Yangyang Qu

Piotr Pander

Oleh Vybornyi

Marharyta Vasylieva

Régis Guillot

Fabien Miomandre

Fernando B Dias

Peter Skabara

Przemyslaw Data

Gilles Clavier

Pierre Audebert
email: audebert@ppsm.ens-cachan.fr

Photoluminescence Turn-on Property by Inverse Electron Demand Diels-Alder Reaction

published or not. The documents may come

INTRODUCTION

The synthesis of 1,2,4,5-tetrazine, or s-tetrazine has attracted a significant interest since the inception of fast bioconjugation. [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF][START_REF] Devaraj | Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging[END_REF] Applications spanning biological imaging and detection, cancer targeting, drug delivery and biomaterials science have been recently demonstrated. [START_REF] Oliveira | Inverse Electron Demand Diels-Alder Reactions in Chemical Biology[END_REF][START_REF] Knall | Inverse Electron Demand Diels-Alder (IEDDA)-Initiated Conjugation: A (High) Potential Click Chemistry Scheme[END_REF][START_REF] He | Water-Soluble Chiral Tetrazine Derivatives: Towards the Application of Circularly Polarized Luminescence from Upper-Excited States to Photodynamic Therapy[END_REF][START_REF] Lang | Genetically Encoded Norbornene Directs Site-Specific Cellular Protein Labelling via a Rapid Bioorthogonal Reaction[END_REF][START_REF] Carlson | BODIPY-Tetrazine Derivatives as Superbright Bioorthogonal Turn-on Probes[END_REF][START_REF] Meimetis | Ultrafluorogenic Coumarin-Tetrazine Probes for Real-Time Biological Imaging[END_REF][START_REF] Wu | In Situ Synthesis of Alkenyl Tetrazines for Highly Fluorogenic Bioorthogonal Live-Cell Imaging Probes[END_REF][START_REF] Uttamapinant | Genetic Code Expansion Enables Live-Cell and Super-Resolution Imaging of Site-Specifically Labeled Cellular Proteins[END_REF][START_REF] Jiménez-Moreno | Vinyl Ether/Tetrazine Pair for the Traceless Release of Alcohols in Cells[END_REF][START_REF] Lee | Monochromophoric Design Strategy for Tetrazine-Based Colorful Bioorthogonal Probes with a Single Fluorescent Core Skeleton[END_REF][START_REF] Wu | Inverse Electron-Demand Diels-Alder Bioorthogonal Reactions[END_REF] Moreover, for the past several decades electron-deficient 1,2,4,5-tetrazine molecules have been found particularly useful in various fields [START_REF] Clavier | S-Tetrazines as Building Blocks for New Functional Molecules and Molecular Materials[END_REF] such as organic electronics (e.g., OPVs, OFETs), [START_REF] Li | Development of a New S-Tetrazine-Based Copolymer for Efficient Solar Cells[END_REF][START_REF] Li | Alternating Copolymers of Dithienyl-s-Tetrazine and Cyclopentadithiophene for Organic Photovoltaic Applications[END_REF][START_REF] Hwang | Stable Solution-Processed Molecular n-Channel Organic Field-Effect Transistors[END_REF] energetic materials, [START_REF] Huynh | Characterization, and Energetic Properties of Diazido Heteroaromatic High-Nitrogen C-N Compound[END_REF][START_REF] Chavez | Azido and Tetrazolo 1,2,4,5-Tetrazine N-Oxides[END_REF] coordination chemistry, [START_REF] Kaim | The Coordination Chemistry of 1,2,4,5-Tetrazines[END_REF][START_REF] Tang | Monochromophoric Iridium(III) Pyridyl-Tetrazine Complexes as a Unique Design Strategy for Bioorthogonal Probes with Luminogenic Behavior[END_REF][START_REF] Yuasa | π-π * Emission from a Tetrazine Derivative Complexed with Zinc Ion in Aqueous Solution: A Unique Water-Soluble Fluorophore[END_REF] electrofluorochromism [START_REF] Audebert | Electrofluorochromism: From Molecular Systems to Set-up and Display[END_REF] and total synthesis of natural products. [START_REF] Hamasaki | Total Synthesis of Ningalin D[END_REF][START_REF] Boger | Asymmetric Total Synthesis of Ent-(-)-Roseophilin: Assignment of Absolute Configuration[END_REF] The most widely used method for the synthesis of 1,2,4,5-tetrazines is a two-step procedure starting from the addition of hydrazine to nitrile precursors, followed by the oxidation of the resulting 1,2-dihydrotetrazine. [START_REF] Clavier | S-Tetrazines as Building Blocks for New Functional Molecules and Molecular Materials[END_REF] In 2012, Devaraj et al. reported a metal-catalyzed one-pot procedure to prepare both symmetrical and unsymmetrical tetrazines from either aromatic or aliphatic nitrile precursors. [START_REF] Yang | Metal-Catalyzed One-Pot Synthesis of Tetrazines Directly from Aliphatic Nitriles and Hydrazine[END_REF] More recently, our group has developed an efficient and metal-free synthetic approach to prepare 3-monosubstituted unsymmetrical 1,2,4,5-tetrazines. [START_REF] Qu | Metal-Free Synthetic Approach to 3-Monosubstituted Unsymmetrical 1,2,4,5-Tetrazines Useful for Bioorthogonal Reactions[END_REF] However, these methods are generally limited in the scope of substrates. The harsh conditions are not compatible with several fragile functional groups such as carbonyls and alkyl halides. [START_REF] Wu | In Situ Synthesis of Alkenyl Tetrazines for Highly Fluorogenic Bioorthogonal Live-Cell Imaging Probes[END_REF] Post-modification on simple tetrazine building blocks has thus been widely used to prepare tetrazines with reactive or complex functional groups, including commonly used fluorophores for bio-imaging application. [START_REF] Oliveira | Inverse Electron Demand Diels-Alder Reactions in Chemical Biology[END_REF][START_REF] Clavier | S-Tetrazines as Building Blocks for New Functional Molecules and Molecular Materials[END_REF][START_REF] Wu | Advances in Tetrazine Bioorthogonal Chemistry Driven by the Synthesis of Novel Tetrazines and Dienophiles[END_REF] Nucleophilic aromatic substitution (SNAr) on readily available 1,2,4,5-tetrazine precursors such as 3,6-dichloro-, 3,6-bis(3,5-dimethyl-1H-pyrazol-1-yl)-or 3,6-dimethylthio-s-tetrazine, has long been regarded as one of the most powerful methods to prepare functionalized 1,2,4,5-tetrazines for various applications. [START_REF] Clavier | S-Tetrazines as Building Blocks for New Functional Molecules and Molecular Materials[END_REF] However, to prepare π-conjugated 1,2,4,5-tetrazines, metal-catalyzed cross-coupling reactions on a simple tertrazine precursor are more useful. Although several examples of metal-catalyzed cross-coupling reactions have been reported on tetrazine, most of them still suffer from low yields and limited scope regarding the tetrazine substrates. [START_REF] Wu | In Situ Synthesis of Alkenyl Tetrazines for Highly Fluorogenic Bioorthogonal Live-Cell Imaging Probes[END_REF][START_REF] Novak | First Cross-Coupling Reactions on Tetrazines[END_REF][START_REF] Leconte | Efficient Palladium-Catalyzed Synthesis of Unsymmetrical (Het)Aryltetrazines[END_REF][START_REF] Bender | Preparation of Unsymmetrical 1,2,4,5-Tetrazines via a Mild Suzuki Cross-Coupling Reaction[END_REF][START_REF] Wieczorek | Rigid Tetrazine Fluorophore Conjugates with Fluorogenic Properties in the Inverse Electron Demand Diels-Alder Reaction[END_REF] This can be explained by the fact that the nitrogen atoms in the tetrazine ring can act as ligands for a metal and deactivate their catalytic activity or the tetrazine core can be reduced by metals followed by the decomposition of the ring. [START_REF] Kaim | The Coordination Chemistry of 1,2,4,5-Tetrazines[END_REF][START_REF] Maji | Metal-Induced Reductive Ring Opening of 1,2,4,5-Tetrazines : Three Resulting Coordination Alternatives , Including the New Non-Innocent 1,2-Diiminohydrazido(2-) Bridging Ligand System[END_REF] Herein we studied an important synthetic tool, Buchwald-Hartwig cross-coupling reaction, for the functionalization of tetrazine compounds. Buchwald-Hartwig cross-coupling reaction is of considerable importance in modern synthetic chemistry, as numerous pharmaceuticals, natural products and novel materials have been synthesized by this methodology. [START_REF] Ruiz-Castillo | Applications of Palladium-Catalyzed C-N Cross-Coupling Reactions[END_REF][START_REF] Heravi | Reaction: An Overview[END_REF] However, there is no detailed study of Buchwald-Hartwig coupling reaction in the tetrazine chemistry to date. The only report describing Buchwald-Hartwig reaction on tetrazine molecules came from our group; however, the scope is limited to only two examples. In addition, the reaction conditions were not optimized and only moderate yields (40% and 47%) were observed after prolonged heating (36 hours and 6 days). [START_REF] Quinton | Triphenylamine/Tetrazine Based π-Conjugated Systems as Molecular Donors for Organic Solar Cells[END_REF] In this report, we present a systematic study of Buchwald-Hartwig coupling reaction applied to tetrazine molecules, and successfully prepared a series of novel donor-acceptor 1,2,4,5-tetrazine molecules in good to high yields (61%-72%). The important factors influencing the Buchwald-Hartwig cross-coupling reaction for tetrazine functionalization are discussed. Those conditions could be of practical importance for researchers performing Buchwald-Hartwig coupling reaction on other difficult substrates. The methodology provided here not only offers a facile and efficient pathway to prepare new donor-acceptor 1,2,4,5-tetrazine molecules, but is also expected to facilitate the future applications of 1,2,4,5-tetrazines as electron-deficient components in organic electronics and as valuable intermediates for the synthesis of natural products.

