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. 2021 May 25;2(2):021301. doi: 10.1063/5.0050850

Azide-based bioorthogonal chemistry: Reactions and its advances in cellular and biomolecular imaging

Samira Husen Alamudi 1,a), Xiao Liu 2, Young-Tae Chang 2,3,2,3,a)
PMCID: PMC10903415  PMID: 38505123

Abstract

Since the term “bioorthogonal” was first demonstrated in 2003, new tools for bioorthogonal chemistry have been rapidly developed. Bioorthogonal chemistry has now been widely utilized for applications in imaging various biomolecules, such as proteins, glycoconjugates, nucleic acids, and lipids. Contrasting the chemical reactions or synthesis that are typically executed in vitro with organic solvents, bioorthogonal reactions can occur inside cells under physiological conditions. Functional groups or chemical reporters for bioorthogonal chemistry are highly selective and will not perturb the native functions of biological systems. Advances in azide-based bioorthogonal chemical reporters make it possible to perform chemical reactions in living systems for wide-ranging applications. This review discusses the milestones of azide-based bioorthogonal reactions, from Staudinger ligation and copper(I)-catalyzed azide-alkyne cycloaddition to strain-promoted azide-alkyne cycloaddition. The development of bioorthogonal reporters and their capability of being built into biomolecules in vivo have been extensively applied in cellular imaging. We focus on strategies used for metabolic incorporation of chemically tagged molecular building blocks (e.g., amino acids, carbohydrates, nucleotides, and lipids) into cells via cellular machinery systems. With the aid of exogenous bioorthogonally compatible small fluorescent probes, we can selectively visualize intracellular architectures, such as protein, glycans, nucleic acids, and lipids, with high specificity to help in answering complex biological problems.

I. INTRODUCTION

Fluorescence-based bioimaging has emerged as one of the utmost techniques for probing biological systems due to its sensitivity and technical simplicity.1 Fluorescent proteins (FPs), as represented by green fluorescent protein (GFP) and its variants, have been widely used to genetically label cellular proteins. This technique is popularly used for investigating protein localization and movement in living organisms, which significantly increases our understanding of protein dynamics and functions in cells and animals.2 However, the big size of FPs may perturb the native functions and biological interactions of the target proteins. Moreover, this genetic tagging tool is not applicable for other target biomolecules, such as glycoconjugates or glycans, nucleic acids, lipids, and metabolites. To this end, a reactivity-based bioorthogonal chemistry approach has been developed.

Bioorthogonal chemistry was introduced by Bertozzi and co-workers in 2003. It refers to the chemistry that does not interfere with native biological processes.3,4 Unlike typical chemical reactions or synthesis that are commonly performed in vitro with organic solvent in a chemical flask, bioorthogonal reactions can occur inside cells in physiological conditions. Functional groups or chemical reporters for bioorthogonal chemistry provide a way to tag target biomolecules without the need of direct genetic encoding. They are abiotic, which means that they do not have counterparts in complex biological systems. This, therefore, makes bioorthogonal chemistry highly selective and specific. As Bertozzi put it in 2005, “Bioorthogonal chemical reporters are non-native, non-perturbing chemical handles that can be modified in living systems through highly selective reactions with exogenously delivered probes.”3 Bioorthogonal chemistry has since made a remarkable scientific impact and opened a path to the development of tagging tools to answer biological questions.

Bioorthogonal labeling technique generally follows a two-step reaction (Fig. 1). First, a substrate for a target (e.g., protein, sugar, nucleic acid, lipid) is functionalized with a bioorthogonal chemical reporter. The substrate, which structurally mimics the natural ones, is then delivered to the target using either intrinsic or engineered biosynthetic pathways without visibly perturbing the target's native functionality. Second, a corresponding bioorthogonal functionalized probe is introduced to allow the detection of the target.

FIG. 1.

FIG. 1.

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Schematic representation for bioorthogonal labeling strategy in cells. Step 1: a substrate or building block of a target is functionalized with a bioorthogonal chemical reporter and delivered to the target using either cellular intrinsic or engineered biosynthetic pathways. Step 2: a corresponding bioorthogonal functionalized probe is introduced to allow detection and visualization of the target.

