2.Introduction

the first part of the study. To our knowledge, only one such example of two biotin tags on the same molecule had been reported for possible multivalent binding.⁴

In this chapter, we will expose the synthesis of these derivatives by detailing the access routes as well as show preliminary data of the study involving these two series of compounds by time-resolved fluorescence spectroscopy through another collaboration with the group of Prof. Eric Vauthey of the Physical Chemistry Department of the University of Geneva. The photophysical results shown in this chapter are the work of Petr Sherin, a post-doctoral associate in the mentioned group.

IV-2.Introduction

Even though biotin-avidin affinity had been discovered earlier, it was only in 1941 that Snell identified the egg-white component that affected dermatitis in rats as being the consequence of this strong interaction.⁵ Another analog which presents structural and functional similarities with avidin is streptavidin available from bacteria of streptomyces avidinii.^8,8

Avidin is a positively charged protein that can be mostly found in raw egg white. It is a highly stable homotetramer of 4 x 128 residues⁶ where each monomer is constructed of eight antiparallel β-strands forming a classical β-barrel.⁷ Four binding sites are present in avidin to bind its ligand biotin in a non-covalent way. This is possible due to no less than 11-hydrogen bonding and 5-hydrophobic interactions that contribute to the stabilization of biotin in its binding pocket. On the other hand, with streptavidin,⁸ the complex formed is stable but in a lesser manner due to “only” eight hydrogen-bonds and four hydrophobic interactions.

The binding sites are usually easily accessible and located near one end of the monomeric subunit, with every subunit containing in the case of avidin four tryptophan (Trp) residues, two of them in the biotin binding site, one in the protein interior and the last one Trp 110 in a solvent-exposed loop which then is able to interact with the biotin in the neighboring monomer.⁶

4 J.-A. Farrera; P. Hidalgo-Fernández; J. M. Hannink; J. Huskens; A. E. Rowan; N. A. J. M. Sommerdijk; R. J. M.

Nolte^b Org. Biomol. Chem., 2005, 3, 2393-2395.

5 Eakin, R. E.; Snell, E. E.; Williams, R. J. J. Biol. Chem. 1941, 140, 535-543.

6 N. M. Green, Adv. Protein Chem. 1975, 29, 85–133. N. M. Green, Methods Enzymol. 1990, 184, 51–67.

7 O. Livnah, E. A. Bayer, M. Wilchek, J. L. Sussman, Proc. Natl. Acad. Sci. USA 1993, 90, 5076–5080.

8 F. Tausig, F. J. Wolf, Biochem. Biophys. Res. Commun. 1964, 14, 205–209. W. A. Hendrickson, A. Pahler, J. L.

Smith, Y. Satow, Y. S. Meritt, R. P. Phizackerley, Proc. Natl. Acad. Sci. USA 1989, 86, 2190–2194.

84 | P a g e  However, streptavidin contains six Trp residues in each subunit, three present at the end of the binding site, two buried in the protein interior and the last one known as Trp 120 in a solvent-exposed loop interacting similarly to Trp 110 in avidin.⁸

Figure IV-1: Structural representation of an avidin-biotin monomer, with the eight strands of the β-barrel labeled. Biotin molecule is depicted in a ball and stick model (left).⁹ Chemical structure of (+)-D-biotin 16

(right).

The ease with which molecules can be functionalized or derivatized with a biotin tag is shown in many reports in the literature. Thanks to its valeric acid function, biotin is flexible enough to survive many conditions and hence be linked to synthetic molecules, without subsequently affecting its binding affinity towards avidin or streptavidin.

Such a matter was put to use in our aim to study protein affinities by forming a series of biotinylated compounds associated with the cationic [4]helicenes studied in the group. The key to the success of this project was evaluated as being the correct selection of the right linking fragments that could be readily introduced on the two studied moieties, i.e. the cation and the biotin.

As linking methodology, we decided to use Copper(I)-catalyzed alkyne azide 1,3-dipolar cycloaddition which was first reported by Huisgen.¹⁰ In view of its measurable success, it has been rebaptized as “click chemistry” by Sharpless in 2001, because it is straightforward, uses       

9 Livnah, O.; Bayer, E. A.; Wilchek, M.; Sussman, J. L. Proc. Nat. Acad. Sci. U.S.A. 1993, 90, 5076-5080.

10 Huisgen, R. "Centenary Lecture - Dipolar Cycloadditions". Proc. Chem. Soc. 1961, 357. R. Huisgen, in 1,3-Dipolar Cycloadditional Chemistry (Ed.: A. Padwa), Wiley, New York, 1984.

readily available materials and affords the compounds effectively, usually in high yield without enormous drawbacks such as toxicity or hazardous side products.

“Click chemistry” will not be detailed in this manuscript as this topic has been reviewed several times over the last few years.¹¹ The triazole moiety has often been said to mimic the electronic properties of a peptide / amide bond (especially the capacity for H-bonding) without the same sensitivity towards hydrolysis, which prompted various biological applications of this reaction.¹² Although two regiosiomers are possible upon thermal 1,3-cycloaddition between alkynes and azides, the use of copper at milder temperatures (RT) allows the selective formation of the 1,4-regioisomer.

Scheme IV-1: Regioisomers obtained by thermal 1,3-cycloaddition between alkynes and azides

Figure IV-2: Topological and electronic similarities between an amide and the 1,2,3-triazole moiety

This reaction is proposed to proceed in a stepwise mechanism, beginning with the formation of a Cu^I acetylide species via π-complexation. Calculations have shown that copper coordination

11 H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed. 2001, 40, 2004-2021. H. C. Kolb, K. B.

Sharpless, Drug Discov. Today 2003, 8, 1128-1137. Bock, V. D.; Hiemstra, H.; Maarseveen, J. H. v. Eur. J. Org.

Chem. 2006, 2006, 51-68. Stanley, L. M.; Sibi, M. P. Chem. Rev. 2008, 108, 2887-2902.

12 For anti-HIV activity: a) R. Alvarez, S. Velazquez, A. San-Felix, S. Aquaro, E. De Clercq, C.-F. Perno, A.

Karlsson, J. Balzarini, M. J. Carmarasa, J. Med. Chem. 1994, 37, 4185–4194; b) S. Velaquez, R. Alvarez, C. Perez, F. Gago, C. De, J. Balzarini, M. J. Camarasa, Antiviral Chem. Chemother. 1998, 9, 481–489

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Zurenko, J. C. Hamel, R. D. Schaadt, D. Stapert, B. H. Yagi, J. Med. Chem. 2000, 43, 953–970.

86 | P a g e  renders the deprotonation of the alkyne much easier without the recourse to a base, by lowering its pKa.¹³ The reaction then continues to yield the desired 1,4-triazole selectively.