RESULTS AND DISCUSSION

We initially chose 3-(4-bromophenyl)-1,2,4,5-tetrazine 1 as the tetrazine substrate, because 3-monosubstituted unsymmetrical tetrazines are useful for bio-orthogonal click chemistry due to their fast cycloaddition reaction rate with strained unsaturated cycloalkanes. Tetrazine 1 was prepared by the metal-free synthetic approach recently developed in our group. [START_REF] Qu | Metal-Free Synthetic Approach to 3-Monosubstituted Unsymmetrical 1,2,4,5-Tetrazines Useful for Bioorthogonal Reactions[END_REF] Molecules with strong electron donating abilities (carbazole, phenoxazine, phenothiazine and 9,10-dihydro-9,9-dimethylacridine) have been chosen for this study, because donor-acceptor molecules are of particular interest in organic electronics, due to the charge transfer (CT) excited states induced by the electron donor-acceptor system. [START_REF] Wong | Purely Organic Thermally Activated Delayed Fluorescence Materials for Organic Light-Emitting Diodes[END_REF][START_REF] Yang | Recent Advances in Organic Thermally Activated Delayed Fluorescence Materials[END_REF] The reaction conditions were optimized by using 1 and carbazole as the starting materials (Table 1). Firstly, the choice of base is crucial for the outcome of this reaction. When strong bases such as t BuONa (pKa = 17) or lithium hexamethyldisilamide (LHMDS, pKa = 26) were used, the tetrazine precursor 1 quickly degraded resulting in no target product (entries 1 and 2). However, when the weak base NEt3 (pKa = 11) was chosen, it did not promote the hydrogen bromide elimination, resulting in no reaction at all (entry 5). Fortunately, weak inorganic carbonate bases (pKa = 10) which could react heterogeneously in non-polar solvent were found to promote the hydrogen bromide elimination successfully (entries 3 and 4), and cesium carbonate (Cs2CO3) was found to be the most reactive resulting in a high yield of the expected product (72%, entry 3). Secondly, the choice of the catalyst and ligand played an important role in the reaction yield. We found out that tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3) was the most effective catalyst tested and 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos) gave much better results than other ligands (P( t Bu)3, 1,1'-Bis(diphenylphosphino)ferrocene (DPPF), entries 6 and 7).

The optimization of the amount of palladium catalyst and ligand loading is summarized in Table S1. It is important to note that excess amount of XPhos relative to Pd2(dba)3 was necessary to achieve a high yield, and 4 equivalents XPhos to Pd2(dba)3 was found to give the best yield. This is probably because excess amount of ligand is able to sufficiently stabilize the palladium catalyst and prevents the binding of catalyst to the nitrogen atoms on tetrazine which potentially deactivates the palladium catalyst. The amount of Pd2(dba)3 and XPhos loading were finally optimized to 3% and 12%, respectively, to give the best yield (72%). Additionally, the influence of the reaction temperature and time was also examined. It was found that both high temperature and a prolonged reaction time are not preferable for this reaction, therefore heating at 100 °C for 2 hours was found to give the best result (Table S2). With these optimized parameters in hand, we intended to extend the scope of this synthetic strategy with other electron rich amines. Three new donor-acceptor 3-monosubstituted unsymmetrical tetrazines TA2-TA4 were successfully prepared in good to high yields (61%-71%) using phenoxazine, phenothiazine or 9,10-dihydro-9,9-dimethylacridine as a donor instead of carbazole (Scheme 1). Moreover, donor-acceptor 3,6-disubstituted symmetrical tetrazines TA5-TA7 were also prepared in good to high yields (61%-69%) by Buchwald-Hartwig coupling reaction using bifunctional tetrazine precursor 2 (Scheme 2). In this case, it was initially found that using the above mentioned optimized conditions only led to a low yield of product (less than 10%), while a large amount of tetrazine precursor 2 was recovered. This suggested that the tetrazine precursor 2 is less reactive than tetrazine 1, therefore the stronger base t BuONa was used to increase the product yields. It was found that tetrazine precursor 2 is more chemically stable to bases compared to tetrazine 1 (tetrazine 2 only degrades slowly in the presence of t BuONa), which is important to obtain a high yield of product. As expected, the yield of the reaction significantly increased (up to 69%) using t BuONa as a base, along with 6 hours of heating time (necessary to consume tetrazine precursor 2). Scheme 1. Synthesis of 3-monosubstituted unsymmetrical tetrazines TA2-TA4.

Scheme 2. Synthesis of 3, 6-disubstituted symmetrical tetrazines TA5-TA7.

The electrochemical properties and photophysical properties of TA1-TA7 are depicted in Table S3 and Figure S1 andS3. Two reversible processes were found for TA2-7 molecules in cyclic voltammetry, which can be ascribed to the oxidation of the donor moieties and the reduction of the tetrazine acceptor, respectively. The oxidation peak of TA1 is irreversible because of the carbazole moiety which is known to be irreversibly oxidized (Figure S3). [START_REF] Karon | Spectroelectrochemical Characterization of Conducting Polymers from Star-Shaped Carbazole-Triphenylamine Compounds[END_REF][START_REF] Pander | Electrochemistry and Spectroelectrochemistry of Polymers Based on D-A-D and D-D-D Bis(N-Carbazolyl) Monomers, Effect of the Donor/Acceptor Core on Their Properties[END_REF] The influence of the donor group on the redox potential of the tetrazine is quite insignificant, this being indicative of a weak conjugation in the ground state between these two moieties.