Over the years, numerous bioorthogonal reactions have been successfully developed. Significant development includes novel genetic encoding techniques that allow more efficient incorporation of bioorthogonal chemical reporters into biological systems; reaction kinetics optimization via catalysts or substrate activation; new reporters such as tetrazines, nitrones, and others; and development of smart probes for robust labeling in living systems.5,13,90

In this review, we will highlight the development of azide-based bioorthogonal reactions due to their extensively wide applications in various research fields, from Staudinger ligation and copper(I)-catalyzed azide-alkyne cycloaddition to strain-promoted azide-alkyne cycloaddition. Importantly, we will outline some applications of azide-based bioorthogonal reactions for cell imaging, with a particular emphasis on metabolic incorporation strategies of tagged biomolecules for applications in imaging intracellular architectures in mammalian cells.

II. AZIDE-BASED BIOORTHOGONAL REACTIONS

Azides are essentially small, adequately polar, abiotic, and unreactive with native biological functionalities. For these qualities, azide has become a prominent bioorthogonal reporter that can be readily incorporated into varied biomolecules.

A. Staudinger ligation

One of the earliest reports of bioorthogonal reaction is the Staudinger ligation.6 Staudinger ligation follows a reaction between azide and triphenylphosphine that results in the selective formation of an amide bond linkage. The pair is reactive under physiological pH, room temperature and aqueous solution.

The amide linkage can be formed within the biological environment. Azide acts as a soft electrophile that then intrinsically favors soft nucleophiles, such as triphenylphosphine, over hard nucleophiles that are generally found in biological systems.7 Importantly, this azide-triphenylphosphine pair is naturally absent from the living system, making the Staudinger ligation selectively reactive yet specifically inert toward other biological functionalities. The Staudinger reaction kicks in after a nucleophilic attack on an azide group by the phosphine to form an aza-ylide intermediate. This eventually yields primary amines after the aza-ylide intermediate is spontaneously hydrolyzed in the presence of water [Fig. 2(A)].8 Modification of this ligation yields an amide bond from an azide and a specifically functionalized phosphine containing a cleavable linkage. This improvement, which is also called “traceless” Staudinger ligation, leaves no phosphine oxide motif in the ligation product [Fig. 2(B)].9,10

FIG. 2.

FIG. 2.

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(A) The Staudinger ligation (B) “Traceless” Staudinger ligation. X is the cleavable linkage.

The Staudinger ligation was quite a breakthrough. It sets a new era in bioorthogonal chemistry and its applications appears in a wide range of both in vitro and in vivo labeling, for instance in labeling nucleic acid11 and cell surface glycans with azide-containing sugar in lysates and live cells.6,12 This also enables covalent modification of the surface glycans with chemical probes.

Despite its advantages, however, Staudinger ligation suffers from a rather slow reaction kinetics (second-order rate constant of 0.0020 M−1S−1).8 In addition, high concentration of the phosphine is required for the reaction. This causes high fluorescence background noise when the system is applied for fluorescence cell imaging because an excess of the phosphine was retained in the cell.14 Moreover, the reaction is not suitable for intracellular imaging due to poor permeability and high susceptibility to oxidation of its arylphospine group.15

B. Copper(I)-catalyzed azide-alkyne cycloaddition

To overcome the slow kinetics of Staudinger ligation for applications in cellular imaging, an alternative bioorthogonal pair of azide-alkyne was introduced. The pair would regiospecifically produce a 1,4-disubstituted-1,2,3-triazole molecule through 1,3-dipolar [3 + 2] cycloaddition. The triazole part is highly stable and is not toxic. Rolf Huisgen, who first introduced the concept of azide-alkyne 1,3-dipolar cycloaddition in the 1950s, pioneered this reaction.16 However, the reaction requires high temperatures and pressures to promote cycloaddition and triazole formation, which are not compatible with biological systems.17 In addition, a mixture of the isomeric products is often observed [Fig. 3(A)].

FIG. 3.

FIG. 3.

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(A) Huisgen's azide-alkyne 1,3-dipolar cycloaddition (B) The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).

