firstly, I would like to acknowledge Dr. Pere Clapés and Dr. Àngels
Manresa for the opportunity to perform this thesis in their research groups.
During these years, they have perfectly supervised my dialy work and I
have learned more than I expected. Moreover, I always could participate in
the working plans and in my opinion, this is especially important for the
formation of PhD students.

The present thesis has involved some collaborations with different
research groups. I would like to thank all the following supervisors in
chronological order for their assistance and their kind predisposition to
collaborate with our group: Josep Carilla and Dr Isabel Haro from IIQAB-
CSIC, Prof. Dr. Mª Asunción Alsina from Dept. Physical Chemistry, Faculty
of Pharmacy, University of Barcelona, Dr. Jaume Comas from Servei
cientifico-técnic, Parc Científic de Barcelona and Prof. Dr. Georg A.
Sprenger from Institut of Microbiology, University of Stuttgart. I would like
to acknowledge specially Dr. Jesús Joglar from IIQAB-CSIC for his helpful
advises in Organic Chemistry.

Biological assays with compounds synthesised in the present thesis
have been carried out by Jordi Calveras and Dr. Fina Casas from IIQAB-
CSIC, Montse Mitjans and Dr. Pilar Vinardell from Dept. Physiology,
Faculty of Pharmacy, University of Barcelona and Dr Jaume Carrió and Dr.
Roser Fisa from Dept. Parasitology, Faculty of Pharmacy, University of
Barcelona. To all of them, I want to acknowledge for their collaboration.

To work in different laboratories for some years has involved having
many colleagues. I have been really lucky because in all these places I have
found very nice people and pleasant environments to work. For all this
reasons I would like to thank Carmen, Laia, Jordi, Xavi, Susana, Carles,
Ari, Sonia, Guillem and Anna from IIQAB-CSIC, Jaume, Éscar, Mònica and
Adrià from Faculty of Pharmacy, University of Barcelona and Tomoyuki
from University of Stuttgart. After doing the PhD, I do not know what I will
do in the future nor where, but I would like to find people like them.

Finally, I would like to thank my family and especially my mother for
their support and comprehension during these years.

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FOREWORD

The present thesis has been performed at the Department of Peptide
and Protein Chemistry IIQAB-CSIC (Barcelona), under the supervision of
Dr. Pere Clapés and at the laboratory of Microbiology, Faculty of Pharmacy,
University of Barcelona, under the supervision of Dr. Àngels Manresa.
Experimental parts of this thesis that have been performed in other
laboratories are the following:

- Physico-chemical analyses using a Langmuir’s film balance at Department
of Physical Chemistry, Faculty of Pharmacy, University of Barcelona,
supervised by Prof. Dr. Mª Asunción Alsina.

- Analyses by differential scanning calorimetry at laboratory of thermal
analyses, IIQAB-CSIC, supervised by Josep Carilla.

- Leakage assay by fluorescent spectroscopy at Department of Peptide and
Protein Chemistry, IIQAB-CSIC, supervised by Dr. Isabel Haro.

- Flow cytometry analyses at Servei cientifico-técnic, Parc Científic de
Barcelona, supervised by Dr. Jaume Comas.

- Overexpression on E. coli and purification of fructose 6-phosphate aldolase
at Institut of Microbiology, University of Stuttgart, supervised by Prof. Dr.
Georg A. Sprenger:

Assays carried out in collaboration with other laboratories have been
the following:

- Assays of inhibition of glycosidases by the iminocyclitols synthesised in
this thesis were carried out by Jordi Calveras and supervised by Dr. Fina
Casas (Research Unit on Bioactive Molecules (RUBAM), IIQAB-CSIC).

- Cytotoxic assays of the bis(PhAcArg) derivatives and of the iminocyclitols
synthesised in this thesis were carried out by Dr. Montse Mitjans and
supervised by Dr. Pilar Vinardell (Department of Physiology, Faculty of
Pharmacy, University of Barcelona).

- Assays of the antileishmanial activity of the bis(PhAcArg) derivatives
synthesised in this thesis were carried out by Dr. Jaume Carrió and
supervised by Dr. Roser Fisa (Department of Parasitology, Faculty of
Pharmacy, University of Barcelona)

Financial support from the Ministry of Science and Education MYCT
projects PPQ2000-1687-CO2-01, PPQ2000-04625-CO2-01, PTR1995-0514-
OP, CTQ-2004-771-CO2-01/PPQ, a CSIC I3P postgraduate scholarship
programme and the COST D25 STSM are acknowledged.

