Schiff Base Hydrolysis Research Articles

Modulating signalling lifetime to optimise a prototypical animal opsin for optogenetic applications

Animal opsins are light activated G-protein-coupled receptors, capable of optogenetic control of G-protein signalling for research or therapeutic applications. Animal opsins offer excellent photosensitivity, but their temporal resolution can be limited by long photoresponse duration when expressed outside their native cellular environment. Here, we explore methods for addressing this limitation for a prototypical animal opsin (human rod opsin) in HEK293T cells. We find that the application of the canonical rhodopsin kinase (GRK1)/visual arrestin signal termination mechanism to this problem is complicated by a generalised suppressive effect of GRK1 expression. This attenuation can be overcome using phosphorylation-independent mutants of arrestin, especially when these are tethered to the opsin protein. We further show that point mutations targeting the Schiff base stability of the opsin can also reduce signalling lifetime. Finally, we apply one such mutation (E122Q) to improve the temporal fidelity of restored visual responses following ectopic opsin expression in the inner retina of a mouse model of retinal degeneration (rd1). Our results reveal that these two strategies (targeting either arrestin binding or Schiff-base hydrolysis) can produce more time-delimited opsin signalling under heterologous expression and establish the potential of this approach to improve optogenetic performance.

Structural basis for the allosteric modulation of rhodopsin by nanobody binding to its extracellular domain

Rhodopsin is a prototypical G protein-coupled receptor (GPCR) critical for vertebrate vision. Research on GPCR signaling states has been facilitated using llama-derived nanobodies (Nbs), some of which bind to the intracellular surface to allosterically modulate the receptor. Extracellularly binding allosteric nanobodies have also been investigated, but the structural basis for their activity has not been resolved to date. Here, we report a library of Nbs that bind to the extracellular surface of rhodopsin and allosterically modulate the thermodynamics of its activation process. Crystal structures of Nb2 in complex with native rhodopsin reveal a mechanism of allosteric modulation involving extracellular loop 2 and native glycans. Nb2 binding suppresses Schiff base deprotonation and hydrolysis and prevents intracellular outward movement of helices five and six – a universal activation event for GPCRs. Nb2 also mitigates protein misfolding in a disease-associated mutant rhodopsin. Our data show the power of nanobodies to modulate the photoactivation of rhodopsin and potentially serve as therapeutic agents for disease-associated rhodopsin misfolding.

A short story on how chromophore is hydrolyzed from rhodopsin for recycling.

The photocycle of visual opsins is essential to maintain the light sensitivity of the retina. The early physical observations of the rhodopsin photocycle by Böll and Kühne in the 1870s inspired over a century's worth of investigations on rhodopsin biochemistry. A single photon isomerizes the Schiff-base linked 11-cis-retinylidene chromophore of rhodopsin, converting it to the all-trans agonist to elicit phototransduction through photoactivated rhodopsin (Rho*). Schiff base hydrolysis of the agonist is a key step in the photocycle, not only diminishing ongoing phototransduction but also allowing for entry and binding of fresh 11-cis chromophore to regenerate the rhodopsin pigment and maintain light sensitivity. Many challenges have been encountered in measuring the rate of this hydrolysis, but recent advancements have facilitated studies of the hydrolysis within the native membrane environment of rhodopsin. These techniques can now be applied to study hydrolysis of agonist in other opsin proteins that mediate phototransduction or chromophore turnover. In this review, we discuss the progress that has been made in characterizing the rhodopsin photocycle and the journey to characterize the hydrolysis of its all-trans-retinylidene agonist.

Schiff base synergized with protonation of PEI to achieve smart antibacteria of nanocellulose packaging films

