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Here, four MOFs, namely Sc-TBAPy, Al-TBAPy, Y-TBAPy, and Fe-TBAPy (TBAPy: 1,3,6,8-tetrakis(p-benzoic acid)pyrene), were characterized and evaluated for their ability to remediate glyphosate (GP) from water. Among these materials, Sc-TBAPy demonstrates superior performance in both the adsorption and degradation of GP. Upon light irradiation for 5 min, Sc-TBAPy completely degrades 100% of GP in a 1.5 mM aqueous solution. Femtosecond transient absorption spectroscopy reveals that Sc-TBAPy exhibits enhanced charge transfer character compared to the other MOFs, as well as suppressed formation of emissive excimers that could impede photocatalysis. This finding was further supported by hydrogen evolution half-reaction (HER) experiments, which demonstrated Sc-TBAPy’s superior catalytic activity for water splitting. In addition to its faster adsorption and more efficient photodegradation of GP, Sc-TBAPy also followed a selective pathway towards the oxidation of GP, avoiding the formation of toxic aminomethylphosphonic acid observed with the other M³⁺-TBAPy MOFs. To investigate the selectivity observed with Sc-TBAPy, electron spin resonance, depleted oxygen conditions, and solvent exchange with D₂O were employed to elucidate the role of different reactive oxygen species on GP photodegradation. The findings indicate that singlet oxygen (¹O₂) plays a critical role in the selective photodegradation pathway achieved by Sc-TBAPy.

 

Red fluorescent proteins (RFPs) represent an increasingly popular class of genetically encodable bioprobes and biomarkers that can advance next‐generation breakthroughs across the imaging and life sciences. Since the rational design of RFPs with improved functions or enhanced versatility requires a mechanistic understanding of their working mechanisms, while fluorescence is intrinsically an ultrafast event, a suitable toolset involving steady‐state and time‐resolved spectroscopic techniques has become powerful in delineating key structural features and dynamic steps which govern irreversible photoconverting or reversible photoswitching RFPs, and large Stokes shift (LSS)RFPs. The pertinentcis‐transisomerization and protonation state change of RFP chromophores in their local environments, involving key residues in protein matrices, lead to rich and complicated spectral features across multiple timescales. In particular, ultrafast excited‐state proton transfer in various LSSRFPs showcases the resolving power of wavelength‐tunable femtosecond stimulated Raman spectroscopy (FSRS) in mapping a photocycle with crucial knowledge about the red‐emitting species. Moreover, recent progress in noncanonical RFPs with a site‐specifically modified chromophore provides an appealing route for efficient engineering of redder and brighter RFPs, highly desirable for bioimaging. Such an effective feedback loop involving physical chemists, protein engineers, and biomedical microscopists will enable future successes to expand fundamental knowledge and improve human health.

 

The development of bioorthogonal fluorogenic probes constitutes a vital force to advance life sciences. Tetrazine‐encoded green fluorescent proteins (GFPs) show high bioorthogonal reaction rate and genetic encodability but suffer from low fluorogenicity. Here, we unveil the real‐time fluorescence mechanisms by investigating two site‐specific tetrazine‐modified superfolder GFPs via ultrafast spectroscopy and theoretical calculations. Förster resonance energy transfer is quantitatively modeled and revealed to govern the fluorescence quenching; for GFP150‐Tet with a fluorescence turn‐on ratio of ∼9, it contains trimodal subpopulations with good (P₁), random (P₂), and poor (P₃) alignments between the transition dipole moments of protein chromophore (donor) and tetrazine tag (Tet‐v2.0, acceptor). By rationally designing a more free/tight environment, we created new mutants Y200A/S202Y to introduce more P₂/P₁ populations and improve the turn‐on ratios to ∼14/31, making the fluorogenicity of GFP150‐Tet‐S202Y the highest among all up‐to‐date tetrazine‐encoded GFPs. In live eukaryotic cells, the GFP150‐Tet‐v3.0‐S202Y mutant demonstrates notably increased fluorogenicity, substantiating our generalizable design strategy.

Ultrafast spectroscopy reveals FRET in action and inhomogeneous populations with different transition dipole moment alignments.

Rational protein design of two new superfolder GFP mutants with record‐high fluorogenicity.

