This work wouldn't be possible without past and present funding and support from @scilifelab.se, @uu.se , @vetenskapsradet.bsky.social , NARSAD, Swedish Brain Foundation.

13/🧵

What if we swap pyrrolidine for a triazole?

Boons et al. showed it makes a UV-excited, blue fluorophore ✨ But we find the photocage is essential: rmv it generates radicals💥

This makes pyrrolidine fusions the first functional heterocyclic-fused photoclick moiety on dimethoxy-DBCO scaffolds

12/🧵

The NMR spectra match the 3 peaks expected for the cis isomer, not trans - confirming that our DBCO adopts the crucial tub-like geometry needed for tetrazine reactivity. 🎯

11/🧵

But how do we know we got the tub-like cis structure DFT predicts? Could it be the trans isomer? 🤔🧪

It doesn’t crystallize easily, so with help from @screspi.bsky.social we used NMR ⚛️ + DFT-predicted 🤖 shifts to confirm ✅

10/🧵

Hold your horses 🐎🐴 couldnt this be explained by solvent effects, the nitrogen exhibiting a protonated state?

To rule this out we performed the reaction in an aprotic solvent (DMSO) supplemented with a non-nucleophilic base (DIPEA) and still observed the same behavior.

9/🧵

Unexpectedly, the Pyrrolidine-fusion retains azide reactivity (SPAAC) but doesn't react with tetrazines (iEDDAC). Contradicting DFT predictions.

8/🧵

Alfred came up with the following synthetic route using a small trick from 1983 paper to generate it:
pubs.rsc.org/en/content/a...

7/🧵

Density Functional Theory (DFT) predicts that fusing 3-, 4-, or 5-membered rings onto the DBCO scaffold could make it reactive toward tetrazines.

Among these, 5-membered heterocycle (Pyrrolidine) is ideal - it not only tunes reactivity but also nitrogen handle for bioconjugation.

6/🧵

Why does this matter? 🤔

DBCOs usually react only with azides, but fusing cycloalkanes can change their geometry, letting tetrazines approach “face-on” ( @klanglab.bsky.social , @dr-dennis.bsky.social ).
rb.gy/8wkpul
This could enable nowash/fluorogenic apps for multiple barcode rounds!

5/🧵

The graphical abstract

Attaching pyrrolidine to a photo-cyclooctyne makes it react only with azides, giving a selective light-activated click. Fusing a triazole creates a fluorophore, but breaking the photocage destroys it ⚡radicals, bright, but no further clicks.💡

4/🧵

Alfred’s work focuses on designing photolabile cyclooctynes - molecules that can be “switched on” with light to react selectively in click chemistry.

The twist? He fused different heterocycles onto the cyclooctyne core to see how it changes their properties. 🔬✨

3/🧵

First, a bit about the cover art 🎨🖼️:

We attached the photoclick moiety to proteins in RPE1 cells 🧫 and used a DMD projector from @EKBTechnologies on a @healthcare.nikon.com Ti2🔬 to selectively irradiate regions.

Activated areas are labeled w fluorophores✨ -we even printed the @rsc.org logo!

2/🧵