Fluorescent Chromophore Research Articles

Facile fabrication of luminescent ultra‐high molecular weight polyethylene fibers based on novel alkyl‐substituted 1,8‐naphthalimide fluorescent dyes

AbstractUltra‐high molecular weight polyethylene (UHMWPE) fiber, with excellent light resistance, and chemical resistance, is one of the most important high‐performance fibers. However, the high hydrophobicity and crystallinity make it difficult to color, restricting the development of colored fluorescent UHMWPE protection materials. In this study, three hydrophobic fluorescent dyes (NC4, NC8, NC16) for fluorescent dyeing of UHMWPE were developed by introducing alkyl substituents of different lengths (butyl, octyl, and hexadecyl) and polar cyano groups onto 4‐NH‐1,8‐naphthalimide fluorescent chromophore. The relationship between the structures and the application performance was studied, and theoretical calculations were combined to explore the influence of the substituents on the spatial and electronic effects of fluorescent dyes. With the extension of the alkyl chain, the dye‐uptake rate and building‐up property was enhanced, and the equilibrium dye‐uptake rates for NC4, NC8, and NC16 were 18.21%, 93.22%, and 62.96%, respectively. NC8, which was proven to have high hydrophobicity and maximum dipole moment, exhibited the highest dye‐uptake rate. The dyed UHMWPE fabrics exhibited strong yellow‐green fluorescence. This study provides a facile strategy for constructing luminescent UHMWPE protection materials.

An intramolecular charge transfer based fluorescent probe for imaging of OCl–

The discovery and utilization of new fluorescent chromophore is indispensable to exploit high performance probes for biological research. Stokes shift is one of the most important properties of chromophore accounting for super-resolution fluorescence imaging. Intramolecular charge transfer (ICT) is one of the fundamental mechanisms for fluorescence that accompanied by large Stokes shifts. Based on the conformational changes between ground and excited states, ICT models can be divided into two types: conformation-steady ICT, whose conformation remains unchanged, and conformation-changeable ICT, which is characterized by the rotation of the chromophore around an axis upon excitation. Herein, we report a new chromophore whose donor and acceptor parts took a butterfly geometry with a dihedral angle of 21° in ground state and a planar conformation upon photo excitation. The bent conformation might be ascribed to the extra conjugated double bond, which made the coplanarity of the chromophore in ground state get worse. The chromophore shows a remarkable Stokes shift over 150 nm and a high fluorescence quantum yieldof 0.62. The limit of detection is 41 nM, which enabled the imaging of basal as well as induced OCl– in different cells. Moreover, the pronounced spectroscopic properties ensure the in vivo monitoring of OCl– in arthritic mice. This finding would shed light on the exploitation of small molecule probes based on new fluorescence chromophore for precise biological imaging.

Self-assembly of green fluorescent protein chromophore analogue to form microfibers:inhibited and disassembled by Hg2+ through Z-E isomerization

A novel analogue of the green fluorescent protein (GFP) chromophore, named p-DBCI, was synthesized via a 2,3-cycloaddition reaction. The compound was structurally characterized using NMR, HRMS, and single crystal X-ray diffraction, confirming its Z-form conformation in both solid and solution phases. While exhibiting low fluorescence quantum yields in solution, p-DBCI showed strong fluorescence emission in the solid state, indicating aggregation-induced emission (AIE) properties. p-DBCI demonstrated a remarkable ability for self-assembly, forming regular microrods with intense fluorescence through a solvent exchange method. The presence of Hg2+ was found to inhibit and disassemble the self-assembly of p-DBCI, leading to fluorescence quenching via Z-E isomerization driven by strong complexation between p-DBCI and Hg2+. The ability of Hg2+ to induce Z-E isomerization in GFP analogs has been rarely reported. Furthermore, the fluorescence intensity of p-DBCI assemblies in water solution exhibited a highly linear relationship (R2= 0.9992) with increasing concentrations of Hg2+, suggesting that p-DBCI could serve as a sensitive probe for quantitatively detecting Hg2+ in aqueous solutions. These findings offer new theoretical guidance for exploring and utilizing the solid fluorescence properties of GFP analogues.

