Synthesis of trans-lipids for biomarkers development · 2013. 9. 25. · “Design, synthesis and preliminary biological evaluation of new HCN blockers” PCT/EP2010/059369 J Med

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


biological samples



Targeting Biology



1

2

3

4

5

1 2 3 4 5

1





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


Targeting Biology



1

3

4

5

1 3 4 5

2

Synthesis

Synthesis of molecular libraries2

2





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





2

4

5

2 4 5

3

Targeting Biology

1

1

Analysis

3

3





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




2

3

5

2 3 5

4

Targeting Biology

1

1

Follow up in biological samples

Recognition in biologicals4

4





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


biological samples

Targeting Biology



JACS 2011, 133, 15184

1 2 3 4 5



biological samples



2

3

4

2 3 4

5

Targeting Biology

1

1



5

5

Ongoing project





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:

Documents

Synthesis of trans-lipids for biomarkers development · 2013. 9. 25. · “Design, synthesis and preliminary biological evaluation of new HCN blockers” PCT/EP2010/059369 J Med