(Figure 1).
The term bioorthogonal chemistry refers to any chemical reaction that can occur in the presence of other rich chemical functionalities found in biological systems without interacting or interfering with native biochemical processes. The bioorthogonally – activated components react specifically and spontaneously only with each other forming the desired conjugate. This bioorthogonal ligation strategy is outlined in(Figure 1)
In order for chemically reactive functional groups to be suitable for bioconjugation, three basic features are of high importance: reactivity, chemoselectivity, and biocompatibility. The first of these – reactivity, is clearly a prerequisite for applications performed under highly dilute conditions, for example, protein – protein conjugations. Protein – protein conjugations are often constrained to low protein concentrations (e.g. <1 mg/mL) due to limited availability of proteins and/or associated cost factors.
Reactivity can be defined by the second order rate constant for the bioorthogonal reactant pairs. The higher the 2nd order rate constant for product formation, the more efficient the conjugation at low reactant concentrations within reasonable time scales, at near neutral pH, and without having to use a large excess of either biomolecule. The relationship between 2nd order rate constants (M–1s–1) for bioorthogonal reactants at 10 μM and the percent conjugate yield over time is illustrated in (Figure 2)
(Figure 2). Simulation of 2nd order reactions at 10 μM reactants
Another important bioconjugation feature for reactants is that they be chemoselective. Namely, selective reactivity with each other while in the presence of a rich milieu of other biological functional groups (e.g. amino (–NH2), carboxyl (–COOH), and/or thiol (–SH)). This important feature is often overlooked, however, lack of selectivity is the driving force behind self–conjugation (homodimer formation) via unwanted and non–selective intramolecular interactions. Lastly, it is important that chemically reactive functional groups be biocompatible.
It is crucial for ligation reactions to proceed under mild aqueous buffer condition compatible with biological molecules, without the need for catalytic reagents (e.g. Cu(I)) that are highly toxic to biological systems. Bioorthogonal chemical functionalities must remain highly reactive but stable during long – term aqueous storage, while remaining inert to high concentrations of other biological functional groups.
Unfortunately, only a few chemical transformations meet these strictly defined conditions: reactivity, selectivity, and biocompatibility. Consequently, only a small subcategory of chemical transformations are suitable for bioorthogonal ligation of highly functionalized biomolecules (e.g. proteins, nucleic acids, and sugars) in aqueous, pH neutral environments at ambient temperatures while still preserving biological function.
The choice for a particular ligation strategy suitable for a specific application is based on different factors. The use of copper–catalyzed azide–alkyne cycloaddition reaction (CuAAC) is limited to applications where the toxicity of copper is not important (e.g. peptide or oligo modification, or cell lysate labeling). In this respect, strain–promoted alkyne–azide cycloaddition reaction (SPAAC) is better suited for bioconjugation since no catalyst or additional reagents are required. However, reaction rates of SPAAC reaction are inherently low (e.g. 0.1 to 0.9 M–1s–1), limiting their use to relatively high concentration applications (e.g. protein–small molecule conjugations, cell labeling).
For applications at low biomolecule concentrations (e.g. < 5 μM), the inverse–demand Diels Alder ligation pair trans–cyclooctene–tetrazine (TCO–Tz) are the pair of choice. The chemoselective TCO–Tz ligation pairs possess ultrafast kinetics (> 800 M–1s–1) unparalleled by any other bioorthogonal ligation pair. The combination of ultrafast kinetics, selectivity, and long–term aqueous stability make TCO–Tz the ideal pair for low concentration applications such as protein–protein conjugations.
(Figure 2). Simulation of 2nd order reactions at 10 μM reactants
Example Application
Five Goat IgG samples (100 μL at 5 mg/mL, 4 mg/mL, 3 mg/mL, 2 mg/mL and 1 mg/mL) were labeled in BupH buffer (pH 7.5) using a 20–fold molar excess of Tetrazine–PEG5–NHS ester. Similarly, 0.1 mL HRP (500 μg) at 5.0 mg/mL in BupH buffer (pH 7.5) was labeled using a 20–fold molar excess TCO-PEG4-NHS ester for 60 min. After removal of excess reagents and determining each protein concentrations 3-fold excess of HRP–TCO was added to IgG–Tetrazine at 5 mg/mL, 4 mg/mL, 3 mg/mL, 2 mg/mL and 1 mg/mL. After 60 minutes, an aliquot (1 μL) from each conjugation reaction was analyzed by SDS–PAGE.
Bioorthogonal Ligation Reactions
Cu – Catalyzed Click Chemistry (CuAAC)
• Reagents are easily accessible
• Wide variety of alkynes and azides are commercially available
• Fast reaction kinetics
• Alkyne – and azide – modified biopolymers are stable in aqueous media for months
• Toxic copper catalyst damages biological systems and affects the function of some proteins
• Reaction requires accessory reagents (e.g. chelating ligands and reducing agents)
Cu – Free Click Chemistry (SPAAC)
• Biocompatible reaction occurs under mild aqueous buffer conditions
• No catalyst or accessory reagents required
• Bioorthogonal pair (DBCO/Azide) exhibit long – term aqueous stability
• Wide variety of azides and cyclooctynes (e.g. DBCO) are commercially available
• Kinetics are fast enough for relatively high concentration applications (e.g., protein – small molecule conjugation, protein – peptide conjugation)
Inverse – Demand Diels Alder Reaction
• Extremely fast kinetics (> 800 M – 1s – 1) permits efficient protein – protein conjugation at < 1 mg/mL in 60 minutes or less
• Biocompatible reaction occurs under mild aqueous buffer conditions at ambient temperature
• No toxic catalyst or accessory reagents required
• Tetrazine – and TCO – modified biopolymers remain reactive in aqueous storage (weeks at 4°C)
• Tetrazines and TCOs are not readily accessible but commercially available.
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