Vol. 8, No. 1 Chemical Ligation Chemical Ligation by Click Chemistry Native Chemical Ligation Staudinger Ligation Diphenylphosphinemethanethiol: efficacious reagent for traceless Organic Azides and Staudinger ligation Azide Sources Functionalized Alkynes 2 Introduction Vol. 8 No. 1 More and more researchers face the task of selectively combining large molecules, attaching molecular probes, or covalently Aldrich Chemical Co., Inc. immobilizing substrates on surfaces. In particular when biopolymers Sigma-Aldrich Corporation and bioconjugates are involved there is an urgent need for mild and 6000 N. Teutonia Ave. biocompatible reaction conditions. A toolbox of several powerful Milwaukee, WI 53209, USA chemical ligation techniques already exists and is continually being expanded. In this issue of ChemFiles, we provide an overview of modern To Place Orders chemical ligation methods and introduce highly innovative and Telephone 800-325-3010 (USA) unique new tools for research at the interface between chemistry FAX 800-325-5052 (USA) and biology. The most prominent chemical ligation techniques (click chemistry, native chemical ligation, and Staudinger ligation) will be discussed. A comprehensive listing of available organic azides and functionalized alkynes rounds off this Customer & Technical Services issue of ChemFiles with valuable building blocks for click chemistry or Staudinger ligation. Customer Inquiries 800-325-3010 If you are unable to find the specific reagent you need, “Please Bother Us.” with your Technical Service 800-231-8327 suggestions at [email protected], or contact your local Sigma-Aldrich® office SAFC™ 800-244-1173 Introduction (see back cover). 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When projects demand custom arrays of reagents, DiscoveryCPR.com Purchaser must determine the suitability of the DiscoveryCPR can meet the challenge. product for its particular use. See reverse side of invoice or packing slip for additional terms and conditions of sale. sigma-aldrich.com All prices are subject to change without notice. ChemFiles (ISSN 1933–9658) is a publication of About Our Cover Aldrich Chemical Co., Inc. Aldrich is a member of the The cover structure depicts diphenylphosphinemethanethiol, the most efficacious Sigma-Aldrich Group. © 2008 Sigma-Aldrich Co. reagent known today to induce traceless Staudinger ligations (Raines ligation reagent). Diphenylphosphinemethanethiol can be obtained easily from the shelf-stable precursor 670359 by removing the acetyl and borane protective groups. 3 Chemical Ligation by Click Chemistry—A “Click” Away from Discovery The traditional process of drug discovery based on natural In an extensive study Finn and co-workers only recently showed secondary metabolites has often been slow, costly, and labor- that tris(2-benzimidazolylmethyl)amines (general structure in intensive. Even with the advent of combinatorial chemistry Figure 2) are the most promising family of accelerating ligands and high-throughput screening in the past two decades, the for the Cu catalyzed azide-alkyne cycloaddition reaction from generation of leads is dependent on the reliability of the individual among more than 100 mono-, bi-, and polydentate candidates.10 reactions to construct the new molecular framework. Under both preparative (high concentration, low catalyst loading) and dilute (lower substrate concentration, higher catalyst Click chemistry is a newer approach to the synthesis of drug- loading) conditions, these tripodal benzimidazole derivatives give like molecules that can accelerate the drug discovery process substantial improvements in rate and yields, with convenient by Click Chemistry Chemical Ligation by utilizing a few practical and reliable reactions. Sharpless and workup to remove residual Cu and ligand. co-workers have defined what makes a click reaction: one that is wide in scope and easy to perform, uses only readily available A new reagent developed by Carolyn R. Bertozzi and co-workers reagents, and is insensitive to oxygen and water. In fact, water is eliminates the toxicity to living cells that is usually associated with in several instances the ideal reaction solvent, providing the best the copper catalyzed Huisgen 1,3-dipolar cycloaddition.11 By using yields and highest rates. Reaction work-up and purification uses a difluorinated