Conventional analytical techniques, including chromatographic and spectroscopic methods, have delivered important insights into acrolein chemistry, but they come with practical burdens. Many require harsh reaction conditions and labor-intensive sample preparation, and they often rely on indirect detection of acrolein-derived protein adducts or acrolein-amine conjugates. In complex biological matrices, these approaches suffer from limited chemical selectivity and reduced sensitivity, making convenient and accurate quantification difficult in both research and clinical settings. Fluorescence-based methods have therefore emerged as attractive alternatives, offering high sensitivity, operational simplicity and the possibility of direct readout.

Existing fluorescent probes, however, still leave room for improvement. Naphthalimide-based probes optimized for well-defined matrices such as food extracts show strong fluorescence and good biocompatibility but depend strongly on pH, solvent composition and incubation time, which restricts their quantitative reliability in complex samples like tobacco smoke. Nanomaterial-based platforms that combine sensing with acrolein scavenging are useful for threshold indication and removal assessment but are poorly suited to precise, reproducible quantification. Activity-based probes that collectively target lipid-derived electrophiles, including acrolein and 4-hydroxynonenal, can map the overall electrophilic environment but cannot selectively measure acrolein alone. These gaps motivated a Japanese research team to refine a distinctly chemical solution.

Writing in the open-access journal Results in Chemistry, Masayuki Takamatsu, Hiroya Tsuchida, Hinako Nabetani, Kazuya Kabayama, Ambara R. Pradipta, Katsunori Tanaka, Koichi Fukase and Atsushi Shimoyama report a second generation of pyrene-based fluorescent probes with enhanced chemoselectivity for acrolein. The work builds on a striking piece of chemistry the group had uncovered earlier: acrolein reacts with biologically relevant amines to form characteristic eight-membered heterocycles through a formal imino [4+4] cycloaddition. That unusual dimerization is the entire engine of the new sensing strategy.

The first-generation probe, Probe A, featured an ethanolamine unit as the reactive handle attached to a pyrene fluorophore. When acrolein triggered dimerization of Probe A, two pyrene units were brought into close proximity, and the monomer emission near 375 nanometers gave way to a red-shifted band near 475 nanometers, the signature of pyrene excimer emission. Pyrene is uniquely suited to this role because two pyrene units held close together in an appropriate excited-state geometry produce exactly this red-shifted excimer glow. Rather than acting as a passive label, pyrene functions as a mechanism-linked reporter: the fluorescence change is tied directly to covalent dimer formation, not to reversible binding of the analyte.

To improve on Probe A, the team designed four new analogues, Probes B through E, by systematically modifying the 1,2-amino alcohol motif that carries the reactive amine. Probe B repositioned the primary amine from a terminal carbon to an internal carbon of the amino alcohol, introducing steric bulk around the reactive site. Probe C replaced the hydroxy group with an additional amino group, creating a 1,2-diamine scaffold. Probes D and E were serine-derived 1,2-amino alcohol analogues chosen for synthetic accessibility and to test how linker length between the amino alcohol and the pyrene unit influences excimer formation. All four probes were synthesized from readily available protected amino acid derivatives using coupling agents such as DMT-MM and standard protection-deprotection chemistry.

Time-course experiments revealed clear structure-activity relationships. When each probe was reacted with one equivalent of acrolein in chloroform and the excimer emission at 475 nanometers monitored over 48 hours, all three of Probe A, Probe B and Probe C plateaued after roughly 40 hours, and mass spectrometry confirmed dimer formation in each case. But the trajectories differed. Probe B, with its internal amine, responded more slowly at first, likely because forming the conjugated imine intermediate with acrolein is less favorable in its altered amine environment. Probe C reached only about 60 percent of the plateau intensity of the other two, apparently because the eight-membered aminal dimers it forms are less stable and partially decompose over time.

The chemoselectivity comparison proved decisive. The team challenged each probe with a panel of structurally related alpha,beta-unsaturated aldehydes: methacrolein, crotonaldehyde and sorbaldehyde, which introduce alpha,beta-substitution and extended conjugation respectively. After 24 hours, the relative preference for acrolein followed the order Probe B greater than Probe D approximately equal to Probe E, far exceeding Probe A. The first-generation probe showed only moderate selectivity because its terminal primary amine readily forms conjugated imines with bulkier unsaturated aldehydes as well, and those dimers also emit excimer fluorescence, masking the acrolein signal. The more sterically hindered amine environments of the second-generation amino alcohol probes discriminated against these larger aldehydes, exactly as the design concept predicted.