All the TA molecules show an absorption band in the visible region with a maximum located near 550 nm and a rather small extinction coefficient (540-700 dm [START_REF] Oliveira | Inverse Electron Demand Diels-Alder Reactions in Chemical Biology[END_REF] •mol -1 •cm -1 ), which is typical of the n-π* transition common to all tetrazine derivatives. [START_REF] Clavier | S-Tetrazines as Building Blocks for New Functional Molecules and Molecular Materials[END_REF] The second band located around 400 nm is probably the charge transfer one as it has been shown with similar derivatives. [START_REF] Quinton | Triphenylamine/Tetrazine Based π-Conjugated Systems as Molecular Donors for Organic Solar Cells[END_REF][START_REF] Quinton | Novel S-Tetrazine-Based Dyes with Enhanced Two-Photon Absorption Cross-Section[END_REF] In addition, all the TA molecules are non-emissive because all the S1 1 nπ* molecules transit into the CT triplet state by intersystem crossing according to the EI-Sayed's rule, and finally relax non-radiatively to the ground state. [START_REF] Baba | Intersystem Crossing in the 1nπ* and 1ππ* States[END_REF][START_REF] Li | Thermally Activated Delayed Fluorescence from 3nπ* to 1nπ* Up-Conversion and Its Application to Organic Light-Emitting Diodes[END_REF] Furthermore, we were interested in applying the inverse Electron Demand Diels-Alder (iEDDA) reaction to the obtained donor-acceptor tetrazine molecules. The Diels-Alder cycloaddition of tetrazines with electron rich dienophiles was first re-ported in 1959. [START_REF] Carboni | Reactions of Tetrazines with Unsaturated Compounds. A New Synthesis of Pyridazines[END_REF] Since then, this chemistry has been utilized in a number of studies for preparing biologically active molecules, including a number of studies in total synthesis of natural products, [START_REF] Hamasaki | Total Synthesis of Ningalin D[END_REF][START_REF] Boger | Asymmetric Total Synthesis of Ent-(-)-Roseophilin: Assignment of Absolute Configuration[END_REF][START_REF] Sakya | Preparation of Novel Tricyclic Diazo Carbapenems: Application of Inverse Electron Demand Diels-Alder Reactions of 3,6-Bis(Methylthio)-l,2,4,5-Tetrazine[END_REF][START_REF] Che | Exo-2-(Pyridazin-4-Yl)-7-Azabicyclo[2.2.1]Heptanes: Syntheses and Nicotinic Acetylcholine Receptor Agonist Activity of Potent Pyridazine Analogues of (±)-Epibatidine[END_REF] as well as the recently developed fast bioconjugation. [START_REF] Oliveira | Inverse Electron Demand Diels-Alder Reactions in Chemical Biology[END_REF][START_REF] Knall | Inverse Electron Demand Diels-Alder (IEDDA)-Initiated Conjugation: A (High) Potential Click Chemistry Scheme[END_REF][START_REF] Wu | Inverse Electron-Demand Diels-Alder Bioorthogonal Reactions[END_REF] Cyclooctyne was chosen as the dienophile to react with tetrazine compounds TA1-TA7, because the triple bond in cyclooctyne allowed us to obtain the pyridazine products directly in one step without the need for further oxidation and tedious purification steps. In addition, the very fast cycloaddition reaction rate of cyclooctyne with tetrazines [START_REF] Sauer | 4,5-Tetrazine: Synthesis and Reactivity in [4+2] Cycloadditions[END_REF] gave molecules DA1-DA7 in almost quantitative yields (Scheme 3, 4). Compounds TA1-TA4 react with cyclooctyne in dichloromethane (DCM) at room temperature within minutes to give DA1-DA4, while the reactions of TA5-TA7 with cyclooctyne were performed at 50 °C for 1 hour to give DA5-DA7. This is in accordance with the reported superior cycloaddition reaction rate of 3-monosubstituted unsymmetrical tetrazines compared to those of 3, 6-disubstituted symmetrical ones. [START_REF] Oliveira | Inverse Electron Demand Diels-Alder Reactions in Chemical Biology[END_REF][START_REF] Knall | Inverse Electron Demand Diels-Alder (IEDDA)-Initiated Conjugation: A (High) Potential Click Chemistry Scheme[END_REF] Scheme 3. Synthesis of pyridazines DA1-DA4.

Scheme 4. Synthesis of pyridazines DA5-DA7.

As mentioned above, all the TA molecules are non-emissive; however, photoluminescence appears upon reaction with cyclooctyne in all cases. The reaction of TA2 with cyclooctyne is exemplified here to show the fluorescence turn-on performance of our donor-acceptor tetrazines (Figure 1). After the addition of cyclooctyne, the red color of tetrazine solution quickly disappeared and nitrogen gas evolved, resulting in a colorless and highly cyan emissive solution (Figure 1c). The X-ray single crystal structures of both TA2 and DA2 are shown in Figure 1b. The fluorescence turn-on property was studied by recording fluorescence spectra during the course of the reaction. The gradual increase in emission intensity indicates the formation of the pyridazine moiety (Figure 2a). The reaction initially proceeded rapidly (90% conversion of TA2 to DA2 in 10 minutes), and completed when it reached a plateau approximately 18 minutes after the addition of cyclooctyne (Figure 2b). The electrochemical and photophysical properties of DA1-DA7 are depicted in Table 2 and Figure S2, S4-S11. Only one redox signal can be found in all DA molecules in cyclic voltammetry, corresponding to the oxidation of the donor moiety (Figure S2) while the parent TA compounds also display the reduction signal of the tetrazine moiety (Figure S1). For all DA molecules, the reduction signal of the acceptor unit was not observed and it is presumed to lie outside the electrochemical window. The electrochemical behavior of DA molecules is very different when comparing the phenoxazine DA2, DA5 and phenothiazine DA3, DA6 derivatives on one hand with the carbazole DA1 and acridine DA4, DA7 derivatives. The first series show a reversible pure electron transfer with a very slight anodic shift of the oxidation potential for the disubstituted compounds vs. the monosubstituted. Conversely, DA1, DA4 and DA7 all display a more complicated response with two successive peaks in forward oxidation and backward reduction. This strongly suggests a radial coupling following the first electron transfer and leading to a dimer or oligomers that precipitate on the electrode surface. Indeed, in absence of heteroatom on the intermediate cycle, the spin density can be delocalized on the terminal positions of the phenyl rings thus favouring the radical coupling. This behavior can also be observed on the parent compounds TA1, TA4 and TA7 (see Figure S1), the oxidation of TA1 being even fully irreversible. The disubstituted compound DA7 behaves similarly to the monosubstituted DA4 with a very slight anodic shift of its oxidation potential as for the first series. Phenoxazine and phenothiazine substituted DA2, DA3, DA5 and DA6 have a lower oxidation potential than the carbazole substituted DA1 and acridine substituted DA4 and DA7 in agreement with the stabilization of the cation radical by the donor effect of the heteroatom.

A clear positive solvatochromic effect was found in fluorescence for DA2, DA3, DA5 and DA6 indicating that the emission originates from a charge transfer (CT) excited state (Figure S6,S7,S9,S10). However, no such effect could be observed for DA1, DA4 and DA7 because these compounds are emissive in DCM but very weakly or not-emissive in other solvents (Figure S5,S8,S11). In addition, the phosphorescence spectra of all the DA compounds were recorded in Zeonex 1% (w/w) at 80 K at a delay of 10 ms after excitation pulse (Figure S5-S11) and data are summarized in Table 2.

All the molecules are luminescent to some extent, but most of them remain purely fluorescent. Interestingly, DA3 and DA6 exhibit clear room temperature phosphorescence (RTP) properties (Figure 3 and Figure S12) due to an efficient intersystem crossing to a long-lived triplet excited state that is promoted by the phenothiazine donor attached to the pyridazine acceptor core. [START_REF] Pander | Thermally Activated Delayed Fluorescence with Narrow Emission Spectrum and Organic Room Temperature Phosphorescence by Controlling Spin-Orbit Coupling and Phosphorescence Lifetime of Metal-Free Organic Molecules[END_REF][START_REF] Pander | Room Temperature Phosphorescence Lifetime and Spectrum Tuning of Substituted Thianthrenes[END_REF] Phenothiazine (due to the involvement of sulfur's atomic orbitals) not only induces higher triplet formation yield, but also increases radiative decay rate of the triplet state. RTP materials have attracted intensive interests because of their relatively long decay lifetimes and large Stoke shifts, which have already been reported to be useful in many fields such as organic light-emitting diodes (OLEDs), [START_REF] Baldo | Highly Efficient Phosphorescent Emission from Organic Electroluminescent Devices[END_REF][START_REF] Liu | Room-Temperature Phosphorescence from Purely Organic Materials[END_REF] bioimaging and sensing [START_REF] Zhao | Phosphorescent Heavy-Metal Complexes for Bioimaging[END_REF] or organic photovoltaics (OPV). [START_REF] Lee | Triplet Exciton and Polaron Dynamics in Phosphorescent Dye Blended Polymer Photovoltaic Devices[END_REF] The photoluminescence spectrum of DA3 in Zeonex 1% (w/w) in vacuum shows an additional signal (λ = 500-550 nm) compared to the spectrum in air-equilibrated condition, which can be attributed to the room temperature phosphorescence (Figure 3b). The time-resolved spectra and photoluminescence decay of DA3 indicate that this additional signal has a long radiative decay lifetime which corresponds to the phosphorescence of the molecule (Figure 3c,3d). The ratio of the room λAbs (nm) (a) λFL (nm) temperature phosphorescence component to the fluorescence one was determined to be 0.68. In addition, the singlet and triplet energy levels were estimated to be 3.16 eV and 2.63 eV respectively (Figure 3c). In conclusion, we have extended the scope of the Buchwald-Hartwig cross-coupling reaction to the functionalization of 1,2,4,5-tetrazine derivatives. We have shown that this reaction can be used for the preparation of symmetrical and more importantly rather fragile unsymmetrical 3-monosubstituted tetrazines with various electron-rich aromatic secondary amines. This synthetic methodology should largely improve the access to various functionalized 1,2,4,5-tetrazines. Donoracceptor tetrazine molecules (TA1-TA7) were thus conveniently prepared in good to high yields (61%-72%) which were shown to act as new tetrazine turn-on luminescent probes by iEDDA reaction with cyclooctyne. Moreover, the cycloaddition products pyridazines DA3 and DA6 have been found to exhibit promising RTP properties.