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ABBREVIATIONS

ANTS = 8-aminonaphtalene-1,3,6-trisulfonic acid
Bis-oxonol = bis-(1,3-diethylthiobarbituric acid)trimethine
Br s = broad singlet
BSA = bovine serum albumin
C3(CA)2 = bis(Nα-caproyl-L-arginine)-1,3-propanediamide dihydrochloride
C3(OA)2 = bis(Nα-octanoyl-L-arginine)-1,3-propanediamide dihydrochloride
Cbz = benzyloxycarbonyl
cfu = colony forming units
CHX = chlorhexidine dihydrochloride
de = diastereoisomeric excess
D = detection (wavelength)
DI = denaturation index
DTAB = dodecyltrimethylammonium bromide
DHA = dihydroxyacetone
DMSO = dimetilsufoxid
DPPC = 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DPPG = 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac(1-glycerol)] sodium salt
DPX = N,N’-p-xylenebis(pyridinium) bromide
DTT = 1,4-dithio-D,L-threitol
Eq = equivalent
F = flow
FC = flow cytometry
FSA = fructose 6-phosphate aldolase
FU = fluorescence units
Gly-3-P = DL-glyceraldehyde-3-phosphate
Glu-6-P-DH = glucose-6-phosphate dehydrogenase
Hepes = 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
HLB = hydrophilic-lipophilic balance
HTAB = hexadecyltrimethylammonium bromide
HPLC = high performance liquid chromatography
HC = concentration that induces the haemolysis of 50 % of the cells
50
I = injection
IC = 50％ inhibitory concentration
50
L/D = lysis/denaturation ratio
LAE = Nα-lauroyl-L-arginine ethyl ester hydrochloride
LAM = Nα-lauroyl-L-arginine methyl ester hydrochloride
LPS = lipopolysaccharides
LUV = large unilamelar vesicles
MIC = minimum inhibitory concentration
MLV = multillamelar vesicle
MRSA = methicillin-resistant S. aureus
Mw = molecular weight
NADP: β-nicotinamide adenine dinucleotide phosphate disodium salt
n = number of methylenes
nd = not determined
nr = no reaction
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PBS = Phosphate buffered saline
P = pressure
PGI = phosphoglucose isomerase
OMPs = outer membrana proteins
PBS = phosphate buffered saline
PI = propidium iodide
RAMA = rabbit muscle aldolase
rpm = revolutions per minute
QACs = quaternary ammonium compounds
QSAR = quantitative structure-activity relationships
SEM = standard error media
SDS-Page = sodium dodecyl sulphate polyacrilamide gel electrophoresis
Spp. = specie (of micro-organism)
Suppl. Mat. = supplementary material
TEM = transmission electronic microscopy
TSA = tryptocase soy agar
TSB = tryptone soy broth
U = enzymatic unit