Acetic acid emerged as a surprising and powerful additive. When the probes were treated with acrolein in the presence of 23 millimolar acetic acid, the emission responses of the serine-derived Probes D and E increased markedly, while those of Probes A and B decreased. The authors propose that acetic acid facilitates activation of the aldehyde or of imine and iminium-type intermediates, promoting dimer formation, while protonation of the more basic amines in Probes A and B suppresses their nucleophilicity. Under the optimized chloroform-acetic acid conditions, Probe D and Probe E both reached a plateau within 24 hours, faster than the first-generation system, and Probe D showed the strongest overall response.

Probe D was accordingly selected as the lead candidate for quantitative evaluation. With the probe fixed at 1.0 millimolar and acrolein varied from 0.033 to 1.1 millimolar, the excimer intensity at 475 nanometers showed an excellent linear relationship after 24 hours, with a coefficient of determination of 0.9985. The authors are careful to note that a formal limit of detection was not calculated, so the lowest concentration examined, 33 micromolar, should not be read as the detection limit; previously reported tethering and ratiometric methods have achieved submicromolar limits, though direct comparison is complicated by very different conditions and media. The contribution here is not record sensitivity but chemoselectivity, synthetic practicality and compatibility with an extraction-based workflow.

That workflow was put to the test with biological samples. The researchers lysed RAW264.7 macrophage-like cells with Triton X-100 and sonication, extracted acrolein into chloroform, and reacted the extract with Probe D under the optimized conditions. When identical amounts of acrolein were extracted from buffer alone or from buffer mixed with cell lysate, comparable excimer intensities resulted, showing that major co-extracted components did not suppress the reaction or the fluorescence readout. The team then treated cells with hydrogen peroxide at 0, 62 and 125 millimolar for three hours to induce oxidative stress, extracted the lysates, and observed a concentration-dependent increase in the red-shifted fluorescence response, consistent with more extractable acrolein-derived reaction products.

The authors are appropriately measured about what this means. The extraction-based experiment does not constitute direct intracellular imaging or real-time cellular sensing, and it does not establish acrolein as the exclusive contributor among all extractable electrophilic components. Still, the demonstration that a simple covalent dimerization reaction, read out through pyrene excimer emission, can survive the jump from pure chloroform solutions to cell-lysate extracts is a meaningful step toward practical acrolein analysis. Further optimization aimed at stabilizing the dimeric products and improving fluorescence efficiency may push sensitivity further. For now, the study shows that rearranging a single amino group within a 1,2-amino alcohol motif, and letting a touch of acetic acid assist the chemistry, can turn an unusual [4+4] cycloaddition into a sharper, more selective fluorescent eye on one of biology’s most damaging aldehydes.

Subject of Research: Development of second-generation pyrene excimer fluorescent probes for chemoselective detection of acrolein via imino [4+4] cycloaddition dimerization

Article Title: An improved pyrene excimer fluorescent probe with enhanced chemoselectivity for acrolein detection

Article References: Takamatsu, M., Tsuchida, H., Nabetani, H., Kabayama, K., Pradipta, A. R., Tanaka, K., Fukase, K., & Shimoyama, A. (2026). An improved pyrene excimer fluorescent probe with enhanced chemoselectivity for acrolein detection. Results in Chemistry, 31, Article 103962. https://doi.org/10.1016/j.rechem.2026.103962

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.103962

Keywords: acrolein, pyrene excimer, fluorescent probe, chemoselectivity, imino [4+4] cycloaddition, 1,2-amino alcohol, serine-derived probe, oxidative stress, lipid peroxidation, cell lysate extraction, fluorescence spectroscopy, Results in Chemistry

A new study published in the open-access journal Results in Chemistry by Menaka Bhuyan and Jubaraj B. Baruah of the Indian Institute of Technology addresses this challenge with an unusual design philosophy. Rather than building a probe that reacts chemically with aldehydes, a strategy that consumes both the analyte and the sensing site, the team engineered two fluorescent molecules that recognize salicylaldehyde purely through non-covalent binding. The probes, named anambenz and animbenz, both carry an anthracene fluorophore, one of the most reliable light-emitting units in fluorescence chemistry, connected through a phenylene diamine-derived linker to an electron-withdrawing benzoyl group bearing a hydrogen-bond acceptor. A planar aryl unit with a hydrogen-bond donor, either an amino or an imino group, sits between the two ends, creating a molecule with three distinct faces above which an aromatic analyte can stack.