In the 2000s, two independent groups (Sharpless and Meldal) discovered that copper (I) dramatically catalyzes the Huisgen reaction and increases the reaction rate to seven orders of magnitude or more.18,19 This reaction was then named copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), commonly referred to as “click chemistry.” While Huisgen's reaction often produces mixture of regioisomers, CuAAC allows the synthesis of specifically 1,4 cycloaddition products [Fig. 3(B)].

This then put a focus on CuAAC as a prototype for click reaction and led to wide applications in both organic synthesis and chemical biology, especially due to the high yield and reaction efficiency in aqueous solvents at room temperature.20,21 However, copper toxicity is a major drawback of CuAAC. Copper (I) can induce severe changes in cellular metabolism and damage cellular functions due to Cu(I)-mediated generation of reactive oxygen species (ROS).22

Many have pursued improvement of the CuAAC reaction to eradicate copper toxicity via either ligand-assisted (TBTA,23 BTTPS,24 THPTA,25 BTTAA26) or chelate-assisted (such as 2-picolylazide27,28 and AIO-129) reactions. These additional substances evidently not only reduced the toxicity level but also accelerated the reaction kinetics. However, additional ligands and chelates may not be particularly convenient for applications in intracellular imaging in living systems.26,30

C. Strain-promoted azide-alkyne cycloaddition

To circumvent the copper toxicity issue,22 a copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) was developed through alkyne substrate activation. SPAAC utilizes a strained cyclic alkyne known as cyclooctyne to enhance the cycloaddition rate (Fig. 4). The faster reaction rate eventually eliminates the requirement for copper catalyst and thus makes SPAAC biologically compatible for labeling biomolecules in living cells.31

FIG. 4.

FIG. 4.

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The strain-promoted azide-alkyne cycloaddition (SPAAC).

The first generation of cyclooctyne, OCT, developed by the Bertozzi group in 2004, has a considerable hydrophobicity but relatively poor water solubility with the second-order rate constant k of 0.0012 M−1s−1.31

In order to improve the physical properties of the cyclooctyne, an aryl-less octyne (ALO)32 and highly water-soluble dimethoxy azacyclooctyne (DIMAC)33 were developed. The study showed that ALO has an improved water solubility but similar kinetic properties to the unmodified cyclooctyne (k = 0.0013 M−1s−1), while DIMAC reasonably has a higher reaction rate (k = 0.0030 M−1s−1).

A substantial rate enhancement was achieved by the addition of an electron-withdrawing fluorine atom at the propargylic position to yield a monofluorinated cyclooctyne (MOFO)32 and difluorinated cyclooctyne (DIFO).34 NOFO, a nonfluorinated version of MOFO, was also synthesized to investigate the fluorination effect. Compared to its nonfluorinated version (NOFO), MOFO demonstrated a better reactivity and about fourfold greater reaction rate (k = 0.0043 M−1s−1), thanks to the electron-withdrawing fluorine atom within the structure.32 Interestingly, DIFO showed a dramatically enhanced reaction rate up to 60-fold.34 Following that, DIFO-fluorophore conjugates were developed for cell imaging purposes of azide-tagged target visualization in live mammalian cells and C. elegans.34

To prepare a readily accessible alkyne without lengthy synthesis routes, Delft group reported a bicyclo[6.1.0]nonyne (BCN) via a straightforward process through cyclopropanation of 1,5-cyclooctadiene.35 With an excellent reaction rate of k = 0.14 M−1s−1, BCN derivatives were applied in the labeling of glycans.35

Boons group reported an alternative cyclooctyne by fusing two benzene rings to the cyclooctyne core, resulting in highly strained dibenzocyclooctyne (DIBO) with increased reactivity (k = 0.057 M−1s−1).36 An analogue of DIBO with an exocyclic amide called aza-dibenzocyclooctyne (DIBAC) showed faster kinetics and higher hydrophilicity than DIBO.37 In the same year, another derivative of DIBO with an amide bond, a biarylazacyclooctynone (BARAC), was reported to have remarkably improved reaction kinetics of k = 0.96 M−1s−1 due to dibenzo system in DIBO and the addition of an sp2-like center in DIBAC.5,38 A summary of the cyclooctyne derivatives and their reaction rate constants for SPAAC is presented in Fig. 5.

FIG. 5.

FIG. 5.