cyclooctyne (Figure 3) instead of the usual terminal benign solvents and avoids chromatography.1 alkyne a rapid cycloaddition reaction takes place even without a catalyst. The ring strain and the electron-withdrawing difluoro Of the reactions comprising the click universe, the “perfect” group activate the alkyne for copper-free click chemistry. This example is the Huisgen 1,3-dipolar cycloaddition of alkynes to method was used to attach fluorescent labels to cells with azide- azides to form 1,4-disubsituted-1,2,3-triazoles (Scheme 1). The containing sialic acid in their surface glycans. Thus, it was possible copper(I)-catalyzed reaction is mild and very efficient, requiring no to study the dynamics of glycan trafficking in living cells over the protecting groups and no purification in many cases.2 The azide course of 24 hours with no indication that the reaction or the and alkyne functional groups are largely inert towards biological labels perturb the process. This is an impressive example of how molecules and aqueous environments, which allows the use of copper-free click chemistry can be used as a biologically friendly the Huisgen 1,3-dipolar cycloaddition in target guided synthesis3 method to label and track biomolecules in living cells. and activity-based protein profiling,4 or the ligation of biopolymers to probes or surfaces.5 For example, Carell and co-workers Sigma-Aldrich® proudly offers a choice of catalysts and ligands demonstrated the labelling of alkyne modified DNA oligomers for the Huisgen cycloaddition reaction. Later sections in this issue with fluorescence probes by click chemistry.6 present a comprehensive overview of available organic azides, azide sources, and alkynes that may be applied in click chemistry. The triazole has similarities to the ubiquitous amide moiety found in nature. Thus triazole formation was used for the otherwise If you want to learn about hot new product additions to the difficult macrocyclization of a cyclic tetrapeptide analog to a click chemistry universe and other innovative areas of chemical potent tyrosinase inhibitor.7 synthesis as soon as they become available, please check our regularly updated product highlights at sigma-aldrich.com/ Additionally triazoles are nearly impossible to oxidize or reduce. chemicalsynthesis. This is a main reason why material science has discovered Huisgen cycloadditions as major ligation tools in diverse areas such as References: (1) For recent reviews, see: (a) Kolb, H. C.; Sharpless, K. B. Drug Discovery polymer science or nanoelectronics.8 Today 2003, 8, 1128. (b) Kolb, H. C. et al. Angew. Chem. Int. Ed. 2001, 40, 2004. (2)(a) Rostovtsev, V. V.; Green, L.G.; Fokin, V.V.; Sharpless, K.B. Angew. Chem. Int. Ed. Using Cu(II) salts with ascorbate has been the method of 2002, 41, 2596. (b) Tornøe, C. W. et al. J. Org. Chem. 2002, 67, 3057. (3)(a) Manetsch, choice for the preparative synthesis of 1,2,3-triazoles, but it is R. et al. J. Am. Chem. Soc. 2004, 126, 12809. (b) Lewis, W.G. et al. Angew. Chem. Int. Ed. 2002, 41, 1053. (4) Speers, A. E. J. Am. Chem. Soc. 2003, 125, 4686. (5) Wolfbeis, problematic in bioconjugation applications. However, O.S. Angew. Chem. Int. Ed. 2007, 46, 2980. (6) Gierlich, J.; Burley, G.A.; Gramlich, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, TBTA (Figure 1), P.M.E.; Hammond, D.M.; Carell, T. Org. Lett. 2006, 8, 3639. (7) Bock, V.D.; Perciaccente, has been shown to effectively enhance the copper-catalyzed R.; Jansen, T.P.; Hiemstra, H.; Maarseveen, J.H. Org. Lett. 2006, 8, 919. (8) Lutz, J.-F. 9 Angew. Chem. Int. Ed. 2007, 46, 1018. (9) Chan, T.R. et al. Org. Lett 2004, 6, 2853. cycloaddition without damaging biological scaffolds. (10) Rodionov, V. O.; Presolski, S. I.; Gardinier, S.; Lim, Y.-H.; Finn, M. G. J. Am. Chem. Soc. 2007, 129, 12696. (11) Baskin, J.M.; Prescher, J.A.; Laughlin, S.T.; Agard, N.J.; Chang, P.V.; Miller, I.A.; Lo, A.; Codelli, J.A.; Bertozzi, C.R. PNAS 2007, 104, 16793. R2 1 mol% CuSO4 N N 2 N N 5 mol% sodium ascorbate N N R 1 N N R H O/tBuOH 2:1 1 R 2 R R rt, 8 h N R = H or -(CH ) CO K Scheme 1 N 2 4 2 N N N N N R Figure 2 N N N N O R F N N N H F N N R = fluorescent dye or biotin Figure 3 Figure 1 Ready to scale up? For competitive quotes on larger quantities or custom synthesis, contact your local Sigma-Aldrich office, or visit safcglobal.com.