EXPERIMENTAL SECTION

General Methods. All chemicals were received from commercial sources and used without further purification. Thin layer chromatography (TLC) was performed on silica gel. Flash column chromatography purification was performed on a Com-biFlash-Rf system with a variable wavelength UV detector. All mixtures of solvents are given in v/v ratio. NMR spectra were recorded on a JEOL ECS (400 MHz) spectrometer. [START_REF] Wu | Inverse Electron-Demand Diels-Alder Bioorthogonal Reactions[END_REF] C NMR spectra were proton decoupled. HRMS spectra were measured either on an UPLC/ESI-HRMS device (an Acquity Waters UPLC system coupled to a Waters LCT Premier XE mass spectrometer equipped with an electrospray ion source), or a Q-TOF mass spectrometer (Q-TOF 6540, Agilent) equipped with an APPI ion source. Melting points (m.p.) were determined on a Reichert Kofler Heizbank melting point apparatus and a KRÜSS melting point meter M5000.

Tetrazine 1, 27 2 41 and cyclooctyne [START_REF] Brandsma | An Improved Synthesis of Cyclooctyne[END_REF] were prepared according to published procedures. X-ray diffraction data for compounds TA2, TA3, DA2 and DA3 were collected by using a VENTURE PHOTON100 CMOS Bruker diffractometer with Micro-focus IuS source Mo Kα radiation. Crystal was mounted on a CryoLoop (Hampton Research) with Paratone-N (Hampton Research) as cryoprotectant and then flashfrozen in a nitrogen-gas stream at 100 or 250 K. The temperature of the crystal was maintained at the selected value by means of an N-Helix to within an accuracy of ±1K. The data were corrected for Lorentz polarization, and absorption effects. The structures were solved by direct methods using SHELXS-97 [START_REF] Sheldrick | Program for Crystal Structure Solution[END_REF] and refined against F 2 by full-matrix least-squares techniques using SHELXL-2018 [START_REF] Sheldrick | A short history of SHELX[END_REF] with anisotropic displacement parameters for all non-hydrogen atoms. All calculations were performed by using the Crystal Structure crystallographic software package WINGX. [START_REF] Farrugia | WinGX suite for small-molecule single-crystal crystallography[END_REF] The crystal data collection and refinement parameters are given in Table ORTEP drawing of the compounds are shown in Figure S13-S16.

CCDC 1937098-1937101 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/Community/Requestastructure.

Photophysics. Zeonex ® 480 blends were prepared from toluene solutions by the drop-cast method and dried in a vacuum (Zeonex ® 480 is a type of Cyclo Olefin Polymer (COP) developed by ZEON CORPORATION). All solutions were investigated at 10 -5 mol dm -3 concentration and were degassed using five freeze/pump cycles. Absorption and emission spectra were collected using a UV-3600 double beam spectrophotometer (Shimadzu), and a Fluoromax fluorescence spectrometer (Jobin Yvon) or QePro fluorescence spectrometer (Ocean Optics). Photoluminescence quantum yields in DCM solution were measured using 9,10-diphenylanthracene in cyclohexane (ΦPL = 0.68) as the standard.

Phosphorescence and prompt fluorescence (PF) spectra and decays were recorded using nanosecond gated luminescence and lifetime measurements (from 400 ps to 1 s) using either third harmonics of a high-energy, pulsed Nd:YAG laser emitting at 355 nm (EKSPLA) or a N2 laser emitting at 337 nm. Emission was focused onto a spectrograph and detected on a sensitive gated iCCD camera (Stanford Computer Optics) of sub-nanosecond resolution. PF/DF time-resolved measurements were performed by exponentially increasing gate and delay times.

Electrochemistry. The electrochemical cell comprised of platinum electrode with a 1 mm diameter of working area as a working electrode, an Ag electrode as a reference electrode and a platinum coil as an auxiliary electrode. Cyclic voltammetry measurements were conducted at room temperature at a potential rate of 50 mV/s and were calibrated against ferrocene/ferrocenium redox couple. All voltammograms were recorded on a CHInstruments Electrochemical Analyzer model 660 potentiostat. Electrochemical measurements were conducted in 1.0 mM concentrations for all cyclic voltammetry measurements. Electrochemical studies were undertaken in 0.1 M solutions of Bu4NPF6, 99% in dichloromethane (DCM) at room temperature.

Synthesis. 9-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-9H-carbazole (TA1)

Procedure A: To a 100 ml two-neck round bottom flask with a stir bar, tetrazine 1 (0.2 mmol, 47.6 mg), carbazole donor (0.24 mmol, 40.1 mg), cesium carbonate (0.4 mmol, 130 mg) and 10 ml of anhydrous toluene were added. The reaction mixture was degassed by bubbling through nitrogen for 15 min under vigorous stirring. Then Pd2(dba)3 (0.006 mmol, 5.5 mg) and XPhos (0.024 mmol, 11.5 mg) were added and degassed for another 15 min. The reaction mixture was heated to 100 °C on a heat-on reaction block under a nitrogen atmosphere and stirred for 2 h. The reaction mixture was then cooled down to room temperature, extracted with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate (MgSO4), filtered and concentrated under reduced pressure. The product was purified using flash column chromatography (Petroleum ether:CH2Cl2= 3:1) to give 46.5 mg of TA1 as a red solid (yield: 72%). [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF]

10-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-10H-phenoxazine (TA2)

TA2 was prepared following Procedure A using phenoxazine as a donor. A deep red solid was obtained after flash column chromatography (Petroleum ether:CH2Cl2 = 3:1) (48.1 mg, yield: 71%). Single crystal of TA2 was grown from slow evaporation of petroleum ether/CH2Cl2 (3:1) mixture. [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF] H NMR (400 MHz, CDCl3, δ): 10.28 (s, 1H), 8.87 (d, J = 8.24 Hz, 2H), 7.63 (d, J = 8.24 Hz, 2H), 6.79-6.59 (m, 6H), 6.05 (dd, J = 7.80 Hz, J = 1.50 Hz, 2H) ppm; [START_REF] Wu | Inverse Electron-Demand Diels-Alder Bioorthogonal Reactions[END_REF]

10-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-10H-phenothiazine (TA3)

TA3 was prepared following Procedure A using phenothiazine as a donor. A deep red solid was obtained after flash column chromatography (Petroleum ether:CH2Cl2 = 3:1) (45.4 mg, yield: 64%). Single crystal of TA3 was grown from slow evaporation of petroleum ether/CH2Cl2 (3:1) mixture. [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF] H NMR (400 MHz, CDCl3, δ): 10.13 (s, 1H), 8.56 (d, J = 8.24 Hz, 2H), 7.37 (d, J = 7.80 Hz, 2H), 7.32 (d, J = 8.24 Hz, 2H), 7.24 (d, J = 7.80 Hz, 2H), 7.17-7.10 (m, 4H) ppm; 13 C{ 1 H} NMR (100 MHz, CDCl3, δ): 166.2, 157.5, 148.9, 141.9, 130.7, 130.2, 128.6, 127.4, 125.8, 125.6, 124.1, 120.1 ppm; HRMS [M+H] + m/z calcd. for [C20H14N5S] + 356.0970, found 356.0977.

10-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-9,9-dimethyl-9,10-dihydroacridine (TA4)

TA4 was prepared following Procedure A using 9,10-dihydro-9,9-dimethylacridine as a donor. A red solid was obtained after flash column chromatography (Petroleum ether:CH2Cl2 = 3:1) (44.4 mg, yield: 61%). [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF] H NMR (400 MHz, CDCl3, δ): 10.28 (s, 1H), 8.89 (d, J = 8.24 Hz, 2H), 7.62 (d, J = 8.24 Hz, 2H), 7.49 (dd, J = 7.80 Hz, J = 1.80 Hz, 2H), 7.05-6.95 (m, 4H), 6.40 (dd, J = 7.80 Hz, J = 1.80 Hz, 2H) ppm; [START_REF] Wu | Inverse Electron-Demand Diels-Alder Bioorthogonal Reactions[END_REF]

3,6-bis(3-(10H-phenoxazin-10-yl)phenyl)-1,2,4,5-tetrazine (TA5)

Procedure B: To a 100 ml two-neck round bottom flask equipped with a stir bar, tetrazine 2 (0.2 mmol, 78.4 mg), phenoxazine donor (0.48 mmol, 87.9 mg), sodium tert-butoxide (0.48 mmol, 46.1 mg) and 15 ml of anhydrous toluene were added. The reaction mixture was degassed by bubbling through nitrogen for 15 min under vigorous stirring. Then Pd2(dba)3 (0.012 mmol, 11 mg) and XPhos (0.048 mmol, 23 mg) were added and degassed for another 15 min. The reaction mixture was heated to 110 °C on a heat-on reaction block under a nitrogen atmosphere and stirred for 6 h. The reaction mixture was then cooled down to room temperature, extracted with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate (MgSO4), filtered and concentrated under reduced pressure. The product was purified using flash column chromatography (Petroleum ether:CH2Cl2= 4:1) to give 78.3 mg of TA5 as a purple solid (yield: 66%). [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF] H NMR (400 MHz, CDCl3, δ): 8.77 (d, J = 8.24 Hz, 2H), 8.69 (t, J = 1.80 Hz, 2H), 7.87 (t, J = 8.24 Hz, 2H), 7.67 (d, J = 8.24 Hz, 2H), 6.77-6.56 (m, 12H), 6.00 (dd, J = 7.80 Hz, J = 1.80 Hz, 4H) ppm; [START_REF] Wu | Inverse Electron-Demand Diels-Alder Bioorthogonal Reactions[END_REF]