Bioimaging application of the designed bioorthogonal protein mutant in live eukaryotic cells.

 

Recent advances in sustainable optoelectronics including photovoltaics, light‐emitting diodes, transistors, and semiconductors have been enabled by π‐conjugated organic molecules. A fundamental understanding of light‐matter interactions involving these materials can be realized by time‐resolved electronic and vibrational spectroscopies. In this Minireview, the photoinduced mechanisms including charge/energy transfer, electronic (de)localization, and excited‐state proton transfer are correlated with functional properties encompassing optical absorption, fluorescence quantum yield, conductivity, and photostability. Four naturally derived molecules (xylindein, dimethylxylindein, alizarin, indigo) with ultrafast spectral insights showcase efficient energy dissipation involving H‐bonding networks and proton motions, which yield high photostability. Rational design principles derived from such investigations could increase the efficiency for light harvesting, triplet formation, and photosensitivity for improved and versatile optoelectronic performance.

 

Strategic incorporation of ameta‐dimethylamino (–NMe₂) group on the conformationally locked green fluorescent protein (GFP) model chromophore (m‐NMe₂‐LpHBDI) has drastically altered molecular electronic properties, counterintuitively enhancing fluorescence of only the neutral and cationic chromophores in aqueous solution. A ~200‐fold decrease in fluorescence quantum yield ofm‐NMe₂‐LpHBDI in alcohols (e.g., MeOH, EtOH and 2‐PrOH) supports this GFP‐derived compound as a fluorescence turn‐on water sensor, with large fluorescence intensity differences between H₂O and ROH emissions in various H₂O/ROH binary mixtures. A combination of steady‐state electronic spectroscopy, femtosecond transient absorption, ground‐state femtosecond stimulated Raman spectroscopy (FSRS) and quantum calculations elucidates an intermolecular hydrogen‐bonding chain between a solvent –OH group and the chromophore phenolic ring –NMe₂and –OH functional groups, wherein fluorescence differences arise from an extended hydrogen‐bonding network beyond the first solvation shell, as opposed to fluorescence quenching via a dark twisted intramolecular charge‐transfer state. The absence of ameta‐NMe₂group twisting coordinate upon electronic excitation was corroborated by experiments on control samples without themeta‐NMe₂group or with bothmeta‐NMe₂andpara‐OH groups locked in a six‐membered ring. These deep mechanistic insights stemming from GFP chromophore scaffold will enable rational design of organic, compact and environmentally friendly water sensors.

 

Fluorescence‐activating proteins (FAPs) that bind a chromophore and activate its fluorescence have gained popularity in bioimaging. The fluorescence‐activating and absorption‐shifting tag (FAST) is a light‐weight FAP that enables fast reversible fluorogen binding, thus advancing multiplex and super‐resolution imaging. However, the rational design of FAST‐specific fluorogens with large fluorescence enhancement (FE) remains challenging. Herein, a new fluorogen directly engineered from green fluorescent protein (GFP) chromophore by a unique double‐donor‐one‐acceptor strategy, which exhibits an over 550‐fold FE upon FAST binding and a high extinction coefficient of approximately 100,000 M⁻¹ cm⁻¹, is reported. Correlation analysis of the excited state nonradiative decay rates and environmental factors reveal that the large FE is caused by nonpolar protein−fluorogen interactions. Our deep insights into structure‐function relationships could guide the rational design of bright fluorogens for live‐cell imaging with extended spectral properties such as redder emissions.

 

The versatile functions of fluorescent proteins (FPs) as fluorescence biomarkers depend on their intrinsic chromophores interacting with the protein environment. Besides X-ray crystallography, vibrational spectroscopy represents a highly valuable tool for characterizing the chromophore structure and revealing the roles of chromophore–environment interactions. In this work, we aim to benchmark the ground-state vibrational signatures of a series of FPs with emission colors spanning from green, yellow, orange, to red, as well as the solvated model chromophores for some of these FPs, using wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with quantum calculations. We systematically analyzed and discussed four factors underlying the vibrational properties of FP chromophores: sidechain structure, conjugation structure, chromophore conformation, and the protein environment. A prominent bond-stretching mode characteristic of the quinoidal resonance structure is found to be conserved in most FPs and model chromophores investigated, which can be used as a vibrational marker to interpret chromophore–environment interactions and structural effects on the electronic properties of the chromophore. The fundamental insights gained for these light-sensing units (e.g., protein active sites) substantiate the unique and powerful capability of wavelength-tunable FSRS in delineating FP chromophore properties with high sensitivity and resolution in solution and protein matrices. The comprehensive characterization for various FPs across a colorful palette could also serve as a solid foundation for future spectroscopic studies and the rational engineering of FPs with diverse and improved functions. 