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Structure of cyclooctyne derivatives for strain-promoted cycloaddition with their respective reaction rate constant: NOFO, ALO, OCT, DIMAC, MOFO, DIBO, DIFO, BCN, DIBAC, and BARAC. Rate reaction was monitored on the reaction cyclooctyne derivatives with benzyl azide in CD3CN.

III. AZIDE-BASED LABELING STRATEGY FOR CELL IMAGING

The unique properties of bioorthogonal chemical reporters led to the development of smart fluorescence probes that allow labeling and imaging of the bioorthogonally tagged biomolecules. The integration of such bioorthogonal reactivity into biomolecules in vivo resulted in wide applications of specific biomolecule imaging in cellular settings. In this section, we will discuss incorporation strategies of the bioorthogonally tagged biomolecules, such as analogues of amino acids, monosaccharides, nucleosides and lipids, into cellular proteins, glycans, nucleic acids, and biolipids respectively. The incorporation is facilitated by cellular biosynthetic machinery systems. After incorporation, exogenous bioorthogonal fluorescent probes can be added to allow chemical ligation and visualization of the biomolecules in cells.

A. Bioorthogonal protein imaging

Proteins are responsible for virtually all of the complex processes of cellular life, from cell structure and organization, signal transduction, immune response to cell maintenance. They are vastly diverse in the functions they serve. To understand and investigate these functions, methods to allow labeling and visualization of proteins are crucial.

Biological systems have evolved to encode two extra rare natural amino acids that can co-translationally be inserted into proteins. The amino acids, selenocysteine (Sec) and pyrrolysine (Pyl), are also dubbed as the 21st and 22nd amino acids in the genetic code.39 Moving forward, incorporation of chemically reactive groups embedded in unnatural amino acids (UAAs) into cellular proteins can be done either in a residue-specific or site-specific manner.40 Site-specific incorporation of the unnatural amino acids directly into proteins, has opened up a new horizon in the field of chemical biology. Pioneering this field is Peter G. Schultz.41–43 This incorporation approach requires the addition of new apparatuses to the cellular machinery that are orthogonal to the endogenous cellular system. It includes: (i) a unique tRNA-codon pair, (ii) an orthogonal aminoacyl-tRNA synthetase (AARS), and (iii) an unnatural amino acid [Fig. 6(A)].

FIG. 6.

FIG. 6.

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(A) A general approach for site-specific incorporation of unnatural amino acid containing bioorthogonal handle in live cells. A new set of components containing a unique tRNA-codon pair, an orthogonal aminoacyl-tRNA synthetase (AARS), and an unnatural amino acid is required. The orthogonal AARS acrylates the orthogonal tRNA with an UAA (red ball). The acylated orthogonal tRNA then inserts the UAA at the position specified by the unique amber codon, which then is incorporated into the growing protein of interest during translation. (B) Schematic illustration of Azi incorporation into specific site of histone H2B protein. Once incorporated, the azide moiety of Azi is labeled by fluorescence probe CO-1 to allow its visualization. (C) Fluorescence imaging of histone H2B in live U-2 OS [see (B)]. U-2 OS cells were co-transfected with plasmids pIre-Azi3 and pmH2B-6-mKate2-16tag to incorporate UAA Azi into H2B-mKate2 at site 16. Afterward, cells were labeled with CO-1 at 37 °C and imaged. The mKate2 (red) signals were detected in nucleolar site only, and colocalized with the CO-1 (green) signals in the merged image. Figure 6(C) was reproduced from S. H. Alamudi et al., Development of background-free tame fluorescent probes for intracellular live cell imaging, Nat. Commun. 7, 11964 (2016); licensed under a Creative Commons Attribution (CC BY) license.

