Since thefirst mass spectrometry experiments, it has been observed that some l-structures may turn into the cyclo ones, likely via atemperature-induced process. In this work, combining several differentexperimental techniques  with tight-binding and ab initio simulations,we provide evidence that, in the case of l-phenylalanyl-l-alanine, an irreversible cyclization mechanism, catalyzedby water and driven by temperature, occurs in the condensed phase.This process can be considered as a very efficient strategy to improvedipeptide stability by turning the comparatively fragile linear structureinto the robust and more stable cyclic one. This mechanism may haveplayed a role in prebiotic chemistry and can be further exploitedin the preparation of nanomaterials and drugs.Dipeptides, the prototypepeptides, exist in both linear ( Their role in proteinsand enzymes and their use in the development of innovative preparationmethods of nanomaterials3 have made these compounds theobject of widespread interest since the 50s of the previous century.Peptides, 2 terminal groups, with theelimination of a water molecule. Cyclo dipeptides consist of a six-memberedring containing two head–tail peptide bonds. The simplest memberis cyclo(glycylglycine) or diketopiperazine, DKP  dipeptide exposed to controlledtemperature in ultrahigh-vacuum (UHV) conditions and consider fewother dipeptide samples to support the data interpretation.Considering that thermalcyclization may have provided an effectivesurvival mechanism for oligopeptides in harsh conditions of the primordialuniverse and can represent a useful resource in the case of thermalprocesses used for nanomaterial production,l- to c-structures, driven by temperature,occurs in the condensed phase. This temperature-induced cyclizationin l-PheAla is also confirmed by a comparative spectroscopicstudy with cyclo-glycylphenylalanine (c-GlyPhe) andcyclo-alanylglycine (c-AlaGly).Our study combines measurements with different techniques and theoreticalsimulations. In detail, we performed time-of-flight mass spectrometry(TOF-MS) of gas-phase, sublimated, samples; thermogravimetric analysis(TGA) of the pristine samples; and infrared (IR) and Raman spectroscopiesof the pristine samples and of the residual ones after the sublimationfor the TOF-MS experiments. The experimental results are supportedand complemented by a multilevel computational protocol rooted ontight-binding and ab initio simulations, including the calculationof IR and Raman spectra, of the reaction paths and barriers of dipeptidecyclization. This study shows that a clear evolution from l-phenylalanyl-l-alanine, cyclo(3S)-3-benzyl-2,5-piperazinedione , linear glycyl-l-alanine ,and linear glycyl-l-phenylalanine  molecules were purchased from Sigma-Aldrich,while cyclo (3S)-3-methyl-2,5-piperazinedione  was purchased fromBACHEM. All species had a purity ≥95% and were used withoutfurther purification.Linear 26 coupled to a VUV rare-gas discharge lamp andan effusive beam of the molecule under investigation. A general descriptionof the apparatus was reported elsewhere.28 Briefly, the instrument was equipped with a channel electron multiplier(CEM) and microchannel plates (MCP) for electron and ion detection,respectively. The detection of a kinetic-energy unselected photoelectronprovided the trigger for the measurement of the time of flight ofthe ions by a TDC card . At variancewith ref , with heating and flow rates of 5 °C/minand 50 mL/min, respectively.–1, respectively.TheIR measurements wereperformed using an FT-IR Shimadzu Prestige 21 equipped with an ATR Golden Gate accessory (Specac), a Michelsoninterferometer, and a DLATGS detector. The acquisition range and theresolution were 4000–450 and 4 cm–1.A Dilor XY spectrometer, with a 514.5 nm excitation wavelengthand a 2.5 mW power, was used to perform Raman spectroscopy. The Ramanspectra were obtained from 5 scans of 240 s in the spectral rangefrom 180 to 3220 cml-PheAla and r-PheAla for short.The IR and Raman spectrawere acquired at room temperature on boththe pristine sample and the residual sample after heating in an ovenat the working temperature during mass spectrometry measurements.In the following, these two samples will be named l-PheAla samples to secure the reproducibilityof the results.The TOF-MS,IR, and Raman measurements were carried out on threedifferent 31 In the case of neutral linear  and cyclo dipeptides, the most stable structureswere used as the initial guess for DFT geometry optimizations. Inthe case of the zwitterionic linear structure (l-zwitterion),the extremely strong interaction between the terminal charged −NH3+ and COO– groups led to theoccurrence of spurious vibrational normal modes. In this case, thestarting configuration of the molecule for DFT calculations was cutout from the hydrated l-PheAla crystal structure.32 DFT calculations were performed using the ORCAsuite of programs.34 Molecular structures were fullyreoptimized using the B3LYP functional35 and the D3BJ dispersion correction.36 Kohn–Sham orbitals were expanded on the def2-QZVPP Gaussianbasis set.37 The corresponding def2/J basiswas also used as an auxiliary basis set for Coulomb fitting in a resolution-of-identity/chain-of-spheres(RIJCOSX) framework. Cyclization mechanisms were investigated by usingthe same multilevel protocol. Reaction paths were first systematicallyexplored by using an automated reaction-path finder based on tight-bindingsimulations.30 Representative paths wereused as the initial guess for climbing image nudged elastic band (CI-NEB)calculations based on density functional theory. As suggested in previouscalculations of symmetric aliphatic dipeptides,38 accurate estimates of reaction barriers along differentcyclization paths were obtained using the M06-2X functional39 and a def2-TZVPP basis set.Theoreticalsimulations of harmonicIR and Raman spectra were performed by following a multilevel protocol,applied to isolated molecules embedded in a dielectric environment.A preliminary screening of molecular configurations was performedusing a conformer-rotamer ensemble sampling tool (CREST), which usesthe GFN2-xTB Hamiltonian as an “engine” for the explorationof potential energy surfaces in order to find low-energy structures.l-PheAla were acquiredby slowly increasing the temperature in steps of 5 °C every 100min. In m/z 18 and 17, related to water and its OH+ fragment, areobserved, while at 130 °C, a series of dipeptide fragments (see m/z ratio is 218 with no trace of the l-PheAla parention (m/z 236) observed. In m/z 18 and 218 ions are reported versustime, while the temperature of the crucible is increased. The waterion yield displays a nonlinear trend with a drastic increase at 85°C  wasnever observed throughout the whole heat-up process.Despite itsintensity being orders of magnitude smaller, the 30–85 °C 3a, with 41 the main features of the mass spectrum in m/z 218) andamine fragments [C7H7CH–NH2]+ and [CH3CH–NH2]+ ;the [C5H7N2O2]+ and its complementary fragment C7H7+ may be attributed to the loss of the Phe and Ala residues, respectively,from m/z 218; the fragment at m/z 99 may be tentatively assigned to afurther CH–CH3 loss from m/z 127.With supportfrom the literature,14 it has been realized thatsome cyclization mayoccur at the typical sublimation temperature13 (120–160 °C). According to these reports, evidence ofthe process is provided by the observation of ions at (i) m/z 18, (ii) m/z corresponding to the [parent ion – water molecule],(iii) m/z corresponding to the aminefragment (RCH=NH2)+ where R is the sidechain of the amino acid, and (iv) other smaller ions produced by thedecomposition of the fragment following an initial elimination ofCO and HNCO, as established from the study of deuterated samples atthe active sites. The features observed in the present mass spectrumare consistent with the findings of ref , while the overall descriptionis reported in the Supporting Information (SI). In the temperature range of 30–200 °C, the TG-DTAdisplays two endothermic peaks in the enthalpy of the system (measuredas heat flow in mW) centered at about 105 and 135 °C, respectively is expected to be lower in UHV conditions.Accordingly, the TOF-MS results in m/z 18 intensity up to T ≈ 85 °C) followed by a structural reorganizationinvolving further release of water (step at T ≈85 °C in the m/z 18 intensity).With the onset temperature of evaporation/sublimation or thermaldecomposition measured in a TG-DTA experiment at ambient pressurebeing higher than the one measured in UHV conditions,46 Hence, to investigate the possibility of a molecular rearrangementoccurring during sublimation, we recorded IR spectra of both l-PheAla and r-PheAla. These spectra, reportedin the upper panel of It is well-known that IR spectroscopy can be used as a diagnostictool of molecular structures and functional groups.r-PheAla shows intense andwell-resolved absorptions in the functional group region of 1300–1700cm–1, while for l-PheAla, onlybroad peaks centered at ≈1600 and 3300 cm–1can be observed. Conversely, the broad spectral feature at ≈3000cm–1 in l-PheAla evolves in a well-resolvedmanifold in r-PheAla.In this regard, only l-PheAla and r-PheAlaare shown in the upper panels of –1) reported in theright-handside. As in the case of the IR measurements, a few differences betweenthese two samples are clearly recognizable. In the r-PheAla Raman spectrum, (i) new intense features appear as a tripletof peaks in the range of 400–500 cm–1 andtwo intense peaks around 760 and 1520 cm–1, while(ii) the features at about 770 and 1540 cm–1 decreasein intensity. In the region of CH stretching modes, there is a closesimilarity between the spectra, supporting the hypothesis that thestructural rearrangement affects only minimally the hydrocarbon partof the molecule.The experimental Ramanspectra of l-PheAla undergoes first asteady loss of water molecules trapped in the sample (m/z 18 and 17 in the mass spectra) and then a suddenburst of water molecules at 85 °C (m/z 218), insteadof the linear one (m/z 236), whichwas never observed through the whole temperature range of mass spectraacquisition.The experimental resultsintroduced in the previous section suggestthat above 65 °C, at 85 °C 2 and 3a.r-PheAla and l-PheAla, with support of theoreticalcalculations. In the calculations, the linear neutral (l-neu), linear zwitterion (l-zwi), and cyclo neutral(c-neu) structures of PheAla, embedded in an implicitdielectric environment to enhance the similarities with the measuredsolid-state samples,47 have been considered,as reported in the inset of l-zwi, l-neu, and c-neu labels,which all refer to the different PheAla species. The comparison reportedin the SI between cyclic PheAla and pristinecyclic GlyPhe and AlaGly species, where some confusion can arise,is made using more precise labels such as c-neu(GlyPhe).To find consistent evidence of this hypothesis,we analyzed indetail the major changes in the IR spectral bands of l-PheAla,the dipeptide is found as a zwitterion or an inner salt, stabilizedby the strong H-bond interaction between −NH3+ and −COO– groups, also involvingthe central −C=O–NH– backbone, as wellas by the presence of crystallized water molecules.32 However, zwitterions are not stable as isolated moleculesin the gas phase and are readily converted into a neutral linear molecule.This same process may happen also on the surface of a solid sample,when heated, and it can be considered as a preliminary step of cyclization,38 even in the bulk, as discussed below. On theother hand, c-PheAla is an educated guess as a resultof a thermal process that eliminates water and makes the parent iondisappear from the measured mass spectrum. For these reasons, ourtheoretical investigation has been extended to the three species l-neutral, l-zwitterion, and c-neutral. A comprehensive theoretical assignment of IR bands is listedin the SI, while the key features willbe discussed here.As a preliminary remark, we note that in l-PheAla and the l-zwitterion simulation, as well as between that of r-PheAla and the c-neutral simulation. Regardingthe region of high-frequency bands involving the C–H, N–H,and O–H stretching modes, the general blueshift of all calculatedbands is due to two concurring effects, namely, the lack of anharmoniccontributions and of intermolecular H-bonds.51 The latter is particularly significantin the case of NH- and OH-containing groups while barely affectingCH-containing groups. Even with these limits, the simulated spectracontain a broad contribution in the region of 2800–3200 cm–1 assigned to CH stretching modes, consistently observedin the experimental results (cf. the same regionof the Raman spectra) as unperturbed by thermal treatment