Microbial growth and reproduction can cause food spoilage. Developing the controlled release packaging films for food is an ideal solution. In this study, polyethyleneimine (PEI) was grafted to cellulose nanofibers (CNF) films by Schiff base, and when the CNF/PEI films were stimulated by pH, PEI released from the CNF/PEI films due to Schiff base hydrolysis, improving the antibacterial efficiency of PEI. Stimulated by acid with pH of 4, the PEI cumulative release rate of the CNF/PEI800 and the CNF/PEI2000 films reached to 92.90 % and 87.28 %, respectively. At the same time, the amino groups of PEI protonated by obtaining H+, the charge density increased, and PEI molecular chains extended, enhancing the antibacterial activity of films. The Zeta potential value on the surface of the CNF/PEI film increased with the decrease of pH value. Schiff base synergized with protonation of PEI to achieve smart antibacteria of CNF packaging films. The antibacterial rates of the film against L. monocytogenes and E. coli were 94.7 % and 90.6 % at pH 4, but 29.5 % and 23.6 % at pH 8, respectively. The developed films also had good barrier properties of oxygen, visible light and mechanical properties, and had an attractive application prospect in food preservation to control release of antibacterial agent.

PH-sensitive self-assembled nanofibers based on electrostatic interaction and Schiff base bonding for controlled release of curcumin

In this study, chitosan-dialdehyde-β-cyclodextrin-curcumin (CS-DCD-CUR) and alginate dialdehyde (ADA) were alternately deposited on the surface of electrospun cellulose acetate (CA) nanofibers driven by Schiff base bonding and electrostatic attraction. With the increasing number of bilayers, the self-assembled nanofibers showed an increased average fiber diameter from 441 to 576 nm and had relatively rough surfaces. The improved thermal stability and tensile strength of self-assembled nanofibers were attributed to the interactions between CS-DCD-CUR and ADA. The self-assembled nanofibers exhibited pH-sensitive release characteristics under acidic conditions due to the weakening of the electrostatic interaction and the hydrolysis of Schiff-base. Moreover, the self-assembled nanofibers could be used for sustained and controlled release of the curcumin within 620 min and maintained the antioxidant activities. The highest radical scavenging activities against ABTS and DPPH radicals of the nanofibers were 24.75% and 83.56%, respectively. These results suggested that the layer-by-layer self-assembled nanofibers provided a pH-responsive sustained delivery system for bioactive substances with potential applications in functional foods. • Electrostaic attraction and Schiff base bonding were employed to drive the self-assembly of the nanofibers. • The self-assembled nanofibers had improved thermal stability and mechanical properties. • The self-assembled nanofibers exhibited a controllable pH-sensitive release manner of curcumin.

Cysteine driven decomposition and quenching of a fluorescent metalloreceptor: Optical detection and mechanistic insight

We herein devised a fluorescent metalloreceptor (ZNQ) for selective recognition of cysteine (Cys) via fluorescence suppression. The metalloreceptor, ZNQ is a Zn2+ complex of Quinolineimines based ligand (CMO). The surgical action of Cys over metalloreceptor (ZNQ) finishes in three steps viz. demetallation, hydrolysis of schiff base (CMO) i.e. ligand itself and finally cyclization of aldehyde. Interestingly this type of synergistic mechanistic approach have never been reported in the literature for the detection of any thiols. The structure of metalloreceptor ZNQ was confirmed by single crystal X-ray diffraction and its interaction with Cys have been explored through various spectroscopic studies. The bioimaging of Zn2+ and Cys were also carried out in the living HeLa cell.

Crystal structures and Full Interaction Maps of proton transfer coordination compounds, templated via Schiff base hydrolysis in situ

The used synthetic procedures was able to control delivery of amine (to generate in situ protonated amine), to prepare coordination compounds with different compositions. The anionic coordination compounds {(H2en)[Zn3(2,5-pdc)4(H2O)4]⋅2H2O}n− 1, (H2en)2[Mn(2,5-pdc)3]⋅2H2O – 2a, (H2en)[Mn(2,5-pdc)2(H2O)2]⋅3H2O – 2b, (H2en)2[Cu2(2,5-pdc)2(μ-2,5-pdc)2]⋅8H2O − 3, {[Cu(en)2(H2O)]⋅[Cu3(2,5-pdc)2(μ-2,5-pdc)2(en)2(H2O)2]∙3H2O} − 4a, (H2en)[Cu(2,5-pdc)2(H2O)]⋅H2O – 4b are co-crystallized with ethylenediammonium, via imine hydrolysis; where 2,5-pdc = 2,5-pyridinedicarboxylate and H2en = ethylenediammonium. We are reporting the first crystal structure of a binary coordination complex involving the 2,5-pdc ligand and Mn2+ metal center with 1:3 stoichiometry (2a). The crystal stability and supramolecular analysis of the proton transfer compounds using Full Interaction Maps for coordination compounds is relatively novel and contributed to study the chemical propensity in the increase of dimensionality from binary and ternary discrete metalloligands. The supramolecular arrangements involves significative hydrogen bonds between coordination complexes and ethylenediammonium cation. The synthetic pathway adopted provide metalloligands open bind sites, potential to be employed in the rational design of to prepare multi-metal coordination compounds (homo or hetero).