Among the total 64 natural genetic codons, 3 of them are stop codons that signal the termination of the protein translation process. One of the stop codons, namely the amber stop codon UAG, can be recognized by a unique suppressor tRNA to insert an unnatural amino acid into a growing protein instead of marking the point at which the synthesis of a protein ends. Among tRNA-AARS pair systems, the discovery of a pyrrolysyl-tRNA synthetase (PylRS)-pyrrolysyl tRNA (tRNApyl) pair has advanced this field. This has been utilized widely to site-specifically incorporate unnatural amino acids for genetic code expansion.40,44

The reaction between UAA containing a bioorthogonal handle and externally added orthogonal fluorescence probe has to proceed under biologically compatible conditions, in which the reactants do not cross-react with the many other functional groups found in living cells.4 The Chin group has reported excellent examples of this bioorthogonal reaction advancement for cellular imaging.45,46 One of the earlier reports on azide-based ligation was the use of alkyne-containing UAA homopropargylglycine (Hpg) for tagging and visualizing newly synthesized proteins for residue-specific labeling, by the Tirrel group.47 A few years later, the same group developed coumarin-conjugated cyclooctyne probes to label azide-tagged protein, which was incorporated with azidohomoalanine Aha, in live mammalian cells through SPAAC.48

Additional challenges for specifically intracellular protein imaging, including the limited availability of cell-permeant fluorescent probes and high fluorescence background from the unreacted ones, still exist. Efforts have been made to tackle these issues. For example, recently, the Chang group developed a new pair of reactive 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-derived probes containing azide and cyclooctyne BCN for SPAAC intracellular site-specific labeling. In the applications, alpha tubulin carrying N-(cyclooct-2-yn-1-yloxy)carbonyl)L-lysine (CoK) was tagged with background-free “tame” azide dye AzG-1. Additionally, the authors also demonstrated the clear imaging of nucleolar protein histone H2B bearing azido UAA p-Azido-L-phenylalanine (Azi), which was labeled with “tame” BCN-bearing BODIPY-derived probe CO-1 [Figs. 6(B) and 6(C)].49

Structures of UAA developed for the use in site-specific labeling through azide-based reactions for cell imaging are summarized in Fig. 7.41,45,49,76–81

FIG. 7.

FIG. 7.

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Structures of unnatural amino acid used for site-specific protein labeling via azide-based bioorthogonal chemistry. 1. p-Azido-L-phenylalanine (Azi), 2. N-ε-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys or o-AzbK), 3. Nε-(2-azidoethoxy)carbonyl-L-lysine (AzK), 4. Nε-(((1R,2R)-2-azidocyclopentyloxy)carbonyl)-L-lysine (ACPK), 5. N- ε-(cyclooct-2-yn-1-yloxy)carbo- nyl)L-lysine (CoK), 6. bicyclo[6.1.0]nonyne (BCNK), 7. cyclooct-2-ynol tyrosine (COY).

B. Bioorthogonal glycan imaging

Glycoconjugates or glycans can be defined as complex carbohydrates that consist of a large number of monosaccharides and are known to play key metabolic and physiological roles in biological systems.50 Glycans displayed on the cell surface hold a unique signature of a certain cell type. The profile of the glycans fluctuates in response to physiological cellular changes and is also associated with diseases.51 Despite its importance, the development of glycan research is lagging behind that of protein or gene research due to its structural diversity and complexity, as well as technical difficulties in engineering glycans using conventional genetic manipulation ways. This is where bioorthogonal chemistry serves as a powerful tool.

Azide as the bioorthogonal chemical reporter is the most influential instrument for glycan research. Thanks to its abiotic property, unique reactivity, and small size,4 azides can conveniently be installed onto monosaccharides to serve as a complementary partner for azide-based bioorthogonal reactions and subsequently be applied to wide glycobiology research topics, particularly glycans imaging.

Among various unnatural monosaccharides, there are three main azido monosaccharides that have been used extensively for glycan metabolic engineering and cellular imaging, namely tetraacetylated N-azidoacetylmannosamine (Ac4ManNAz), tetraacetylated N-azidoacetylglucosamine (Ac4GlcNAz), and tetraacetylated N-azidoacetylgalactosamine (Ac4GalNAz) [(Fig. 8(A)]. These azido analogues can be recognized and well-tolerated by cell biosynthesis machinery and eventually transported into Golgi compartments to be finally incorporated into complex glycans [(Fig. 8(B)]. ManNAz is metabolized through sialic acid pathways for N-linked glycosylation (to produce azido sialic acid),6,12 while GlcNAz and GalNAz are metabolically incorporated into O-linked cellular glycans.52–56

FIG. 8.

FIG. 8.

