Synthesis of trans-lipidsfor biomarker development
Bologna – September 25th, 2012
Coffee Talks @ ISOF
Michele Melchiorre
CNR – ISOFBologna
Education & previous experiences
• Mar 2006: Laurea degree in Chemistry and Pharmaceutical Sciences at the Alma Mater Studiorum – University of Bologna. Supervisor: Prof. M. Roberti
• Jan 2007 – Dec 2009: PhD student in Chemistry and Pharmaceutical Sciences at the University of Florence. Supervisor: Prof. M.N. Romanelli
• 2009: 10-month training period at Harvard University (Cambridge, MA) Supervisors: Prof. S.L. Schreiber, Dr. D.W. Young
Expanding the potential of enyne metathesis chemistry for high skeletal diversity
“Synthesis of a small library of terphenilic derivatives with a potential pro-apoptotic activity”
Synthesis of new fluorinated catalysts in a flow-chemistry projectProf. Klavs Jensen and Dr. Kevin Nagy
Education & previous experiences
• Feb 18th, 2010: PhD defense
• Mar 2010 - Jul 2010: post-doc fellow at the Italian Institute of TechnologyMed Chem Team. Director: Prof. D. Piomelli. Supervisor: Dr. T. Bandiera
• Sep 2010 - present: post-doc fellow at ISOF-CNR. BioFreeRadicals groupSupervisors: Dr. C. Chatgilialoglu, Dr. C. Ferreri
“Design, synthesis and preliminary biological evaluation of new HCN blockers”PCT/EP2010/059369J Med Chem 2010, 53, 6773Br J Pharmacol 2012, 166, 602
Present research
- synthesis of molecular libraries for lipid modification by free radicals
- lipidomic analysis
- biomarkers development
Organic synthesis
Finding a biomarker: a multidisciplinary approach
Analytical protocols
Biomimeticchemistry
Possiblebiomarkers
identification
Biology
Biomarkersvalidation
GenomicsEpigenomicsProteomics
MetabolomicsLipidomics
ClinicalResearchLibraries of
modifications
Reaction mechanisms
Radiation chemistry
Bio-inspired methods
Physical organic chemistry
Route toward biomarker development
Synthesis AnalysisFollow up in
biological samples
Synthesis of molecular libraries
Full characterization by means of several analytical techniques
Recognition in biologicals
Targeting Biology
Validation & possible
pathological correlation
1
2
3
4
5
Identification of a possible mechanism of transformationmediated by free radicals
1 2 3 4 5
Life scienceapplication
Synthesis AnalysisFollow up in
biological samples
Synthesis of molecular libraries
Recognition in biologicals
Targeting Biology
Validation & possible
pathological correlation
1
2
3
4
5
1 2 3 4 5
1
Life scienceapplication
Full characterization by means of several analytical techniques
Identification of a possible mechanism of transformationmediated by free radicals
Route toward biomarker development
Drugs
Oxidativeprocesses
Electrontransport
Environmentpollution
Inflammation
NO •
Radiations
FREERADICAL EXCESS
ROS,RNS,RSS
R•
R•+
R•–
CELLDAMAGE
LIPIDSperoxidationisomerization
AMINO ACIDSPROTEINS
oxidationracemization
CARBOHYDRATESoxidationmutation
NUCLEOTIDESDNA
oxidationmutation
AntioxidantNetworkEnzymesVitamines
Free Radical Stress 1
CELLDAMAGE
LIPIDSperoxidationisomerization
CARBOHYDRATESoxidationmutation
AMINO ACIDSPROTEINS
oxidationracemization
NUCLEOTIDESDNA
oxidationmutation
Damage preventionand
intervention strategies Membrane lipids
Free Radical Stress
Chatgilialoglu et al. Chimia 2012, 66, 368
1
Cell membrane and phospholipids
Natural unsaturated fatty acids have the CIS geometry
geometrical isomerscis trans
SFA MUFA PUFA
1
Fatty acid geometry
- Bent structures of cis fatty acids regulate membrane properties(e.g. permeability and fluidity)
- Desaturase enzymes provide only cis double bonds during lipid biosynthesis
- Biological activities are based on the cis geometry
CIS phospholipid
PUFA lipid transformations
Porter, N.A.Acc. Chem. Res. 1985, 19, 262
Chem. Rev. 2011, 111, 5944
1
PUFA
PUFA lipid transformations
Chatgilialoglu, C.; Ferreri, C.Acc. Chem. Res. 2005, 38, 441
1
PUFA & MUFA
Step-by-step radical isomerization
Free radical isomerization 1
CIS
MONO-TRANS
DI-TRANS
LIPOSOME MODEL
Biomimetic mechanism
The process is regiospecific due to thenon-equivalent reactivity of the double bonds !
1
a) JACS 2001, 123, 4459. b) JACS 2004, 126, 1063.
What happens in membranes?