What Is Hidden Behind Schiff Base Hydrolysis? Dynamic Covalent Chemistry for the Precise Capture of Sialylated Glycans.

The aberrant expression of sialylated glycans (SGs) is closely associated with the occurrence, progression, and metastasis of various cancers, and sialylated glycoproteins have been widely used as clinical biomarkers for cancers. However, the identification and comprehensive analysis of SGs are exceptionally complex, which urgently need an innovative and effective method of capturing SGs from biosamples prior to MS analysis. Here, we report that a novel dynamic covalent chemistry strategy based on Schiff base hydrolysis can be applied to the precise capture of SGs. The prepared glucopyranoside-Schiff base-modified silica gel displays extraordinary enrichment selectivity (even at a ratio of 1:5000 with interference), high adsorption capacity (120 mg·g-1), and satisfying enrichment recovery (95.5%) toward sialylated glycopeptides, contributing to a highly specific, efficient, mild, and reversible SG capturing approach that can remarkably promote the development of glycoproteomics and sialic acid sensing devices and can be considerably promising in cancer biomarker discovery. Meanwhile, the facile hydrolysis characteristic of our Schiff base material completely subverts conventional knowledge of enrichment materials, the chemical stability of which is usually regarded as a prerequisite. Importantly, we find an exciting story hidden behind the Schiff base hydrolysis reaction, which demonstrates the unique advantage of dynamic covalent chemistry in glycoproteomics and biomolecule sensing.

A “turn-on” fluorescent and colorimetric chemodosimeter for selective detection of Au3+ions in solution and in live cellsviaAu3+-induced hydrolysis of a rhodamine-derived Schiff base

A chromogenic and “off–on” fluorogenic chemodosimeter (L) based on a naphthalene–rhodamine B derivative was designed, synthesized and characterized for the selective and sensitive detection of Au3+ions in mixed acetonitrile aqueous media.

Study on the Structure of Cu/ZrO2 Catalyst and the Formation Mechanism of Disodium Iminodiacetate and Sodium Glycine

A new method to prepare Cu/ZrO2 catalysts by reducing CuO/ZrO2 with hydrazine hydrate is reported, and the prepared catalysts were used to synthesize disodium iminodiacetate by diethanolamine dehydrogenation. Hydrazine hydrate can rapidly reduce the CuO/ZrO2 precursor powder in an alkaline environment at a moderate temperature. The ratio of Cu0/Cu+ at the Cu/ZrO2 surface was controlled by the amount of hydrazine hydrate and the reduction reaction time. The formation mechanism of disodium glycine as the main byproduct and iminodiacetate were deduced by investigating the product yield, the reaction time, and the presence of acetaldehyde in the evolved gas. It has been shown that the ratio of Cu0/Cu+ in Cu/ZrO2 significantly affects the dehydrogenation of diethanolamine into disodium iminodiacetate. Cu0 and Cu+ are the catalytic activity centers in the dehydrogenation of diethanolamine which respectively produce intermediate aldehydes and an ester via nucleophilic addition reactions. The formation mechanism of sodium glycinate is related to the tautomerism of intermediate products and Schiff base hydrolysis. The formation mechanism of disodium iminodiacetate and sodium glycine producing by the dehydrogenation of diethanolamine over the Cu/ZrO2 catalysts which were prepared by a new reduction method.

