Cyclopentadiene as a Multifunctional Reagent for Normal- and Inverse-Electron Demand Diels–Alder Bioconjugation

Abstract

Optimizing the Diels–Alder (DA) reaction for aqueous coupling has resulted in practical methods to link molecules such as drugs and diagnostic agents to proteins. Both normal electron demand (NED) and inverse electron demand (IED) DA coupling schemes have been employed, but neither mechanism entails a common multipurpose reactive group. This report focuses on expanding the bioconjugation toolbox for cyclopentadiene through the identification of reactive groups that couple through NED or IED mechanisms in aqueous solution. Dienophiles and tetrazine derivatives were screened for reactivity and selectivity toward antibodies bearing cyclopentadiene amino acids to yield bioconjugates. Twelve NED dienophiles and four tetrazine-based IED substrates were identified as capable of practical biocoupling. Furthermore, tetrazine ligation to cyclopentadiene occurred at a rate of 3.3 ± 0.5 M–1 s–1 and was capable of bioorthogonal transformations, as evidenced by the selective protein labeling in serum. Finally, an antibody–drug conjugate (ADC)-bearing monomethyl auristatin E was prepared via tetrazine conjugation to cyclopentadiene. The resulting ADC was stable and demonstrated potent activity in vitro. These findings expand the utility of cyclopentadiene as a tool to couple entities to proteins via dual DA addition mechanisms.

Design, Synthesis, and Application of a Water‐soluble Photocage for Aqueous Cyclopentadiene‐based Diels‐Alder Photoclick Chemistry in Hydrogels

“…The great utility of our photocaged cyclopenta-diene as a bioorthogonal ligation partner can be realized, however, using alternative patterning agents. Notably, the bioorthogonal IEDDA reaction between cyclopentadiene and tetrazine derivatives for bioconjugation has recently been reported [58] and highlights the potential of this diene for bioorthogonal photopatterning applications. Importantly, unlike thiol-ene chemistry which is traditionally used for preparing patterned hydrogels, [6] our DA patterning platform circumvents the use of a radical propagating click chemistry providing several advantages: 1) access to patterning photosensitive species (cyanine dyes) through a two-step patterning process 2) orthogonality with photoinduced and free radical polymerization conditions, and 3) future access to radical sensitive hydrogel scaffolds [21] and biomolecules.…”

Evolution of Pyrrolysyl-tRNA Synthetase: From Methanogenesis to Genetic Code Expansion

“…232,233 Here, GCE appears to be a straightforward solution to address both the heterogeneous product-formation and off-target toxicity issues, offering the potential to design ADCs with new modalities (improved properties). GCE has indeed emerged as a technology for creating ADCs with enhanced characteristics, introducing novel possibilities with examples such as ncAAs 12, 234,235 338, 236,237 337, 238 70, 239 13, 63, 65, and 68 (all 240 ). The concept of using an antibody or nonantibody binding scaffold to direct a payload to a specific target can be mixed and matched.…”

“…In contrast, one of the most common drawbacks unrelated to the antibody size is associated with current conjugation chemistry, which can lead to the premature release of the toxic payload, causing off-target toxicity and decreasing the therapeutic window. , Here, GCE appears to be a straightforward solution to address both the heterogeneous product-formation and off-target toxicity issues, offering the potential to design ADCs with new modalities (improved properties). GCE has indeed emerged as a technology for creating ADCs with enhanced characteristics, introducing novel possibilities with examples such as ncAAs 12 , , 338 , , 337 , 70 , 13 , 63 , 65 , and 68 (all).…”

Diels–Alder Photoclick Patterning of Extracellular Matrix for Spatially Controlled Cell Behaviors

“…[ 20 ] The photo‐uncaging of Cp in this reaction proceeds without the production of free radicals, and Cp has shown promise as a bio‐orthogonal click handle. [ 21 ] Additionally, the use of maleimide as the dienophile partner enables our strategy to interface with a number of commercially available maleimide‐bearing labeling agents and biologically relevant peptides.…”