Cyclooctynes — Strain-Promoted Click Reagents

Cyclooctynes are strained cyclic alkynes that react with azides via strain-promoted azide-alkyne cycloaddition (SPAAC) without the need for a copper catalyst. The ring strain inherent in the eight-membered ring (~18 kcal/mol) significantly lowers the activation energy barrier for the [3+2] cycloaddition with azides, enabling copper-free click chemistry. This breakthrough, pioneered by Carolyn Bertozzi[4], opened the door to bioorthogonal labeling in living systems where copper toxicity is prohibitive.

Cyclooctyne ring structures including DBCO and BCN enabling strain-promoted click chemistry
Strained cyclooctynes like DBCO and BCN enable copper-free SPAAC reactions
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Chemical Properties

Core structure Cyclooctyne (8-membered ring with internal triple bond) Ring strain ~18 kcal/mol (parent cyclooctyne)[4] Key advantage Eliminates need for copper catalyst — fully biocompatible Reaction product Triazole (regioisomeric mixture, both 1,4- and 1,5-) Selectivity React selectively with azides; stable toward biological thiols, amines, and nucleophiles

SPAAC — Strain-Promoted Azide-Alkyne Cycloaddition

R–N3 (Azide)
+
Cyclooctyne
→
Fused Triazole

No catalyst required, aqueous media, 37 °C or RT, biocompatible

Comparison of Major Cyclooctynes

Variant Full Name Ring Strain Rate k (M−1s−1)[9] Key Feature
OCT Cyclooctyne ~18 kcal/mol ~2.4 × 10−3 Simplest cyclooctyne; baseline reactivity
DIBO Dibenzoazacyclooctyne Higher ~5.8 × 10−2 First biologically applied strained variant (Bertozzi, 2004)
DBCO / DIBAC Dibenzocyclooctyne High ~0.3–0.8 Most commercially popular; excellent availability
BCN Bicyclo[6.1.0]nonyne Moderate ~0.1 Compact, symmetric; suitable for dual SPAAC/IEDDA
BARAC Biarylazacyclooctynone Highest ~1.0 Fastest SPAAC reagent; Bertozzi lab
DIMAC Dimethoxyazacyclooctyne Moderate ~0.03 Improved water solubility; reduced background

Cyclooctyne Variants

Detailed Descriptions

OCT — Cyclooctyne (Parent Compound)

The parent cyclooctyne possesses ~18 kcal/mol of ring strain and reacts with azides at a rate of ~2.4 × 10−3 M−1s−1. While this is sufficient for some labeling applications, the relatively slow kinetics and poor aqueous solubility of OCT itself have driven the development of enhanced cyclooctyne variants.

Advantages Simplest structure; well-understood reactivity Disadvantages Slow kinetics; poor water solubility; limited commercial availability of derivatives

DIBO — Dibenzoazacyclooctyne

DIBO was the first cyclooctyne variant successfully applied to biological labeling (Bertozzi, 2004). Fusion of two aromatic rings onto the cyclooctyne framework increases ring strain and reactivity approximately 25-fold over OCT. DIBO enabled the first live-cell SPAAC labeling of azido-sugars on cell surfaces.

Advantages ~25× faster than OCT; proven in live-cell labeling; historically significant Disadvantages Synthesis is multi-step; limited commercial availability of derivatives; hydrophobic

DBCO / DIBAC — Dibenzocyclooctyne

DBCO (also called DIBAC) is the most commercially popular cyclooctyne. Fusing two benzene rings onto the cyclooctyne framework dramatically increases ring strain and reaction rates (~0.3–0.8 M−1s−1). Its excellent commercial availability in numerous functionalized forms (amine, NHS ester, PEG conjugates, fluorophores) has made it the go-to reagent for SPAAC experiments.

Advantages Excellent commercial availability; fast kinetics; wide range of derivatives; stable in aqueous media Disadvantages Relatively hydrophobic; can exhibit non-specific binding; larger molecular footprint than BCN

BCN — Bicyclo[6.1.0]nonyne

BCN features a fused cyclopropane ring that provides moderate ring strain. Its compact, symmetric structure makes it attractive for applications where minimal steric perturbation is important. BCN can serve as both a SPAAC reagent (with azides) and a dienophile in IEDDA reactions with tetrazines, enabling orthogonal labeling strategies.

Advantages Compact and symmetric; dual reactivity (SPAAC + IEDDA); reduced hydrophobicity; commercially available Disadvantages Slower SPAAC kinetics than DBCO; moderate reactivity

BARAC — Biarylazacyclooctynone

BARAC is the fastest SPAAC reagent developed to date, with a second-order rate constant of ~1.0 M−1s−1. Developed by the Bertozzi group, its enhanced reactivity stems from additional ring strain introduced by the biaryl and lactam moieties. However, its complex synthesis and limited commercial availability have restricted widespread adoption.

Advantages Fastest SPAAC kinetics; excellent for rapid labeling at low concentrations Disadvantages Difficult multi-step synthesis; limited commercial availability; photodegradation concerns

DIMAC — Dimethoxyazacyclooctyne

DIMAC was designed to address the hydrophobicity problem common to most cyclooctynes. The incorporation of methoxy groups improves water solubility and reduces non-specific binding to cellular components, making DIMAC advantageous for applications requiring low background labeling.

Advantages Improved water solubility; reduced non-specific binding; lower background Disadvantages Moderate reactivity (~0.03 M−1s−1); limited commercial availability

Applications

In Vivo Imaging

SPAAC with cyclooctynes enables labeling in live animals without copper toxicity. Azido-sugars are metabolically incorporated into cell-surface glycans and then visualized by SPAAC with cyclooctyne-fluorophore conjugates in zebrafish, mice, and other model organisms.

In vivo

Cell-Surface Labeling

Metabolic oligosaccharide engineering (MOE) followed by SPAAC labeling enables visualization and proteomic identification of specific glycan populations on live cells without fixation or permeabilization.

Cell surface

Antibody-Drug Conjugates

Site-specific introduction of azide handles on antibodies followed by SPAAC conjugation with cyclooctyne-drug payloads produces homogeneous ADCs with defined drug-to-antibody ratios.

ADCs

Dual-Color Labeling

Orthogonal bioorthogonal reactions (e.g., SPAAC + CuAAC, or SPAAC + IEDDA) enable simultaneous labeling of two distinct targets in the same sample using cyclooctynes with different reaction partners.

Multiplexing
Tip — Choosing a Cyclooctyne

DBCO is the most widely used cyclooctyne for SPAAC due to excellent commercial availability and a wide range of functionalized derivatives (NHS esters, amines, maleimides, fluorophores, PEG conjugates). For applications requiring faster kinetics, consider BARAC or DIBO. For reduced hydrophobicity, BCN or DIMAC may be preferable. Always match the cyclooctyne reactivity to your azide concentration — faster reagents are essential when azide density on the target is low.