A HIGHLY SELECTIVE FLUORESCENT SENSOR FOR DETECTION OF TRIVALENT METAL IONS BASED ON A SIMPLE SCHIFF-BASE

A simple and easily synthesized fluorescent sensor L, based on condensation reaction of 1-Naphthylamine and terephthalaldehyde, was synthesized and characterized systemically. Both UV-vis and fluorescence spectroscopic studies indicated that the sensor L showed good selectivity toward trivalent metal ions (such as Al3+, Fe3+, Cr3+, La3+ and Ce3+) over other monovalent or divalent metal ions. The sensing mechanism was suggested that the trivalent ion promoted the hydrolysis of Schiff base. Meanwhile, took the common trivalent ions Al3+, Fe3+ and Cr3+ as examples, limit of detection (LOD) values of sensor L were calculated to be 1.23, 0.94 and 1.14 nM for Al3+, Cr3+ and Fe3+, respectively, suggesting very high sensitivity towards the cations which could make it attractive in trivalent metal ions detection.

Optimization of Enzyme Mechanism along the Evolutionary Trajectory of a Computationally Designed (Retro-)Aldolase.

De novo biocatalysts have been successfully generated by computational design and subsequent experimental optimization. Here, we examined the evolutionary history of the computationally designed (retro-)aldolase RA95. The modest activity of the starting enzyme was previously improved 105-fold over many rounds of mutagenesis and screening to afford a proficient biocatalyst for enantioselective cleavage and synthesis of β-hydroxyketones. Using a set of representative RA95 variants, we probed individual steps in the multistep reaction pathway to determine which processes limit steady-state turnover and how mutations that accumulated along the evolutionary trajectory influenced the kinetic mechanism. We found that the overall rate-limiting step for aldol cleavage shifted from C-C bond scission (or an earlier step in the pathway) for the computational design to product release for the evolved enzymes. Specifically, interconversion of Schiff base and enamine intermediates, formed covalently between acetone and the catalytic lysine residue, was found to be the slowest step for the most active variants. A complex hydrogen bond network of four active site residues, which was installed in the late stages of laboratory evolution, apparently enhances lysine reactivity and facilitates efficient proton shuffling. This catalytic tetrad accounts for the tremendous rate acceleration observed for all steps of the mechanism, most notably Schiff base formation and hydrolysis. Comparison of our results with kinetic and structural studies on natural aldolases provides valuable feedback for computational enzyme design and laboratory evolution approaches alike.

Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state

Here, we describe two insights into the role of receptor conformational dynamics during agonist release (all-trans retinal, ATR) from the visual G protein-coupled receptor (GPCR) rhodopsin. First, we show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, we observe that this equilibrium can be shifted by either promoting the active-like population or increasing the agonist concentration. Second, we find that during decay of the signaling state an active-like, yet empty, receptor conformation can transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion is based on time-resolved, site-directed fluorescence labeling experiments that show a small, but reproducible, lag between the retinal leaving the protein and return of transmembrane helix 6 (TM6) to the inactive conformation, as determined from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissociation, either by addition of hydroxylamine or introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations show that rhodopsin can bind its agonist in equilibrium like a traditional GPCR, provide evidence that an active GPCR conformation can persist even after agonist release, and raise the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.

Coumarin-decorated Schiff base hydrolysis as an efficient driving force for the fluorescence detection of water in organic solvents.

A coumarin based Schiff base was found to be an excellent indicator of moisture, via rapid in situ hydrolysis. A structure-relationship examination of a small library of Schiff bases revealed the critical importance of hydrogen bond acceptors in close proximity to the imine bond, and this observation was further supported by theoretical calculations as well as the solid state structure analysis. The most sensitive compound demonstrated a limit of detection and quantification of 0.18% and 0.54% v/v water in DMSO, respectively.

A fluorescent turn-on probe for detection of HSO4− ion based on hydrolysis of BODIPY-derived Schiff base with chromogenic and fluorogenic dual signals

A novel colorimetric fluorescent chemosensor for HSO4− using BODIPY-based Schiff base (L) has been developed. L is capable of showing high selectivity and sensitivity toward HSO4− over a wide range of other interfering anions. In presence of HSO4−, L shows a significant blue-shift in the absorption spectra from 523 to 497nm and an increase of the ratio of the absorbance at 497 and 523nm (A497/A523) over 150-fold, which induces color changes from pink to yellow. Non-fluorescent L upon interaction with HSO4− ions displays a 114-fold fluorescence enhancement at 533nm, resulting in strong green emission. The binding mode of L with HSO4− has been investigated by Job's plot, 1H NMR and HRMS measurements. The sensing mechanism is suggested to proceed via a hydrolysis process. In addition, the cell imaging experiments demonstrate that L can be successfully applied as a bioimaging agent for monitoring HSO4− in HeLa cells.