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(A) Structures of common unnatural monosaccharides for azide-based labeling: N-azidoacetylmannosamine (Ac4ManNAz), peracetylated N-(4-pentynoyl)mannosamine (Ac4ManNAl, N-azidoacetylglucosamine (Ac4GlcNAz), N-azidoacetylgalactosamine (Ac4GalNAz). (B). A schematic illustration of the incorporation of unnatural monosaccharides through metabolic oligosaccharide engineering technology. Unnatural monosaccharides can be incorporated into glycan through cellular biosynthetic machinery and labeled with corresponding bioorthogonal chemical probes for glycan visualization in cells.

Since its introduction in the 2000s, the Bertozzi group and others have tremendously advanced methods to image glycans in living cells, even animals. The labeling was done either through Staudinger ligation, CuAAC, or SPAAC. So far, this labeling approach has been utilized to image different glycans on the cell surface. Some recent advancements include the hybridization chain reaction (HCR)-based approach to image protein-specific GalNAcylation (specific sialylation) on cancer cells by first incorporating Ac4GalNAz into glycans on the cell surface and then imaging them by DIBO-modified Förster resonance energy transfer (FRET) donor probe fluorescence probe.57 Using controlled bleaching of fluorescent probes and single-molecule technique via super-resolution microcopy, Ovryn group successfully imaged N-linked sialic acids and O-linked N-acetyl galactosamine on the membrane of cancer cells by metabolically incorporated Ac4GalNAz and alkyne-bearing monosaccharide Ac4ManNAl58 into N- and O-linked glycans, and then labeled them with Alexa 488 azide.59 Another effort using HCR amplification-based approach was also reported recently by developing a DBCO-nanoassembly probe, which was assembled with two Texas Red (TR)-labeled DNA hairpin probes, to enhance visualization of azide-bearing galactosamine and sialic acid on the cell surface.60 It is also noteworthy that the development of highly permeable cyclooctyne probes has led to the visualization of intracellular glycoconjugates at the early stage of their biosynthetic pathway in the native condition, before they are expressed on the cell surface.56 Through multi-labeling approach via SPAAC, the authors demonstrated the profile dynamic of both extracellular and intracellular different glycoconjugates (Fig. 9).56

FIG. 9.

FIG. 9.

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(A) Multi-labeling of both extracellular and intracellular azide-tagged glycoconjugates in various live cell lines using AlexaFluor 488-DIBO (green) and BODIPY-BCN probes (blue), respectively. Scale bar 50 μm. Reproduced with permission from S. H. Alamudi et al., Chem. Sci. 9, 2376–2383 (2018). Copyright 2018 Royal Society of Chemistry.

C. Bioorthogonal nucleic acid imaging

Besides proteins and glycans, nucleic acids also play a central role in cellular functions and processes. Deoxyribonucleic acid (DNA) is the central information storage system that carries the genetic information from one cell to the next generation of the cell. Ribonucleic acid (RNA) is central for transcription and translation to convert the information stored in DNA into proteins.

Study of nucleic acids and their non-coding derivatives, such as miRNA61 and eRNA,62 is an increasingly developing topic, and investigation of their specific cellular roles in their native environment is of great interest.

As discussed before, bioorthogonal labeling, particularly for live cellular imaging, has predominantly been developed for proteins and glycans. To the best of our knowledge, it has only been recently applied for nucleic acids, and even so, there are not many reports found in the current literature. Factors such as poor stability of the chemically modified nucleic acid building blocks in the cellular environment, their lack of permeability to cross the plasma membrane, their incompatibility with DNA or RNA polymerase, as well as potential inhibition of cellular metabolism by the modified building blocks, may contribute to the challenge of nucleic acid labeling in living cellular settings.63

In general, incorporation of modified oligonucleotides into the cellular system can be done in two ways: (1) delivery of presynthesized modified oligonucleotides, which was synthesized either on solid phase (SPS) using a synthesizer or in vitro enzymatic approach via primer extension (PEX) and other enzymatic oligonucleotide extensions (EX) by polymerase chain reaction (PCR) technique, into cells via transfection; and (2) metabolic labeling of modified building blocks into nascent nucleic acids through cellular endogenous enzymatic system (Fig. 10).64 Albeit challenging, the latter is preferred because the reactive functional group is incorporated into nascent cellular nucleic acids by means of natural biosynthesis and thus is not a hassle in the preparation of the functional group-containing nucleic acids.