The synthetic route to the two probes is elegantly economical. Both compounds arise from the condensation of 3,4-diaminobenzophenone with 9-anthracenecarboxaldehyde, but divergent reaction conditions steer the chemistry toward different products. Refluxing in methanol for 24 hours delivers anambenz in an 80 percent yield, retaining a free amino group on the central ring. Heating the same starting materials in dimethylformamide with acetic acid for two days at 120 degrees Celsius instead promotes cyclization, furnishing the imino-containing animbenz in 60 percent yield. The structures were confirmed by proton and carbon-13 nuclear magnetic resonance spectroscopy, infrared spectroscopy, and high-resolution mass spectrometry, and the crystal structure of anambenz, solved by single-crystal X-ray diffraction, revealed a U-shaped geometry in which the benzoyl carbonyl projects away from the anthracene ring, with the crystal packing governed by nitrogen-hydrogen to nitrogen hydrogen bonds and weak carbon-hydrogen to pi contacts.

In dimethylformamide solution, anambenz absorbs strongly at 345 nanometers and emits a broad, featureless band at 475 nanometers when excited at that wavelength, while animbenz absorbs at the same wavelength but emits at 425 nanometers with the characteristic vibrational structure of anthracene derivatives. Both probes display solvatochromic behavior, with their absorption and emission shifting across solvents, and both suffer fluorescence quenching in protic media, with water quenching more strongly than ethanol, which in turn quenches more than methanol. Crucially, the emission of each probe in dimethylformamide remained stable for over an hour, establishing a reliable baseline for analytical work.

The decisive experiment came when the researchers exposed each probe to a panel of thirteen aromatic aldehydes, including 4-hydroxybenzaldehyde, 2,4,6-trihydroxybenzaldehyde, 2-carboxybenzaldehyde, 2-hydroxynaphthaldehyde, 2,3-dihydroxybenzaldehyde, nitro-substituted benzaldehydes, 4-bromobenzaldehyde, 4-methoxybenzaldehyde, and 4-(diethylamino)salicylaldehyde. Against this crowded field, salicylaldehyde stood alone: it completely quenched the emission of both probes, while the other aldehydes produced either partial, substrate-specific changes or no effect at all. Some near-misses were instructive. 2,3-Dihydroxybenzaldehyde actually enhanced the fluorescence of anambenz and shifted its emission peak to 480 nanometers, and 4-(diethylamino)salicylaldehyde caused partial quenching with a shift to 507 nanometers, but only salicylaldehyde silenced the anthracene emission entirely.

Quantitative titrations revealed Stern-Volmer plots that were linear, pointing to dynamic quenching arising from collisions between excited probe molecules and the analyte rather than the formation of stable ground-state complexes. Time-correlated single-photon counting added a deeper layer of insight. Anambenz alone decays through a tri-exponential profile dominated by a 4.93-nanosecond pathway accounting for 85.54 percent of the emission. Upon adding salicylaldehyde, that dominant fast pathway collapses, and the profile reorganizes so that nearly half the emission follows an ultrafast 0.02-nanosecond route while a new 7.74-nanosecond component emerges, evidence that the interaction redirects the excited-state dynamics. Animbenz, by contrast, shows a single 1.67-nanosecond lifetime that barely changes to 1.68 nanoseconds in the presence of the aldehyde, a signature of purely dynamic quenching that helps explain why its detection limit, 4.8 times ten to the minus two molar, is far weaker than the 181 micromolar limit achieved by anambenz.