of the sample.On the other hand, the NH contribution appears as a doublet in thesimulated l-zwitterion (3440 and 3510 cm–1) and in the measurement of l-PheAla (≈3300and 3346 cm–1) and as a singlet in the simulated c-neutral (3570 cm–1) and the measured r-PheAla (3315 cm–1). The OH band at 3610cm–1 suggests the presence of trapped water in l-PheAla, rather than the −COOH group of the simulated l-neutral molecule, as discussed in more detail below. Particularlyrelevant to the present discussion is the C=O stretching region,which is primarily affected by the formation of the second intramolecularpeptide bond. The spectrum of r-PheAla is indeedcharacterized by a strong band at ≈1660 cm–1, matching well the one assigned to the asymmetric stretching ofthe two C=O bonds at 1684 cm–1 in the simulatedspectrum of the c-neutral. Unfortunately, the c-PheAla sample is not available commercially, making adirect comparison of our TOF-MS and IR spectra impossible. However,measurements and simulations of IR spectra of the most closely resemblingsamples commercially available, i.e., c-AlaGly and c-GlyPhe, show striking similaritieswith r-PheAla. As shown in Figure S2 (SI), the simulated IR spectra of these cyclic dipeptidesare characterized by the same fingerprints of those simulated for c-neutral PheAla and measured for r-PheAla.A comparison among the three simulated IRspectra and the experimentalones is reported in l-PheAla IR spectrum, the weak contributionat 1676 cm–1 corresponds to the C=O stretchingof the NH–C=O group in the l-zwitterion,while the broader peak at a lower wavenumber contains (among others)a strong contribution assigned to the asymmetric stretching of theCOO– group. Therefore, the l-neutralconfiguration of the molecule seems to be definitely ruled out, havingthe contribution of the −COOH group falling at 1776 cm–1, i.e., well above the NH–C=Ogroup (1681 cm–1) and unmatched by any measuredline. The frequencies and assignments discussed above are reportedin –1 confirms the identification of the l-PheAla and r-PheAla samples with the l-zwi and c-neu structures, respectively,as detailed in Figure S2.Regarding the l-PheAlaand r-PheAla, consistent with the predictions for l-zwi and c-neu structures, respectively,confirmed that a structural rearrangement occurred in the solid phasedue to the thermal treatment. This structural rearrangement is fullycompatible with a temperature-driven cyclization in the condensedphase. This result is also consistent with the cyclo peptide formationpreviously reported13 in the temperaturerange of 170–215 °C and with the mass spectra of dipeptides.14The analysis of vibrational IR bands in l-PheAla and r-PheAla samples, shown in Tables S1–S3) through the comparison withsimulations. In these spectra, the characteristic Raman lines of the[phenyl–CH2]– group at 1605, 1206, 1029,and 1001 cm–1 have been assigned by means of a closecomparison between the two PheAla measurements in c-GlyPhe (containing [phenyl–CH2]) and c-AlaGly (not containing this group), which are shown inthe SI . The simulations of the Raman spectrum also offerthe possibility to unequivocally assign the contribution listed inthe following. The strongest contribution at 1001 cm–1, accompanied by a weaker satellite at 1029 cm–1, is assigned to a CC stretching of the phenyl ring . Weaker contributions around 1605 and 1200 cm–1 are assigned to a different CC stretching of thephenyl ring  and to more localizedvibrations involving the −CH2– moiety connectedto the phenyl ring .We remark the fact that these experimental bands are insensitive tothe thermal treatment and can therefore be safely assigned to theside [phenyl–CH2]– group, practically unaffectedby cyclization. Once assigned the precise fingerprint of [phenyl–CH2], we can analyze a few specific differences between the l-PheAla and r-PheAla spectra  and to the out-of-planeCC bending of the phenyl ring (502 cm–1). Only thislast contribution survives in the l-PheAla spectrum,assigned to the same phenyl mode at 506 cm–1 inthe l-zwi calculation.Finally,the triplet band in the 400–500 cm–1, rich in several well-resolved bands, is verydescriptive of the different CH stretching modes in the molecule.However, these features barely change between linear and cyclo structures:the region above 3030 cm–1 (above 3150 cm–1 in Raman simulations) is assigned to phenyl stretching modes, whilethe region below is assigned to methyl, methylene, and methyne stretchingmodes. A complete assignment of these bands in the calculated spectraof c-neu and l-zwi configurationsis also reported in the SI .As anticipated in theprevious section, the region of 2800–3200cmr-PheAla are fully compatible with those observedfor similar cyclo species, the Raman spectra of the pristine c-GlyPhe and c-AlaGly samples have beenmeasured and compared with the simulated c-neu(GlyPhe)and c-neu(AlaGly) spectra. The corresponding results,reported in the SI, support once againthe hypothesis that l-PheAla has undergone a cyclizationprocess.As in the case of IR spectra, to confirm that the features observedin l-PheAla (7.6%) expectedfor a 100% efficient “intramolecular” peptide bond formation,which releases a water molecule for each linear dipeptide, which undergoescyclization. It follows that in the TOF-MS measurements, the surgeof the water peak at about 85 °C corresponds to the water releasedue to peptide bond formation, which then results in the appearanceof the m/z 218 (c-PheAla parent ion), while there is no trace of the parent ion ofthe l-PheAla species (m/z 236). As opposed to previous works on mass spectra ofdipeptides,16 in this specific dipeptide and experimental conditions,the cyclization process involves ≈100% of the sample, and wenever observed the l-PheAla parent ion.After this fine spectroscopic comparison, we can reinterpretTGAresults on more solid ground: the mass loss of ≈(6 ± 2)%observed at 120 °C in the TGA is consistent with the mass ratioof the water molecule and l-PheAlaoccurs in the condensed phase, we now focus on the mechanism leadingto such cyclization. Let us reconsider first the spectroscopic resultsdiscussed in detail above. Starting from about 65 °C, we observewater released from the pristine sample, up to the substantial burstat 85 °C. Interstitial water stabilizes the zwitterionic crystalstructure of PheAla, and its initial slow release favors a rearrangementin which linear neutral molecules may tend to coil, especially onthe surface of the sample where the zwitterion is only partially screenedby surrounding molecules and water molecules are steadily extractedfrom the sample. In this phase of the heating process, protons mayeven rearrange in the molecular network to form neutral linear peptides.However, the lack of any fingerprint of l-neutralmolecules in the IR and Raman spectra of both l-PheAlaand r-PheAla samples indicates that such a configurationcan be only an intermediate stage between 65 and 85 °C, precedingthe actual cyclization. The large production of water molecules at85 °C indicates a bulk process compatible with the cyclizationof the majority or even all the molecules.Onceestablished that the cyclization of Building on thisground, the cyclization has been further investigatedby simulations based on density functional theory. We note that thedescription of a reaction coordinate involving a dipeptide in a solid-stateenvironment, characterized by strong, anisotropic H-bonds betweenmolecules, and also involving water molecules as possible playersis out of reach for accurate, quantitative calculations. Our forcedchoice has been to model the process by considering a single moleculeembedded in an isotropic dielectric environment, which can stabilizethe initial zwitterion configuration. The significant effect of watermolecules on the process has been taken into account by calculatingreaction barriers in the presence or absence of a single water molecule.et al.(2–C(OH)2–NHintermediate (path 2). A third, intermolecular path (path 3 below)is characterized instead by the active participation of a single watermolecule, serving as a bridge for proton transfers through the formationof transition states involving six-membered rings, separated by thesame locally stable CH2–C(OH)2–NHintermediate of path 2.Recently, Li et al. investigSI. The same approach has then been applied to the l-PheAla dipeptide, and the results for the three paths are shownin Our computational approach has beenfirst tested in the case ofthe cyclization of the GlyGly dipeptide, yielding results consistentwith those previously obtained in ref , as disc3+ to COO–, with a small barrier of 0.1 kcal/mol  and other fragments attributable to its fragmentationhave been observed. The comparison of the measured IR and Raman spectraof the pristine l-PheAla and r-PheAlasamples with the simulated spectra of the l-zwitterion, l-neutral, and c-neutral PheAla structuresallowed us to establish that the pristine sample is in its zwitterionicform and that all the main spectroscopic features of r-PheAla are fully compatible with those typical of a cyclo dipeptidespecies. Such a compatibility is confirmed by further comparison withthe IR and Raman spectra of commercially available, very similar compoundslike c-GlyPhe and c-AlaGly. Allthese pieces of evidence lead to the sound statement that a thermallyinduced cyclization occurs before sublimation of l-PheAla in vacuum, i.e., a massive process involvingthe bulk occurs in the condensed phase. A theoretical investigationof the cyclization reaction mechanism indicates that even a singlewater molecule plays an essential catalytic role to lower reactionbarriers, which are otherwise too high to be compatible with a thermallyinduced cyclization process. Preliminary results also suggest a complementaryrole of cooperative peptide–peptide and water–peptideintermolecular interactions in the further lowering of the energybarrier. Since the investigated mechanism does not require the presenceof activating agents or chemical precursors but only a single watermolecule to halve the potential energy barrier, it provides an effectivestrategy to protect peptides, converting them from the fragile, bycomparison, l-structure into the more robust andstable c-structure, thus preserving the amino acidsequence from further decomposition in harsh environments. Hence,if the l-PheAla dipeptide can be generated abioticallyby coupling the two constituent amino acids, then this cyclizationprocess may occur spontaneously on interstellar objects like cometsand carbonaceous chondrites experiencing thermal alterations duringtheir travel in space, as well as in the early Earth after the interactionwith the primordial terrestrial environment.A combined experimental and theoretical study has been carriedout to shed light on the thermally induced cyclization of the l-dipeptides in thesolid phase may lead to the formation of peculiar self-assembled 2Dnanostructures, while usually, the linear molecules tend to form amorphousfilms under the same conditions. In light of further development ofinnovative techniques for the preparation of nanomaterials based onoligopeptides, the ability to handle thermally induced cyclizationprocesses is useful to control the properties of such nanomaterialsvia the growth of specific nanostructures triggering different self-assemblymechanisms, corresponding to different starting peptides.Finally, the cyclizationof


