Mutually Orthogonal Bioorthogonal Reactions: Selective Chemistries for Labeling Multiple Biomolecules Simultaneously

Abstract

Bioorthogonal click chemistry has played a transformative role in many research fields, including chemistry, biology, and medicine. Click reactions are crucial to produce increasingly complex bioconjugates, to visualize and manipulate biomolecules in living systems and for various applications in bioengineering and drug delivery. As biological (model) systems grow more complex, researchers have an increasing need for using multiple orthogonal click reactions simultaneously. In this review, we will introduce the most common bioorthogonal reactions and discuss their orthogonal use on the basis of their mechanism and electronic or steric tuning. We provide an overview of strategies to create reaction orthogonality and show recent examples of mutual orthogonal chemistry used for simultaneous biomolecule labeling. We end by discussing some considerations for the type of chemistry needed for labeling biomolecules in a system of choice.

Keywords: Bioconjugation; Chemical Biology; Click chemistry; In vivo chemistry; Protein Engineering.

Fig. 1

Schematic representation of bioorthogonal reactions often used in bioconjugation. A [3 + 2] dipolar cycloadditions. (i) copper-catalyzed azide-alkyne cycloaddition (CuAAC), (ii) strain-promoted azide-alkyne cycloaddition (SPAAC), (iii) strain-promoted alkyne-nitrone cycloaddition (SPANC), and (iv) mesoionic compounds. B Inverse electron demand Diels–Alder reaction. (i) IEDDA with strained alkene, (ii) IEDDA with cycloalkyne, and (iii) IEDDA with non-strained alkene. C Boronic ester condensation. D [4 + 1] cycloaddition with isonitrile. (i) Isonitrile-tetrazine ligation and (ii) isonitrile-chlorooxime ligation. E Phosphine ligations. (i) Staudinger-Bertozzi ligation and (ii) cyclopropenone-phosphine ligation

Fig. 2

A Strategies to increase reaction rates of strained alkynes. Conformational constraint, ring contraction, heteroatom insertion, ring expansion and electronic modifications [28]. B Reactions of azide, nitrones, and mesoionic heterocycles with cyclooctynes. C Overview of the structures of common strained alkynes

Fig. 3

Mutual orthogonal labeling through steric tuning. A Inverse-electron demand (IED) mechanism occurs with BCN but not DBCO. B A duel-labeling experiment with two SPAAC reaction. DBCO-SiR preferentially reacts with the primary azide while BCN-BODIPY can react with the remaining tertiary azide [43]. C DTO reacts with BCN but not DBCO owing to steric hindrance

Fig. 4

The reactivity of tetrazine scaffolds based on the electronic effect of the substitution pattern. Electron-withdrawing groups increase the reactivity and kinetics. Tetrazines with increased reactivity show decreased stability under physiological conditions [–50]

Fig. 5

Matrix depicting reaction kinetics between several types of tetrazines and triazines with dienophiles [56, 60, 62, 64, 65]

Fig. 6

1- or 3-substitution on cyclopropenes strongly influences the capability to undergo click reactions with tetrazines [57]

Fig. 7

Development of mutually orthogonal reactions that arise from intrinsic reactivities and steric factors. A The reactivity trend of 5-phenyltriazine, 6-phenyltriazine, and 3,6-diphenyltetrazine with TMTH. 5-substituted triazines react with TMTH, even in the presence of tetrazine and Cp. B Dual orthogonal protein labeling through two IEDDA reactions by reacting Nluc-triazine and GFP-Cp with TMTH and 3,6-Pyr-tetrazine. The general approach for developing mutual orthogonal reaction by balancing electronic interactions (number of nitrogen atoms in the heterocycle) with steric effects [60]

Fig. 8

Overview of the A Bertozzi-Staudinger ligation, B “traceless” Staudinger ligation with the iminophosphorane intermediate, C the Staudinger-phosphonite, D Staudinger-phosphite, and E cyclopropenone-phosphine ligations

Fig. 9

The reactions between tetrazines and tertiary and primary isonitriles create stable and unstable conjugates respectively

Fig. 10

Photocaged click reagents. A CpO-caged bicyclononynes are light-induced for tetrazine ligation using visible light [95]. B Modular caging strategy with bulky Cp “reactivity cages’’ where attachment of photolabile groups at the cyclopropene nitrogen prevents ligation [97]. C Tetrazine oxidation via excitation of a photocatalyst. D) Photodecaging of a protected tetrazine at 405 nm