FIG. 10.

FIG. 10.

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Incorporation of bioorthogonally modified nucleic acid building blocks into cells. (A) Delivery of presynthesized modified oligonucleotides, which was synthesized either on solid phase using a synthesizer or in vitro enzymatic approach via primer extension (PEX) and other enzymatic oligonucleotide extensions (EX) by polymerase chain reaction (PCR) technique, for intracellular transport via transfection. Once incorporated inside the cells, exogenous complement bioorthogonal probe is added for azide-based nucleic acid labeling. (B) Metabolic labeling of modified building blocks into nascent nucleic acids through cellular endogenous enzymatic system. Bioorthogonally modified nucleosides or nucleotides are uptaken by cells and converted into active nucleotides and thus incorporated into nascent DNA/RNA. Subsequently, exogenous complement bioorthogonal probe is added for azide-based nucleic acid labeling.

The earliest known report on the in vivo fluorescent labeling of DNA inside the cells was by the incorporation of a thymidine analogue, 5-bromo-2′-deoxyuridine (BrdU), in the 1970s.65 Instead of a bioorthogonal fluorescent probe, the successful incorporation was detected using fluorescent antibodies that bind to the BrdU moieties.65 However, the high cytotoxicity of BrdU66 and the use of antibodies restrict its applications. Decades later, terminal alkyne-containing analogue 5-ethynyl-2′-deoxyuridine (EdU) was developed for labeling of cellular DNA through CuAAC.67 The study showed that EdU is efficiently incorporated into the DNA of proliferating mammalian cells by feeding the cells with EdU-containing cell media. After cellular uptake and incorporation into nucleic acids, the use of fluorescent azide of tetramethylrhodamine and fluorescein allow for its detection in live cells. Ever since, this method has been popularly applied in a wide range of nucleic acid research.

Due to the still toxic properties of EdU that can induce DNA damage signaling (DDS),68 the EdU was modified to reduce cell damage by at least two ways:

  • (i)

    derivatization on the base moiety, for instance: purine analogues of 7-deaz a-7-ethynyl-2′-deoxyadenosine (EdA) and 7-deaza-7-ethynyl-2′-deoxyguanosine (EdG)69 and pyrimidine analogue of 2′-deoxycytidine (EdC);70

  • (ii)

    replacing terminal alkyne with either azides, for instance 5-azido-2′-deoxyuridine AdU, and 5-(azidomethyl)-2′-deoxyuridine AmdU71 or the recently reported one with cyclooctyne carboxymethylmonobenzocyclooctyne derivative of 2′-deoxyuridines, COMBOdU,72 for a more biocompatible labeling via SPAAC.

The cyclooctyne-building block, however, may possess significant disadvantages due to its steric size and the corresponding lipophilicity.73 Azide-building blocks, on the other hand, are smaller, but it may be tricky to incorporate them into RNA and DNA. The Luedtke group reported that AmdU exhibited much higher stability than AdU under the physiological environment with no detectable decomposition after 64 h at 37 °C. Staining of AmdU-modified cellular DNA was demonstrated in tandem reactions by both SPAAC and CuAAC using BCN-AlexaFluor 488 and AlexaFluor 594 alkyne/Cu(I), respectively, in Hela cells.71

Another method to detect nucleic acids indirectly is to utilize azide-modified nucleic acid-binding ligands. G-quadruplexes (G4s) are a series of biologically stable nucleic acid structures formed by consecutive guanine-rich sequences.82 Much effort has been made for the development and target identification of G4 ligands. Several fluorescent probes have been developed by conjugating fluorophores to G4 ligands.83,84 However, the covalent addition of a fluorophore may dramatically change the nature of the ligand. Therefore, to maintain the property of the G4 ligand, the Nagasawa group modified a hexaoxazole-macrocycle derivative (L2H2–6OTD-Az) with a small azide moiety for bioorthogonal labeling of G4.74 The introduction of the azide group has almost no influence on the ligand activity compared to the mother compound (L2H2–6OTD). The BCN moiety-containing probe CO-1 was then introduced for intracellular click reaction and live-cell imaging. The target of the G4 ligand was identified as RNA G4 by post-binding visualization.