The incorporation of noncanonical amino acids (ncAAs) into fluorescent proteins is promising for red-shifting their fluorescence and benefiting tissue imaging with deep penetration and low phototoxicity. However, ncAA-based red fluorescent proteins (RFPs) have been rare. The 3-aminotyrosine modified superfolder green fluorescent protein (aY-sfGFP) represents a recent advance, yet the molecular mechanism for its red-shifted fluorescence remains elusive while its dim fluorescence hinders applications. Herein, we implement femtosecond stimulated Raman spectroscopy to obtain structural fingerprints in the electronic ground state and reveal that aY-sfGFP possesses a GFP-like instead of RFP-like chromophore. Red color of aY-sfGFP intrinsically arises from a unique “double-donor” chromophore structure that raises ground-state energy and enhances charge transfer, notably differing from the conventional conjugation mechanism. We further developed two aY-sfGFP mutants (E222H and T203H) with significantly improved (∼12-fold higher) brightness by rationally restraining the chromophore's nonradiative decay through electronic and steric effects, aided by solvatochromic and fluorogenic studies of the model chromophore in solution. This study thus provides functional mechanisms and generalizable insights into ncAA-RFPs with an efficient route for engineering redder and brighter fluorescent proteins. 

Proton transfer processes of organic molecules are key to charge transport and photoprotection in biological systems. Among them, excited-state intramolecular proton transfer (ESIPT) reactions are characterized by quick and efficient charge transfer within a molecule, resulting in ultrafast proton motions. The ESIPT-facilitated interconversion between two tautomers (PS and PA) comprising the tree fungal pigment Draconin Red in solution was investigated using a combination of targeted femtosecond transient absorption (fs-TA) and excited-state femtosecond stimulated Raman spectroscopy (ES-FSRS) measurements. Transient intensity (population and polarizability) and frequency (structural and cooling) dynamics of –COH rocking and –C=C, –C=O stretching modes following directed stimulation of each tautomer elucidate the excitation-dependent relaxation pathways, particularly the bidirectional ESIPT progression out of the Franck–Condon region to the lower-lying excited state, of the intrinsically heterogeneous chromophore in dichloromethane solvent. A characteristic overall excited-state PS-to-PA transition on the picosecond timescale leads to a unique “W”-shaped excited-state Raman intensity pattern due to dynamic resonance enhancement with the Raman pump–probe pulse pair. The ability to utilize quantum mechanics calculations in conjunction with steady-state electronic absorption and emission spectra to induce disparate excited-state populations in an inhomogeneous mixture of similar tautomers has broad implications for the modeling of potential energy surfaces and delineation of reaction mechanisms in naturally occurring chromophores. Such fundamental insights afforded by in-depth analysis of ultrafast spectroscopic datasets are also beneficial for future development of sustainable materials and optoelectronics. 

Amine groups are common constituents of organic dyes and play important roles in tuning fluorescence properties. In particular, intensive research works have demonstrated the tendency and capabilities of amines in influencing chromophore brightness. Such properties have been explained by multiple mechanisms spanning from twisted intramolecular charge transfer (TICT) to the energy gap law and beyond, which introduce additional nonradiative energy dissipation pathways. In this review, we aim to provide a focused overview of the mechanistic insights mainly for the TICT mechanism, accompanied by a few other less common or influential fluorescence quenching mechanisms in the amine-containing fluorescent molecules. Various aspects of current scientific findings including the rational design and synthesis of organic chromophores, theoretical calculations, steady-state and time-resolved electronic and vibrational spectroscopies are reviewed. These in-depth understandings of how the amine groups with diverse chemical structures at various atomic sites affect excited-state nonradiative decay pathways will facilitate the strategic and targeted development of fluorophores with desired emission properties as versatile chemosensors for broad applications.