Deciphering paleoclimate on Mars has been a driving goal of Martian science for decades. Most research has addressed this issue by studying Mars' large polar layered deposits (PLDs) as a paleoclimate proxy, but the certainty to which we know the link between climate and orbit is debated. Here, we instead consider the record of other, smaller ice deposits located within craters separated from the PLDs using images from NASA's High Resolution Imaging Science Experiment camera and signal processing techniques. We show that the climate record in Burroughs Crater  contains robust evidence of orbital forcing, with periodicities that have wavelengths of 15.6 and 6.5 m. The ratio of these dominant wavelengths is 2.4, the same as the ratio between the periods of Mars' obliquity changes and orbital precession. This result suggests orbital control of recent Mars climate, and would imply an average ice accumulation rate of 0.13 mm/yr over 4.5 Myr in this region. The icy record in Burroughs crater shows Martian climate is strongly controlled by astronomical forcingThe average ice accumulation rate in this region was 0.13 mm/yrThe deposit in Burroughs crater represents a younger, more easily decipherable climate record than that in the SPLD Here, we consider ice deposits located within impact craters that are smaller than, and separate from, the PLDs. We argue that these crater deposits represent records in which orbital signals could be more straightforward to identify because they are at lower latitudes than the PLDs and thus might be more sensitive to obliquity variations. These deposits may also represent relatively young stratigraphy that has undergone fewer cycles of accumulation and ablation compared to the PLDs. Previous work has identified 18 crater ice deposits in the northern hemisphere , we found a spectral structure similar to a lag‐1 autoregressive process, a type of red noise relevant in climate records  consistency of visible layers around the perimeter of the deposit in CTX images, (b) the detection of internal layering in SHARAD radargrams, and (c) the significant matches between different pieces of Burroughs stratigraphy quantified by dynamic time warping is that local visible layer exposures and the subsequent paleoclimate analysis on them are representative of the Burroughs ice deposit as a whole.4.2Why were we able to identify a ratio of periodicities that exactly matched orbital periodicities in the Burroughs ice deposit, when similar studies of the PLDs (Becerra et al., We found that a statistically significant match does not exist between visible layers at Burroughs crater and layers exposed in the SPLD, using the same 90% threshold described in Section −11 yr−1 km−2, and the ice deposit has total surface area approximately 4,300 km2. The probability p that a surface area A which experiences cratering rate N does not form any craters over a time t is given by:To constrain the surface age of the Burroughs ice deposit, we analyzed the size‐frequency distribution of superposed craters. We searched THEMIS and CTX images and determined there are no craters with diameter 300 m or greater superposed on the Burroughs ice deposit. Inferred cumulative cratering rates (Hartmann, We propose that a hypothesis advanced by Becerra et al.  is corre5We conclude that an ice deposit in Burroughs crater reveals that recent Martian climate has been strongly orbitally controlled. The average accumulation rate at Burroughs has been 0.13 mm/yr, which implies that the visibly exposed stratigraphy formed over 1.4 Myr if no major hiatuses are present in the record. If the exposed stratigraphy is representative of the entire Burroughs deposit, then the ice formed over 4.5 Myr. Finally, if the most recent layer formed near the present‐day as suggested by the superposed cratering record, then the age of the deposit is about 4.5 Ma, which corresponds to a large change in Mars' obliquity. Ultimately, confirmation of absolute ages will require sample analysis either in situ or via sample return, but our results at least show that the onset of ice formation is consistent with strong control by obliquity variation at around 4–5 Ma.Our work shows that non‐PLD ice deposits on Mars represent valuable, readable records of orbitally forced paleoclimate. In particular, south polar climate over the last few Myrs may be recorded most effectively not in the current SPLD, but in separate crater ice deposits. We thus argue that the crater ice deposits are particularly well suited for studying the climate of the late Amazonian. We speculate that lower latitude deposits like that in Burroughs are more readily deposited and removed during orbital cycles compared to the NPLD and SPLD, as non‐PLD reservoirs may act as fundamentally different sources and sinks of ice and dust than the PLDs (Levrard et al., Supporting Information S1Click here for additional data file.


As impairments of prediction may underlie MMN reduction in patients with schizophrenia, an aberrant hierarchical temporal-frontal network might be related to this pathological condition.Sensory prediction is considered an important element of mismatch negativity (MMN) whose reduction is well known in patients with schizophrenia. Omission MMN is a variant of the MMN which is elicited by the absence of a tone previously sequentially presented. Omission MMN can eliminate the effects of sound differences in typical oddball paradigms and affords the opportunity to identify prediction-related signals in the brain. Auditory predictions are thought to reflect bottom-up and top-down processing within hierarchically organized auditory areas. However, the communications between the various subregions of the auditory cortex and the prefrontal cortex that generate and communicate sensory prediction-related signals remain poorly understood. To explore how the frontal and temporal cortices communicate for the generation and propagation of such signals, we investigated the response in the omission paradigm using electrocorticogram (ECoG) electrodes implanted in the temporal, lateral prefrontal, and orbitofrontal cortices of macaque monkeys. We recorded ECoG data from three monkeys during the omission paradigm and examined the functional connectivity between the temporal and frontal cortices by calculating phase-locking values (PLVs). This revealed that theta- (4–8 Hz), alpha- (8–12 Hz), and low-beta- (12–25 Hz) band synchronization increased at tone onset between the higher auditory cortex and the frontal pole where an early omission response was observed in the event-related potential (ERP). These synchronizations were absent when the tone was omitted. Conversely, low-beta-band (12–25 Hz) oscillation then became stronger for tone omission than for tone presentation approximately 200 ms after tone onset. The results suggest that auditory input is propagated to the frontal pole  Such predictive coding is thought to be implemented within hierarchically organized neural circuits. Predictive signals are hypothesized to be sent via top-down connections, whereas prediction error signals derived from mismatches between predictions and actual sensory inputs are returned via bottom-up connections  framework. MMN is a negative deflection of the auditory event-related potential (ERP) elicited by an abrupt change in a sound stimulus after prior repetition of that sound . MMN redPrediction occurs in the absence of the expected tone stimulus . PreviouAuditory MMN is localized to the temporal and frontal cortices. This has been confirmed in various ways, including source estimation in human , 18 and Despite these works, we do not know how prediction signals are communicated between the temporal and frontal cortices, or how prediction errors are computed using predictive and sensory inputs. This is because it is difficult to quantify information propagation in most human recordings, and it is challenging to interpret any observed connectivity. Recent studies have found that feedforward and feedback connections were processed using distinct frequency band channels , 25. ThuMacaca fuscata) weighing 6.0 kg (monkey J), 5.3 kg (monkey D), and 4.9 kg (monkey N) were used. After training the monkeys to sit calmly in a dedicated chair, they were implanted with subdural ECoG electrode sheets in the temporal cortex (160 channels), lateral prefrontal cortex (64 channels), and orbitofrontal cortex (OFC: 32 channels) of the left hemisphere  was controlled using the Benjamini/Hochberg approach.We recorded ECoG data from 256 channels for each monkey during auditory stimulus presentation or omission. ECoG signals were filtered from 0.3 to 500 Hz and digitized at 1 kHz using dedicated hardware . We used EEGLAB software  for offlTo evaluate functional connectivities between electrodes, we computed phase-locking values (PLVs). A PLV denotes the degree of phase difference consistency across trials and shows event-related phase coherence between two data time series . The PLVn denotes the number of trials, e denotes the natural logarithm, i denotes the imaginary unit, and p denotes the phase angle difference in radians of each t trial. To avoid synchrony attributable to noise, we re-referenced all data by subtracting the mean response across each 32-channel connector from the raw response, followed by notch filtering (48–52 Hz) prior to calculating PLVs. We focused on theta- (4–8 Hz), alpha (8–12 Hz), low-beta- (12–25 Hz), high beta- (25–35 Hz), and gamma- (35–60 Hz) band PLVs.where Figures 3, 4). To examine the significance of the PLV difference between tone presentation and omission, we used a circular analog of the Kruskal-Wallis test (circ_cmtest) to determine whether the median phase angle between tone presentation and omission was significantly different for each frequency band . To summarize the areal difference and time course of PLV difference between tone presentation and omission, we calculated the proportion of significant electrodes within a 10 ms time window averaged across 16 channels for 3 representative areas . All statistical analyses were performed using custom scripts written in MATLAB R2014a (MathWorks).We calculated PLVs between 2 temporal seed electrodes and 96 frontal electrodes (2 × 96 electrode pairs). For the anterior temporal cortex, we identified several electrodes that showed greater ERP response during tone omission than during tone presentation. Of these, the electrode located near the edge of the lateral sulcus  was selected as the seed electrode. For the posterior temporal cortex, the electrode placed near A1 (presumably ML) was selected as the seed electrode. We used the Circular Statistics Toolbox for Matlab to statistically test the significance of PLVs and the PLV difference between tone presentation and omission . To examncy band . The PLVt-test, p < 10−6, FDR corrected). Similar patterns were observed for the frontal pole, ventro-lateral prefrontal cortex (VLPFC), OFC, and anterior temporal cortex in the three monkeys.We first calculated ERPs associated with tone presentation and omission by averaging the responses to all event onsets. t-test, p < 10−6, FDR corrected). A late (from 200 ms) gradual increase was evident in the anterior and posterior temporal cortices , which was also slightly evident in the prefrontal cortices. The early positive deflection in the frontal pole was observed in all three monkeys, whereas it was observed in the anterior temporal cortex and OFC in two of the three monkeys . The late gradual increase was observed in all three monkeys . These results suggest that the absence of a predicted auditory input elicits PRSs across a widespread network that includes both the temporal and frontal cortices. As an early positive deflection was observed, we hypothesized that PRSs might be generated in the anterior temporal cortex that is communicated to the frontal pole and OFC.However, the ERPs to tone omission did not exhibit any sharp deflection, the timing of which varied among the cortical areas. An early (0–100 ms) positive deflection was first observed in the anterior temporal cortex, followed by the frontal pole and OFC . The existence of theta-low-beta frequency PLV during omission suggests that it represents a communication relevant to the PRSs. No specific pattern was observed in the high-frequency range. For comparison, vice versa. For the anterior temporal seed, tone presentation PLVs were larger than tone omission in the theta- to high-beta range around 100 ms after tone onset  prediction and prediction error. The omission paradigm allowed us the possibility to distinguish the two because it eliminates the effects of sound differences in typical oddball paradigms. In general terms, responses to only tone presentation may principally be sensory inputs, those to both tone presentation and omission may be predictions, and those to only tone omission may be prediction errors. Gradual positive deflections in the ERPs were observed at both tone presentation and omission and are considered to contain prediction signals. Conversely, the beta-band PLV between the anterior auditory cortex and the frontal pole was larger at tone omission compared with presentation and is considered to contain prediction error signals. In reality, it is difficult to conclude that a response is attributable to one condition and not another. For example, responses to both tone presentation and omission may be caused by sensory inputs and prediction error instead of predictions. This is particularly true for heterogeneous signals, such as those observed in ECoG and EEG. Thus, although the omission paradigm renders it possible to distinguish prediction from prediction error signals, signal assignments are necessarily tentative. Indeed, only a few studies have successfully isolated prediction signals. Ohmae et al.  found prIn this study, we aimed to obtain prediction-related signals that may be related to neuropsychiatric disorder . TherefoIn conclusion, we found communication of PRSs between the anterior temporal cortex and the frontal pole in the omission paradigm, a variant of the typical oddball MMN paradigm. These results may not necessarily extrapolate to humans and thus patients with schizophrenia. However, auditory prediction is considered as one of the important elements of MMN whose reduction is well known in patients with schizophrenia . FurtherThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Animal Care and Use Committee: Niigata University, Tamagawa University, and University of Yamanashi.TSU constructed the electrodes. TMA, KKAW, and IH performed the surgery. YS, TSA, MI, TMI, and HK collected the data. YS, MT, KKI, and TU analyzed the data. YS, MT, and TU interpreted the results and wrote the manuscript.YS, MT, KM, KKAS, and TU designed the study. KM, IH, KKAS, and TU supervised all aspects of data collection, analysis, and interpretation. All authors commented on the manuscript and contributed to and approved the final manuscript.This study was supported by a Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) grant from the Japan Agency for Medical Research and Development (AMED) (No. 15653128 to KKAS and KM and JP19dm0207069 to KKAS and TU).