Proton NMR titrations confirmed that the ground-state interaction between anambenz and salicylaldehyde is genuinely feeble: the phenolic hydroxyl of the aldehyde shifted by a mere 0.009 parts per million, the imine proton of the probe moved by the same tiny amount, and the amino protons were untouched. The selectivity therefore cannot rest on strong binding. To understand what does control it, the team turned to molecular modeling. Density functional theory calculations at the B3LYP level with a 6-311+G(d,p) basis set, followed by noncovalent interaction region analysis using the Multiwfn program, showed that salicylaldehyde positions itself above the anthracene ring of both probes in an orientation that enables an OH-pi interaction between its hydroxyl group and the pi cloud of the fluorophore. The structurally similar 4-hydroxybenzaldehyde, lacking the ortho hydroxyl geometry, ends up either perpendicular to the anthracene unit or displaced to a remote end of the probe, unable to make the crucial contact.

Time-dependent DFT calculations, performed with the dielectric constant of dimethylformamide to mimic the experimental solution, reinforced the picture. The calculated absorption maxima of the salicylaldehyde assemblies carried lower oscillator strengths than those of the 4-hydroxybenzaldehyde assemblies, 0.22 versus 0.24 for animbenz and 0.41 versus 0.43 for anambenz, meaning a reduced probability of the radiative transition and hence stronger quenching. Frontier orbital analysis showed why the geometry matters so much: in the salicylaldehyde assemblies, both the highest occupied and lowest unoccupied molecular orbitals localize on the anthracene portion, whereas the 4-hydroxybenzaldehyde assemblies distribute the orbitals differently across the imine and carbonyl regions. The precise way an analyte hovers over the probe, in other words, rewrites the electronic structure of the whole assembly and with it the fluorescence outcome.

How does this approach compare with existing salicylaldehyde sensors? The literature includes metal-organic frameworks based on europium and zinc, a terbium coordination polymer, a europium zirconium oxo-cluster, and organic turn-on probes, with reported detection limits spanning from 0.259 micromolar for a zinc benzimidazolate framework up to 58.2 micromolar and beyond. Several of these systems are more sensitive than the new probes, and a terbium polymer even allows naked-eye detection, but that color change is not specific, since benzaldehyde triggers it too, and amine-based turn-on probes suffer interference from other aldehydes. The distinctive achievement of anambenz and animbenz is selectivity through molecular recognition: only salicylaldehyde, of thirteen aldehydes tested, quenches the emission completely, and competitive experiments showed that the quenched state cannot be reversed by adding 4-hydroxybenzaldehyde, while salicylaldehyde retains its quenching power even in the presence of that competitor. The probes also functioned in solutions containing milk, hinting at practical utility in food and product screening.

The authors are candid about the limits. A detection limit of 181 micromolar falls well short of the concentrations implied by the predicted no-effect threshold, so these probes are not yet suited to environmental screening at trace levels. Where they could shine instead is industrial spill alerting and process control, situations in which salicylaldehyde is present at high concentrations and in which distinguishing it from structurally related aldehydes matters more than ultimate sensitivity. More broadly, the study demonstrates a design principle with wide reach: by arranging a fluorophore, a hydrogen-bond donor, and a hydrogen-bond acceptor on a single scaffold, and by letting subtle differences in pi-stacking and OH-pi geometry dictate the excited-state fate, chemists can build sensors that discriminate between molecules as similar as two hydroxybenzaldehydes without ever touching them chemically. As the authors conclude, probes that modulate emission through molecular recognition offer a large and largely unexplored space for developing high-efficiency, non-invasive detection of reactive compounds in the world around us.

Subject of Research: Selective fluorescence detection of salicylaldehyde using anthracene-based molecular recognition probes

Article Title: Efficient and selective detection of salicylaldehyde

Article References: Bhuyan, M., & Baruah, J. B. (2026). Efficient and selective detection of salicylaldehyde. Results in Chemistry, 31, Article 103952. https://doi.org/10.1016/j.rechem.2026.103952

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.103952

Keywords: salicylaldehyde, fluorescent probe, anthracene, fluorescence quenching, molecular recognition, OH-pi interaction, DFT calculations, Stern-Volmer, detection limit, aromatic aldehydes, Results in Chemistry, sensor chemistry

The research team, led by Setiasih Setiasih and colleagues at Indonesia’s National Research and Innovation Agency (BRIN) and partner institutions, published their findings in Results in Chemistry. Their strategy centered on a single deceptively simple question: what exactly is inside rose myrtle leaves, and how does the answer change depending on how you extract it? To find out, they harvested leaves from an experimental garden in Riau Province, dried them at 40 degrees Celsius for three days, and ground them into a flour. That flour was then subjected to maceration with three solvents spanning the polarity spectrum: 96 percent ethanol as a polar solvent, ethyl acetate as a semipolar solvent, and n-hexane as a nonpolar solvent. Each extract was subsequently analyzed by liquid chromatography coupled to high-resolution mass spectrometry, or LC-HRMS, at BRIN’s Advanced Characterization Laboratories.

