Stretching the Bisalkyne Raman Spectral Palette Reveals a New Electrophilic Covalent Motif

Abstract

Small heteroaryl‐diyne (Het‐DY) tags with distinct vibrational frequencies, and physiologically relevant cLog P were designed for multiplexed bioorthogonal Raman imaging. Pd−Cu catalyzed coupling, combined with the use of Lei ligand, was shown to improve overall yields of the desired heterocoupled Het‐DY tags, minimizing the production of homocoupled side‐products. Spectral data were in agreement with the trends predicted by DFT calculations and systematic introduction of electron‐ rich/poor rings stretched the frequency limit of aryl‐capped diynes (2209–2243 cm−1). The improved Log P of these Het‐DY tags was evident from their diffuse distribution in cellular uptake studies and functionalizing tags with organelle markers allowed the acquisition of location‐specific biological images. LC–MS‐ and NMR‐based assays showed that some heteroaryl‐capped internal alkynes are potential nucleophile traps with structure‐dependent reactivity. These biocompatible Het‐DY tags, equipped with covalent reactivity, open up new avenues for Raman bioorthogonal imaging.

Visualization of Modified Bisarylbutadiyne-Tagged Small Molecules in Live-Cell Nuclei by Stimulated Raman Scattering Microscopy

“…This change in the peak height, or Raman activity, is consistent with previous results showing that the addition of nucleophilic moieties to diyne reduces the number of conjugated C�C triple bonds from two to one. 32 In contrast to JQ1-MPDY, JQ1-BADY in cells showed a spectral peak identical to that in DMSO, suggesting that the bioreactivity of BADY was lower than that of MPDY. To verify this, we examined the reactivity of JQ1-BADY with L-cysteine.…”

“…To reduce the deviation in lipophilicity while maintaining high Raman activity, we modified the BADY tag structure by replacing the two phenyl rings flanking the diyne with nitrogen-containing six-membered heteroaromatic rings to reduce the lipophilicity of JQ1-BADY. 32 We chose pyridine and pyridazine for each heteroaromatic ring. Pyridazine benefits from its double hydrogen bonding ability, high dipole moment, and weak basicity.…”

“…47 We hypothesized that nucleophilic moieties are added to the tag. 32 To test this, we investigated the reactivity of JQ1-MPDY with L-cysteine possessing a nucleophilic thiol group. We compared the SRS spectra for JQ1-MPDY mixed with and without L-cysteine in culture medium and DMSO.…”

“…To obtain tags with higher biostability, BADY modifications that account for the electron density of the tag are needed, such as the introduction of a 5membered instead of a 6-membered heteroaromatic ring to compensate for the electron deficiency. 32 Importantly, any modifications should ensure that the Raman activity remains sufficiently high for intracellular observations. In addition, to verify the effectiveness of the improved tag, we compare the efficacies of the tagged drug and the parent drug, which will be our future work.…”

“…In click chemistry, drug derivatives with small clickable moieties are incorporated into cells and conjugated to subsequently added fluorescent dyes via bio-orthogonal reactions to reveal the localization of the drug derivatives to target organelles. − However, it usually involves cell fixation and membrane permeabilization, potentially obscuring the original distribution in living cells. A combination of stimulated Raman scattering (SRS) microscopy and alkyne tags has been used to analyze intracellular drug concentrations. − SRS microscopy offers high detection sensitivity and compatibility with live-cell observations. − Alkyne tags have relatively low molecular weights and bio-orthogonal Raman spectra. − This combination, especially using bisarylbutadiyne (BADY) as an alkyne tag with strong Raman activity, has been used to visualize the intracellular behavior of small molecules. − However, non-negligible deviations in physicochemical properties between BADY-tagged and parent small-molecule drugs were recently reported . These physicochemical properties can affect the subcellular distribution, suggesting that BADY tagging disturbs the original intracellular distribution of parent drugs.…”