Design and Synthesis of Novel Squaraine-Based Fluorescent Probe for Far-Red Detection of Chymotrypsin Enzyme.

Chymotrypsin, a crucial enzyme in human digestion, catalyzes the breakdown of milk proteins, underscoring its significance in both health diagnostics and dairy quality assurance. Addressing the critical need for rapid, cost-effective detection methods, we introduce a groundbreaking approach utilizing far-red technology and HOMO-Förster resonance energy transfer (FRET). Our novel probe, SQ-122 PC, features a unique molecular design that includes a squaraine dye (SQ), a peptide linker, and SQ moieties synthesized through solid-phase peptide synthesis. Demonstrating a remarkable quenching efficiency of 93.75% in a tailored H2O:DMSO (7:3) solvent system, our probe exhibits absorption and emission properties within the far-red spectrum, with an unprecedented detection limit of 0.130 nM. Importantly, our method offers unparalleled selectivity towards chymotrypsin, ensuring robust and accurate enzyme detection. This pioneering work underscores the immense potential of far-red-based homo-FRET systems in enabling the sensitive and specific detection of chymotrypsin enzyme activity. By bridging the gap between cutting-edge technology and biomedical diagnostics, our findings herald a new era of enzyme sensing, promising transformative advancements in disease diagnosis and dairy quality control.

Photophysical Characterization and Biointeractions of NIR Squaraine Dyes for in Vitro and in Vivo Bioimaging.

The increasing demand for reliable near-infrared (NIR) probes exhibiting enduring fluorescence in living systems and facile compatibility with biomolecules such as peptides, antibodies or proteins is driven by the increasing use of NIR imaging in clinical diagnostics. To address this demand, a series of carboxy-functionalized unsymmetrical squaraine dyes (SQ-27, SQ-212, and SQ-215) along with non-carboxy-functionalized SQ-218 absorbing and emitting in the NIR wavelength range were designed and synthesized followed by photophysical characterization. This study focused on the impact of structural variations in the alkyl chain length, carboxy functionality positioning, and spacer chain length on dye aggregation and interaction with bovine serum albumin (BSA) as a model protein. In phosphate buffer (PB), the absorption intensity of the dyes markedly decreased accompanied by pronounced shoulders indicative of dye aggregation, and complete fluorescence quenching was seen in contrast to organic solvents. However, in the presence of BSA in PB, there was a enhancement in absorption intensity while regaining the fluorescence coupled with a remarkable increase in the intensity with increasing BSA concentrations, signifying the impact of dye-BSA interactions on preventing aggregation. Further analysis of Job's plot unveiled a 2:1 interaction ratio between BSA and all dyes, while the binding studies revealed a robust binding affinity (Ka) in the order of 107/mol. SQ-212 and SQ-215 were further tested for their in vitro and in vivo imaging capabilities. Notably, SQ-212 demonstrated nonpermeability to cells, while SQ-215 exhibited easy penetration and prominent cytoplasmic localization in in vitro studies. Injection of the dyes into laboratory mice showcased their efficacy in visualization, displaying stable and intense fluorescence in tissues without toxicity, organ damage, or behavioral changes. Thus, SQ-212 and SQ-215 are promising candidates for imaging applications, holding potential for noninvasive cellular and diagnostic imaging as well as biomarker detection when coupled with specific vectors in living systems.

Antiplasmodial and Antimalarial Activity of 3,5-Diarylidenetetrahydro-2H-pyran-4(3H)-ones via Inhibition of Plasmodium falciparum Pyridoxal Synthase.

A series of 22 different 3,5-diarylidenetetrahydro-2H-pyran-4(3H)-ones (DATPs) were synthesized, characterized, and screened for their in vitro antiplasmodial activities against chloroquine (CQ)-sensitive Pf3D7, CQ-resistant PfINDO, and artemisinin-resistant PfMRA-1240 strains of Plasmodium falciparum. DATP 19 (3,5-bis(4-hydroxy-3,5-dimethoxybenzylidene)tetrahydro-2H-pyran-4(3H)-one) was found to be the most potent (IC50 1.07 µM) against PfMRA-1240, whereas 21 (3,5-bis(3,4,5-trimethoxybenzylidene)tetrahydro-2H-pyran-4(3H)-one) showed IC50 values of 1.72 and 1.44 µM against Pf3D7 and PfINDO, respectively. Resistance indices (RI) as low as 0.2 to 0.5 for 10 (3,5-bis(4-nitrobenzylidene)tetrahydro-2H-pyran-4(3H)-one) and 20 (3,5-bis(3-nitrobenzylidene)tetrahydro-2H-pyran-4(3H)-one), and <1 for most other DATPs reveals their greater potency against resistant strains than the sensitive one. The single-crystal XRD data for DATP 21 are reported. In silico support was obtained through docking studies. Killing all three strains within 4-8 h, these DATPs showed rapid kill kinetics toward the trophozoite stage. Furthermore, DATP 18 (3,5-bis(quinolin-4-ylmethylene)tetrahydro-2H-pyran-4(3H)-one) inhibited PfPdx1 enzyme activity with IC50 20.34 µM, which is about twofold lower than that (IC50 43 µM) for an already known inhibitor 4PEHz. At an oral dose of 300 mg/kg body weight, DATPs 19 and 21 were found to be nontoxic to mice, and at 100 mg/kg body weight, DATP 19 was found to suppress parasitaemia, which led to an increase in median survival time by three days relative to untreated control mice in a malaria curative study.

Anti-cancer activity of heteroaromatic acetals of andrographolide and its isomers.

Acetals (2a-d, 3a-d, and 6a-d) of andrographolide (1), 14-deoxy-12-hydroxyandrographolide (4), and isoandrographolide (5) were synthesized using benzaldehyde and heteroaromatic aldehydes. All the synthesized derivatives were characterized using 1H-NMR, 13C-NMR, mass spectrometry, UV, and IR. The compound 6d was characterized via a single-crystal X-ray diffraction study. All the compounds were tested against 60 cell lines of NCI. The acetals (2a-d) of andrographolide (1) exhibited better activity than the acetals (3a-d, and 6a-d) of 12-hydroxyandrographolide (4) and isoandrographolide (5). Preliminary studies suggested that acetals synthesized using benzaldehyde improved anticancer activity. Compound 2a showed the highest growth inhibition of 90.97% against the leukaemia cancer cell line CCRF-CEM. Andrographolide and seven selected compounds were tested against the MDA-MB-231 breast cancer cell line. Compound 3b showed the best activity with an IC50 value of 3 µM among all the tested compounds. Furthermore, this compound 3b was subjected to cell cycle analysis and protein expression confirming apoptosis through the disruption of the mitochondrial potential membrane (Δψ m).

Synthesis of novel spirobibenzopyrans as potent anticancer leads inducing apoptosis in HeLa cells.

Spirobibenzopyrans are an unexplored class of therapeutics. We report the anticancer activity of novel spirobibenzopyrans, synthesized by a one-pot reaction and extensively characterized. Structure of one of the spirobibenzopyran has been determined by the single crystal XRD technique. The in vitro anticancer activity of these derivatives across the NCI 60-cell line panel was evaluated and for the first time their mechanism of action against HeLa cells was probed via cell morphology analysis and cell cycle analysis. They were determined to be apoptosis inducers with cell cycle arrest in G0/G1 and S phase suggesting CDK-4 protein inhibition and the inhibition of DNA replication. The DNA inhibition was studied and confirmed using the alkaline comet assay for the compound CHX-4MO-SAL showing S phase inhibition. Further, conformity with the in silico Lipinski's score signify the potential of spirobibenzopyrans as anticancer leads.