The choice of solvent turned out to matter enormously. LC-HRMS profiling tentatively identified 191 distinct compounds in the ethanol extract, 226 in the ethyl acetate extract, and 206 in the n-hexane extract. Ethanol preferentially pulled out polar molecules, yielding a profile dominated by flavonoid glycosides, phenolic acids and related derivatives, including myricitrin, myricetin, quinic acid derivatives and multiple dihydroxybenzoic acids. These hydroxyl-rich compounds dissolve readily in polar solvents through hydrogen bonding and are classic free-radical scavengers, which helps explain the high antioxidant activity long associated with the plant. The ethyl acetate extract displayed the greatest metabolite diversity of all, dominated by pentacyclic triterpenoids such as ursolic acid, asiatic acid, oleanolic acid, arjungenin and glycyrrhetinic acid derivatives, alongside phenolics including ellagic acid, gallic acid, myricetin and taxifolin. The n-hexane extract, by contrast, was rich in lipophilic sesquiterpenes such as caryophyllene oxide, alpha-farnesene and curcumene, together with assorted triterpenoid derivatives.

The analytical power of the method lies in its ability to go beyond a simple mass readout. Using higher-energy collisional dissociation fragmentation, the researchers could watch molecules break apart in characteristic ways, dramatically raising confidence in their identifications. Myricitrin, for example, produced a precursor ion at mass-to-charge ratio 463.0881 that fragmented to give a product ion at 316.0224, corresponding to the loss of its rhamnose sugar and formation of the myricetin aglycone. Oleanolic acid showed a precursor at 455.3534 with a diagnostic fragment at 411.3472 reflecting loss of carbon dioxide, while caryophyllene oxide in the n-hexane extract yielded sequential ring-cleavage fragments at 161.0962 and 109.1014. The authors are careful to stress that this was qualitative screening rather than quantitative metabolomics, conducted without biological replicates, so peak areas reflect relative signal intensity within a run rather than absolute concentrations. Even so, the picture that emerges is of a plant whose chemistry shifts meaningfully with every solvent a chemist chooses.

With the chemical inventory in hand, the team turned to biological testing. Using the agar disc diffusion method, they challenged four bacterial strains with each extract at concentrations of 25, 50 and 80 percent: Escherichia coli ATCC 25922 and Salmonella typhimurium ATCC 14028 as Gram-negative representatives, and Staphylococcus aureus ATCC 25923 and Listeria monocytogenes ATCC 19115 as Gram-positive representatives. The result was a clean split. Every extract, regardless of solvent, inhibited both Gram-positive species and failed entirely to inhibit either Gram-negative one. Chloramphenicol, the reference antibiotic, produced a 14.82 millimeter zone against S. aureus, larger than the 7.11 to 7.70 millimeter zones produced by the extracts, but the n-hexane extract at 80 percent actually outperformed chloramphenicol against Listeria, generating a 7.17 millimeter zone compared with the antibiotic’s 5.23 millimeters.

The selectivity is rooted in bacterial architecture. Gram-negative bacteria wrap themselves in an outer membrane studded with lipopolysaccharides, an exceptionally effective permeability barrier that excludes hydrophobic molecules and is reinforced by selective porins and efflux pumps that expel antimicrobials before they reach lethal intracellular concentrations. Gram-positive bacteria, lacking that outer membrane, rely on a thick but porous peptidoglycan layer some 20 to 80 nanometers deep, through which small plant metabolites can diffuse relatively freely toward the cytoplasmic membrane. The researchers also observed that solvent type shaped potency: ethanol extracts, rich in phenolics and flavonoids, were most effective against S. aureus, while n-hexane extracts, loaded with membrane-disrupting terpenoids, were the champions against Listeria, a reminder that even within the Gram-positive category, membrane composition and defensive machinery differ enough to matter. The findings align with earlier reports that rose myrtle extracts and the pure compound rhodomyrtone, an antibiotic candidate with activity comparable to vancomycin, are most effective against Gram-positive organisms.