3,6-bis(3-(10H-phenothiazin-10-yl)phenyl)-1,2,4,5-tetrazine (TA6)

TA6 was prepared following Procedure B using phenothiazine as a donor. A red solid was obtained after flash column chromatography (Petroleum ether:CH2Cl2 = 4:1) (76.3 mg, yield: 61%). [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF] H NMR (400 MHz, CDCl3, δ): 8.74-8.67 (m, 4H), 7.81 (t, J = 8.24 Hz, 2H), 7.66 (d, J = 8.24 Hz, 2H), 7.11 (dd, J = 7.80 Hz, J = 1.80 Hz, 4H), 6.97-6.85 (m, 8H), 6.44 (dd, J = 7.80 Hz, J = 1.80 Hz, 4H) ppm; [START_REF] Wu | Inverse Electron-Demand Diels-Alder Bioorthogonal Reactions[END_REF] (3-(9,9-dimethylacridin-10(9H)-yl)phenyl)-1,2,4,5-tetrazine (TA7) TA7 was prepared following Procedure B using 9,10-dihydro-9,9-dimethylacridine as a donor. A red solid was obtained after flash column chromatography (Petroleum ether:CH2Cl2 = 4:1) (89.9 mg, yield: 69%). [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF]

Procedure for synthesis of DA1-DA4

To a 50 mL round bottom flask equipped with a stir bar, 0.1 mmol of TA1-TA4, and 10 mL of DCM were added. Then 4 equivalents of cyclooctyne (50 µL) was added with stirring at room temperature. The color of the solution turned from red to colorless after several minutes and N2 gas evolved. The reaction mixture was allowed to stir for another 20 min. All the volatiles were evaporated under vacuum and the residue was purified using flash column chromatography. (5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-1-yl)phenyl)-9H-carbazole (DA1) A white solid was obtained after flash column chromatography (Petroleum ether:EtOAc = 2:3) (39.6 mg, yield: 98%). [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF] (5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-1-yl)phenyl)-10H-phenoxazine (DA2) A white solid was obtained after flash column chromatography (Petroleum ether:EtOAc = 2:3) (41.5 mg, yield: 99%). Single crystal of DA2 was grown from slow evaporation of petroleum ether/EtOAc (2:3) mixture. [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF] (5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-1-yl)phenyl)-10H-phenothiazine (DA3) A light yellow solid was obtained after flash column chromatography (Petroleum ether:EtOAc = 2:3) (42.6 mg, yield: 98%). Single crystal of DA3 was grown from slow evaporation of petroleum ether/EtOAc (2:3) mixture. [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF] (5,6,7,8,9,10-hexahydrocycloocta[d]pyridazin-1-yl)phenyl)-9,9-dimethyl-9,10-dihydroacridine (DA4) A white solid was obtained after flash column chromatography (Petroleum ether:EtOAc = 2:3) (44.1 mg, yield: 99%). [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF]

9-(4-

Procedure for synthesis of DA5-DA7

To a 50 mL round bottom flask equipped with a stir bar, 0.1 mmol of TA5-TA7, and 15 mL of DCM were added. Then 4 equivalents of cyclooctyne (50 µL) was added with stirring at room temperature. The reaction mixture was heated to 50 °C on a heat-on reaction block and allowed to stir for 1 hour. The color of the solution turned from red to colorless indicating the completion of the reaction. The reaction mixture was then cooled down to room temperature. All the volatiles were evaporated under vacuum and the residue was purified using flash column chromatography. -5,6,7,8,9,10-hexahydrocycloocta[d]pyridazine (DA5) A light yellow solid was obtained after flash column chromatography (Petroleum ether:EtOAc = 2:1) (65.8 mg, yield: 97%). [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF] H NMR (400 MHz, CDCl3, δ): 7.75 (t, J = 8.24 Hz, 2H), 7.66 (d, J = 8.24 Hz, 2H), 7.52 (s, 2H), 7.48 (d, J = 8.24 Hz, 2H), 6.74-6.56 (m, 12H), 6.03 (d, J = 7.80 Hz, 4H), 2.84 (s, br, 4H), 1.60 (s, br, 4H), 1.41 (s, br, 4H) ppm; [START_REF] Wu | Inverse Electron-Demand Diels-Alder Bioorthogonal Reactions[END_REF] 1,4-bis (3-(9,9-dimethylacridin-10(9H)-yl)phenyl) -5,6,7,8,9,10-hexahydrocycloocta[d]pyridazine (DA7) A white solid was obtained after flash column chromatography (Petroleum ether:EtOAc = 2:1) (72.1 mg, yield: 99%). [START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF]

1,4-bis(3-(10H-phenoxazin-10-yl)phenyl)

ASSOCIATED CONTENT

Figure 1 .

 1 Figure 1. a) iEDDA reaction of TA2 with cyclooctyne. b) X-ray crystallographic structure of the initial tetrazine (left) and the product (right). c) Photos of the initial compound (left) and the adduct product under 365 nm UV-irradiation (right).

  (a) Absorption maxima recorded in DCM (1×10 -5 M) at room temperature. (b) Emission maxima recorded in DCM (1×10 -5 M) at room temperature. (c) Phosphorescence maxima recorded in Zeonex 1% (w/w) at 80 K with a delay of 10 ms after excitation pulse. (d) Photoluminescence quantum yield recorded in DCM at room temperature. (Standard: 9,10-diphenylanthracene in cyclohexane, ΦPL = 0.68) (e) Onset of the oxidation peak recorded in DCM / 0.1 M Bu4NPF6 supporting electrolyte at room temperature by cyclic voltammetry. (f) Optical energy gap in DCM calculated form the onset of the absorption spectra. (g) Highest occupied molecular orbital energy estimated from the onset of the oxidation peak, EHOMO /eV = -(Eox /V + 5.1). (h) Lowest unoccupied molecular orbital energy estimated using optical band gap ELUMO = EHOMO + Eg.

Figure 2 .

 2 Figure 2. a) Fluorescence spectra recorded during the course of the iEDDA reaction of TA2 with cyclooctyne in DCM at room temperature. b) Plot of fluorescence intensity (integration of the area of each spectrum) as a function of time.

Figure 3 .

 3 Figure 3. a) Chemical and crystal structure of DA3. b) Photoluminescence spectra of DA3 in Zeonex 1% (w/w) in vacuum (red) and air-equilibrated (blue) conditions at room temperature. c) Time-resolved spectra of DA3 in Zeonex 1% (w/w) at room temperature in vacuum. The blue spectrum corresponds to the fluorescence and the red one to the phosphorescence. d) PL decays of DA3 in Zeonex 1% (w/w) at room temperature in vacuum.

  C{ 1 H} NMR (100 MHz, CDCl3, δ): 166.1, 158.0, 144.2, 144.1, 133.8, 132.0, 131.6, 131.2, 123.5, 122.1, 115.9, 113.5 ppm; HRMS [M+H] + m/z calcd. for [C20H14N5O] + 340.1198, found 340.1192; m.p. 243 °C.

  C{ 1 H} NMR (100 MHz, CDCl3, δ): 166.2, 158.0, 146.4, 140.6, 131.9, 131.1, 131.0, 126.6, 125.5, 121.4, 114.6, 36.3, 31.2 ppm; HRMS [M+H] + m/z calcd. for [C23H20N5] + 366.1719, found 366.1725.

  C{[START_REF] Blackman | Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity[END_REF] H} NMR (100 MHz, CDCl3, δ): 160.4, 144.0, 141.0, 139.7, 139.0, 134.3, 131.7, 131.5, 131.2, 129.6, 123.4, 121.6, 115.6, 113.4, 30.4, 27.5, 25.9 ppm; HRMS [M+H] + m/z calcd. for [C46H37N4O2] + 677.2917, found 677.2923; m.p. ˃260 °C. 1,4-bis(3-(10H-phenothiazin-10-yl)phenyl)-5,6,7,8,9,10-hexahydrocycloocta[d]pyridazine (DA6) A light yellow solid was obtained after flash column chromatography (Petroleum ether:EtOAc = 2:1) (69.3 mg, yield: 98%). 1 H NMR (400 MHz, CDCl3, δ): 7.73 (t, J = 8.24 Hz, 2H), 7.63 (d, J = 8.24 Hz, 2H), 7.54 (s, 2H), 7.51 (d, J = 8.24 Hz, 2H), 7.04 (dd, J = 7.80 Hz, J = 1.80 Hz, 4H), 6.93-6.79 (m, 8H), 6.36 (d, J = 7.80 Hz, 4H), 2.85 (s, br, 4H), 1.60 (s, br, 4H), 1.40 (s, br, 4H) ppm; 13 C{ 1 H} NMR (100 MHz, CDCl3, δ): 160.5, 144.1, 141.3, 140.6, 139.7, 131.2, 131.0, 130.4, 128.9, 127.1, 127.0, 122.9, 121.0, 116.8, 30.4, 27.5, 25.9 ppm; HRMS [M+H] + m/z calcd. for [C46H37N4S2] + 709.2460, found 709.2485; m.p. 250 °C.

Table 1 . Optimization of the reaction conditions (a) .

 1

	Entry	Pd, Ligand	Base	Yield (%) (b)
	1	Pd2(dba)3, Xphos	t BuONa	0
	2	Pd2(dba)3, Xphos	LHMDS	0
	3	Pd2(dba)3, Xphos	Cs2CO3	72
	4	Pd2(dba)3, Xphos	K2CO3	35
	5	Pd2(dba)3, Xphos	NEt3	0
	6	Pd2(dba)3, P( t Bu)3	Cs2CO3	15
	7	Pd2(dba)3, DPPF	Cs2CO3	10
	8	Pd(OAc)2, Xphos	Cs2CO3	56
	(a) All reactions were carried out on a 0.2 mmol scale in 10 ml of toluene. Pd = palladium catalyst. (b) Yields (isolated) based on the tetrazine precursor 1.

Table 2 . Photophysical and electrochemical properties of DA1-DA7.

 2

  H NMR (400 MHz, CDCl3, δ): 10.27 (s, 1H), 8.89 (d, J = 8.24 Hz, 2H), 8.17 (d, J = 7.80 Hz, 2H), 7.88 (d, J = 8.24 Hz, 2H), 7.56 (d, J = 7.80 Hz, 2H), 7.46 (t, J = 7.80 Hz, 2H), 7.34 (t, J = 7.80 Hz, 2H) ppm; 13 C{ 1 H} NMR (100 MHz, CDCl3, δ): 166.1, 158.0, 142.6, 140.3, 130.10, 130.06, 127.4, 126.4, 124.1, 120.8, 120.7, 110.0 ppm; HRMS [M+H] + m/z calcd. for [C20H14N5] + 324.1249, found 324.1253; m.p. 180 °C.

  C{ 1 H} NMR (100 MHz, CDCl3, δ): 163.6, 144.1, 140.6, 135.8, 135.0, 134.1, 132.4, 131.1, 128.2, 123.5, 121.9, 115.8, 113.4 ppm; HRMS [M] + m/z calcd. for [C38H24N6O2] + 596.1961, found 596.1947; m.p. ˃260 °C.

  H NMR (400 MHz, CDCl3, δ): 8.81 (d, J = 8.24 Hz, 2H), 8.67 (t, J = 1.80 Hz, 2H), 7.90 (t, J = 8.24 Hz, 2H), 7.66 (d, J = 8.24 Hz, 2H), 7.49 (dd, J = 7.80 Hz, J = 1.80 Hz, 4H), 7.03-6.91 (m, 8H), 6.34 (dd, J = 7.80 Hz, J = 1.80 Hz, 4H), 1.72 (s, 12H) ppm; 13 C{ 1 H} NMR (100 MHz, CDCl3, δ): 163.7, 142.6, 140.8, 136.4, 134.9, 132.1, 131.4, 130.4, 127.9, 126.6, 125.5, 121.0, 114.1, 36.2, 31.3 ppm; HRMS [M+H] + m/z calcd. for [C44H37N6] + 649.3080, found 649.3093; m.p. ˃260 °C.

  H NMR (400 MHz, CDCl3, δ): 8.96 (s, 1H), 8.17 (d, J = 8.24 Hz, 2H), 7.78-7.68 (m, 4H), 7.51 (d, J = 7.80 Hz, 2H), 7.44 (t, J = 7.80 Hz, 2H), 7.32 (t, J = 7.80 Hz, 2H), 2.94-2.85 (m, 4H), 1.90-1.80 (m, 2H), 1.76-1.66 (m, 2H), 1.54-1.40 (m, 4H) ppm; 13 C{ 1 H} NMR (100 MHz, CDCl3, δ): 161.3, 151.8, 141.6, 140.8, 138.9, 138.3, 137.0, 130.8, 126.9, 126.2, 123.7, 120.5, 120.3, 109.9, 31.3, 30.6, 29.7, 26.8, 26.1, 25.6 ppm; HRMS [M+H] + m/z calcd. for [C28H26N3] + 404.2127, found 404.2127; m.p. 251 °C (dec.).

	10-(4-

  6H), 1.71-1.65 (m, 2H), 1.55-1.40 (m, 4H) ppm; 13 C{ 1 H} NMR (100 MHz, CDCl3, δ): 161.3, 151.8, 141.7, 141.5, 140.9, 138.8, 137.9, 131.8, 131.4, 130.2, 126.5, 125.4, 120.8, 114.2, 36.1, 31.3, 31.2, 30.4, 29.7, 26.8, 26.1, 25.5 ppm; HRMS [M+H] + m/z calcd. for [C31H32N3] + 446.2596, found 446.2606; m.p. 194 °C.

	H
	NMR (400 MHz, CDCl3, δ): 8.97 (s, 1H), 7.77 (d, J = 8.24 Hz, 2H), 7.54-7.44 (m, 4H), 7.05-6.91 (m, 4H), 6.37 (d, J = 7.80 Hz, 2H),
	2.94-2.84 (m, 4H), 1.89-1.80 (m, 2H), 1.71 (s,

  H NMR (400 MHz, CDCl3, δ): 7.78 (t, J = 8.24 Hz, 2H), 7.70 (d, J = 8.24 Hz, 2H), 7.52 (t, J = 1.80 Hz, 2H), 7.50-7.43 (m, 6H), 7.03-6.90 (m, 8H), 6.39 (d, J = 7.80 Hz, J = 1.80 Hz, 4H), 2.94-2.82 (m, 4H), 1.70 (s, 12H), 1.60 (s, br, 4H), 1.38 (s, br, 4H) ppm; 13 C{ 1 H} NMR (100 MHz, CDCl3, δ): 160.5, 141.2, 141.02, 140.99, 139.4, 132.2, 131.6, 131.3, 130.2, 129.3, 126.6, 125.3, 120.8, 114.2, 36.1, 31.3, 30.3, 27.5, 25.9 ppm; HRMS [M+H] + m/z calcd. for [C52H49N4] + 729.3957, found 729.3975; m.p. 221 °C.

ACKNOWLEDGMENT

The research leading to these results has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 674990 (EXCILIGHT).

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website. Supporting data for optimization of reaction conditions; supporting photophysical and electrochemical data and spectra; crystallographic data and ORTEP drawings of TA2, TA3, DA2 and DA3; 1 H and [START_REF] Wu | Inverse Electron-Demand Diels-Alder Bioorthogonal Reactions[END_REF] C NMR spectra of all reported compounds found in the SI.

AUTHOR INFORMATION Corresponding Author

* E-mail: audebert@ppsm.ens-cachan.fr

Author Contributions

The manuscript was written through contributions of all authors.

Notes

Any additional relevant notes should be placed here.


Rashmi Rani

Sofia De

Sousa Coutinho

Stephane Holé

Brigitte Leridon
email: brigitte.leridon@espci.fr

Colossal dielectric constant in K 2 Ti 2 O 5

Keywords: Colossal dielectric constant, Potassium dititanate, Ionic conductivity, Electrical properties, Ceramics

Potassium dititanate (K 2 Ti 2 O 5 ) ceramics are shown to exhibit solid electrolyte properties with a colossal dielectric constant (up to 10 7 ) together with a high ionic conductivity (up to 10 -5 S cm -1 ) at room temperature. Compared to Rb 2 Ti 2 O 5 crystals and ceramics, this is 2 orders of magnitude below but uses more abundant and eco-friendly compounds.

For both materials, the dielectric constant and the ionic conductivity as a function of temperature and frequency seem to be linked together. The 2-order-of-magnitude difference can be attributed to a c-axis variation in the structure. K 2 Ti 2 O 5 material can be interesting for electronic applications as a supercapacitor compound.

Introduction

Since the discovery of potassium dititanate K 2 Ti 2 O 5 by Andersson in 1960, number of studies were focused on formation and growth of K 2 Ti 2 O 5 using methods from conventional solid state, flux or hydrothermal [START_REF] Andersson | Five coordinated titanium in K 2 Ti 2 O 5[END_REF][START_REF] Masaki | Hydrothermal synthesis of potassium titanates in Ti-KOH-H 2 O system[END_REF][START_REF] Uchida | Synthesis of a New potassium titanate, KTiO 2 (OH) by hydrothermal oxidation of Ti[END_REF][START_REF] Bao | Study on the Formation and Growth of Potassium Titanate Whiskers[END_REF]. Later on, attention has been given to ion-exchange syntheses of K 2-x H x Ti 2 O 5 and K 2 Ti 2 O 5 .xH 2 O from K 2 Ti 2 O 5 [START_REF] Masaki | Hydrothermal synthesis of potassium titanates in Ti-KOH-H 2 O system[END_REF][START_REF] Kudo | Photochemical host-guest interaction in Tb 3+ and Eu 3+ ionexchanged K 2-x H x Ti 2 O 5 layered oxides[END_REF]. Systematic research were dedicated to modulations of morphologies, sizes, water contents, and crystallinity of K 2 Ti 2 O 5 and corresponding reaction conditions [6,[START_REF] Bao | Low-Temperature Controllable Calcination Syntheses of Potassium Dititanate[END_REF].

The crystal structure of K 2 Ti 2 O 5 possesses (Ti 2 O 5 ) 2-layers separated by potassium ions in which titanium atoms are five-fold coordinated and at the center of oxygen pyramids.These pyramids are partly edge-shared and partly corner shared and the structure is lamellar with C2/m space group [START_REF] Andersson | Crystal structure of K 2 Ti 2 O 5[END_REF]. It has been used in various applications such as an ion-exchanger, highly active photocatalyst and photoluminescence [START_REF] Uchida | Synthesis of a New potassium titanate, KTiO 2 (OH) by hydrothermal oxidation of Ti[END_REF][START_REF] Shibata | Photocatalytic activities of layered titanium compounds and their derivatives for H 2 evolution from aqueous methanol solution[END_REF][START_REF] Clearfield | Role of ion exchange in solid-state chemistry[END_REF][START_REF] Kudo | Photoluminescence of layered alkali-metal titanates (A 2 Ti n O 2n+1 , A = Na, K) at 300 and 77 K[END_REF].

In the present paper we aim at investigating the electrical properties of K 2 Ti 2 O 5 in comparison with the exceptional properties of the parent compound Rb 2 Ti 2 O 5 such as 10 9 dielectric constant and 0.1-C cm -1 electrical polarization [START_REF] Federicci | The crystal structure of Rb 2 Ti 2 O 5[END_REF][START_REF] Federicci | Rb 2 Ti 2 O 5 : Superionic conductor with colossal dielectric constant[END_REF] . The fact that K is more abundant and eco-friendly than Rb makes it an ideal candidate for supercapacitors.

Material and methods

K 2 Ti 2 O 5 crystals were prepared by conventional solid state route method. Starting materials are high purity metal oxide or nitrate powder, KNO 3 (> 99.9%) and TiO 2 (> 99.8%). Powders were weighed according to the stoichiometric proportion for the required compositions and mixed for 30 min using acetone. They were then calcined at 930 • C for 3 h in a platinum crucible and finally crystals were obtained after a slow cooling down process at 0.3 • C/min. In order to obtain sample pellets suitable for electrical measurements, as-grown crystals were ground and then pressed in a 13-mm-diameter die under 7 tons/cm 2 at room temperature. Gold electrodes were evaporated on both sides of the sample pellets.

The crystal structure was controlled by X-ray diffraction using a PhilipsX'pert-MPD, employing CuKα radiation under 50 kV and 40 mA. Samples were scanned at 0.03 • /min for 2θ from 10 • to 60 • . Fig. S1 [15] shows a typical diffraction pattern of K 2 Ti 2 O 5 which is consistent with previously reported diffractograms [START_REF] Andersson | Crystal structure of K 2 Ti 2 O 5[END_REF].

Dielectric constant measurements were performed with Solartron 1296 Dielectric interface and 1260 impedance/gain-phase analyser both automated by SMART software.

The sample temperature was varied using a Quantum Design Physical Properties Measurement System (PPMS). Real and imaginary parts of equivalent capacitance were measured as a function of temperature and frequency from 10 -1 Hz to 10 6 Hz under 100-mV ac voltage and the equivalent dielectric constant was inferred from the capacitance considering area and thickness of sample pellets.

Results

Maps of real ( r ) and imaginary ( r ) parts of complex permittivity r are displayed in logarithmic scale in Fig. 1a and 1b respectively. Above 200 K, the dielectric permittivity increases to colossal values, as high as 10 7 at 300 K and 10 -1 Hz. Fig. 1c shows real and imaginary parts of r as a function of frequency at three different temperatures. At 400 K and 300 K, r and r follow similar behaviour and are decreasing with frequency, while at 170 K they are much smaller and around the system detection level. The black solid lines represent respectively the slope corresponding to the Warburg effect, when both r and r vary as ω -1/2 , and to the conduction effect when r varies as ω -1 see [START_REF] Federicci | Rb 2 Ti 2 O 5 : Superionic conductor with colossal dielectric constant[END_REF] and references therein. It appears that the system is dominated by Warburg behaviour at low frequency and ion conduction at high frequency.

Permittivity of K 2 Ti 2 O 5 ceramics was also measured at various bias voltage ranging from -10 V to +10 V. The corresponding results measured at 100 Hz for 100-mV amplitude are shown in Fig. 2. Permittivity exhibits strongly nonlinear behavior and depends on the bias voltage sweeping direction. Two symmetrical broad peaks in the graph are reminiscent of the strong remanent polarization, evocative of a ferroelectric-like behaviour.

However, the material is not ferroelectric, due to the lack of spontaneous symmetry break-ing inside the unit cell. Due to macroscopical inversion symmetry breaking of the system due to the accumulation of ions at the blocking interface, the overall response of the sample is ferroelectric. Such behaviour was already reported in single crystals of the parent compound Rb 2 Ti 2 O 5 [START_REF] Sousa Coutinho | Virtual cathode induced in Rb 2 Ti 2 O 5 solid electrolyte[END_REF]. Ionic conductivity of K 2 Ti 2 O 5 ceramic taken at 1 kHz where r slope is -1 is plotted in Fig. 3 as a function of temperature. It can be seen that the conductivity reaches its highest value, 10 -5 S cm -1 , at around 300 K. The conductivity increases by 5 orders of magnitude from 150 to 300 K and decreases by 2 orders of magnitude from 300 K to 400 K. It is worth noting that K 2 Ti 2 O 5 ceramics and Rb 2 Ti 2 O 5 crystals behave qualitatively the same however the value of conductivity is two orders of magnitude lower in K 2 Ti 2 O 5 and the maximum is found at around 270 K in Rb 2 Ti 2 O 5 crystals [START_REF] Federicci | Rb 2 Ti 2 O 5 : Superionic conductor with colossal dielectric constant[END_REF].

Discussion

Previous work [START_REF] Federicci | Rb 2 Ti 2 O 5 : Superionic conductor with colossal dielectric constant[END_REF] reported colossal dielectric permittivity up to 10 9 in Rb 2 Ti 2 O 5 single crystals due to ionic diffusion processes. This mechanism was attested by the presence of a Warburg element in the dielectric permittivity at low frequency, where r and r vary as ω -1/2 . In the present case, curves at 300 K exhibit a low frequency dependence consistent with Warburg diffusion and a high frequency behaviour consistent with conduction.

But while the qualitative behaviour of permittivity is the same, the highest value is two orders of magnitude below the one of Rb 2 Ti 2 O 5 single crystals and the maximum value is still attained at 300 K rather than at 270 K. In order to understand the difference, we measured Rb 2 Ti 2 O 5 ceramics and the results are displayed in [15]. The maximum value is still attained at 270 K and permittivity is around 10 9 at 0.1 Hz which indicates that the observed difference is due to K 2 Ti 2 O 5 material itself and not to sample ceramic nature.

The behaviour of ionic conductivity is also qualitatively similar to the one reported in Rb 2 Ti 2 O 5 crystals, though the maximum value is obtained at 300 K and is two orders of magnitude lower than in Rb 2 Ti 2 O 5 . While we do not have a definite explanation for the decreasing in conductivity above 300 K, we suspect that it is related to thermally activated phonons slowing down the mobile ions.

