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A COMBINED EXPERIMENTAL AND DFT INVESTIGATION OF ISOMERIC HEPARIN
DISACCHARIDES METAL COMPLEXES
Daniel Ortiz, Jean-Yves Salpin, Jeanine Tortajada, Al Mokhtar Lamsabhi, Otilia Mo, Manuel Yanez
To cite this version:
Daniel Ortiz, Jean-Yves Salpin, Jeanine Tortajada, Al Mokhtar Lamsabhi, Otilia Mo, et al.. A COM-BINED EXPERIMENTAL AND DFT INVESTIGATION OF ISOMERIC HEPARIN DISACCHA-RIDES METAL COMPLEXES. World Association of Theoretical and Computational Chemists, Jul 2011, Santiago de Compostela, Spain. 303, pp.1274 - 1286, 2002. �hal-00619208�
Motivations :
Why Heparin disaccharides?Heparin (HP) glycosaminoglycans (GAGs)1, an anticoagulant drug, are recognized
to be a biologically important polysaccharide, and have been involved in many biological processes such as blood coagulation, cell-cell and cell-matrix interaction inflammatory processes, cell growth, lipid transport and metabolism.
Why is it important to study the interaction between HP and metal cations?The effect of metal ions on protein-carbohydrate complexes is largely unknown. Heparin-biomolecule interaction can be influenced by the binding of metal ions to these complexes2. For example, it has been reported that physiological Ca2+
induces conformational changes in heparin that are necessary for the interaction between the anticoagulant Heparin and Annexin V, a protein proposed to play an important role in the inhibition of blood coagulation3. It is therefore a
Calcium-dependant interaction.
1
LAMBE UMR 8587, Laboratoire Analyse et Modélisation pour la Biologie et l’Environnement, Université d’Evry val d’Essonne, 91025 Evry (France).
2Department of Chemistry C‐9, Universidad autónoma de Madrid (UAM) Cantoblanco, 28049‐ Madrid (Spain).
A COMBINED EXPERIMENTAL AND DFT INVESTIGATION OF ISOMERIC HEPARIN
DISACCARIDES METAL COMPLEXES
D. Ortiz
1
, J-Y. Salpin
1
, J. Tortajada
1
, AM. Lamsabhi
2
, O. Mó
2
, M. Yáñez
2
What is our strategy?Experimentally our aim was to study (Ca(II-H))+ and (Ca(II-A))+ complexes by tandem
ESI/MS. Once generated in the gas phase, ions then undergo a fragmentation process by Collision Induced Dissociation (CID). The Ca2+ cation induces different conformational
changes in both isomers, resulting in completely different fragmentation pathways. Theoretically our aim is to explain this Metal-HP interaction by DFT calculations and delineate mechanisms of dissociation accounting for the experimental data.
Why II-A and II-H isomers?Without metal there is no difference in the MS/MS spectra of these two isomers. Only
0,2A
2 fragmentation is observed. With Ca2+ dissociation pattern changes drastically.
Interaction between Acetyl/Ca2+ must be important in the dissociation process.
O O COOH HO OH O OH OH NH2 OSO3 O O COOH HO OH O OH OH NHAc OSO3 0,2 A 2* 0,2 X 1*
II‐H
II‐A
MS/MS results:
Experiments were carried out on a LTQ Orbitrap XL mass spectrometer coupled to an ESI source. Nitrogen gas was used as collision gas
.
Computational results:
The geometries were optimized using the density functional theory (DFT) with the B3LYP hybrid functional and 6-311G** basis set. Refined relative energies were obtained at the 6-311++G(3df,2p) level.
Without Ca2+ all the calculated 50 conformers, for each disaccharide, are very close in
energy (<50 KJ/mol). Nevertheless, when (Ca(HP))+ is formed it is observed an increase
in relative energies between conformers (>50KJ/mol).
High Binding Energy (BE) values (~1400 KJ/mol) are obtained. As deduced from the conformers calculation and the BE values, the metal complex stabilizes strongly one structure. It seems safe to deduce that both sugars lose partially their possibilities to change structurally. Biologically, this consideration could be critical in order to explain the
strong interactions aforementioned. Analytically, when (Ca(HP))+ is formed, the
molecule loses it flexibility due to the fixation structure effect and therefore it is noticed a decrease in the number of fragments.
II‐H
II‐A
References:
1. I. Capila and R.J. Linhardt, Angew. Chem. Int. Ed., 391 (2002).
2. Y. Seo, M.R. Schenauer, J. A. Leary, Int. J. Mass spectrom, 303, 191-198 (2011).
3. I.Capila, M.J. Hernaiz, Y.D. Mo, T.R. Mealy, B. Campos, J.R. Dedman, R.J. Lindhardt, B.A. Seaton, Structure, 9, 57-64, (2001).
4. O.M.Saad, J.A. Leary, J. Am. Soc. Mass Spectrom, 15 1274-1286 (2004).
Choosing the fragmentation pathway
Leary et al.4 delineates mechanisms of dissociation for isomeric HP without metal
based upon CID experiments and H/D exchange. Another mechanism has been tested in this work but those pathways remain the most favorable ones.
0,2A
2
Dissociation
The acetyl group in II-A blocks the R1 (Rearrangement) step. Nevertheless, it is still possible to transfer the proton through the acetyl carbonyl group. The energy associated (PT2 barrier) is however bigger (163 KJ/mol) than for II-H (-55 KJ/mol).
0,2X
1
Dissociation
Starting from the initial structures, and using the dissociation mechanism given by Leary et al. shown below, the same methodology will be employed in order to unravel the 0,2 X 1 fragmentation pattern. , Ca2+ O COOH HO OH O O OH NHR OH OSO3 , Ca2+ O COOH HO OH OH SO3 O H PT1 O COOH HO OH O OH O NHR OH OSO3 O COOH HO OH OH SO3 OH RHN O H O COOH HO OH OH SO3 O RHN OH H RHN OH D1 R1 PT2 II‐A II‐H 164 141 ‐9 * 85 * Transition state ‐55 163 24 255 ‐22 * ‐19 8 , Ca2+ O COOH HO OH O O OH NHR OH OSO3 O COOH HO OH O O OH NHR OH OSO3 OH O O OH NHR OH OSO3 O COOH HO (Ca((II-H))+ 140 200 260 320 380 440 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative A bundance 397.08 456.08 426.58 376.17 438.08 340.08 (Ca(II-H))+ CID 12 0,2 A 2 * -SO3 - H2O 0,2 X1 ((Ca(II-H))2- 0.2A 2)+ : 150 200 250 300 350 m/z 400 450 500 550 600 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative A bundance 382.08 399.75 480.00 418.08 364.08 498.08 (Ca((II-A))+ -H2O 0,2 X 1 * +H2O -H2O (Ca(II-A))+ CID 15 -SO3 Energy values are in KJ/mol