The second half of the study addresses climate. Ruminant livestock account for roughly a quarter of anthropogenic greenhouse gas emissions measured by methane’s contribution, with enteric fermentation in the digestive tract responsible for 80 to 89.5 percent of that methane and manure contributing the rest. Methanogenic archaea in the rumen generate the gas, and feed additives that suppress them are among the most actively pursued mitigation strategies. Because flavonoids, tannins and saponins have documented anti-methanogenic effects, the team tested the ethyl acetate extract, the richest in bioactive triterpenoids and phenolics, in an in vitro rumen fermentation system using Brachiaria ruziziensis grass as the basal feed, at inclusion levels of 1 to 4 percent, with methane measured by gas chromatography over 48 hours.

The outcome was striking in one particular way. Total gas production was unaffected across all treatment levels, meaning fermentation efficiency was preserved, but methane output fell steadily as the extract dose increased. The methane percentage of total gas dropped from 7.72 percent in the untreated control to 3.77 percent at the 4 percent inclusion level, a statistically significant reduction, and methane volume declined as well with a P value of 0.056, right at the threshold of significance. The chemical fingerprint offers a plausible mechanism in several complementary parts. Triterpenoids such as ursolic acid are membrane-active against rumen protozoa, which maintain physical symbioses with methanogenic archaea and supply them with hydrogen; suppressing protozoa often drags methane down with it. Phenolic compounds can selectively inhibit methanogens while redirecting metabolic hydrogen toward propionate synthesis, an energetically favorable route for the animal. Terpenoids such as caryophyllene oxide may interfere with membrane integrity and ion gradients in anaerobic microbes. The authors also cite prior molecular docking work showing that rose myrtle compounds bind strongly to methyl coenzyme M reductase, the enzyme at the heart of methanogenesis, with rhodomyrtial A achieving a predicted binding affinity of minus 10.4 kilocalories per mole.

The researchers are appropriately measured about what these results do and do not prove. The antibacterial assay was a preliminary screen, and they recommend follow-up with minimum inhibitory and bactericidal concentration determinations, plus in vivo testing, to establish therapeutic potential; earlier work suggests possible veterinary applications including mastitis treatment in dairy cows. Similarly, they emphasize that the methane findings provide a chemical rationale rather than a demonstrated field benefit, and that in vivo feeding trials will be required to confirm efficacy and safety. Nevertheless, the study is the first LC-HRMS-based phytochemical characterization of rose myrtle in the context of ruminant nutrition, and it reveals an intertwined network of triterpenoids, phenolics, flavonoids and lipophilic terpenoids whose combined effects may prove more stable than any single isolated compound. In an era when agriculture is under intense pressure to cut emissions without compromising animal productivity, a shrub that grows wild across tropical Asia, fights Gram-positive pathogens in the laboratory, and measurably dampens methane in simulated rumen fluid is exactly the kind of natural lead worth chasing.

Subject of Research: Phytochemical characterization of Rhodomyrtus tomentosa leaves and their antibacterial and methane-mitigating bioactivities

Article Title: LC–HRMS-based phytochemical characterization and biological activities of Rhodomyrtus tomentosa leaves extracts: Antibacterial and methane mitigation potentials

Article References: Setiasih, S., Anggraeny, Y. N., Puspito, S., Widodo, S., Wardi, W., Prihandini, P. W., Antonius, A., Shiddieqy, M. I., Sasongko, W. T., Istiqomah, N., Haryanto, B., Kurniawati, A., Prihartini, I., Wulansari, W. I., & Ernawati, A. (2026). LC–HRMS-based phytochemical characterization and biological activities of Rhodomyrtus tomentosa leaves extracts: Antibacterial and methane mitigation potentials. Results in Chemistry, 30, Article 103812. https://doi.org/10.1016/j.rechem.2026.103812

Image Credits: AI Generated

DOI: 10.1016/j.rechem.2026.103812

Keywords: Rhodomyrtus tomentosa, rose myrtle, LC-HRMS, phytochemical profiling, antibacterial activity, triterpenoids, flavonoids, enteric methane, ruminant nutrition, feed additives, methanogens, Results in Chemistry