Cholesteryl esters: “geometrical” radical stress markers
Cholesteryl Linoleate 9c,12c-18:2
Cholesteryl Arachidonate 5c,8c,11c,14c-20:4
1
Cholesteryl esters: biosynthesis
Connection with membrane phospholipid turnover
Close relationship with the onset of atherosclerosis and vascular inflammation
1
CETP
Lipoproteins(HDL, LDL, VLDL)
Phospholipids
LCAT
Cholesteryl esters
LCAT: Lecithin Cholesterol Acyl TransferaseCETP: Cholesteryl Ester Transfer Protein
Target: cholesteryl esters in human plasma
- target for lipidomics
- generation of diffusible thiyl radicals by different in vivo mechanisms (biomimetic chemistry with S-containing compounds)
- cholesteryl ester content (ca. 100-150 mg/mL) rich in PUFA moieties(>50% linoleic acid) deriving in their turn from membrane phospholipid
exchange via the LCAT enzymatic activity
human plasma
blood cells
Repair in the case of thiols
1
AnalysisFollow up in
biological samples
Recognition in biologicals
Targeting Biology
Validation & possible
pathological correlation
1
3
4
5
1 3 4 5
2
Synthesis
Synthesis of molecular libraries2
2
Life scienceapplication
Full characterization by means of several analytical techniques
Identification of a possible mechanism of transformationmediated by free radicals
Route toward biomarker development
Synthesis of mono-trans PUFA cholesteryl esters 2
Synthesis of mono-trans PUFA cholesteryl esters
Radical isomerization followed by Ag/TLC purification
y. 80%
y. 30%
2
SynthesisFollow up in
biological samples
Synthesis of molecular libraries
Recognition in biologicals
Validation & possible
pathological correlation
2
4
5
2 4 5
3
Targeting Biology
1
1
Analysis
3
3
Life scienceapplication
Full characterization by means of several analytical techniques
Identification of a possible mechanism of transformationmediated by free radicals
Route toward biomarker development
Analytical characterization of the molecular library 3
monotrans PUFA cholesteryl ester library
GCderivatization to fatty
acid methyl ester (FAME) fraction
RamanC=C trans @ 1670 cm-1
IRunconjugated
trans C=C @ 966 cm-1
NMR1H and 13C
ANALYSIS
mono-trans PUFA cholesteryl esters: NMR analysis
1H - 400 MHz - CDCl3 13C - 100 MHz - CDCl3
cisCh ARA
monotrans Ch LIN
monotrans Ch ARA
ppm1.952.052.15
ppm128.0129.0130.0131.0
cisCh LIN
3
NMR Information applicable in metabolomics
Synthesis Analysis
Synthesis of molecular libraries
Validation & possible
pathological correlation
2
3
5
2 3 5
4
Targeting Biology
1
1
Follow up in biological samples
Recognition in biologicals4
4
Life scienceapplication
Full characterization by means of several analytical techniques
Identification of a possible mechanism of transformationmediated by free radicals
Route toward biomarker development
Isolation of cholesteryl esters from human plasma
centrifugation
Cholesteryl esters fraction
Total lipids fraction
Human Plasma*
Human Blood
lipid extractionCHCl3 - MeOH (2/1)
4
Prep-TLC purification(10% of diethyl ether in hexanes)
vibrationalspectroscopy
GCanalysis
INDIRECTMETHOD
DIRECTMETHOD
* in collaboration with Lipinutragen s.r.l.
RECOGNITION
Recognition of mono-trans cholesteryl esters in human plasma: vibrational spectroscopy
a) the analysis is directly performed on the lipid extracts
without derivatization reactions
b) cis and trans geometrical configurations have
distinct vibrational bands
c) reported as official methods for trans unsaturated FA
determination in edible oils and fats
4
Raman spectroscopy: an emerging technique
The 1750-1600 cm-1 Raman region
curve fitting analysis
ringchain
Trans chain 1670 - 1672Ring 1667 - 1669Cis chain 1655 - 1659Oxidized 1644
Armida Torreggiani
4
Recognition of mono-trans cholesteryl esters in human plasma: GC analysis
TRANSESTERIFICATION 1M NaOH in benzene/methanol (2/3 v/v)90 min, RT, under Argon, no light
GC injection
4
Cholesteryl esters fraction
FAME (fatty acid methyl esters)
GC analysis
FAME
18:2 cis ω620:4 cis ω6
tot. 18:2 mono-transtot. 20:4 mono-trans
other trans
% rel (n=15)
50.1 ± 2.46.1 ± 1.70.6 ± 0.20.3 ± 0.10.7 ± 0.3
8.7 ± 2.2 nM4.1 ± 1.3 nM
Synthesis AnalysisFollow up in
biological samples
Targeting Biology
Validation & possible
pathological correlation
JACS 2011, 133, 15184
1 2 3 4 5
Route toward biomarker development
Synthesis AnalysisFollow up in
biological samples
Synthesis of molecular libraries
Recognition in biologicals
2
3
4
2 3 4
5
Targeting Biology
1
1
Validation & possible
pathological correlation
5
5
Ongoing project
Life scienceapplication
Full characterization by means of several analytical techniques
Identification of a possible mechanism of transformationmediated by free radicals
Route toward biomarker development
Medical significance
Does cholesteryl esters content change in specific pathological conditions?
5
Ongoing project in collaboration with Lipinutragen s.r.l.
OBESITY(n=16)
DIABETES(n=4)
CARDIOVASCULARDISEASES
(n=13)
DYSLIPIDEMICDISORDER
(n=7)
HEALTYSUBJECT
(n=9)
CHOLESTERYLESTERS
Biomarker validation: a collaborative strategy 5
Ongoing projects
BioFreeRadicalsISOF- CNR
LIPINUTRAGENS.r.l.
Univ.Montpellier
France
Hebrew Univ.Israel
Naples Univ.
RBI-ZagrebCroatia
Antalya Univ.Turkey
Univ.Paris-Sud
France
RASRussia
CM1201
Future work
• Trans cholesteryl esters in pathologies
• Protocols for FAST detection
(high-throughput analysis by Raman)
• Library of hydroperoxide isomers of cholesteryl esters for
evaluating the competition between peroxidation and
isomerization
Acknowledgment
Valentina SundaSimone Deplano
PRIN-MIUR2009K3RH7N_002 (2011-2013)
COST
• ChemBioRadical (2007-2011)Action: CM0603• Biomimetic Radical Chemistry (2012-2016) Action: CM1201
Chrys Chatgilialoglu
Carla Ferreri
Annalisa Masi
Anna Sansone
Michael Terzidis
Armida Torreggiani
Partnership:
Financial support: