DOI 10.1515/ntrev-2012-0083 Nanotechnol Rev 2013; 2(2): 215–227 Review Maha K. Rahim , Rajesh Kota , Sumi Lee and Jered B. Haun * Bioorthogonal chemistries for nanomaterial conjugation and targeting Abstract: Bioorthogonal chemistries are covalent reaction 1 Introduction pairs that proceed in the presence of biological compo- nents with complete specificity. A suite of reactions has Nanomaterials continue to generate significant interest been described to date that provides scientists and engi- in biomedical applications owing to their unique proper- neers with diverse operational characteristics for different ties. For example, nanocarriers can provide protection, applications. Nanomaterials in particular have benefitted high cargo capacity, controlled release, and attractive from these new capabilities, resulting in improved cou- pharmacokinetics/biodistribution [1] . Moreover, nanocrys- pling efficiencies and multifunctionality. In this review, tals exhibit unique physical properties such as paramag- we will discuss the application of bioorthogonal chemis- netisim [2, 3] , semiconductor fluorescence [4 – 6] , plasmon tries to different nanomaterial systems, highlighting the resonance [7] , and photoluminescence upconversion [8, 9] advantages and limitations for use in bioconjugation. We that can be harnessed as exquisitely sensitive, stable, and/ will also describe how recent improvements in the reac- or multiplexed detection signals. Finally, biomolecules can tion speed of catalyst-free bioorthogonal chemistries have be conjugated to the surface of all types of nanomaterials enabled the successful coupling of nanomaterials directly to impart efficient and multivalent adhesion for molecular to live cells. Using a recently developed reaction pair, targets. Owing to these attributes, targeted nanomaterials tetrazine and trans -cyclooctene, the direct covalent cou- present attractive diagnostic and therapeutic capabilities. pling to cells has been shown to occur on time-scales that However, to maximally exploit their potential, effective are relevant for biological studies and diagnostic applica- bioconjugation strategies are needed that are simple, fast, tions and can even amplify nanomaterial binding greater broadly applicable, have a small footprint, and are suit- than tenfold relative to traditional immunoconjugates. able for complex biological environments. This powerful technique still maintains exquisite specific- Bioorthogonal chemistries are a class of reactions ity, however, yielding robust results in clinical diagnos- whose cognate partners do not interfere with the func- tic applications using human tissue and blood samples. tional groups endogenous to cells. This includes standard Future work will likely focus on further advancement of protein functional groups such as primary amine, thiol, the in situ amplification technique, such as increasing carboxylic acid, and hydroxyl groups, as well as meta- nanomaterial binding, enabling multiplexed detection bolic intermediates like aldehydes and ketones [1] . These through the use of orthogonal reaction systems and exten- recently developed chemistries offer greater control and sion to applications in vivo . flexibility for manipulating biological systems and, thus, are ideal for elucidating the biological processes or engi- Keywords: amplification; bioorthogonal chemistry; neering-specific interactions within a complex cellular or molecular detection; nanomaterials; tetrazine. tissue environment. In this review, we discuss the development of *Corresponding author: Jered B. Haun , Department of Biomedical bioorthogonal coupling reactions and highlight their Engineering and Chao Family Comprehensive Cancer Center, use for probing biomolecule targets with nanomate- University of California Irvine, 3107 Natural Sciences II, Irvine, CA rial sensors in biological systems. We describe how the 92697, USA, e-mail: [email protected] growing suite of bioorthogonal chemistries is suited for Maha K. Rahim, Rajesh Kota and Sumi Lee: Department of Biomedical Engineering and Chao Family Comprehensive Cancer different types of nanomaterials and targeting appli- Center , University of California Irvine, 3107 Natural Sciences II, cations. Specifically, bioorthogonal chemistries have Irvine, CA 92697, USA enabled more efficient and diverse modifications of nanomaterials and subsequent targeting to biomole- cules. This includes recent applications in which the 216 M.K. Rahim et al.: Bioorthogonal chemistries for nanomaterials reactions, themselves, have been used for coupling these reasons, the CuAAC reaction has become a valuable nanoparticles to live cells in situ (i.e., direct labeling by technique for surface modifications and protein labeling. covalent reaction). Finally, we discuss the clinical trans- The CuAAC has also been referred to as “ click chemistry ” lation of bioorthogonal chemistry-based cell diagnostics. or the “ click reaction ” . Unfortunately, the CuAAC has Future work in this area will improve the sensitivity of limited applicability for living systems because the Cu(I) nanoparticle detection platforms as well as utilize mul- catalyst is cytotoxic. Another limitation is that the Cu(I) tiple bioorthogonal chemistries simultaneously or in affects the fluorescence yield of fluorescent proteins such concert with classic chemistries for multiplexing capa- as GFP. More directly relevant to this review, the CuAAC is city. Another promising advancement is the translation not compatible with quantum dots because the Cu(I) cat- of bioorthogonal chemistries to in vivo systems for detec- alyst nonreversibly quenches fluorescence [15] . Finally, tion or drug delivery. care must be taken when achieving the reducing condi- tions required for Cu(I) because they can easily affect acid-labile species such as iron oxide nanocrystals. As an alternative to metal catalysis, the Bertozzi 2 History of development group demonstrated that the introduction of ring strain into the alkyne moiety can effectively drive cycloaddi- Taking inspiration from the classical Staudinger reaction, tion, but with a slower reaction rate [16] . In this cata- Saxon and Bertozzi [10] designed a chemoselective liga- lyst-free approach, the bioorthogonal cycloaddition of tion between an azide and phosphine. Unlike the origi- geometrically strained alkyne, cyclooctyne, reacts with nal reduction reaction whose intermediate hydrolyzes to azide to form a triazole. This reaction is stable under form a primary amine and phosphine oxide, the modified physiological conditions, and cytotoxic effects were not Staudinger reaction introduces an electrophilic trap on observed after labeling azido sugars with a biotin-con- the phosphine reactant. As a result, a stable amide bond jugated cyclooctyne. Though improved biocompatibility forms after hydrolysis. Representing both the origins and is certainly an advantage of the strain-promoted reac- future application goals for this reaction, it was named tion, the slower reaction rate relative to CuAAC is limit- the Staudinger ligation. Utility in a biological setting ing for some applications. The slow reaction has been was first demonstrated using azido-modified sugars that addressed to some degree by modifying cyclooctyne were metabolically incorporated onto cellular surfaces. with substituent groups such as monofluoriniated [17] Azide moieties were then specifically labeled by covalent and difluorinated cyclooctyne [18] . The addition of reaction with a biotinylated phosphine and subsequent electron-withdrawing fluorine groups adjacent to the binding with fluorescein isothiocyanate (FITC)-avidin alkyne encourages reaction with azide. Cyclooctyne vari- [10] . Owing to the small size of the azide reactant, modi- ations, such as 4-dibenzyocylooctynols (DIBO) [19] and fications do not perturb the biomolecule. In addition, biarylazacyclooctynones (BARAC) [20] , introduce aro- azide is entirely absent from biological systems. These matic rings to impose higher ring strain and, thereby, advantages, along with the chemoselectivity of the reac- increase the reactivity of the alkyne. The improved tion, have resulted in extensive use of the Staudinger liga- reaction rates observed with DIBO-azide cycloaddition tion for labeling biomolecules [11] . Despite its effective- prompted Boons et al. to utilize DIBO for a strain-pro- ness, the Staudinger ligation is limited by its relatively moted nitrone cycloaddition [21] . Nitrone cycloadditions, slow reaction kinetics and susceptibility to phosphine though similar, offer faster kinetics than their azide oxidation [11, 12] . counterparts on the order of sixfold. The copper-catalyzed Huisgen cycloaddition was To meet the needs of the most demanding biological developed to address the kinetic limitations of the applications, the groups led by Fox and Weissleder further Staudinger ligation, while still making use of azide as a accelerated catalyst-free capabilities by developing a set reactant. This 1,3 dipolar cycloaddition yields a triazole of reactions between 1,2,4,5-tetrazines (Tzs) and strained linkage by reacting an azide with a terminal alkyne [13] . alkenes that proceeds via inverse-electron-demand Diels- Without a catalyst, this reaction only proceeds at high tem- Alder cycloaddition [22 – 25] . The strained dienophiles that peratures. However, when catalyzed by reduced copper, have been tested include norbornene, cyclooctyne, cyclo- or copper(I), the reaction proceeds at an accelerated rate propene, and trans-cyclooctene (TCO),