Institut de Biologie de Lille

Biomolecules and Micro-nanotechnologies laboratory

Team Leader : Oleg Melnyk, DR2, CNRS

Oleg Melnyk graduated as an engineer in chemistry in 1989 from Ecole Nationale de Chimie de Paris. He received his Ph.D. degree in 1994 from the University of Paris VI. During the thesis he developed a novel total synthesis of Cortisone. He did post-doctoral training from 1994 to 1996 in Lille in the group of Pr A. Tartar. During this period, he worked on the synthesis of b-turn mimetics and their incorporation into small structured proteins. He’s working since 1996 as a CNRS researcher in Lille and created his own group in 2002. He is member of the scientific board of Bioconjugate Chemistry since 2004 and member of the scientific board of Groupement Français des Peptides et Protéines (GFPP, see the web site http://gfpp.free.fr)

oleg.melnyk ibl.fr
Phone : +33 3 20 87 12 14
Fax : +33 3 20 87 12 35

Scientific members of the laboratory of biomolecules and micro-nanotechnologies

Presentation

The group “Chemistry of Biomolecules” is involved in two research programs which are linked together. The first research program is focused on the discovery of novel chemical methods for the convergent synthesis of complex molecular architectures or proteins. These methods, which are called ligations in the field of biomolecules and in particular peptide chemistry, allow the site-specific chemoselective ligation of fully deprotected fragments. Our main goal is to find chemical methods permitting the formation of native peptide bonds between two fully deprotected peptides. These methods are used for the total synthesis of proteins that cannot be produced by conventional chemical or recombinant techniques. The group has a long experience in peptide chemistry.

 

Novel ligation methods for protein synthesis

The chemical synthesis of proteins is a great challenge for organic chemists. Synthesis of peptides of moderate size (up to 50 amino acids) is now easily feasible due to robust and efficient chemical methods for peptide bond formation using protected amino acids, the development of efficient solid supports which can be swelled in a variety of solvents and to the development of automation. The peptide is synthesized on the solid phase by stepwise coupling of the amino acids from the C-terminus to the N-terminus. At the end of the peptide elongation, the peptide is cleaved from the solid support to be purified. It can be easily understood that stepwise methods generate at each chemical step some impurities so that the amount of impurities grows as the power of the number of steps. Consequently, the yield of the desired peptide drops with peptide length. If the mean coupling yield for amino acids is X, the final yield for a peptide composed of n amino acids is Xn. Even if X is as high as 0.98, the yield for a very long peptide is generally low. For a 100 amino acids peptide (0.98)100 equal to 13%!
These considerations demonstrate that proteins cannot be usually synthesized using conventional stepwise solid phase methods and that there is a need for chemical methods allowing the convergent synthesis of large polypeptides by linking together short peptide fragments in a controlled manner (Fig. 1). The great challenge is the necessity to form a native peptide bond using unprotected peptide fragments and in aqueous solution (for solubilising peptide fragments).

Fig. 1. Chemoselective formation of a native peptide bond in aqueous solution is needed to assemble proteins.

Some methods already exist. Chemoselective ligation methods such as native chemical ligation[1] or Staudinger ligation[2] allow the assembly of proteins of moderate size (150 amino acids) and provide ready access to natural as well as modified proteins. However, native chemical ligation requires a cystein at the ligation site. Our goal is to design and develop novel chemical method for chemoselective native peptide bond formation that can be complementary to existing methods. This research activity is linked to the Peptide Synthesis Platform at IBL headed by O. Melnyk.

([1]) a) Dawson P. E., Muir T. W., Clark-Lewis I., Kent S. B. H. Science 1994, 266, 776-779. b) Dawson, P. E. Methods Enzymol. 1997, 287, 34-45. c) Hackeng, T. L.; Griffin, J. H.; Dawson, P. E. Proc. Natl. Acad. Sci. 1999, 96, 10068-10073.

([2]) a) Saxon, E., Bertozzi, C. R. Science 2000, 287, 2007-2010. b) Nilsson B. L., Kiessling L. L., Raines R. T. Org. Lett. 2000, 2, 1939-1941. c) Nilsson, B. L.; Hondal, R. J.; Soellner, M. B.; Raines, R. T. J. Am. Chem. Soc. 2003, 125, 5268-5269.