Structures of the bioorthogonally modified building blocks discussed here are summarized in Fig. 11.

FIG. 11.

FIG. 11.

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Structures of unnatural building blocks used for azide-based nucleic acid labeling in cells.

D. Bioorthogonal lipid imaging

Biolipids are a big family of intracellular hydrophobic macromolecules, including fatty acids, sterols, glycerides, and phospholipids. Lipids serve as essential building blocks of biological membranes and the main form of energy storage as triglycerides.85,86 Furthermore, evidence shows that lipids such as steroid hormones are also closely involved in the cell signaling.87,88

Numerous studies have been conducted to understand the biological functions and metabolism of lipids. Due to the development of bioorthogonal click reaction, the imaging of the lipid metabolism process in live cells has been achieved recently by modifying lipid molecules with azido moiety both exogenously and endogenously (Fig. 12).

FIG. 12.

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Structures of unnatural lipids used for azide-based lipid labeling in cells.

Sphingosine is a commonly used targeting moiety for Golgi apparatus. By adding an azidoacetyl group to the amine of sphingosine, the Chang group applied the azide-modified Sphingo-Az and BCN-containing probes CO-1, COA-1, and COC-1 for intracellular labeling of Golgi apparatus via SPAAC.49,56 Additionally, the authors have also achieved dual-color labeling of Golgi apparatus and other organelles in live cells.56 Another study of using exogenously azido-modified lipid was performed by Fabrias group.75 The authors developed an azide-labeled N-octanoyl-18-azidodeoxysphinganine (RBM5-177) as a targeting reporter and utilized CO-1 as its fluorescent probe partner to monitor the acid ceramidase activity specifically in intact cells.

Endogenous azide-labeling was first reported by the Baskin group.89 Phosphatidylcholine (PC) can be hydrolyzed by Phospholipase D (PLD) enzymes to generate phosphatidic acid (PA) and then interact with primary alcohols to produce phosphatidyl alcohols through a transphosphatidylation reaction. By treating cells with 3-azido-1-propanol, PC was finally converted to phosphatityl azidoalcohol (PAA) by PLD enzymes. PAA was then fluorescently tagged using the strain-promoted azide-alkyne cycloaddition with CO-1, enabling the visualization of PLD activity.

IV. CONCLUSIONS AND OUTLOOK

Bioorthogonal chemistry has progressed immensely since its introduction in the early 2000s. The development of new reactive groups, particularly the azide-based functional groups, have enabled scientists to conduct controlled chemistry in physiological conditions within living cells.

This review is intended to provide an introductory overview of the development of azide-based bioorthogonal chemistry and the diverse applications of this highly selective and biocompatible chemistry for labeling and imaging varied biomolecules, such as tagged proteins, glycoconjugates, nucleic acids and lipids, in mammalian cells using corresponding bioorthogonal fluorescent probes. It should be noted the there are other excellent examples on this wide topic and omissions of such in this review are unintentional.

Although significant achievements have been accomplished, there are still challenges to overcome in this field, such as the need for faster reaction rate and higher selectivity, more efficient metabolic incorporation of tagged biomolecule building blocks, and smarter fluorescent probes to allow clear imaging with higher resolution in intracellular compartment under native conditions. Nevertheless, we believe that the increasing developments and applications of azide-based bioorthogonal chemistry will add versatility of this tool to further assist in answering complex biological and even medical problems.

AUTHORS' CONTRIBUTIONS

All authors contributed equally to this manuscript. All authors reviewed the final version of this manuscript.

ACKNOWLEDGMENTS

This work was supported by IBS-R007-A1. S.H.A. thanks Dr. Maulana Bachtiar for his assistance in proofreading the manuscript.

The authors declare no conflicts of interest.

This article does not contain any studies with human participants or animals performed by any of the authors.

Contributor Information

Samira Husen Alamudi, Email: mailto:samirahusenalamudi@gmail.com.

Young-Tae Chang, Email: mailto:ytchang@postech.ac.kr.

DATA AVAILABILITY

Data sharing is not applicable to this article as no new data were created or analyzed in this article.

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