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The “marine ice-sheet instability” hypothesis continues to be used to interpret the observed mass loss from the Antarctic and Greenland ice sheets. This hypothesis has been developed for conditions that do not account for feedbacks between ice sheets and environmental conditions. However, snow accumulation and the ice-sheet surface melting depend on the surface temperature, which is a strong function of elevation. Consequently, there is a feedback between precipitation, atmospheric surface temperature and ice-sheet surface elevation. Here, we investigate stability conditions of a marine-based ice sheet in the presence of such a feedback. Our results show that no general stability condition similar to one associated with the “marine ice-sheet instability” hypothesis can be determined. Stability of individual configurations can be established only on a case-by-case basis. These results apply to a wide range of feedbacks between marine ice sheets and atmosphere, ocean and lithosphere. Using theoretical, numerical and data analyses, this study finds that there are no general stability conditions for marine ice sheets if feedbacks caused by interactions of ice sheets with atmosphere, ocean and lithosphere are taken into account. This hypothesis proposed by Weertman5, suggests that a marine ice sheet that resides on a bed below sea level and slopes towards its interior is “inherently unstable”. He further suggested that “A stable ice sheet can occur if the bed slopes away from the center of the ice sheet.” Weertman arrived to this conclusion by considering an unconfined marine ice sheet flowing into an unconfined ice shelf that gains its mass by accumulating snow at its surface with the accumulation rate constant in space and time. In later studies11, a similar ice-sheet configuration was considered and the same stability condition was derived by means of a linear stability analysis12.The observed mass loss from the Antarctic and Greenland ice sheets associated with the retreat of their grounding lines13; one of them is a feedback between the net accumulation/ablation rate and the ice-sheet height16. The net accumulation/ablation rate at a given location on the surface of the ice-sheet is determined by the difference between snow accumulation and surface melting (ablation). If the annual snow precipitation exceeds annual surface melt  this location experiences net accumulation; if the reverse is true, it experiences net ablation. Both processes, snow precipitation, and surface melting, depend on in situ atmospheric conditions. Snow precipitation depends on the atmospheric moisture content and the surface ablation rate is determined by the surface energy balance. The latter involves a number of processes, e.g., absorption of short- and long-wave radiation, reflection due to albedo, wind, and many others. Both the snow precipitation rate and ablation rate strongly depend on the surface elevation17. Such a dependence produces a feedback between the surface elevation and the net accumulation/ablation rate15.Ice sheets interact with all other components of the Earth system—atmosphere, ocean, sea ice, lithosphere, land surface, and biosphere. These interactions give rise to a number of feedbacks18 have considered the effects of a feedback between the surface elevation and behavior of land-based ice sheets, all existing studies of stability of marine ice sheets21 did not take into account feedbacks between the external conditions and the ice-sheet characteristics. It is, thus, not a priori clear whether the results of these studies remain valid in the presence of feedbacks.While several studiesHere we show that the previously derived stability conditions of unconfined marine ice sheets that disregard the feedback between net accumulation/ablation rate and the surface elevation do not generally hold if the feedback is taken into account. Furthermore, there is no general stability condition which is determined by properties of steady-state configurations at the grounding line. Stability of specific configurations can be determined only individually, on a case-by-case basis. These results apply to other feedbacks in interactions of marine ice sheets with other components of the climate system—ocean (the dependence of sub-ice-shelf melting on ice thickness), continental crust and lithosphere .17. Ablation depends on the (near) surface temperature in two ways—the latter determines whether surface melting occurs (if the surface temperature is above the freezing point), and it determines the magnitude of the surface melting, the ablation rate. Although there are many complex processes that govern interactions between the ice-sheet surface and atmosphere, the atmospheric surface temperature can be used as a sole proxy parameter to estimate both precipitation and surface melting, and consequently the net accumulation/ablation rate.Precipitation is determined by the atmospheric moisture content. A good proxy for the moisture content is the saturation vapor pressure, governed by the Clausius-Clapeyron equation, which in turn, depends on the surface temperatureAtmospheric temperature is a strong function of elevation: it decreases according to the lapse rate as elevation increases. As a result, the net accumulation/ablation rate implicitly depends on the ice-sheet surface elevation via the surface temperature.23. In the absence of the direct continent-wide long-term observations, this has made results of these models’ simulations a useful tool for analysis of the polar atmospheric conditions. For example, results of simulations of the regional atmospheric model, MAR, have been used to construct a parameterization of changes in the net accumulation/ablation due to changes in the surface elevation of Greenland Ice Sheet based on a probabilistic approach24.Recent developments in the regional atmospheric modeling for polar regions have allowed for more accurate representation of the near-surface atmospheric processes25 and Greenland26 are used to construct an empirical relationship between the net accumulation/ablation rate and the surface temperature. The simulated accumulation/ablation rates, T is described by Eq. . Consequently, considering the accumulation/ablation rate of Antarctica and Greenland together, and simulated for the high-end emission scenario, allows construction of an empirical relationship for a wider range of surface temperatures and surface elevations that can simultaneously capture the net accumulation and ablation regimes.Results of MAR simulations Fig. b show a Tsl makes the empirical relationship  bed. According to both a stability condition derived by Schoof12 and a more complex one that accounts for the bed curvature and the gradient of the accumulation/ablation rate 20, this grounding line position is stable.A steady-state configuration of an unconfined marine ice sheet can be described by a solution of the problem  describeionships , is show20 that do not account for feedbacks.These stability analyses, however, have been performed under the assumption that entclass1pt{minimaentclass1pt{minima20 that do not account for feedbacks. This experiment illustrates that it is the presence of the feedback that changes stability of the grounding line from stable if However, if entclass1pt{minima20 for the case of the net accumulation/ablation rate being a function of the position, 20, and considers small, time-dependent perturbations around the steady-state solutions, e.g., 27 and is determined by the sign of the largest eigenvalue—either the fastest growing, in the case of an unstable grounding line position, or the slowest decaying in the case of a stable grounding line position20. In the case of To shed light on such a different behavior of the grounding lines of ice sheets, which accumulation/ablation rates vary either with the surface elevation or only with the distance along the ice sheet, we analyze a simplified problem  used to tary Eq.  indicatementclass2pt{minimResults of a numerical solution of the linear-perturbation eigenvalue problem are illustrated in Fig. Tsl this formulation can be an efficient, physically based parameterization of the net accumulation/ablation for paleo-climate simulations.In order to investigate how feedbacks may affect stability conditions of marine ice sheets, we have constructed an empirical relationship between the surface temperature, which varies linearly with elevation according to the lapse rate, and the net accumulation/ablation rate. The constructed formulation uses the fundamental relationships between the atmospheric surface temperature and precipitation (Clausius-Clapeyron equation) and the surface temperature and ice-sheet surface melting . Expressed in terms of temperature at the sea level, Numerical and analytical analyses of the marine ice-sheet stability performed with this empirical relationship produce results markedly different from those of previous analyses that did not account for any feedbacks. Stability conditions derived for the cases with no feedbacks between the ice-sheet characteristics  and other parameters  do not hold in general if such feedbacks are present. Moreover, in the demonstration of this considered here, there are no general stability conditions that depend on characteristics of the steady-state configurations at the grounding line. In the presence of feedbacks, stability of a particular ice-sheet steady-state configuration can be determined only by either numerically simulating evolution of this configuration perturbed from its steady state, or by solving a perturbation eigenvalue problem to determine the largest eigenvalue, which sign indicate stability  or instability .S), together with the characteristics of the steady-state configuration, determine whether this steady-state configuration is stable or unstable. Changes in net accumulation/ablation rate caused by changes in surface elevation due to small perturbations from steady-state configurations could suppress or amplify these perturbations, and consequently stabilize or destabilize the grounding line.A physical interpretation of these results is the following: If the net accumulation/ablation rate depends on the surface elevation then the specific details of such a dependence , basal conditions  and feedbacks between the ice sheet configuration and atmosphere, ocean, and lithosphere interactions.A number of processes that have feedbacks with the ice-sheet characteristics are in play on the Antarctic and Greenland ice sheetsa1, a2, T0, and σ are fitting parameters and TS is the temperature at the surface elevation S, which isTsl is the temperature at sea level and Γ is the lapse rate. In this study, we use the dry adiabatic lapse rate Γ = 9.8 ∘C/km. In general, surface temperature at the sea level depends on insolation, which is function of latitude. In this study, we disregard this dependence and treat sea-level surface temperature Tsl as constant.The fitting (green) line in Fig. Tsl controls whether an ice sheet can experience net ablation or it experiences only net accumulation. The steady-state configuration shown in Fig. Tsl = −4 ∘C; the corresponding net ablation/accumulation rate shown in Fig. Tsl = −10 ∘C, a steady-state configuration computed with Eqs.  is the depth-averaged ice velocity, h(x) ice thickness, B(x) is bed elevation (negative below sea level and positive above sea level), S = h + B is the surface elevation, An−1/ is the ice stiffness parameter (assumed to be constant), n is the exponent of Glen’s flow law, g is the acceleration due to gravity, C and m = 1/n, xd and xg are positions of the ice divide (taken here to be xd = 0) and the grounding line, respectively, and TS described by  with  instead entclass1pt{minimaSupplementary InformationPeer Review File