Microarrays

As peptide chemists we collaborate with biologists to study the biological activity or recognition properties of peptides. Microarrays are useful tools in this field because they allow the high throughput study of many different biomolecular interactions (peptide-protein, peptide-enzymes, polysaccharides-proteins such as growth factors…), and thus yield a large number of data very rapidly.
This part of our activity involves a strong collaboration with IEMN (Dr Didier Stiévenard), IRI (Dr Rabah Boukheroub, Dr Arndt Benecke), USTL (CIB, Clarisse Dhaenens) and University of Lille 2. This work is financed by the European Community, FEDER and la Région Nord Pas de Calais.
This research activity is linked to the Peptide Polysaccharide Microarray Platform at IBL headed by O. Melnyk.

Our group develop novel microarray methodologies, from novel surface chemistries to novel application of microarrays. For exemple, we have recently a novel microarray method to study the interaction between polysaccharides and growth factors. In the figure below, we have microarrayed a collection of polysaccharides of high MW ( 40 kD) prepared by chemical synthesis to study their ability to bind PDGF-BB, a growth factor involved in woud healing. The microarray was incubated with the growth factor, an antibody against PDGF-BB and then streptavidine labelled with a fluorescent dye. Fig. A and B correspond to the microarray incubated with or without the growth factor respectively. The fluorescence intensity is shown in a false-color scale.

A

B

The microarrays were further incubated successively with biotinylated anti-PDGF-BB antibodies and with streptavidin labeled with CY3 derivative’s (fluoroprobe 547, Interchim) . Detection was performed at 532 nm at L 80, PMT 85 scanner sensitivity.
Such microarray technologies are currently used for the discovery of molecules that are able to interfere with growth factors involved in the development of cancer.

Major publications

“Peptide-protein microarrays for the simultaneous detection of pathogen infections”
Duburcq, X.; Olivier, C.; Malingue, F.; Desmet, R.; Bouzidi, A. ; Zhou, F. ; Auriault, C. ; Gras-Masse, H. ; Melnyk, O.
Bioconjugate Chem. 2004, 15, 307-316.

"Polypeptide semicarbazide glass slide microarrays : characterization and comparison with amine slides in serodetection studies”
Duburcq, X.; Olivier, C.; Desmet, R.; Halasa, M.; Carion, O.; Grandidier, B.; Heim, T.; Stiévenard, D.; Auriault, C.; Melnyk, O.
Bioconjugate Chem. 2004, 15, 317-325.

“A novel a,a’-diaminoacetic acid derivative for the introduction of the a-oxo aldehyde functionality into peptides”

Far, S.; Melnyk, O.
Tetrahedron Lett., 2004, 45, 1271-1273.

“Solid phase synthesis of mandelic acid-derived thioethers by a-keto carbocation trapping”
Fruchart, J.-S.; Behr, J.-B.; Melnyk, O.

Tetrahedron Lett., 2004, 45, 1381-1383.

“Combined Nanogap Nanoparticles Nanosensor for Electrical Detection of Biomolecular Interactions Between Polypeptides”

Haguet, V.; Martin, D.; Marcon, L.; Heim, T.; Stiévenard, D.; Olivier, C.; El-Mahdi, O.; Melnyk, O.
Applied Phys. Lett., 2004, 84, 1213-1215.

“Design, synthesis and antimalarial activity of a glyoxylylhydrazone library”
Ryckebusch, A.; Fruchart, J.-S.; Cattiaux, L.; Rousselot-Paillet, P.; Leroux, V. ; Melnyk, O. ; Grellier, P. ; Mouray, E. ; Sergheraert, C. ; Melnyk, P.

Bioorg. Med. Chem. Lett. 2004, 14, 4439-4443.

“Synthesis of glyoxylyl peptides using a phosphine labile a,a’-diaminoacetic acid derivative”

Far, S; Melnyk, O.
Tetrahedron Lett. 2004, 45, 7163-7165.

“Preparation of Alpha-Oxo Semicarbazone Oligonucleotide Microarray”
Hot, D.; Olivier, C.; Ollivier, N.; Huot, L.; Gouyette, C.; Lemoine, Y.; Melnyk, O.

Current Protocols in Nucleic Acid Chemistry, Unit 12.6.1-12.6.19, 2004.

2005

“Synthesis of glyoxylyl peptides using an Fmoc-protected a,a’-diaminoacetic acid derivative”
Samia Far and Oleg Melnyk
Journal of Peptide Science, 2005, 11, 424-430.

“Semicarbazide-functionalized Si(111) surfaces for the site-specific immobilisation of peptides”

Y. Coffinier, C. Olivier, A. Perzyna, B. Grandidier, X. Wallart, J.-O. Durand, O. Melnyk, and D. Stiévenard
Langmuir, 2005, 21, 1489-1496.

“Anchorage of synthetic peptides onto liposomes via hydrazone and alpha-oxo hydrazone bonds. preliminary functional investigations.”

