Bioorthogonal Chemistry

Introduction

Bioorthogonal chemistry is a set of reactions that can take place in biological environments without affecting biomolecules or interfering with biochemical processes. For this purpose, the reaction must meet the following requirements: fast, efficient, and specific.

• pH: The reaction must occur at the temperatures and pH of physiological environments. 

• Efficient: The reaction must provide products selectively and in high yields and must not be affected by water or endogenous nucleophiles, electrophiles, reductants, or oxidants found in complex biological environments. 

• Fast: the reaction must be fast, even at low concentrations, and must form stable reaction products. 

• Specific: The reaction should involve functional groups not naturally present in biological systems


History

Rate constants comparisons


Figure 1. Chemical structures of various cyclooctyne derivatives in order of reactivity toward azides, rate constants (k2, M1 ·s1 ) for each of the derivatives are given in parentheses.

Fig. 2 Second order rate constants of selected tetrazines with TCO in PBS at 37 °C and corresponding stability assessed in PBS at 37 °C for 10 h. NA, not assessed

 

Strengths and weaknesses of bioorthogonal reactions

Table 1. Summary of Strengths and Weaknesses of Bioorthogonal Reactions

 

biorthogonal reactions advantages disadvantages
Staudinger ligation Azides and phosphines are biocompatible, stable amide linkages Slow reactions, phosphines prone to oxidation
CuAAC (azide + alkyne) Fast reactions, k ~10-100(M-1S -1 ) with 20uM Cu(I). Good regioselectivity Despite some ligands such as THPTA to stabilize copper catalysts, copper toxicity remains a concern
SPAAC (azide + DBCO) No use of copper catalysts k~ 1-60 (M-1S -1 ) 1.Reactions slower than CuAAC, bulky cyclooctynes difficult to incorporate into biomolecules
2.Preferred solvent: ethanol (10-40%) or DMSO(up to 60%)/ PBS buffer
3. pH < 5.5 rxn slow down because low stability DBCO?
4. thiol, sodium azide reacts with DBCO
IEDDA (TCO + Tz) Very fast reactions, k ~ 1-106 (M-1S -1 ) TCO has lower stability in aqueous environments

• CuAAC: Copper-Catalyzed Azide–Alkyne Cycloaddition; 

• SPAAC: strain-promoted azide–alkyne cycloaddition; 

• IEDDA: inverse electron demand Diels–Alder reaction

Categories: Protocols