Modulating Lectin Inhibition with N-Glycosyl-1,2,3-triazole Scaffolds

I. Azcune, E. Balentova, M. Sagartzazu-Aizpurua, J. I. Santos, J. I. Miranda, R. M. Fratila and J. M. Aizpurua

Eur. J. Org. Chem. 2013, 2434 - 2444

Literature Highlights 16th May 2013

S. Staderini

Supervisor: Prof. A. Marra

Introduction: Carbohydrates Roles

More than 50% of the proteins present in humans are glycosylated

Involved in:• Cell – cell recognition events• Neuronal development• Inflammatory processes• Protein – folding and conformation• Tertiary structure stabilization• Proteolytic stability enhancement

Development of synthetic glycopeptides and peptidomimetics 1. To achieve a better understanding of their mechanism of action 2. To design more efficient drugs against carboyhdrate – based

metabolic disorders

D. P. Gamblin, E. M. Scanlan, B. G. Davis, Chem. Rev. 2008, 109, 131-163L. Cipolla, F. Peri, Mini-Rev. Med. Chem. 2011, 11, 39-54

Introduction: Sialyl LewisX (sLeX)

N. Kaila, B. E. Thomas IV, Med. Res. Rev, 2002, 22, 566-601P. Sears, C. – H. Wong, Angew. Chem. 1999, 111, 2446

Fisiology1. sLeX adheres recognition domain

(CRD) of E and P selectins2. Rolling, tethering and

extravasation of leukocytes in the inflammatory cascade are regulated

Pathology1. Excessive and/or irregular

infiltration of leukocytes2. Chronic inflammatory diseases:

RA, psoriasis3. Acute diseases : stroke,

reperfusion injuries, myocardial infarction

Main Goal: Inhibit sLeX – selectin interaction

sLeX mimetics and selectins antagonists are effective

therapeutics versus these disorders

Introduction: Sialyl LewisX (sLeX)

N. Kaila, B. E. Thomas IV, Med. Res. Rev, 2002, 22, 566-601C. – H. Wong, F. Moris – Varas, S. - C. Hung, T. G. Marron, C. – C. Lin, K. W. Gong, G. Weitz – Schimdt, J. Am. Chem. Soc. 1997, 119, 8152-8158

Starting PointTwo pharmacophores: fucose moiety and COO- of sialyc acid.Metabolically labile O-glycosidic backbone.

Main issues to achieve:• Reduce biodegradability• Increase bioavailability• Absence of chiral centers• Easy availability from simple

synthetic routes Good activity, bad pharmacology

parameters

Target: Tuning sLeX Mimetics flexibility

Tuning the flexibility of the peptidomimetics scaffold can be considered a design variable for modulating the mimetic – lectin interaction.

III

IV

V

VI

Synthesis: CuAAC cycloaddiction approach

Glycopeptides III

Reagents and conditions: a) EEDQ, CH2Cl2, -10 °C to RT, 16 h;b) 20% piperidine in DMF, 30 min, RT;c) Ac2O, K2CO3, RT; d) CuSO4·5H2O, Na ascorbate,

tBuOH/H2O/THF, 1:1:1, 16 h, RT

Glycopeptides IV

IV

Previously reported: C. Palomo, J. M. Aizpurua, E. Balentová, I. Azcune, J. I. Santos, J. Jiménez-Barbero, J. Cancada, J. I. Miranda, Org. Lett. 2008, 10, 2227–2230.

Synthesis: CuAAC cycloaddiction approach

J. T. Lundquist, J. C. Pelletier, Org. Lett. 2002, 4, 3219–3221

Glycopeptides V

Reaction conditions: a) CuI, DIPEA, TBTA, MeCN,RT, 16 h; b) CsF, MeCN,RT to 35 °C, 1–3 h; c) CuSO4·5H2O, Na ascorbate, tBuOH/H2O, 1:1, 16 h, RT

Synthesis: CuAAC cycloaddiction approach

Glycopeptides VI

VI

Previously reported:J. M. Aizpurua, I. Azcune, R. M. Fratila, E. Balentova, M. Sagartzazu-Aizpurua, J. I. Miranda, Org. Lett. 2010, 12, 1584–1587.

The design of glycopeptidomimetics III–VI follows an increasing conformational constraint criterion ranging from flexible to rigid. Moreover, to minimize biodegradability and to avoid epimerization at α carbon atoms, the natural O-glycosidic bond was replaced by 1,2,3 triazole linkages and the number of epimerizable centers was reduced.

H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001, 113, 2056; Angew. Chem. Int. Ed. 2001, 40, 2004–2021.

Evaluation of Binding Affinity

• Final product obtained by deprotections (Pd/H2 and MeONa in MeOH)• Experiments toward fucose-specific Ulex Europaeus lectin I (UEL-I)• In silico evaluation of binding affinity using:

o a combination of NMR techniques (ROESY, STD-NMR)o molecular dynamic modeling (NAMD, AMBER) o docking simulations (AutoDock 3.0)

Molecular dynamics simulations

All these data are checked also by ROESY NMR spectra

Docking calculations

STD-NMR experiments

Selective saturation macromolecule protons (UEL-I). Due to effective spin diffusion saturation quickly propagates across the entire receptor. If the smaller molecule ligand binds the receptor, saturation will also spread onto the ligand. The result will be that intensity of the ligand signal will be attenuated. Subtraction of resulting spectrum from the reference spectrum without saturation yields the STD spectrum containing only signals of the binding ligands.

STD-NMR experiments

• STD effects are observed for all six analogues• Fucose exhibit the strongest signals• Carboxylic group interaction can’t be detected (D2O)• Central parts of the scaffolds do not take part in the interaction• Rigid analogues have high value for fucose signals and very low intensities

for amino-acids ones• 14b shows the triazole protons in contact with the surface, 14a shown

them far away from protein surface.

STD-NMR experiments

• STD effects are observed for all six analogues• Fucose exhibit the strongest signals• Carboxylic group interaction can’t be detected (D2O)• Central parts of the scaffolds do not take part in the interaction• Rigid analogues have high value for fucose signals and very low intensities

for amino-acids ones• 14b shows the triazole protons in contact with the surface, 14a shown

them far away from protein surface.

Conclusions & Comments

• Two new families of “dipeptide-like” molecular scaffolds have been developed.• A library of UEL-I binding molecules with different grades of flexibility is now

available for studies.• Simple synthesis methodologies have been used (CuAAC) for and controlled

stereochemistry has been achieved.• Lot of data have been collected regarding different conformers stability (Mol.

Dynamics), different complexes energies (Docking) and geometry of complexes (STD-NMR).

• The semi-rigid conformations are able to modulate their conformation to the “best-fitting” position.

• Mono- and bis-triazole can be useful as universal peptidomimetics.

• Lack of in vivo or in vitro studies: for a starting point it’s good to use calculations and in silico data, but in a long-term perspective can be interesting to test these molecules.

• CuAAC reaction: Cu toxicity drive to cell apoptosis, will they consider that problem? Purification system must be extremely efficient to move to in vivo or in vitro.

• STD – NMR studies are focused on the fucose motif and forget completely to consider the second pharmacophore, the acid.