6-Methoxy-2-Naphthoate as a Standard Chromophore for Chiroptical Studies by Fluorescence-Detected Exciton-Coupled Circular Dichroism.

This study investigated the applicability of fluorescent chromophores for exciton-coupled circular dichroism (ECCD) exploiting fluorescence-detected circular dichroism (FDCD). FDCD had been previously reported useful in allowing the sensitive detection of ECCD in favorable conditions. However, fluorescence detection may prevent applications of the combined method especially when solutions are polarized in emission. Even without polarization of emission, FDCD deviates from circular dichroism (CD) in some cases when the fluorophore of interest interacts with nonfluorescent chromophore. Herein, it was confirmed that employing 6-methoxy-2-naphthoate always yielded interpretable exciton-coupled FDCD spectra even when coupling with nonfluorescent p-substituted benzoates. The 6-methoxy-2-naphthoate chromophore (6-MN) is prescribed in special cases when only a small amount of sample is available for determining the absolute stereochemistry by the CD exciton chirality method observed by FDCD.

Fluorescence of Intrinsic Milk Chromophores as a Novel Verification Method of UV-C Treatment of Milk.

The European Food Safety Authority (EFSA) has approved the use of a 1045 J/L UV-C dose as an adjunct to pasteurization to increase the shelf life and vitamin D3 content of milk. However, there are no verification methods analogous to the alkaline phosphatase test for pasteurized milk to ensure that the desired UV-C dose has been correctly applied. The aim is to develop a real-time in-line detector based on fluorescence spectroscopy. In this study, 22 different UV-C doses (ranging from 0 to 2000 J/L) were applied to milk to assess the impact of photooxidation on intrinsic photosensitive chromophores. Fluorescence spectroscopy (90°-angle) was employed as the method of analysis for monitoring the changes in the fluorescence spectra of chromophores in milk without sample pretreatment. Three important chromophore areas (CAs) were identified: CA 1 (riboflavin), CA 3 (vitamin A and dityrosine) and CA 4 (tryptophan), with statistically significant changes at around 1045 J/L and 1500 J/L. The findings of our preliminary study support our hypothesis that the fluorescence of intrinsic chromophores can be used as verification of the applied UV-C dose.

Robust Luminescent Pyrene-Based Metal-Organic Framework Hydrogel as a pH-Responsive Fluorescence Emitter for Sensitive Immunoassay of Cardiac Troponin I.

Despite many luminescent advantages including outstanding absorption coefficient and high quantum yield, pyrene and its derivatives have been suffering from a dramatic aggregation-caused quenching (ACQ) effect. Although the dramatic ACQ effect of pyrene-based fluorophores has been restrained in pyrene-doped metal-organic frameworks (MOFs), the low loading of fluorescent (FL) units substantially impedes the improved luminescent behaviors. Herein, pyrene-based MOFs hydrogel was synthesized with a high loading of pyrene as the unique organic linker blocks instead of a dopant in MOFs. The gel matrix contributed to rigidifying the location of the FL emitters and achieving intensive FL emission and high luminescent stability and therefore efficiently overcoming the ACQ effect. Furthermore, the protonation of pyrene in the MOFs hydrogel remarkably decreased the luminescent intensity, which endowed the FL hydrogel with highly pH-responsive activity in the broad range (pH 4-10). Interestingly, glucose oxidase was immobilized into ZIF-8 as a highly efficient luminescent quencher, which contributed to catalyzing the form of gluconic acid and thus drastically quenching the FL signal of the MOFs hydrogel. Furthermore, the emitter-quencher pair of pyrene-based MOFs hydrogel and glucose oxidase was successfully employed to develop an ultrasensitive FL immunoassay platform for cardiac troponin I (as a model analyte). The limit of detection for cardiac troponin I was 5.2 pg/mL (3σ). The proof-of-principle study demonstrated the thrilling auxiliary effect of tailorable MOFs hydrogel on boosting the feasibility of aqueous insoluble FL chromophores for trace analysis.

GFP Chromophore Integrated Conjugated Microporous Polymers toward Bioinspired Photocatalytic CO2 Reduction to CO.