Bourel-Bonnet L, Pecheur EI, Grandjean C, Blanpain A, Baust T, Melnyk O, Hoflack B, Gras-Masse H.
Bioconjug Chem. 2005 Mar-Apr;16(2):450-7.

“Hybrid bioorganic-inorganic materials prepared by site-specific ligation of peptides to functionalized polydisperse silica particules”
Pascal Joly, Nicolas Ardès-Guisot, Samiran Kar, Michel Granier, Jean-Olivier Durand, Oleg Melnyk
Eur. J. Org. Chem. 2005, 2473–2480.

“A Novel Phosphoramidite for the Synthesis of a-Oxo Aldehyde-Modified Oligodeoxynucleotides”
Samia Far, Catherine Gouyette, Oleg Melnyk,
Tetrahedron 2005, 61/25, 6138-6142.

“Fmoc solid-phase synthesis of peptide thioesters using an intramolecular N,S acyl shift”
Nathalie Ollivier, Jean-Bernard Behr, Ouafâa El-Mahdi, Annick Blanpain and Oleg Melnyk
Org. Lett. 2005, 7, 2647-2650.

2006

"Polysaccharide microarrays for polysaccharide-platelet-derived growth factor interaction studies”
Olivier Carion, Julien Lefebvre, Guy Dubreucq, Latifa Dahri-Correia, José Correia and Oleg Melnyk

Chembiochem. 2006 May;7(5):817-26.

“Semicarbazide functionalized silicate nanoparticles for peptide ligation”

Tristan Doussineau, Jean-Olivier Durand, Ouafâa El-Mahdi, Céline Maillet, Oleg Melnyk Christophe Olivier, Monique Smaïhi
Eur. J. Inorg. Chem. 2006, 2766-2772.

“Detecting the chemoselective ligation of peptides to silicon with the use of cobalt-carbonyl labels”
Christophe Olivier , Aurore Perzyna, Yannick Coffinier, Bruno Grandidier, Didier Stievenard, Oleg Melnyk, Jean-Olivier Durand 
Langmuir. 2006 Aug 1;22(16):7059-65.

"Determination of glyoxylyl-peptide concentration using oxime chemistry and RP-HPLC analysis”

Jean-Michel Garcia, Samia Far, Eric Diesis and Oleg Melnyk

J. Pep. Science 2006, 12, 734-8.

“Fluidics of a nanogap”

M. Brinkmann, R. Blossey, L. Marcon, D. Stiévenard, Y. F. Dufrêne, and O. Melnyk
Langmuir, 2006, 23, 9784-9788.

2007

"Ti-Cp functionalization by deposition of organic/inorganic silica nanoparticles”
C. Roux, F. Chai, N. Ollivier, S. Winter, O. Melnyk, H. F. Hildebrandt
Biomolecular Engineering, 2007, 24, 549-554.

O. Melnyk and H.F. Hildebrandt are both corresponding authors.
C. Roux, thèse en co-tutelle (O. Melnyk et H.F. Hildebrandt)

”**“Chemicals micropatterning of polycarbonate for site-specific peptide immobilization and biomolecular interactions

V. Souplet, O. Carion, C. Maillet, C. Olivier, N. Médard, J.-O. Durand and O. Melnyk*
ChemBioChem, 2007, 8, 315-322.

“Covalent linking of peptides onto oxygen-terminated boron-doped diamond surfaces”

Yannick Coffinier, Sabine Szunerits, Bernadette Marcus, Rémi Desmet, Oleg Melnyk, Léon Gengembre, Edmond Payen, Didier Delabouglise and Rabah Boukherroub
Diamond and related materials, 2007, 16, 892-898.

"Peptide immobilization on amine-terminated boron-doped diamond surfaces"

Coffinier, Y.; Szunerits, S.; Jama, C.; Desmet, R.; Melnyk, O.; Marcus, B.; Gengembre, L.; Payen, E.; Delabouglise, D.; Boukherroub R.
Langmuir, 2007, 23, 4494-4497.

"Imaging of protein layers with an optical microscope for the characterization of peptide microarrays"

Souplet, V.; Desmet, R.; Melnyk, O.

J. Pept. Sci., 2007, 451-7.

“Electrical detection of human immunoglobulins G from human serum using a microbiosensor”

Marcon, L.; Stiévenard, D.; Melnyk O.

Biosensors and Bioelectronics, 2007, 23, 81-7.

“Reaction of isocyanate-functionalised silicon wafers with complex amino compounds”
Perzyna, A.; Dal Zotto, C.; Durand, J.-O.; Granier, M.; Smietana, M.; Melnyk, O.; Stará, I.-G.; Starý, I.; Klepetárˇová, B.; Sˇ aman, D.

