DOI: 10.1002/cbic.200700559 Surface Modification of Tobacco Mosaic Virus with “Click” Chemistry Michael A. Bruckman, [a] Gagandeep Kaur, [a] L. Andrew Lee, [a] Fang Xie, [a] Jennifer Sepulveda, [a] Rebecca Breitenkamp, [b] Xiongfei Zhang, [b] Maisie Joralemon, [b] Thomas P. Russell, [b] Todd Emrick, [b] and Qian Wang* [a] Cu I -catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction, [1,2] renascent of the well-known Huisgen reaction, [3] has recently flourished with applications in organic synthesis, drug discovery, polymer and materials science, and biotech- nology. [4–7] The high reaction yield, simple reaction and purifi- cation conditions, wide range of solvent and pH stabilities, and functional group tolerance make the CuAAC reaction a proto- typical “click chemistry”, [8] ideal for incorporating functionalities onto desired scaffolds. Over the years it has been widely em- ployed to construct and functionalize polymeric and polyvalent display systems, including polymers, [9–16] dendrimers, [17,18] nano- particles, [19] and surfaces, [20–22] where an extremely high reac- tion efficiency for every unit reaction is desirable. [23,24] In partic- ular, as organic azides and alkynes are almost unreactive with biomolecules and water, CuAAC reactions have been employed in derivatizing biomacromolecules, [25,26] viruses, [27–29] and cells [30–32] with high efficacy under mild reaction conditions. Recently tyrosine residues have been considered as a partic- ularly attractive target for chemoselective modification of pro- teins because of its subabundant distribution. Francis and co- workers have reported a number of transformations, ranging from the Mannich-type reaction to a transition metal mediated allylation reaction to a diazonium-coupling reaction, which can efficiently target the phenolic group of tyrosine residues at physiological conditions. [33–36] To overcome the sluggish reactiv- ity with electron-enriched diazonium salts, a sequential reduc- tion/oxidation/Diels–Alder reaction was developed to break the limitation of functionalities being incorporated. [36] In this communication, we report that CuAAC reactions can be com- bined with a diazonium-coupling reaction to quantitatively functionalize tyrosine residues with a wide array of starting materials. Tobacco Mosaic Virus (TMV) is a classic example of rodlike plant viruses consisting of 2130 identical protein subunits ar- ranged helically around genomic single RNA strand. The length of TMV, that is, 300 nm, is defined by the encapsulated genom- ic RNA that stabilizes the coat protein assembly. The polar outer and inner surfaces of TMV have been exploited as tem- plates to grow metal or metal oxide nanowires, [36–43] and con- ductive polymers have been coated on 1D assembled TMV to produce conductive nanowires. [44,45] TMV based materials have recently shown great potential with applications in nanoelec- tronics and energy harvesting devices. [46–48] In addition, it has been reported that tyrosine residues (Y139) of TMV are viable for chemical ligation using the electrophilic substitution reac- tion at the ortho position of the phenol ring with diazonium salts. [17] This reaction is very efficient, yet has two distinct dis- advantages for broader applications. First, it is difficult to syn- thesize desired starting materials; and second, the reaction is not compatible with acid-labile functional groups and suitable for electron-deficient anilines only. To embrace the structural diversity of various starting materials, TMV offers an ideal poly- valent display system which allows us to test the efficiency of CuAAC reaction in combining with the tyrosine ligation reac- tion. As shown in Scheme 1, TMV was first treated with the diazo- nium salt generated from 3-ethynylaniline 1 in situ adapted from the protocol reported by Francis and co-workers. [36] MALDI-TOF MS analysis indicated that > 95% of the capsid monomers were converted into alkyne derivatives 2 (Figure 1) despite the absence of a strong electron withdrawing group in the diazonium reagent. [49] Encouraged by this result, the CuAAC reactions between 2 and azides were explored. For bio- conjugation reactions using CuAAC, the Cu I catalysts are either generated directly by addition of Cu I salts, [28,31] or in situ from soluble Cu II sources and a reducing agent, such as a copper wire, phosphines, thiols, or ascorbate. [27] Multidentate heterocy- clic ligands are often required for enhancing the reaction effi- ciency. [27–29] Upon screening a series of reaction conditions, we found that the combination of CuSO 4 /sodium ascorbate (NaAsc) gave the best results. Whereas it is destructive to most other protein complex systems, [27] ascorbate is evidently benign to TMV and has no impact on its structural integrity. 3-Azido-7-hydroxy-coumarin a was first employed as the ACHTUNGTRENNUNGazido counterpart in the reaction, which could be easily moni- tored by UV-visible absorption at 340 nm (Figure 1B). [50] As a general protocol, 2 (2mgmL À1 ) and a (3mm) were added to a solution of CuSO 4 (1mm) and NaAsc (2 mm) in Tris buffer (10 mm, pH 7.8) with 20% DMSO (used to increase the solubili- ty of the azide component). After incubation for 18 h at room temperature, the viral particles were separated from the small molecules by sucrose gradient sedimentation. The integrity of TMV was confirmed by TEM and size-exclusion chromatogra- phy (SEC) analysis (data not shown). A strong absorption at 340 nm indicated the successful attachment of coumarin motifs (Figure 1B). MALDI-TOF MS analysis indicated a near quantitative transformation of surface alkynes to triazoles as shown in Figure 1A. [a] M. A. Bruckman, G. Kaur, L. A. Lee, F. Xie, J. Sepulveda, Dr. Q. Wang Department of Chemistry and Biochemistry and Nanocenter University of South Carolina 631 Sumter Street, Columbia, South Carolina 29208 (USA) Fax: (+ 1)803-777-9521 E-mail: wang@mail.chem.sc.edu [b] R. Breitenkamp, X. Zhang, M. Joralemon, Dr. T.P. Russell, Dr. T. Emrick Polymer Science and Engineering Department, University of Massachusetts Conte Center for Polymer Research, Massachusetts 01003 (USA) ChemBioChem 2008,9,519–523 # 2008 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 519