Caged cyclopropenes for controlling bioorthogonal reactivity

Abstract

Bioorthogonal ligations have been designed and optimized to provide new experimental avenues for understanding biological systems. Generally, these optimizations have focused on improving reaction rates and orthogonality to both biology and other members of the bioorthogonal reaction repertoire. Less well explored are reactions that permit control of bioorthogonal reactivity in space and time. Here we describe a strategy that enables modular control of the cyclopropene-tetrazine ligation. We developed 3-N-substituted spirocyclopropenes that are designed to be unreactive towards 1,2,4,5-tetrazines when bulky N-protecting groups sterically prohibit the tetrazine's approach, and reactive once the groups are removed. We describe the synthesis of 3-N spirocyclopropenes with an appended electron withdrawing group to promote stability. Modification of the cyclopropene 3-N with a bulky, light-cleavable caging group was effective at stifling its reaction with tetrazine, and the caged cyclopropene was resistant to reaction with biological nucleophiles. As expected, upon removal of the light-labile group, the 3-N cyclopropene reacted with tetrazine to form the expected ligation product both in solution and on a tetrazine-modified protein. This reactivity caging strategy leverages the popular carbamate protecting group linkage, enabling the use of diverse caging groups to tailor the reaction's activation modality for specific applications.

Caged Cyclopropenes with Improved Tetrazine Ligation Kinetics

“…Having the C3-nitrogen atom in the spirocyclic cyclopropene system is key to the activation strategy as it serves as an anchor for linking photo- or enzyme-labile cages, thereby hindering IEEDA tetrazine–cyclopropene ligation . However, such C3-nitrogen-containing cyclopropenes are rare and difficult to synthesize and generally also require installation of an inductively electron-withdrawing group at the C3 position.…”

“…For example, the nonactivatable spirocyclic cyclopropene scaffold in which a cyclopropene and a cyclobutane are fused together at the cyclopropene C3 is known, but our attempts to install a nitrogen atom at C3 resulted in ring-opening isomerization of cyclopropenes to form alkynes or allenes . Conversely, installing the C3-difluoro inhibits such ring-opening isomerization . These first-generation caged cyclopropenes have excellent modularity of activation but exhibit relatively slow ligation kinetics with s -tetrazines ( k 2 = 0.0004 M –1 s –1 ) in a buffered solution at neutral pH.…”

Lipidated cyclopropenes via a stable 3-N spirocyclopropene scaffold

“…2A). 26 These caged cyclopropenes were inspired by the >7500-fold difference in reaction rate between C3 mono-substituted and C3 di-substituted cyclopropenes. 25,27 Essentially, a carbamate-caged 3-N spirocyclopropene mimics the unreactive C3 di-substituted cyclopropene, whereas the uncaged, 3- N spirocyclopropene mimics the more reactive C3 di-substituted cyclopropene, with the addition of an electron withdrawing group at C3 critical to prevent the ring opening isomerization to an allene or alkyne.…”

“…The one exception is our recently reported orthoformate strategy that installs an ester in one step from the corresponding cyclopropene. 26 Here, we explored a variety of electrophiles in an effort to expand the scope for installation of carboxylic acid functionality on 15 (Scheme S3 in the ESI). For example, we attempted to install an alcohol using oxirane or paraformaldehyde after treatment of the cyclopropene with n -BuLi, but this produced only unreacted starting material.…”

“…Finally, the lipidated-cyclopropene was Boc-deprotected to obtain free amine C1-ceramidated 3-N spirocyclopropene 18 in 22% yield. Importantly, the low yields of 18 are due to difficulties inherent to the purification of this charged lipid, not decomposition, because similar cyclopropene scaffolds, including the Boc- and Nvoc-protected variants of 18 , have survived acidic HPLC and deprotection conditions unscathed 26 . Separately, the Nvoc photocaged version was prepared directly without purifying the Boc-deprotected intermediate 18 to obtain 19 in 89% yield over two steps.…”