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CONTENTS Page
1. Introduction…………………………………………………………….….......9
1.1. Bacteria and antibacterial agents…………………………………...........11
1.1.1. Bacterial cell biology…………………………………………..…..11
1.1.2. Microbial growth control……………………………………........15
1.1.3. Mode of action of cationic biocides………………………..……..20
1.2. Amino acid-based surfactants: classification and structure…..………23
1.2.1. Arginine-based surfactants: preparation and properties…....25
1.3. Iminocyclitols………………………………………………………………....32
1.3.1. Structure and biological activity of iminocyclitols…………....32
1.3.2. Chemoenzymatic synthesis of iminocyclitols……………….....35
1.4. Techniques employed to study the interaction of biocides with
membrane models…………………………………………………………………..39
1.4.1. Differential scaning calorimetry (DSC)………………………....41
1.4.2. Fluorescence spectroscopy: ANTS/DPX leakage assay .…......43
1.4.3. Langmuir film balance………………………………………........44
1.5. Techniques employed to study the antimicrobial action..……………...47
1.5.1. Minimum inhibitory concentration (MIC)……………………...48
1.5.2. Flow cytometry (FC) ……………………..………………………..48
1.5.3. Viable cell counts………………………………………………..…50
1.5.4. Transmission electronic microscopy (TEM)…………………....51
1.5.5. Atomic absorption spectrophotometry……………………….....51
References…………………………………………………………………………...51
2. Objectives……………………………………………………………………...59
3. Results and discussion..........................................................................63
3.1. Interaction of arginine-based surfactants with membrane models…..65
3.1.1. Antimicrobial activity…………...…………………………………66
3.1.2. Interaction of arginine-based surfactants with
multilamellar vesicles.…………………………………………...……...67
3.1.3. Interaction of C (CA) and CHX with
3 2
unilamellar lipid vesicles…………...…………………………………….73
3.1.4. Interaction of arginine-based surfactants with
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lipid monolayers.…….……………………………………………………..75
References…………………………………………………………………...85
3.1.5. Supplementary information……………………………..............88
3.2. Comparative study of antibacterial activity of C (CA) and CHX
3 2
against Staphylococcus aureus and Escherichia coli………………………..107
3.2.1. Flow cytometry analyses and viability reduction..…………..108
3.2.2. Transmission electronic microscopy (TEM)…………………..114
3.2.3. Potassium leakage………………………………………………..116
References………………………………………………………………….118
3.3. Chemoenzymatic synthesis and biological properties
bis(phenyl-acetylarginine) amphiphilic derivatives…………………...……121
3.3.1. Chemoenzymatic synthesis……………………………………..121
3.3.2. Biological properties……………………………………………..125
References…………………………………………………………………133
3.4. Chemoenzymatic synthesis and biological properties of D-fagomine
and N-alkyl-D-fagomine derivatives…………………………………………..138
3.4.1. Chemoenzymatic synthesis…………………………………….138
3.4.2. Biological properties…………………………………………….144
References………………………………………………………………...147
4. Experimental section…………………………………………………….151
4.1. Materials…………………………………………...……………………….153
4.2. Instruments…..……………...……………………………………….……154
4.3. Methods……………………………………………………………………..157
4.3.1. Preparation of multilamellar vesicles (MLVs)……………...157
4.3.2. Differential scanning calorimetry (DSC)…………………….157
4.3.3. Leakage of encapsulated material in LUVs……...………….157
4.3.4. Surface activity…………………………………………………..159
4.3.5. Penetration kinetics at constant area……………….……….159
4.3.6. Compression isotherms…………………………………………159
4.3.7. Minimum inhibitory concentration (MIC)………...…...........160
4.3.8. Exposure of micro-organisms to biocides…………….…........161
4.3.9. Flow cytometry: staining procedure……………….….………162
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4.3.10. Bacterial count…………………………………………………...162
4.3.11. Transmission electron microscopy (TEM)……….…………..163
4.3.12. Potassium leakage………………………………………..……..163
4.3.13. Papain immobilization……………………………………...…..163
4.3.14. Production and purification of FSA…………………….….…164
4.3.15. Haemolysis assays………………………………………………168
4.3.16. Enzymatic inhibition assays………………………………......169
4.3.17. Assay of antileishmanial activity…………………….……….170
4.4. Preparation of bis(phenylacetylarginine) derivatives…………………171
4.4.1. Synthesis of 1,14-diaminotetradecane………………………....171
4.4.2. Synthesis of Nα-phenylacetyl-L-arginine methyl ester
hydrochloride (PhAc-Arg-OMe·HCl) (1) ………….……………………172
4.4.3. Synthesis of bis(Nα-phenylacetyl-L-arginine)-α,ω-dialkylamide
dihydrocloride (bis(PhAcArg)) (3a-e)…………………………………...173
4.4.4. Purification by chromatography…………………………….…..178
4.5. Preparation of D-fagomine and N-alkyl-D-fagomine derivatives…......179
4.5.1. Synthesis of o-iodoxybenzoic acid (IBX) ………..........….........179
4.5.2. Synthesis of N-benzyloxycarbonyl-3-amino-1-propanal (4)....180
4.5.3. Syntheis of (3S,4R)-6-[(benzyloxycarbonyl)amino]-5,6-
dideoxyhex-2-ulose (5)………………………………………...…………..181
4.5.4. Synthesis of D-fagomine [(2R,3R,4R)-2-hydroxymethyl-
piperidine-3,4-diol] (6) …………………………………………….…......182
4.5.5. Synthesis of N-alkyl-D-fagomine derivatives (7a-f)..……...…184
References…………………………………………………………………………..187
5. Concluding remarks………………………………………………………..189
6. Annexes…………................................................................................…...193
6.1. NMR spectra…….....................................................................….……...195
6.2. Publications……………………………………...………………....………...213
6.2.1. Publication I…………………………………...…...….…………...213
6.2.2 Publication II…………………………………...…....……...……...223
6.2.3. Publication III….……………………...…….…..……...………....233
6.2.4. Publication IV……………...………………….………...………....243
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