D-arabinitol (DAB) or 1,4-dideoxy-1,4-imino-L-arabinitol (LAB) moiety held together via linkers of variable lengths containing a 1,2,3-triazole ring and 3, 4, or 7 CH2 groups. The heterodimers were tested as inhibitors of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE). The enantiomeric heterodimers with the longest linkers exhibited the highest inhibition potencies for AChE (IC50 = 9.7 nM and 11 nM) and BuChE (IC50 = 8.1 nM and 9.1 nM). AChE exhibited the highest enantioselectivity (ca. 4-fold). The enantiomeric pairs of the heterodimers were found to be inactive (GI50 > 100 µM), or to have weak antiproliferative properties (GI50 = 84–97 µM) against a panel of human cancer cells.The copper-catalysed azide-alkyne cycloaddition was applied to prepare three enantiomeric pairs of heterodimers containing a tacrine residue and a 1,4-dideoxy-1,4-imino- A more surprising observation (given that the quaternary ammonium group of ACh participates in a cation–π interaction with a Trp residue in CAS) from the modelling studies was that when heterodimers 6 and 8 bind to AChE in their protonated states (on the 1-DNJ nitrogen atom), the positive charged nitrogen atom is not necessarily involved in cation − π interactions with the aromatic residues of the enzyme,The ester group of ACh is held in place for hydrolysis by the catalytic triad in the active gorge of AChE by the aid of cation − π interactions with a Trp residue in CAS9a and 9b, 10a and 10b, and 11a and 11b (D-arabinitol (DAB) (12a) constitutes the iminosugar moiety in 9a, 10a, and 11a, whereas non-natural 1,4-dideoxy-1,4-imino-L-arabinitol (LAB) (12b) is the iminosugar moiety in 9b, 10b, and 11b. Because both iminosugars,11a and 11b against a panel of six cancer cell lines.Thus far, five papers have been published, which demonstrate the potential of iminosugars as ChE inhibitors and 11b  and the 254 plates using UV light (λ = 254 nm) for detection. Silica gel NORMASIL 60® 40–63 µm was used for silica flash columns. A Bruker Avance NMR spectrometer was used to record 1H-NMR spectra (400.13 MHz) and 13C-NMR spectra (100.61 MHz) in CDCl3, CD3OD, or D2O. Chemical shifts (δ) are reported relative to residual DMSO , residual CHCl3 in CDCl3 , residual CD3OD , residual D2O  and TMS as an internal standard in CDCl3. High-resolution mass spectra (HRMS) were recorded on a Qexactive spectrometer in positive electrospray ionisation (ESI) mode.Dichloromethane (DCM), methanol (MeOH), acetone, dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) were dried over 4 Å molecular sieves (oven dried). Petroleum ether (PE) from the 40–65 °C fraction was used for silica flash columns. All reactions were carried out under Ar atmosphere if not otherwise specified. Reactions performed at room temperature (rt) refer to the temperature range of 20 to 22 °C. TLC analyses were performed on Merck silica gel 60 F19b  or 19a , azide 13 (0.98 equiv.), and copper(II) sulphate pentahydrate (0.30 equiv.) in anhydrous DMF in an aluminium foil covered round bottom flask was degassed and introduced an argon atmosphere before the addition of sodium ascorbate (0.60 equiv.). After addition, the mixture was kept stirring for 48 h at rt. The solvent was then removed under reduced pressure and the residue obtained was purified by silica gel column chromatography .A mixture of 20a–22a  or 20b–22b  in anhydrous CH2Cl2 under an argon atmosphere at −78 °C was slowly added BCl3 . After addition, the mixture was kept stirring at −78 °C for 2 h and then at 0 °C overnight. The volatiles were then removed under reduced pressure and the concentrate underwent purification by gradient silica gel chromatography (MeCN/H2O/NH4OH 190:10:1 → 180:20:1) (column 1). The corresponding HCl salt was dissolved in MeOH (2 ml) and NH4OH (0.5 ml) and kept stirring for 48 h. The solvent was removed under reduced pressure and the resulting residue was purified by gradient silica gel chromatography (column 2) .To a mixture of 9a–11a and 9b–11b against cholinesterases (AChE from Electrophorus electricus and BuChE from equine serum) was accomplished following minor modifications of the Ellman assayTM VarioskanTM LUX microplate reader and Greiner F-bottom 96-well plates were used. Cornish-Bowden plots (1/V vs. [I] and [S]/V vs. [I]) were used for the visualisation of the mode of inhibition. Calculation of the kinetic parameters  was accomplished using a nonlinear regression analysis (least squares fit) implemented in GraphPad Prism 8.01 software; such parameters were in turn used for calculating the inhibition constants of the mixed inhibitors using the following equations:Measuring of the inhibition activity of compounds Interactions of enzymes with compounds were analysed by computational docking using Molecular Operating Environment (MOE) software . Crystallographic structures of human AChE and human BuChE was obtained from Protein Data Bank  screening protocol9a, 10a, and 11a commenced from L-xylose (14a), which was converted into 2,3,5-tri-O-benzyl-L-xylofuranose (15a) by following a reported three step procedure  mesylation to provide compound 16a16a was treated with F- ions it cyclized into nitrone 17a,O-silyl group. Tetra-O-benzylated DAB 18a was obtained in 82% yield when nitrone 17a was reduced first by sodium borohydride and followed by zinc in acetic acid18a underwent N-propargylation to form alkyne 19a when it was treated with propargyl bromide. This alkyne underwent copper-catalysed azide – alkyne cycloaddition13a13b13c20a, 21a, and 22a, respectively. In the final step, heterodimers 20a, 21a, and 22a underwent BCl3 promoted de-O-benzylation to generate target compounds 9a, 10a, and 11a, respectively.The synthesis of heterodimers rocedure 40. The o9b, 10b, and 11b were performed in the same way as for 9a, 10a, and 11a by replacing L-xylose (14a) with D-xylose (14b) in the first step  of eeAChE and eqBuChE are presented in 50 values2a) and tacrine as positive references.The minimum inhibitory concentrations of the enantiomeric pairs 9a–11a (incorporating a DAB moiety) and 9b–11b (incorporating a LAB moiety) displayed IC50 values from the submicromolar concentration range down to the nanomolar concentration range for the inhibition of eeAChE and eqBuChE. The only exception was 9b, which exhibits a IC50 value of 1480 nM for the inhibition of eeAChE. Thereby, 9b was the only compound in the testing series that is a less potent AChE inhibitor than (-)-galantamine, which is in in current use against ADBoth series of stereoisomers eeAChE and eqBuChE in which a longer linker provided higher inhibition potencies. This was demonstrated by the result that 11a  is a ca. 43-fold more potent eeAChE inhibitor than 9a  and a 55-fold more potent eeAChE inhibitor than 10a . A similar trend was observed when the enantiomer of 11a, namely, 11b  was compared with 9b  and 10b  for the inhibition of the same enzyme as 11b is a ca. 138- and 14-fold stronger inhibitor than 9b and 10b, respectively. Six CH2-groups between the 1,2,3-triazole and tacrine moiety was also most favourable for the inhibition of eqBuChE as 11a  is a roughly 11-fold stronger inhibitor than 9a  and a 20-fold stronger inhibitor than 10a . Likewise, 11b  is a 22- and 29-fold stronger eqBuChE inhibitor than 9b  and 10b , respectively.The length of the linker between the tacrine ring and iminosugar moiety had a significant impact on the inhibition potency of both eeAChE and eqBuChE was observed for the three pairs of enantiomeric inhibitors included in this study. In addition, no preferential inhibitory activity trend was found for the enantiomers incorporating a DAB or LAB moiety. For instance, 9a is a ca. 4-fold more potent eeAChE inhibitor than its enantiomer 9b, whereas 10b is a ca. 4-fold more potent eeAChE inhibitor than its enantiomer 10a. For the enantiomeric pair 11a and 11b, we observed essentially equal eeAChE inhibitory activities. These observations indicate that the impact on the eeAChE inhibitory potency of our heterodimers by switching between a DAB and LAB moiety is small compared to the contribution from the tacrine ring.No obvious enantioselectivity of eeAChE and eqBuChE by heterodimer 11b were investigated by using the Cornish-Bowden method, that is, by creating two plots (1/V vs. [I] and [S]/V vs. [I]) for the inhibition of both enzymes  are presented in 9a and 9b, and 10a and 10b are presented in Figure SI2 and Figure SI3, respectively. A common trend for all energetically preferred binding poses is that the tacrine moiety and the iminosugar moiety bind to the active site and PAS, respectively. Such preferred binding pose is not very surprising given that X-ray analysis has shown that tacrine is bound to the active site of AChEThe preferred binding poses for enantiomers rhAChE are observed in both 11a  compared to its antipode 11b   and between 10a and 10b  when they are bound to rhAChE. The hydroxyl groups of 9a showed interactions with Tyr341 and Arg296 meanwhile there is an arene cation interaction between one of the hydroxyl groups in 9b and Trp286 . Hydrogen bond interaction between Ser293 and the iminosugar moiety is observed for 10a but is lacking in its antipode 10b .Hydrogen bonding interactions between one of the hydroxyl groups of the iminosugar moiety and Ser293 in both 11a  and 11b 11a  . Slight 50 values  in our series.We found that our measured IC0 values  for the energies , which p11a and 11b to human butyrylcholinesterase (hBuChE) are presented in 9a and 9b, and 10a and 10b to the same enzyme are presented in Figures SI5 and SI6, respectively. The number of CH2-groups between the tacrine ring and 1,2,3-triazole ring appears to control whether the iminosugar moiety is bound to the active site or PAS. In fact, the tacrine ring of 9a, 9b, 11a, and 11b is accommodated in the active site whereas their iminosugar moiety is bound to PAS. For heterodimers 10a and 10b the binding scenarios are different, as the tacrine ring is accommodated in PAS and the iminosugar moiety in the active site. As for the inhibition of eeAChE, even though the calculated binding energies in 11a and 11b to be the most potent BuChE inhibitors, they fail in predicting the relative IC50 values for the whole testing series.The most energetically favourable binding poses of enantiomers 50). The antiproliferative activity of a compound is only significant when GI50 ˂ 100 μM. The measured GI50 values demonstrated that those heterodimers with two CH2-groups (9a and 9b) and three CH2-groups (10a and 10b) between the tacrine and 1,2,3-triazole rings display no significant antiproliferative activity (GI50 > 100 μM). 11a and 11b on the other hand that contain six CH2-groups between the tacrine and 1,2,3-triazole rings display GI50 values below 100 μM for the inhibition of A549 cancer cell growth  for the inhibition of eeAChE and eqBuChE. The following three observations: (1) 9a is a ca. 3.5-fold stronger eeAChE inhibitor than 9b, (2) 10a is a ca. 3.5-fold less potent eeAChE inhibitor than 10b, and (3) 11a and 11b are essentially equipotent eeAChE inhibitors, show that eeAChE exhibits no consequent preference for any of the enantiomeric heterodimers, which include a DAB or LAB moiety. These observations can either be interpreted as the tacrine moiety contributes much more to the eeAChE inhibitory potencies than the DAB or LAB moieties or that the LAB and DAB moieties have similar contribution to the inhibition potencies when they are connected to a tacrine ring. However, the latter interpretation is to some extent contradicted by the modelling studies, which show that the DAB and LAB moieties display different interaction modes with the enzymes.In contrast to the enantiomeric pairs 7 in Figure 211a and 11b with the longest linkers exhibited the highest inhibition potencies. From modelling studies for the binding to BuChE, it appeared that the length of the linker between the tacrine ring and DAB or LAB moiety controls whether the tacrine ring binds to the active site of PAS. On the other hand, because the trend of the measured IC50 values do not parallel the calculated binding energies, it is possible that the title compounds are not bound in their most energetically favourable poses when they inhibit the enzymes in our testing series.Like in our earlier studies when we connected an iminosugar to a tacrine ring to obtain ChE inhibitors of type Click here for additional data file.


Porphyromonas gingivalis is considered the major etiological agent of this disease, having a plethora of virulence factors, including, lipopolysaccharides (LPS), hemolysins, and proteinases. Antimicrobial peptides are one of the main components of the innate immune response that inhibit the growth of P. gingivalis. The aim of this study was to analyze the antimicrobial activity of cystatin C and to assess the effect on the inflammatory and anti-inflammatory cytokines, the production of reactive oxygen species, and in the release of nitric oxide by human gingival fibroblasts incubated with P. gingivalis in the presence and absence of cystatin C.Periodontal disease is considered one of the most prevalent chronic infectious diseases, often leading to the disruption of tooth-supporting tissues, including alveolar bone, causing tooth mobility and loss. P. gingivalis ATCC 33277 was exposed to cystatin C for 24h and co-cultured with human gingival fibroblasts (HGFs) ATCC CRL-2014. The effect of cystatin on growth of P. gingivalis and HGFs was evaluated. Pro-inflammatory  and anti-inflammatory (IL-10) cytokines were determined by ELISA in the supernatants of HGFs incubated with P. gingivalis exposed to cystatin C. Additionally, nitrites and reactive oxygen species (ROS) production were evaluated.P. gingivalis without affecting HGFs. Incubation of HGFs with P. gingivalis led to a significant increase of TNF-α and IL-1β. In contrast, HGFs incubated with P. gingivalis exposed to cystatin C showed a decreased production of both cytokines, whereas IL-10 was enhanced. Incubation of HGFs with P. gingivalis led to an increase of nitric oxide (NO) and ROS production, which was reduced in the presence of the peptide.Cystatin Cinhibited the growth of P. gingivalis. Knowledge on the antimicrobial and immunomodulatory properties of cystatin C could aid in the design of new therapeutic approaches to facilitate the elimination of this bacterium to improve the treatment of periodontal disease.Cystatin C inhibits the growth of P. gingivalis and decreases the inflammatory cytokines, ROS, and NO production during infection of HGFs with  Porphyromonas gingivalis is a periodontopathogen bacterium implicated as a major, etiological agent in periodontitis , where they participate in the repair of periodontal tissues during inflammatory periodontal diseases . GF alsoPorphyromonas gingivalis increases their superoxide concentrations after the exposure to human gingival fibroblasts (HGFs)  . Thus, ts (HGFs) .α and IL-1β are the major secreted pro-inflammatory cytokines, that are important markers of periodontitis progression and severity. They are also the main inducers of effector molecules that cause the breakdown of periodontal tissues , which are involved in the extracellular matrix degradation and, in turn, bone resorption and periodontal tissue destruction  generation in periodontal tissue  was significantly enhanced, these results indicate that oxidative stress can be induced during periodontitis  and lysine-specific (Kgp) proteinases. Additionally, these cysteine proteases exert potent immunomodulatory effects on human gingival fibroblasts. The main causative factor of tissue damage involved in the disease progression, could be the gingipains of the bacterium ,even though P. gingivalis is considered an opportunistic pathogen. Thus, control of proteolytic enzymes of P. gingivalis could represent an interesting target for the treatment of periodontitis  are part of the innate defense system in the oral cavity, where cystatins play an important role. Cystatin C belongs to the type 2 family of the cystatin superfamily, it is ubiquitously distributed in plants and animals . In the P. gingivalis in order to be able to evaluate its potential therapeutic use against one of the main etiological agent causing periodontitis, as well as its potential impact on the severity of periodontal disease.The aim of this study was to assess the effect that cystatin C exerts on cytokine production, NO and ROS production by human gingival fibroblasts incubated with 3 cells per cm2 and cultured in 75 cm2 culture flasks in a water saturated atmosphere at 37 °C and 5% CO2 and maintained in Dulbecco’s modified Eagle high glucose medium , supplemented with 10% fetal bovine serum , containing 10 U penicillin/25 µg streptomycin/mL (Sigma Aldrich). The fibroblasts were cultured to confluence, at a density of 2.5 × 105 cells/mL, washed twice with phosphate-buffered saline, and dissociated with 0.25% trypsin and 1 mM EDTA for 5 min at 37 °C, 5% CO2 . The cells were used at passages 3–7.Human gingival fibroblasts (HGFs)  were seeded at a density of 5 × 10P. gingivalis strain ATCC 33277 was cultured in brain-heart-infusion and in broth-heart-brain extract  containing 5 µg/mL of hemin  and 1 µg/mL of menadione (Sigma-Aldrich) under anaerobiosis using the anaerobic BBL-GasPak jar system (BD Biosciences) at 37 °C for 24 h.4, 1.7 mM KH2PO4, 8.3 mM Na2HPO4, 10 mM glucose, and 1.1 mM CaCl2, pH 7.3). Bacterial growth was monitored spectrophotometrically  at 675 nm. The bacterial density was visually adjusted to a turbidity of 0.5 McFarland   buffer  . EthicalPichia Pastoris  and reconstituted in Tris Base NaCl Buffer (pH 7.4). Minimum inhibitory concentrations (MIC) of Cystatin C was determined using the microdilution method in 96-well microtiter plates   containing KRG Buffer was placed in each well. Subsequently, different cystatin C concentrations  were incubated with the bacteria, for 1, 12, 24, and 48 h, under anaerobiosis conditions, at 37 °C. After the incubation period, 20 µL of Presto Blue Cell Viability Reagent  per well were added. The plates were incubated for 30 min at 37 °C in the dark. Finally, the plates were read in a microplate reader (Multiskan SkyHigh Microplate Spectrophotometer), at a 675 nm wavelength.Lyophilized Cystatin C was obtained from ciences) . Briefly5 cells/well in 24-well plates for 24 h, at 37 °C with 5% CO2. Different concentrations of Cystatin C  were added and incubated for 24 h. After incubation time, 25 µl of XTT/PBS solution (4 mg/4 mL) were added per well, for 40 min at room temperature, in the dark. Subsequently, microplate plates were read at a wavelength of 450 nm in a microplate spectrophotometer (Multiskan SkyHigh Microplate Spectrophotometer).HGFs were seeded at a density of 1 × 105/well, were cultured in a Costar® 24-well plate  in D-MEM medium at 37 °C in an atmosphere of 5% CO2. After the incubation period, fresh medium without antibiotics was added to HGFs, before they were treated with P. gingivalis. HGFs were stimulated with bacteria, at multiplicities of infection (MOI) of 1:100 for 24 h, and with cystatin C at a concentration of 0.3 µg/mL at 37 °C for 24 h, to perform cytokine assays, and evaluate ROS, and NO. Control groups include HGFs without stimulation or stimulated with LPS and peptidoglycans.Human gingival fibroblasts, at a seeding density of 5 × 10P. gingivalis (MOI 1:100) and /or cystatin C at a concentration of 0.3 µg/mL at 37 °C for 24 h. Control groups included HGFs without stimulation or stimulated with LPS 100 ng/mL , or with peptidoglycan 10µg/mL . ELISAs were performed to determine TNF-α, IL-1 β, and IL-10, using the Ready-Set-Go! ELISA kits , according to the manufacturer’s protocol. Dilutions were prepared in dilution buffer. Briefly, 96-well flat-bottom plates  were coated with anti-human TNF-α, IL-1 β, or IL-10 monoclonal antibodies . After blocking with the assay solution (PBS−0.5% casein diluted in 1 M NaOH) overnight at 4 °C to avoid non-specific binding, 100 µL of standard TNF-α, IL-1 β, or IL-10  of supernatants were added. The microplate was washed to remove unbound enzyme-labeled antibodies. The amount of horseradish peroxidase bound to each well was determined by the addition of a substrate solution. The reaction was stopped by the addition of sulfuric acid and the plates were read at 405 nm .For cytokine assays, HGFs were incubated with The cytokine concentration was calculated by regression analysis from a standard curve. The detection limit of the assay was 15 to 2000 pg/mL.P. gingivalis and/or cystatin C at 37 °C was assayed by measuring the accumulation of nitrate in culture supernatants. Briefly, HGFs were stimulated with P. gingivalis (MOI 1:100) and with 0.3 µg of cystatin C, at 37 °C for 24 h. Thereafter, 100 µL of Griess reagent  (Sigma Aldrich) were added at equal volumes of culture supernatants in a 96- well plate  and left at room temperature for 30 min. The absorbance of these supernatants was read at 550 nm (Multiskan SkyHigh Microplate Spectrophotometer) and the nitrate concentrations were calculated from a standard curve established with serial dilutions of NaNO2 (Sigma-Aldrich) in the culture medium. Control groups included HGFs without stimulation or stimulated with LPS or peptidoglycan.The NO production by HGFs incubated with 5), infected with P. gingivalis (MOI 1:100) and stimulated with 0.3 µg/mL of cystatin C at 37 °C for 24 h. The cells were incubated with 100 µg/ mL of 2,7 dichlorodidrofluoroescein diacetate (H2-DCFDA) [2 µM/mL] for 30 min in the dark at room temperature. Cells were rinsed twice with PBS, pH 7.2 and detached from the wells with 0.25% Trypsin/EDTA (Sigma Aldrich). The samples were resuspended in PBS, pH 7.2, with 1% FBS and analyzed on a FACS Canto II BD Biosciences flow cytometer. Data analysis was performed using FlowJo software (USA). Control groups included HGFs without stimulation or stimulated with LPS or peptidoglycan.HGFs were seeded on 24-well plates  at a density of .Experimental and control conditions were statistically compared for significance using analysis of variance (ANOVA), followed by Benferroni correction. The predetermined level of significance was P.gingivalis was analyzed in a time and dose-dependent manner as shown in . Inhibition of bacterial growth (83.3%) was observed after 48 h of culture (p < 0.05). At a concentration of 0.9 µg/mL a marked growth inhibition was observed throughout the incubation time. All the analyzed concentrations of cystatin C showed no effect on the viability of HGFs cell, as illustrated in  for 24 h. P. gingivalis induced the production of 1000 pg/ mL and 750 pg/mL of TNF-α and IL-1 β, respectively, when compared to the control group (p = 0.0001) (α ( p = 0.0001) and IL-1 β (p < 0.05) productions, compared to HGFs. In contrast, no changes were observed in IL-10 production by HGFs incubated with P. gingivalis alone, when compared to controls, whereas cystatin C stimulated de production and secretion of IL-10 (500 pg/mL). Furthermore, the co-incubation of P. gingivalis with cystatin C significantly increased the production of IL-10 (900 pg/mL), when compared with the control group and with HGFs infected with the bacterium (p = 0.0001), (TNF- 0.0001)  and 2B. 0.0001), . These rP. gingivalis, compared to the controls (p = 0.0001). No significant differences were observed in the production of ROS in HGFs incubated with cystatin C (p > 0.05) , (A significant increase was observed in the production of ROS and NO in HGFs incubated with  > 0.05) . In cont 0.001), .P. gingivalis, when compared with the control group (p = 0.0001). Yet when HGFs were incubated with P. gingivalis and cystatin C, a decrease of NO (3 µM) (p = 0.001) was observed with regard to the incubation with P. gingivalis alone  was observed after the incubation of HGFs with is alone .P gingivalis, which contributes to the development of chronic periodontitis. The immunological responses occurring in HGFs after the infection with this key periodontal pathogen were evaluated. P. gingivalis exhibits a variety of virulence factors that enable it to colonize oral soft tissues and evade immune responses. It has been demonstrated that P. gingivalis triggers and suppresses the immune responses in HGFs, suggesting that the pathogenic effects of P. gingivalis are mainly related to the action of gingipains, which participate in the inflammatory and immune response of HGFs , which constitute its main virulence factors. Two gingipains are specific for Arg-Xaa peptide bonds (HRgpA and RgpB), whereas Kgp cleaves after a Lys residue , belongs to the IL-1 family and plays an important role against microbial infections and participates regulating innate immune and inflammatory responses. The upregulation of IL-1 β during P. gingivalis infection suggests that IL-1 β is a critical cytokine in the host’s defense against P. gingivalis infection during the initial phases of inflammation  Cell viability of HGFs. The results are expressed as mean ± SD of the average of five independent assays. Statistical differences are expressed as (*) p < 0.05 when compared to non-treated control P. gingivalis bacteriaEffect of cystatin C on a) Click here for additional data file.


Inhibiting the activity of tyrosinase is a very effective and safe way to prevent enzymatic browning in food and to resist pests in agriculture. Tea polyphenols (TPs), regarded as safe and non-toxic food additives, have been reported due to their potential inhibitory capability against tyrosinase, but their ambiguous inhibitory mechanisms have severely limited their application. In the present work, fluorescence spectroscopy, cyclic voltammetry (CV), oximetry and molecular simulation approaches were employed to shed light on the underlying inhibitory mechanisms of TPs with different structures including (+)-catechin, (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG) against tyrosinase. Fluorescence spectra show that the three TPs are capable of binding tyrosinase with a molar proportion of 1 : 1. The analysis of CV curves and oxygen utilization suggests that these three TPs can be oxidized by tyrosinase, revealing that these three TPs are suicide inhibitors of tyrosinase. Furthermore, ECG and catechin make tyrosinase irreversibly inactivated due to their catechol group (ring B) being catalyzed by tyrosinase through a cresolase-like pathway, while EGCG inhibits the activity of tyrosinase by competing with or delaying the oxidation of substrate. Molecular simulations further confirm that ring B of ECG and catechin makes a significant contribution to tyrosinase inhibitory activities, and has a direct interaction with the coupled binuclear copper ions in the optimal orientation required by the cresolase-like pathway. Revealing the inactivation mechanism of TPs on inhibiting the activity of tyrosinase. Although a plenty of natural tyrosinase inhibitors have been discovered, the understanding of the inhibition mechanism of tyrosinase inhibitors is still deficient, which have severely limited their application in food industry and agriculture and the development of new inhibitors.8–11Tyrosinase (EC 1.14.18.1) containing a catalytic center with bi-copper ions coordinated by six histidine residues, can catalyze the oxidation of both monophenol (cresolase or monophenolase activity) and diphenol (catecholase or diphenolase activity) to the corresponding 12 are the largest groups in natural tyrosinase inhibitors which have been discovered,13–17 up to now. Flavonoids were usually subdivided into six major groups including flavanols, flavones, flavonols, flavanones, isoflavones and anthocyanidins.18 The position and number of hydroxyl group on functional moiety of flavonoids have a significant effect on their inhibitory activity and associated mechanism. Some flavonols with a 3-hydroxyl-4-keto moiety, such as kaempferol, quercetin and morin, exhibit the inhibitory activity toward tyrosinase by chelating with the copper ions and occupying the catalytic center,15,19 but most of flavonols have weaker inhibitory activity than kojic acid.20 Two isoflavone metabolites (8-hydroxydaidzein and 8-hydroxygenistein), deemed as the suicide substrate of tyrosinase, can make tyrosinase irreversibly inactivated by substrate-like interaction.14 Tea polyphenols (TPs), such as (+)-catechin (catechin), (−)-epicatechin gallate (ECG) and (−)-epigallocatechin-3-O-gallate (EGCG), which belong to flavanols, frequently present in human diet due to their strong antioxidant activity. The report has shown that they can competitively inhibit the monophenolase activity of tyrosinase in vitro.21 Particularly, EGCG can penetrate the cell membrane and suppress the activity of tyrosinase in B16 murine melanoma cell.22 Although the inhibitory activity of these TPs against tyrosinase has been reported, their inhibitory mechanisms are still elusive.Flavonoids, widely used as dietary supplements to functional foods,In this work, we managed to reveal the inhibitory mechanisms of TPs , kojic acid (KA), catechin, ECG, and EGCG were purchased from Sigma-Aldrich. Nitric acid and dimethyl formamide (DMF) were purchased from Beijing Chemical Works. Multiwalled carbon nanotubes (MWCNTs) were purchased from the Nanotech Port Co. Ltd . Before use, the MWCNTs were acidified by nitric acid.23Tyrosinase  from mushroom, Spectrophotometer reader  and High Performance Liquid Chromatograph  were used to measure the inhibitory activity of the tyrosinase inhibitors. CV experiments were performed in a standard three-electrode electrochemical cell with a CHI 660E electrochemical analyzer . Oxygen consumptions of tyrosinase interacting with TPs were monitored with YSI 5000 dissolved oxygen meter .2.2i.e., catechin, ECG, and EGCG, and a positive control, i.e., KA, against diphenolase activity of tyrosinase was measured with l-DOPA as a substrate using the reported spectroscopy method.13,24 Assays were conducted in a 96-well micro-plate and a spectrophotometer reader was used to determine the absorbance.25 Briefly, 6 μL of 1000 unit per mL tyrosinase solution was combined with potassium phosphate buffer (pH 6.5) and 1 mM KA or TPs . After pre-incubating at 25 °C for 5 min, 150 μL of 1 mM l-DOPA aqueous solution was added, followed by incubation at 25 °C for 10 min, again, before the concentration determination of DOPA quinone at 475 nm absorbance. The total volume of each reaction system was 300 μL. As an exception, the tyrosinase inhibitory activity of catechin was measured with HPLC rather than spectroscopy, as it possessed an overlap adsorption band with DOPA quinone which will interfere the analysis of inhibitory activity.26 The inhibitory activity was expressed by IC50 value, which is equal to the concentration of inhibitor at 50% inhibitory rate according to the following equation:25A is the optical density (OD475) of potassium phosphate buffer; B, C and D are the OD475 of potassium phosphate buffer with tyrosinase, with both TPs and tyrosinase, and with TPs, respectively.The inhibitory activity of the TPs, 2.327 Briefly, 10 mM TP was titrated into 3 mL potassium phosphate buffer (pH 6.5) containing 10 units tyrosinase under continuous stirring and nitrogen exists. After 5 minutes, tyrosinase was excited at 274 nm and the emission spectrum over the range of 280 nm to 400 nm was detected through a 3 nm slit. The emission spectrum of the tyrosinase solution was also directly measured as a background.The fluorescence spectrum of the TP-TYR complex was measured according to our previous work.2.4−1, and then 5 μL of this dispersion was dropped onto the polished surface of the GC electrode and allowed to dry naturally for 12 h. The dried electrode was submerged in 3 mg mL−1 tyrosinase dissolved in 10 mM potassium phosphate buffer (pH 4.5) for 24 h.The acidified MWCNT was suspended in DMF at a concentration of 200 mg mL−1.CVs were performed in an electrolyte of 10 mM TP and 10 mM potassium phosphate buffer (pH 6.5) in the range from −0.2 V to 0.4 V at a scan rate of 0.05 V s2.528 Briefly, 0.4 mL of 8.2 mM TP was added to 19.2 mL phosphate buffer (pH 6.5). After equilibrating at 30 °C for 5 min, 0.4 mL of 500 unit per ml tyrosinase was injected to TP solution under continuous stirring. The decrease of oxygen was monitored with the dissolved oxygen meter. In order to evaluate the remaining activity of tyrosinase, l-DOPA was added to the above mixture of tyrosinase and TP after 5 minutes if no oxygen decreasing was observed, followed by monitoring the secondary oxygen utilization in the oxidation reaction of l-DOPA catalyzed by the remaining tyrosinase. The concentration of l-DOPA was 194 μM.The oxygen consumption in the process of tyrosinase interacting with TPs was monitored with the reported method.2.6https://www.molinspiration.com) followed by optimization using B3LYP29–31 with a 6-31G  basis set implanted in Gaussian 09. The crystal structure of tyrosinase with tropolone (PDB ID: 2Y9X)32 was taken from the Protein Data Bank (PDB).33 Ligands and crystal water molecules were removed and missing hydrogen atoms were added using Schrödinger software.34 The protonation and disassociation of chargeable residues  and N- and C-terminus residues were set at pH 7.0.35,36 Partial charges in the six histidine residues  and two copper ions in the catalytic center were set according to the report of Choi et al.37 All residues of the protein were parameterized with the AMBER-03 force field.38 The cluster of bi-copper ions coordinated by six histidine residues were firstly optimized using B3LYP with a 6-31G  basis set, and then the parameters of bonds, angles and dihedral formed between the histidine residues and copper ions were evaluated using the Visual Force Field Derivation Toolkit (VFDT).39 A semi-flexible docking was performed by AutoDock Vina40 to identify binding poses of TPs with tyrosinase, where tyrosinase was treated as a rigid body and all rotatable bonds in the ligand were sampled. Optimal binding sites were searched in a grid box of 30 × 30 × 30 Å3 centered on the bi-copper center of tyrosinase.The TP structures were generated using Molinspiration Galaxy 3D Structure Generator v. 2013.02 beta  method42 and was further decomposed into the van der Waals (ΔEvdW), electrostatic (ΔEele), nonpolar solvation (ΔGnonpolar) and polar solvation (ΔGpolar). The conformational entropy (−TΔS) was calculated by a normal-mode analysis.43 More detailed simulation information can be found in our previous papers.44,45The optimal binding modes were further refined with fully flexible atomic molecular dynamics (MD) simulation using NAMD (v. 2.9).3.3.150 value of the TPs and KA (acted as positive control) was measured with l-DOPA as a substrate and listed in 50 values were: ECG > KA > catechin > EGCG. The structural difference between ECG and EGCG is less of hydroxyl group on ring B of ECG, the tyrosinase inhibitory activity of ECG was 6-fold stronger than that of EGCG. This suggested that the catechol group (with two hydroxyl group) on the TPs was crucial in enhancing its tyrosinase inhibitory activity. We speculated that the catechol group (ring B) should have a direct interaction with a coupled binuclear copper site in the catalytic center. Moreover, we found that the pyrogallol group (ring G) would be favorable to the tyrosinase inhibitory activity of TPs by comparing ECG and catechin. The important role of the pyrogallol group in tyrosinase inhibitory activity of compounds has also been reported by Kim and Uyama.18The IC3.2Fluorescence spectroscopy was used to further detect the interaction between tyrosinase and TPs by monitoring the quenching of the intrinsic residues in tyrosinase. The fluorescence emission spectra of tyrosinase excited at 274 nm wavelength were collected with different concentration gradients of TPs and depicted in 46 To clarify the quenching mechanism of the interaction between tyrosinase and TPs, we analyzed the fluorescence quenching data using the Stern–Volmer equation.47F0 and F are the relative fluorescence intensities before and after the addition of inhibitor, respectively; κq is the bimolecular quenching rate constant; τ0 is the fluorescence lifetime at 10−8 s for most biological macromolecules,48 [Q] is the concentration of inhibitor and KSV is the Stern–Volmer quenching constant. The Stern–Volmer quenching constant (KSV) determined by linear regression of a curve of F0/F versus [Q] was plotted in KSV value of ECG with less one hydroxyl group on ring B is much higher than that of EGCG. Good linear relationships in three Stern–Volmer plots of TPs are generally the indication of a single type of quenching manner. The calculated κq values by KSV/τ0 are 0.48 × 1012, 4.10 × 1012 and 3.13 × 1012 M−1 for catechin, ECG and EGCG, respectively. The κq values of tyrosinase quenching procedure caused by three TPs were higher than the limiting diffusion collision quenching constant of the biomolecules (2.0 × 1010 M−1 s−1) by two order of magnitude, suggesting that the quenching process of tyrosinase did not follow dynamic manner,49 but was a static one.48,50 For static quenching mechanism, the binding site number (n) and association constant (KA) can be obtained by the following equation:49Fluorescence quenching mechanism is usually divided into static or dynamic manner.KA) are in the order of EGCG (3.07 × 105) > ECG (0.53 × 105) > catechin (0.04 × 105), being significant inverse relation with their inhibitory activities. This may suggest that the inhibitory mechanism of TPs on tyrosinase is not apparently competitive with its substrate binding into the catalytic site of tyrosinase.The binding sites number of each TP with tyrosinase approximately equals to 1  approach, which has been extensively applied to determine the antioxidant potential of flavonoids.56–5951,60–62 Thus, we speculated that three TPs could interact with tyrosinase with substrate-like manner and inhibit tyrosinase activity by reducing tyrosinase. As shown in ortho-diphenols) catalyzed by tyrosinase, because meta-diphenols, i.e. ring A, have been found to have higher oxidation potentials than ortho-diphenols.56 Kilmartin et al.57 reported that the oxidization peak of meta-diphenols on ring A of flavonoids (such as catechin) was higher than 0.7 V. By comparing the CV curves of ECG with the one of catechin, it can be concluded that the first peak of ECG (0.27 V) corresponds to the oxidation of the catechol group (ring B), contained by both catechin and ECG. The difference between the CV curves of ECG and EGCG can identify that the second peak originates from the oxidation of the pyrogallol group (ring G) of both ECG and EGCG, while the first peak of EGCG (0.21 V) is caused by the oxidation of its pyrogallol group (ring B with three hydroxyl groups). The lower oxidization peak potential of the pyrogallol group compared with the catechol group has also been found by Furuno.63 It should be noted that the first peak of ring B of EGCG has a much lower oxidation potential than that of ECG, indicating it should possess much stronger reductive activity. Thus, the order of ability in reducing tyrosinase should be EGCG > catechin ≈ ECG. However, this order is inversely correlated with their order of tyrosinase inhibitory activity. One possible explanation is that the oxidation of catechin, ECG and EGCG catalyzed by tyrosinase should undergo different reaction pathways, leading to the different inhibitory mechanisms.A few of investigations have reported28Ut is the oxygen consumption at time t; E0 is the initial amount of tyrosinase; k1 and k2 are the oxidation rate and the inactivation rate of tyrosinase, respectively. As listed in k2) of 0.018 and 0.016, respectively, while the inactivation rate of EGCG is ∼4 times lower than catechin. The order of tyrosinase inactivation rate by TPs is in according with their inhibitory activity, indicating that whether inactivating tyrosinase more or not is the vital factor for the inhibitory activity of TPs. Furthermore, the catecholase activities of residual tyrosinase were evaluated by the oxygen utilization of l-DOPA. l-DOPA catalyzed by the residual tyrosinase in each TP solution. The slope (K) of oxygen consumption curve is positive proportional to the amount of residual tyrosinase.64 Obviously, the remaining quantity of tyrosinase for EGCG is largest, while the residual amount is least for ECG. This indicates that the catechol group (ring B with ortho-dihydroxy groups) on ECG or catechin plays crucial roles in inhibiting tyrosinase activity. Therefore, we proposed that the catechol group (ring B) of ECG and catechin resulted in the inactivation of tyrosinase by reducing tyrosinase with Quintox mechanism through cresolase-like catalytic pathway, as reported by Land et al.28 In this inactivation pathway, the catechol is converted to an intermediate by deprotonation and reductive elimination along with the leaving of forming Cu(0) from catalytic center, leading to the irreversible inactivation of tyrosinase.28 However, the oxidation of ring B on EGCG or ring G on both ECG and EGCG  catalyzed by tyrosinase should be competitive with the natural substrate (such as l-DOPA) through the diphenolase-like catalytic pathway , which is the molar proportion of inhibitors and inactive enzyme, is usually used to describe the inactivation capability of substrate with inactivating ability.14,65 As shown in ESI Fig. 3To further reveal tyrosinase inhibitory mechanisms of TPs, we monitored oxygen utilization against time starting from the injecting of TPs into tyrosinase solution using oximetry. The corresponding time-dependent curves of oxygen utilization are depicted in 3.4i.e., ΔEvdW, ΔEele, ΔGnonpolar and ΔGpolar. The entropic contributions (−TΔS) were calculated using normal mode analysis43 based on five conformations. KA) determined by fluorescence quenching. Although EGCG possesses the highest binding ability with tyrosinase, the weakest ability of inactivating tyrosinase than both catechin and ECG results in its lowest tyrosinase inhibitory activity. It was obvious that ECG with the pyrogallol group (ring G) exhibited much stronger binding with tyrosinase than catechin due to the lower binding free energies. This is consistent with the measured inhibitory activity. The larger van der Waals contribution for binding free energy of ECG compared with catechin also explained why the pyrogallol group could enhance the tyrosinase inhibitory activity of TPs very well. The weaker binding ability of catechin with tyrosinase than ECG may be the main reason of its lower inactivation efficiency against tyrosinase. In addition, it can be noted that the pyrogallol group (ring G) also enhances the binding free energy or associated constants (KA) of EGCG with tyrosinase, but EGCG has weaker tyrosinase inhibitory activity than catechin due to the lack of the catechol group on EGCG, which gives rise to the inactivation of tyrosinase. Moreover, we also found that ring B of ECG and EGCG had higher possibility to closely contact with the catalytic center of tyrosinase than ring G, indicating that their tyrosinase inhibitory activities mainly originated from the contributions of ring B. Further, geometrically, the orientation of ring B of ECG and catechin was approximately orthogonal to the line connecting the coupling copper ions with ∼90° included angles  is necessary to inhibit tyrosinase activity with high efficient. TPs have potent quenching effect on the intrinsic fluorescence of tyrosinase with static quenching manner and interact with tyrosinase with the molar proportion of 1 : 1. Cyclic voltammetry profiles and oxidization kinetics of tyrosinase suggest that catechin, ECG and EGCG inhibit the activity of tyrosinase, but with different inhibitory mechanisms. The high inhibitory activities of ECG and catechin were accomplished mainly by making tyrosinase inactivate through cresolase-like catalytic pathway, while EGCG is by competing with or delaying the oxidation of substrate mainly through diphenolase-like catalytic pathway. Molecular simulations further verified the speculated two types of inhibitory mechanisms.There are no conflicts to declare.RA-008-C7RA12749A-s001