The copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC) is the most prominent example of a group of reactions named Click-Reactions.

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1 Introduction: The copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC) is the most prominent example of a group of reactions named Click-Reactions. According to the 2001 Nobel laureate K. Barry Sharpless these reactions are characterized by high yields, mild reaction conditions, and by their tolerance of a broad range of functional groups. Typically, the reactions require simple or no workup or purification of the product. The most important characteristic of the CuAAC Click- Reaction is its unique bioorthogonality, as neither azide nor terminal alkyne functional groups are generally present in natural systems. Labeled Nucleic Acids (NAs) such as oligonucleotides, DNA or RNA, are of daily use for different standard laboratory techniques like PCR assays, FISH experiments or microarrays. State-of-the-art labeling technologies suffer from poor incorporation rates of the label and of instabilities of intermediate products. The new proprietary labeling technology of baseclick GmbH, based on the CuAAC Click-Reaction overcomes this weakness and enables the NA labeling in an unprecedented, highly efficient and site specific way. Below the Click-Chemistry principle applied to DNA. The Click-Reaction minimizes the amount of marker to be used during the labeling reaction significantly, increasing the production efficiency while decreasing the lost of high valuable material.

2 Click on DNA : Nucleic acid (NA) labeling works through the incorporation of baseclick alkynephosphoramidites or alkyne-dntps into NAs followed by the labeling reaction with label azides (single- or multi-labeling). Highly efficient labeling with BaseClickTM-KITS: BaseClickTM-KITs contain all the necessary reagents to label - single and multiple labeling - alkyne-modified oligonucleotides and alkyne-modified PCR-products with marker azides of choice via CuAAC Click-Reaction. Single labeling: Purified oligonucleotides bearing a single alkyne moiety are usually modified with 2 to 5 equivalents of the corresponding label-azide (e.g. fluorescent-dye azides). After the addition of precomplexed Cu(I) / ligand, complete conversion to the labeled oligo is observed in a time span between 30 min and 4 hours. After a simple precipitation step, labeled oligonucleotides can be recovered in near quantitative yields.

3 Multiple labeling: The Click-Reaction enables the multiple postsynthetic labeling of alkyne modified NA. Complete high-density functionalization of several alkyne moieties within the NA e.g. oligonucleotides and PCR fragments - can be achieved without the formation of by-products. Alkyne-modified oligos can be directly purchased from baseclick GmbH or from other baseclick licensed companies such as Integrated DNA Technologies (IDT), Metabion, Ella-Biotech and IBA. Click on Proteins: Protein glycosylation is essential for the proper function of the respective proteins. In the case of glycosylated protein therapeutics, such as erythropoietin, the activity of the protein strongly depends on glycosylation patterns. The synthesis and production of glycosylated proteins to either elucidate their biochemical function or to bring a new therapeutic to the market is still a formidable challenge. Mutant or modified aminoacyl-trna synthetases (aars) have been used to charge non-natural amino acids such as azido-homoalanine (Aha) to the corresponding trna, which incorporates them into polypeptides or proteins during recombinant synthesis. For example, methionine (Met) has been replaced effectively by Aha in proteins by the native methionyl trna synthetase of E. coli. Further developments have been done to specifically incorporate a vast number of Click-Reaction components into practically any protein. Professor T. Carell (LMU, Munich) efficiently used the MmPylRS/MmtRNACUA system to insert three genetically encoded alkyne amino acids into proteins showing that the alkyne functionalities in the protein can be used to link sugar moieties to the protein by the CuAAC Click-Reaction. Below is shown the Click-Reaction between triple alkyne modified YFP and sugar azides 3 6.

4 Once the azido or alkyne function has been built into the protein sequence, conjugation with a large number of diverse partners including PEG-polymers, dyes, cofactors, antibodies, nucleic acids, small molecules, toxins, additional proteins and peptides opens a wide field with many different applications from therapeutics to diagnostics. Summary: The baseclick patented method allows the highly efficient labeling and multi-labeling of nucleic acids via CuAAC Click- Reaction. Thus, nucleic acids can be easily labeled with various molecules, amongst them dyes, quenchers, affinity tags, but also peptides, lipophilic groups, nanoparticles, enzymes, antibodies, solid phases and many more. Moreover genetically encoded proteins containing alkyne amino acids have shown that the alkyne functionalities can be used to link sugar moieties to the protein by the CuAAC Click- Reaction paving the way to new efficient glycosilation methods. Advantages of the baseclick platform: Labeling by simple, efficient Click-Reaction High labeling yield (quantitative) Allows introduction of sensitive labels Highly modular system (combinatorial approach) Orthogonal to conventional methods Bioorthogonal reaction Broad range of marker- and dye-azides Adaptable to linking beads, microarrays, nanoparticles, proteins etc. About us: baseclick GmbH is a start-up company funded and founded by BASF Venture Capital GmbH. It holds the intellectual property for the technologies and offer this for licensing the technology, purchasing of our products, and cooperation for the further development of our technologies. The company has its seat in Tutzing, south of Munich, close to the biotech campus in Munich-Martinsried. The IP was generated at the Ludwig Maximilians University of Munich (LMU) in the group of Prof. Thomas

5 Carell and BASF SE. Two former members of his group joined the company from the beginning. References: P. M. E. Gramlich, C. T. Wirges, A. Manetto, T. Carell. Postsynthetic DNA Modification through the Copper-Catalyzed Azide-Alkyne Cycloaddition Reaction. Angew. Chem. Int. Ed. 2008, 47, P. M. E. Gramlich, S. Warncke, J. Gierlich, T. Carell. Click-Click-Click: Single to Triple Modification of DNA. Angew. Chem. Int. Ed K. L. Kiick, E. Saxon, D. A. Tirrell, C. R. Bertozzi, Proc. Natl. Acad. Sci. USA 2002, 99, 19. E. Kaya, K. Gutsmiedl, M. Vrabel, M. Müller, P. Thumbs, T. Carell. Synthesis of Threefold Glycosylated Proteins using Click Chemistry and Genetically Encoded Unnatural Amino Acids. ChemBioChem., 2009, 10, Granted patents: WO2006/117161, New labeling strategies for the sensitive detection of analytes Patent applications: WO2008/952775, Click Chemistry for the production of reporter molecules Licenses: Baseclick GmbH holds a worldwide non-exclusive license for the research market of the "Click Chemistry" patent from "The Scripps Research Institute", WO03/101972, Copper-catalyzed ligation of azides and acetylenes. Contact : baseclick GmbH Bahnhofstrasse Tutzing, Deutschland Phone: +49(0) Fax: +49 (0) info@baseclick.eu