The development of highly active, durable, and low-cost metal-free catalysts for the photocatalytic CO2 reduction reaction (CO2RR) is an efficient and environmentally friendly solution to address significant problems like global warming and high energy demand. In the present study, we have demonstrated the design and synthesis of a donor-acceptor based conjugated microporous polymer (CMP), TPA-GFP, by integrating an electron donor, tris(4-ethynylphenyl)amine (TPA), with a green fluorescent protein chromophore analogue (Z)-4-(2-hydroxy-3,5-diiodobenzylidene)-1-(4-iodophenyl)-2-methyl-1H-imidazol-5(4H)-one (o-HBDI-I3) (GFP). In comparison to nondonor 1,3,5-triethynylbenzene (TEB) based TEB-GFP CMP, photocatalytic CO2 reduction using donor-acceptor based TPA-GFP CMP displays a 3-fold increment of CO production yield with a maximum CO yield of 1666 μmol g-1 at 12 h. Further, the CO selectivity increases significantly from a mere 54% in TEB-GFP to an impressive 95% in TPA-GFP. The impressive CO2 reduction efficiency and selectivity for TPA-GFP can be attributed to the efficient light-harvesting capability and facile charge separation and migration through donor-acceptor building units of the CMP. The mechanistic aspect of the photocatalytic CO2 reduction process is explored using in situ DRIFTS and DFT calculation, and a plausible photocatalytic mechanism is proposed.

Ratiometric fluorescent probe with AIE characteristics for hypochlorite detection and biological imaging

It is very important and highly valuable to detect ClO− in samples and living cells with accuracy and speed. In this work, a novel fluorescent probe NA was prepared from 4-bromo-1,8-naphthalic anhydride by acylation reaction and Suzuki coupling reaction and used for the detection of ClO−. Thiomethyl serves as the recognition group for probe NA, while naphthalimide serves as fluorescent chromophore. The probe exhibited an extremely pronounced blue shift from yellow to blue fluorescence within 1 min after the addition of hypochlorite (ClO−). The probe demonstrates high sensitivity to ClO− with a limit of detection (LOD) of 1.22 µM. Also, probe NA demonstrates excellent selectivity and immunity to interference. Additionally, simple fluorescent test strips containing probe NA were prepared in this study, enabling rapid detection of ClO− in water samples. And NA had been effectively used to image endogenous and exogenous ClO−fluorescence in living cells. The results suggest that probe NA has significant potential for portable detection and biological applications.

A Practical and Modular Method for Direct C-H Functionalization of the BODIPY Core via Thianthrenium Salts.

Direct structural modification of small-molecule fluorophores represents a straightforward and appealing strategy for accessing new fluorescent dyes with desired functionalities. We report herein a general and efficient visible-light-mediated method for the direct C-H functionalization of BODIPY, an important fluorescent chromophore, using readily accessible and bench-stable aryl and alkenylthianthrenium salts. This practical approach operates at room temperature with extraordinary site-selectivity, providing a step-economical means to construct various valuable aryl- and alkenyl-substituted BODIPY dyes. Remarkably, this protocol encompasses a broad substrate scope and excellent functional-group tolerance, and allows for the modular synthesis of sophisticated symmetrical and asymmetrical disubstituted BODIPYs by simply employing different combinations of thianthrenium salts. Moreover, the late-stage BODIPY modification of complex drug molecules further highlights the potential of this novel methodology in the synthesis of fluorophore-drug conjugates.

Structural and functional assessment of keratin extracted from chicken feathers using microwave-assisted l-cysteine method

Extracting and applying waste feather keratin are important environmental challenges. This study utilized microwave-assisted l-cysteine treatment on chicken feathers, pH adjustments varied reducing capacity of l-cysteine, while modulated microwave power explored synergistic extraction effects on properties of keratin. The highest yield of regenerated keratin was achieved at an l-cysteine concentration of 15 g/L, pH 12, and a microwave power of 400 W. Micromorphological and particle size results revealed that the keratin particle size increased with pH dependency, the highest proportion of particle size values in keratin increasing from 164 nm in L1 (extracted at pH 11) to 220 nm in L5 (extracted at pH 13). Increasing the intensity of both treatments led to a transition of the secondary structure of regenerated keratin from α-helix to β-sheet, but the increasing reducibility of the system resulted in a decrease in the content of tryptophan residues in keratin, whereas the increase in microwave power led to the masking of some fluorescent chromophores within the keratin. The regenerated keratin demonstrated low crystallinity, and it may have undergone refolding and aggregation under microwave-assisted highly reducing extraction conditions. The foaming properties indicated that the highly reducing extraction system significantly decreased foam stability from 95.4% for L1 to 19.8% for L5 (p < 0.05) and improved foaming ability to keratin. Considering the widespread applications of keratin, this study provides a comprehensive characterization of regenerated keratin under different extraction conditions, aiming to offer experimental materials and extraction conditions tailored to specific requirements in the field of biomaterials.

Ultrafast Hot Exciton Nonadiabatic Excited-State Dynamics in Green Fluorescent Protein Chromophore Analogue.

The ultrafast high-energy nonadiabatic excited-state dynamics of the benzylidenedimethylimidazolinone chromophore dimer has been investigated using an electronic structure method coupled with on-the-fly quantitative wave function analysis to gain insight into the photophysics of hot excitons in biological systems. The dynamical simulation provides a rationalization of the behavior of the exciton in a dimer after the photoabsorption of light to higher-energy states. The results suggest that hot exciton localization within the manifold of excited states is caused by the hindrance of torsional rotation due to imidazolinone (I) or phenolate (P) bonds i.e., ΦI- or ΦP-dihedral rotation, in the monomeric units of a dimer. This hindrance arises due to weak π-π stacking interaction in the dimer, resulting in an energetically uphill excited-state barrier for ΦI- and ΦP-twisted rotation, impeding the isomerization process in the chromophore. Thus, this study highlights the potential impact of the weak π-π interaction in regulating the photodynamics of the green fluorescent protein chromophore derivatives.

Molecular composition of Beijing PM2.5 Brown carbon associated with fluorescence revealed by gas chromatography time-of-flight mass spectrometry and parallel factor analysis

Brown carbon (BrC), the important component of carbonaceous aerosol, attracted much attention for its complex chemical composition which affect its light absorption, fluorescent properties, and atmospheric reactions. In this study, we revealed for the first time the molecular composition of non-polar/low-polar organic matters contributing to fluorescence of Beijing PM2.5 from 2016 to 2018. Firstly, excitation-emission matrix (EEM) spectra of the 122 PM2.5 samples were analyzed and supported by gas chromatography-time of flight mass spectrometry (GC-ToF-MS) and partial least squares regression model, 400 BrC molecules associated with fluorescence were screened out among which 44 were the newly ones. Secondly, six chromophores (C1–C6) were identified by applying parallel factor analysis (PARAFAC) model, which represented categories of compounds consistent with the ToF results. Then, the specific molecule composition of each chromophore was achieved by combining GC-ToF-MS and EEM-PARAFAC. For example, indeno[1,2,3-cd]pyrene is a new fluorescent molecule which contributed high in C3 (high oxygenated humic-like chromophore) and C5 (fluorescein chromophore). The results can, on one side, enrich the database of fluorescent molecular composition, which turns out to be similar as that of light-absorption; on the other side and more importantly, provide chemical characteristics of BrC fluorescent chromophores on the level of molecule.

Enzyme-responsive aptasensor based on the functionalized fluorescent protein chromophore derivative for the detection of alkaline phosphatase activity

Fluorescent proteins (FPs) based sensing strategies have been widely developed in bioanalytical applications. However, it still remains a challenge to realize the efficient and precise analysis. Recently, FPs chromophore and its derivatives with favorable optical properties are well-designed to simulate and replace the function of FPs by specifically binding to the selected fluorescent light-up aptamers (FLAPs), it is worthy note that there is still a lack of systematic approaches to remold these FPs mimics with enzyme-responsive functions. Taking alkaline phosphatase (ALP) as a research model, we here propose an ALP-responsive fluorescent DNA aptasensor (p-DAA) by utilizing a truncated Lettuce (tLet-10) FLAP and a phosphorylated DFHBI-derivative (ALP-DFSS) for achieving ALP activity analysis. Taking the HBI-chromophore as the remolding molecular template, ALP-DFSS has been successfully synthesized and characterized to response to ALP activity. With phosphate group substitution in ALP-DFSS, the decayed fluorescence of p-DAA can be observed by forming the complex of tLet-10/ALP-DFSS. While the turn-on fluorescence of p-DAA will be obtained by the ALP-triggered dephosphorylation process and further exhibit the label-free, fast-response, specific, and sensitive detection performances for both evaluating ALP activity and the inhibition efficiency of ALP inhibitor. Moreover, p-DAA allow the spiked ALP analysis in serum samples and is also applied for realizing the visual differentiation between normal cell line and cancer cell line by responding the endogenous ALP, which holds the great potential in guiding the chromophores functionalization and exploring aptasensing related biological research.

Lytic transglycosylase Slt of Pseudomonas aeruginosa as a periplasmic hub protein.

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 μM) include PBPs (PBP1a, KD = 0.07 μM; PBP5 = 0.4 μM); other lytic transglycosylases (SltB2, KD = 0.09 μM; RlpA, KD = 0.4 μM); a type VI secretion system effector (Tse5, KD = 0.3 μM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 μM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.

SPECTRAL PROPERTIES OF CHLOROPHYLLS IN EDIBLE PLANTS INTENDED FOR DIAGNOSTICS

Abstract. This study examined the absorption and fluorescence spectra of chlorophylls and other pigments extracted in ethanol solutions from common edible plant species. Chlorophyll-a was the dominant fluorescent chromophore in all plant varieties. The fluorescence curves for all extracted solutions exhibit after optimizing by concentration dilution an increase in the fluorescence with a typical prominent leading Gaussian peak at a wavelength of 674 nm. This peak is followed by a decreasing shoulder extending into the near-infrared range beyond 724 nm. The dilution of the solutions with ethanol reduces the optical absorption from the other pigments and shows simultaneously an improved resolution of the chlorophyll-a spectral absorption peaks. This investigation provides new insights into the optical absorbance and other photo-excited processes that impact the fluorescence of chlorophylls, such as agglomeration, photobleaching and photo-aggregation. The findings may be used for biodiagnostics, fluorescent quantum dots and bio-sensors.

Seasonal characterization of chemical and optical properties of water-soluble organic aerosol in Beijing

Water-soluble organic aerosol (WSOA) plays a crucial role in altering radiative forcing and impacting human health. However, our understanding of the seasonal variations of WSOA in Chinese megacities after the three-year clean air action plan is limited. In this study, we analyzed PM2.5 filter samples collected over one year (2020−2021) in Beijing to characterize the seasonal changes in the chemical and optical properties of WSOA using an offline aerosol mass spectrometer along with spectroscopy techniques. The mean mass concentration of WSOA during the observation period was 8.84 ± 7.12 μg m−3, constituting approximately 64–67 % of OA. Our results indicate the contribution of secondary OA (SOA) increased by 13–28 % due to a substantial reduction in primary emissions after the clean air action plan. The composition of WSOA exhibited pronounced seasonal variations, with a predominant contribution from less oxidized SOA in summer (61 %) and primary OA originating from coal combustion and biomass burning during the heating season (34 %). The mass absorption efficiency of WSOA at 365 nm in winter was nearly twice that in summer, suggesting that WSOA from primary emissions possesses a stronger light-absorbing capability than SOA. On average, water-soluble brown carbon accounted for 33–48 % of total brown carbon absorption. Fluorescence analysis revealed humic-like substances as the most significant fluorescence component of WSOA, constituting 82 %. Furthermore, both absorption and fluorescence chromophores were associated with nitrogen-containing compounds, highlighting the role of nitrogen-containing species in influencing the optical properties of WSOA. The results are important for chemical transport models to accurately simulate the WSOA and its climate effects.

Synthesis, antiproliferative, and molecular docking of novel donor-π-acceptor benzothiazole conjugates based fluorescent chromophores

Four targeting fluorescent 2-(N,N-dimethyl/diphenyl-aminophenyl)-5-substituted-benzothiazole conjugates 3a, 3b, 6a and 6b were synthesized. Insight into the photophysical characteristics of the synthesized conjugates, as well as measurements of absorbance and fluorescence maxima in diverse organic solvents, showed conjugate 3b ranged from 302 to 324 nm, whereas the fluorescence maxima ranged from 312 nm (CH2Cl2) to 465 nm. The conjugates' cytotoxicity was evaluated across anticancer cell lines, and the inhibitory action of these conjugates against VEGFR-2 was assessed using an antiphosphotyrosine antibody, with 3b and 6b conjugates showing potent cytotoxic activity against MCF-7 cells with IC50 values of 7.06 ± 0.29 and 8.82 ± 0.37 μM, respectively. Meanwhile, conjugate 6b had the greatest potency of the conjugates for VEGFR-2 inhibition, with an IC50 of 0.23 ± 0.20 μM. Though the binding’s interactions were stimulated using molecular docking, A higher binding score and a variety of interaction types for conjugate 3b, temporarily, indicated a stronger interaction. Moreover, the ADME study was performed and showed that conjugates 3b and 6b exhibited enhanced lipophilicity and reduced solubility, which may influence their pharmacokinetic behavior.

Using Protein Design and Directed Evolution to Monomerize a Bright Near-Infrared Fluorescent Protein.

The small ultrared fluorescent protein (smURFP) is a bright near-infrared (NIR) fluorescent protein (FP) that forms a dimer and binds its fluorescence chromophore, biliverdin, at its dimer interface. To engineer a monomeric NIR FP based on smURFP potentially more suitable for bioimaging, we employed protein design to extend the protein backbone with a new segment of two helices that shield the original dimer interface while covering the biliverdin binding pocket in place of the second chain in the original dimer. We experimentally characterized 13 designs and obtained a monomeric protein with a weak fluorescence. We enhanced the fluorescence of this designed protein through two rounds of directed evolution and obtained designed monomeric smURFP (DMsmURFP), a bright, stable, and monomeric NIR FP with a molecular weight of 19.6 kDa. We determined the crystal structures of DMsmURFP both in the apo state and in complex with biliverdin, which confirmed the designed structure. The use of DMsmURFP in in vivo imaging of mammalian systems was demonstrated. The backbone design-based strategy used here can also be applied to monomerize other naturally multimeric proteins with intersubunit functional sites.

Modifying Fluorescent Protein Chromophore to Detect Protamine and Application in Real Serum Samples

AbstractAs effective methods to accurately and rapidly detect protamine remains a major challenge, here we present a fluorescent protein chromophore‐based probe (IDL‐FP) utilizing twisted‐intramolecular charge transfer (TICT) to detect protamine. The introduction of an electron‐donor group 4‐dimethylaminobenzaldehyde extends the emission wavelength of IDL‐FP to the red region. The deprotection of t‐butyloxy carbonyl groups can then generate an electronegative carboxyl group, which promotes the binding to protamine via electrostatic attraction. Thus, IDL‐FP is sensitive and specific towards protamine and can serve as a highly selective detector for protamine with a detection limit as low as 8.87 ng/mL. As IDL‐FP exhibited good stability in physiological pH environment, we successfully apply IDL‐FP to detect spiked‐in protamine in human serum. In summary, probe IDL‐FP is a promising tool for the quantitative determination of protamine.

Quantum dynamics of excited state proton transfer in green fluorescent protein.

Photoexcitation of green fluorescent protein (GFP) triggers long-range proton transfer along a "wire" of neighboring protein residues, which, in turn, activates its characteristic green fluorescence. The GFP proton wire is one of the simplest, most well-characterized models of biological proton transfer but remains challenging to simulate due to the sensitivity of its energetics to the surrounding protein conformation and the possibility of non-classical behavior associated with the movement of lightweight protons. Using a direct dynamics variational multiconfigurational Gaussian wavepacket method to provide a fully quantum description of both electrons and nuclei, we explore the mechanism of excited state proton transfer in a high-dimensional model of the GFP chromophore cluster over the first two picoseconds following excitation. During our simulation, we observe the sequential starts of two of the three proton transfers along the wire, confirming the predictions of previous studies that the overall process starts from the end of the wire furthest from the fluorescent chromophore and proceeds in a concerted but asynchronous manner. Furthermore, by comparing the full quantum dynamics to a set of classical trajectories, we provide unambiguous evidence that tunneling plays a critical role in facilitating the leading proton transfer.