Fluorescent “turn-on” detecting CN− by nucleophilic addition induced Schiff-base hydrolysis

A new chemosensor Sz based on Schiff-base group as recognition site and naphthalene as the fluorescence signal group was designed and synthesised. It could fluorescent “turn-on” detect cyanide (CN−) via a novel mechanism of nucleophilic addition induced Schiff-base hydrolysis. Adding the CN− into the solution of Sz could induce Sz to emit blue fluorescence at 435nm instantly. Moreover, Sz could also colorimetric detect CN−. Upon the addition of CN−, the Sz showed dramatic color change from yellow to colorless. These sensing procedures could not be interfered by other coexistent competitive anions such as F−, AcO−, H2PO4− and SCN−. In addition, Sz showed high sensitivity for CN−, the detection limits is 3.42×10−8M of CN−, which is far lower than the WHO guideline of CN− in drinking water (less than 1.9×10−6M). The CN− test strips based on Sz could act as a convenient CN− test kits.

A Rational Designed Dual‐channel Chemosensor for Mercury Ions Based on Hydrolysis of Schiff Base

AbstractA bilateral Schiff base is reported for the colorimetric and fluorometric dual‐channel sensing of Hg2+ ions by taking advantage of the hydrolysis of carbon‐nitrogen double bond, altering an ICT state mechanism and then Hg2+ ions coordinating with amino moieties of 1,5‐DAN and leading to the aggregation of 1,5‐DAN. Meanwhile, it formed a stable neutral complex of amino‐Hg‐amino. In addition, test strips based on L were fabricated, which also exhibited a good selectivity to Hg2+ as in solution. This work provides a novel approach for the selective recognition of mercury ions. Notably, the color changes are very significant and all the recognition processes can be observed by the naked eyes. We believe the test strips can act as a convenient and efficient Hg2+ test kit.

Detection of HSO₄⁻ ion based on the hydrolysis of diketopyrrolopyrrole-derived Schiff base with chromogenic and fluorogenic dual signals.

A new diketopyrrolopyrrole-based Schiff base L was synthesized and its anion sensing behavior was explored. L showed exclusive response toward HSO4(-) ion and also distinguished HSO4(-) from other anions by color changes (from dark red to orange) and 21 fold fluorescence enhancement at 370 nm in aqueous solution (THF/H2O = 8/1, v/v). The sensing mechanism was suggested to proceed via a hydrolysis process. The results provided colorimetric and fluorimetric assays to selectively detect the presence of a HSO4(-) over a wide range of other interfering anions. The results could potentially be used as a dual colorimetric-fluorescent probe for monitoring HSO4(-) levels in physiological and environmental systems.

A highly selective and femto-molar sensitive fluorescence ‘turn-on’ chemodosimeter for Hg2+

A selective fluorescence ‘turn-on’ chemodosimeter N,N′-bis-(4-cyanobenzylidene)-2,4,6-trimethylbenzene-1,3-diamine (3) based on a Schiff base for femto-molar detection of the Hg2+ has been described. It presents the highest level of detection limit for Hg2+ through Schiff base hydrolysis.

A new selective colorimetric and fluorescent chemodosimeter for [formula omitted] based on hydrolysis of Schiff base

Two new receptors 1 and 2 were prepared, and their chromogenic and fluorogenic behaviors toward various anions were investigated. Receptors 1 and 2 show exclusive response toward HSO4- ion and also distinguish HSO4- from other anions by different color changes in aqueous solution (CH3CN/H2O=4/1, v/v). Between them receptor 1 selectively exhibits a pronounced HSO4--induced fluorescence enhancement. The detection limit for the HSO4- ion was determined as (0.24±0.03μM). Thus, the receptor 1 can be used as a colorimetric and fluorescent sensor for the determination of HSO4- ion. The sensing mechanism has been suggested to proceed via a hydrolysis process. The hydrolysis product has been isolated and further identified by 1H NMR spectroscopy, ESI-MS analysis and X-ray diffraction.