Fig. 11

Mutual orthogonal labeling with visible light initiated photoclick cycloaddition. A UV-induced reaction of 2,5-diphenyl tetrazoles with alkenes. B Sterically shielded sulfonated tetrazoles allows selective reaction with BCN. C Visible-light-induced [4 + 2] cycloaddition between PQ and VE, where PQ is exposed to visible light, becomes excited (PQ*), and transfers electrons to VE through photoinduced electron transfer (PeT). This electron transfer leads to the formation of a highly reactive 1,6-biradical intermediate. The intermediate then undergoes intramolecular radical recombination, forming [4 + 2] cycloadduct with the phenanthrodioxine (PDO) framework [104]. D Two mutual orthogonal labeling reactions of BSA-VE with either LYSO-MF or LYSO-azide. The first orthogonal fluorescent labeling was achieved between reaction pairs BSA-VE and LYSO-MF through the reaction with PQ-TAMRA and tetrazole, respectively (d, top)—the second reaction of BSA-VE and LYSO-azide through the reaction with PQ-TAMRA and DBCO-Cy5 (d, bottom) [103]

Fig. 12

Selective photoclick reactions based on the wavelength. At 285 nm 2,5-diphenyl tetrazoles can react with N-ethylmaleimide to form the pyrazoline ligation product, while at 382 nm o-methyl benzaldehyde can react with N-ethylmaleimide

Fig. 13

A Sequential orthogonal click chemistry in which order of addition or activation of reactants is crucial. B Mutual orthogonal approach in which all reactants are present simultaneously

Fig. 14

Mutual orthogonal triple labeling of antibody–drug conjugates by adopting three reaction pairs proceeding through distinct mechanistic pathways. Three trastuzumab antibodies functionalized with either 2-fPBA, azide, or TCO reacted with fluorophores containing the complementary functional groups, α-amino-hydrazide-RED, DBCO-TAMRA, and Tetrazine-BODIPY. The mutual orthogonality of the boronic acid condensation, SPAAC, and IEDDA reactions enable simultaneous triple labeling [87]

Fig. 15

CRACR-mediated labeling of 5-HTP, SPAAC-labeling of pAzF, and IEDDA labeling of CpK, achieving mutual orthogonal triple labeling in proteins [115]

Fig. 16

Mutual orthogonal triple labeling of proteins through electronic tuning of the alkyne. The SNO-OCT derivatives were designed with substitution patterns to tune the alkyne electronics tailored toward type I and III dipoles. The boronic acid condensation with α-amino hydrazides, which is mechanistically different, was included as a third mutual orthogonal reaction [62]

Fig. 17

One-pot strategy with triple mutual orthogonal labeling based on dispersion forces between the isocyano group and tetrazine substituents. Three proteins, ovalbumin (OVA), bovine serum albumin (BSA), and lysozyme (LYSO), were modified with bioorthogonal handles azide, 3,6-bis-tert-butyl tetrazine (DTTz), and MeTzCOOH, respectively. The functionalized proteins were reacted with three fluorophores with complementary reactive groups, DBCO-AF405, TCO-BODIPY red, and tertiary isonitrile-BODIPY green [83]

Fig. 18

Mutual orthogonal chemistry between tert-butyl tetrazine conjugated to BSA and isonitrile-bodipy and tetrachlorocyclopentadienone ethylene ketal conjugated to OVA and BCN-SiR

Fig. 19

Proximity enhanced click chemistry. A VBA uniquely react with tetrazines containing substituents that coordinate the boron atom. B An aptamer conjugate specifically binds to the target receptor and brings the DBCO in close proximity of an azide. C anti-CD19 CAR T cells can bind their target CD19 expressing cells, thereby bringing their metabolically labelled glycoproteins in close proximity, facilitating the click reaction

Fig. 20

The estimated time needed to reach t_1/2 of the reaction under ideal conditions, at 5 µM, 500 nM, and 10 nM. The reaction rates (K in Mol⁻¹ s⁻¹) for TCO-Tetrazine (~ 20,000) [48], DBCO-Iminosydnone [40], BCN-dipyridyl tetrazine [50], strained alkyne-nitrone [29], and DBCO-azide [21] click reactions were adapted from literature