Eur. J. Org. Chem., 2007, 4032-4037.

“Parallel synthesis of a lipopeptide library by hydrazone-based chemical ligation”

Dubs, P. ; Bourel-Bonnet, L. ; Subra, G. ; Blanpain, A. ; Melnyk, O. ; Pinel, A.-M. ; Gras-Masse, H. ; Martinez, J.
J. Comb. Chem., 2007, 9, 873-81.

2008

“Fundamental studies in nanosciences at the institute of electronics, microelectronics, and nanotechnology (IEMN)”
Allan, G.; Barbet, S.; Coffinier, Y.; Delerue, C.; Deresmes, D.; Diarra, M.; Diesinger, H.; Grandidier, B.; Marcon, L.; Mélin, T.; Melnyk, O.; Stiévenard, D.; Wirtz, L.; Zdrojek, M.
Int. J. Nanotechnol., 2008, 5, 631-648.

“Current based antibodies detection from human serum enhanced by secondary antibodies labelled with gold nanoparticles immobilized in a nanogap”
Marcon, L.; Melnyk, O.; Stiévenard, D.
Biosensors and Bioelectronics, 2008, 23, 1185-1188.

“Biomolecule and Nanoparticle Transfer on Patterned and Heterogeneously Wetted Superhydrophobic Silicon Nanowire Surfaces”
Piret, G.; Coffinier, Y.; Roux, C.; Melnyk, O.; Boukherroub, R.
Langmuir, 2008, 24, 1670-1672

“Electrical detection of antibodies from human serum based on the insertion of gold-labeled secondary antibodies into micro or nanogaps”
Marcon, L.; Stiévenard, D.; Melnyk, O.
Colloidal Nanoparticles in Biotechnologies Elaissari, A. Ed. Published by John Wiley & Sons, US, 2008, 329-351.

“Semicarbazide/a-oxo aldehyde site-specific ligation chemistry. From peptide microarrays to the micropatterning of polycarbonate or titanium oxide using silica nanoparticles”
El-Mahdi, O. ; Souplet, V. ; Carion, O. ; Roux, C. ; Garcia, J.-M. ; Maillet, C. ; Olivier, C. ; Durand, J.-O. ; Melnyk O.
Colloidal Nanoparticles in Biotechnologies Elaissari, A. Ed. Published by John Wiley & Sons, US, 2008, 299-327.

"Peptide microarrays on bisphenol A polycarbonate"
Souplet, V.; Roux, C.; Melnyk, O.
Peptide Microarrays, Humana Press, 2008, Marina Cretich and Marcella Chiari Eds., chapter book, in press.

“PASE: a web-based platform for peptide/protein microarray experiments"
Pamelard, F.; Even, G.; Apostol, C.; Preda, C.; Dhaenens, C.; Desmet, R.; Melnyk, O.
Peptide Microarrays, Humana Press, 2008, Marina Cretich and Marcella Chiari Eds., chapter book, 2008 in press.

“Characterization of Nanogap Chemical Reactivity Using Peptide-Capped Gold Nanoparticles and Electrical Detection”
Marcon, L.; Stiévenard, D.; Melnyk, O.
Bioconjugate Chem., 2008, 802-805.

“Chemoselective peptide ligation using phenylthiocarbamate chemistry”
Besret, S.; Ollivier, N.; Blanpain, A.; Melnyk, O.
J. Pep. Sci., 2008, 1244-1250.

“Towards thrombosis-targeted Zeolite nanoparticles for laser-polarized ¹²⁹Xe MRI”
Lerouge, F.; Melnyk, O.; Durand, J.-O.; Raehm, L.; Berthault, P.; Huber, G.; Desvaux, H.; Constantinesco, A.; Choquet, P.; Detour, J.; Smaïhi, M.
J. Chem. Mat., 2008, submitted.

“Self-assembly of silicon nanowires assisted by collagen”
Salhi, B.; Vaurette, F.; Grandidier, B.; Stiévenard, D.; Melnyk, O.; Coffinier, Y.; Boukherroub, R.; Nanoletters, 2008, submitted.

“Methylated peptide microarrays for the study of antibody responses to methylated proteins: the mycobacterial heparin-binding haemagglutinin”
Desmet, R.; Diesis, E.; Drobecq, H.; Roux, C.; Rouanet, C.; Chemlal, K.; Debrie, A.-S.; Hougardy, J.-M.; Mascart, F.; Locht, C.; Melnyk, O.
J. Proteomic Research, in preparation