While the work reported in [START_REF] Federicci | Rb 2 Ti 2 O 5 : Superionic conductor with colossal dielectric constant[END_REF] had enabled to clearly establish the ionic origin for the permittivity, more recent work has provided an additional mechanism which explains its colossal value [START_REF] Sousa Coutinho | Virtual cathode induced in Rb 2 Ti 2 O 5 solid electrolyte[END_REF]. Indeed, at odds with previous assumptions, only negative charges are found to accumulate at the anode in Rb 2 Ti 2 O 5 while the cathode is virtually shifted into the material, due to the presence of an insulator/metal transition [START_REF] Sousa Coutinho | Virtual cathode induced in Rb 2 Ti 2 O 5 solid electrolyte[END_REF]. Such mechanism could lead to the existence of an ultrathin insulating layer of ionic charges at the anode, yielding colossal equivalent permittivity. The present results seem to indicate that mechanisms at play in K 2 Ti 2 O 5 are similar, but that two-order-of-magnitude-smaller ionic conductivity produces a two-order-of-magntitude-smaller equivalent permittivity, further sustaining the assumption that ionic conductivity is responsible for colossal polarization.

Finally, the highest ionic conductivity is shifted from 270 K in Rb 2 Ti 2 O 5 to 300 K in K 2 Ti 2 O 5 . In any case this points to the effect of the structure itself on the ionic diffusion processes. Indeed, though (Ti 2 O 5 ) 2-planes are quite similar in the case of Rb 2 Ti 2 O 5 and K 2 Ti 2 O 5 , the distance between the planes in c-axis as inferred from X-ray diffraction is decreased by ∼ 5.3% in K 2 Ti 2 O 5 [15].

Conclusion

We have evidenced colossal permittivity (up to 10 7 ) in potassium dititanate at low frequency, and ionic conductivity of the order of 10 -5 S cm -1 at 300 K. The overall frequency behaviour of permittivity and conductivity is consistent with previous measurements in the Rb 2 Ti 2 O 5 parent compound and points out to the same mechanism which is

Figure 1 :Figure 2 :

 12 Figure 1: Dielectric constant of K 2 Ti 2 O 5 (a) Re( r ) and (b) Im( r ) as a function of temperature and frequency with V ac = 100 mV. (c) Re( r ) red dots and Im( r ) (blue dots) curves vs frequency at three different temperatures: 170 K, 300 K, 400 K. Black solid lines indicate respectfully a -1/2 slope (Warburg regime) and a -1 slope (conduction regime)

Figure 3 :

 3 Figure 3: Ionic conductivity extracted from the value of Im ( r ) at 1 kHz and plotted as a function of temperature.


Indra Taufiq

Rukmana

Gabriela Moran

Rachel Méallet-Renault

Gilles Clavier

Tadashi Kunieda

Misato Ohtani

Taku Demura

Ryohei Yasukuni
email: r-yasukuni@ms.naist.jp

Yoichiroh Hosokawa1 Affiliations

Photoinjection of fluorescent nanoparticles into intact plant cells using femtosecond laser amplifier

The introduction of nanoparticles to intact plant cells is promising as a transporting technique of a wide range of functional molecules. Among various molecular delivery methods, femtosecond laser photoinjection possesses target selectivity at a single cell level and is potentially applicable for many types of materials. However, for plant cells, the vacuoles' turgor pressure and the thick cell wall limit the application of photoinjection to only small objects. In this work, we overcome these limitations by employing a single pulse irradiation from a femtosecond laser amplifier. After laser irradiation on intact tobacco BY-2 cells, 80 nm fluorescent nanoparticles dispersed in a cell culture medium were successfully injected into their cytoplasm. This breakthrough would lead to a vast utilization of nanoparticles containing functional molecules for single cell manipulation in plant physiological study and genetic engineering. Such an injection was observed even when the laser pulse was focused neither on the cell wall nor on the cell membrane, but beside the cells. With these results, we suggest pore formation on the cell membrane by instantaneous deformation induced by an intense femtosecond laser pulse as an injection mechanism of nanoparticles. Reported photomechanical effects of the amplified femtosecond laser on the permeability of the biological membrane would offer new perspectives in biophotonics.

I. INTRODUCTION

The introduction of nanoparticles to intact plant cells is a promising technique in agriculture and plant physiological study. Thanks to unique characteristics of nanoparticles such as accumulation behavior in a specific area and release control of encapsulated molecules, they take a role as transporters for a wide range of functional substances from small agrochemicals (fertilizers, pesticides, herbicides, etc.) to large proteins and DNA. [1][START_REF] Siddiqui | Nanotechnology and Plant Sciences[END_REF][START_REF] Sanzari | [END_REF] The introduction of nanoparticles into intact plant cells is generally managed either by diffusion through the plant cell wall or by active uptake mediated by cellular processes.4-6 The introduced nanoparticles are then transported to other plant organs. However, the applicability of such passive methods is highly material dependent. As a physical approach, particle bombardment also allows delivery, through cell walls, of gold or tungsten nanoparticles coated with genetic molecules (DNA, RNA, etc.).6,7 The main drawbacks are low introduction efficiency and damage of target samples or delivered molecules.8-11 Herein, our attention is directed to a laser-assisted injection technique called photoinjection. In this method, molecules are injected through a pore formed by laser irradiation on a cell. Photoinjection possesses target selectivity at a single cell level with high throughput and it is applied to various materials.12-16 These features are significantly important to reveal spatiotemporally complex plant systems such as cell-cell communications.17

In animal cells, photoinjection of various kinds of functional molecules including large DNA plasmids and their expression is achieved by employing the femtosecond (fs) laser, which permits highefficiency and high-precision cells and tissues-processing. [18][19][20][21][22] On the other hand, the application of photoinjection to plant cells is restricted. Indeed, unlike animal cells, turgor pressure originated from the osmotic flow of water to plant vacuoles keeps inner pressure at a higher level that prevents injection through the molecular diffusion process. Another limitation is the presence of thick and rigid cell walls. The cell wall blocks large molecules typically larger than 5 nm. 23,24 In the previous reports, although small molecules were successfully injected into intact plant cells by fs laser irradiation under osmotic pressure control, injection of larger molecules than about 5 nm had not yet been attained. 23,24 We have recently applied single pulse irradiation from fs laser amplifier with higher energy than the previous photoinjection reports. 25 With this fs laser irradiation scheme, large 2 MDa molecules were injected into a target tobacco BY-2 (TBY-2) cell. In addition, the photoinjection efficiency was enhanced by adjunctive enzyme treatment, which increased permeability of the cell wall. Although their injection mechanism by the intense fs laser pulse was not fully understood, they showed the potential of injecting larger substances into single plant cells with cell walls.

In this work, we examined the applicability of the photoinjection using the fs laser amplifier to nanoparticles into a single TBY-2 cell. The photoinjection efficiency of the nanoparticles was investigated from the aspect of cellular conditions and laser irradiation parameters. Moreover, the photoinjection mechanism of nanoparticles was discussed based on a photomechanical effect, which is effectively induced by using the fs laser amplifier.

II. METHODS

A. Preparation of tobacco BY-2 cells

Wild-type TBY-2 cells were prepared using the slightly modified method from previous reports. [25][26][27] The TBY-2 cell culture medium contains 4.6% (w/v) of Murashige-Skoog (MS) basal medium (Ducheva Biochemie) containing 3% (w/v) of sucrose (Sigma-Aldrich), 0.2 g/l of KH2PO4 (Sigma-Aldrich), 1 mg/l of thiamine-HCl (Sigma-Aldrich), 100 mg/l of myo-inositol (Sigma-Aldrich), and 0.1 mg/l of 2,4-dichlorophenoxyacetic acid (Sigma-Aldrich). The cultured cells were incubated on a rotary shaker (130 rpm) in a dark room at 27 °C. Every 7 days, 2 ml of the cell suspension was transferred into 95 ml of new medium for sub-culturing. Two-day old cultured cells were taken for the photoinjection experiments by centrifuging 1 ml of the cell suspension at 1000 rpm for 1 min, and the cell pellet was re-suspended in 1 ml of the same culture medium and used as an untreated sample. The osmotic pressure was controlled by addition of mannitol with a concentration of 0.5M in the final culture medium. The TBY-2 cell sample in this condition is indicated as "mannitol treated."

Adjunctive enzyme treatment was used for partial degradation of the cell wall. The normal culture medium was substituted with 1 ml of an enzyme solution after the centrifugation. The enzyme solution contained 0.05M MES buffer [2-(N-morpholino)ethanesulfonic acid] (Sigma-Aldrich) at pH 5.8%, 0.5% (w/v) of cellulase Onozuka RS (Serva Electrophoresis), 0.05% (w/v) of pectolyase Y-23 (Serva Electrophoresis), and 0.3M mannitol (Sigma-Aldrich). The cell suspension in the enzyme solution was kept in a dark room at 27 °C for 10 min, rinsed five times with 1 ml of 0.4M mannitol solution, and then re-suspended in 1 ml of the same culture medium containing 0.5M mannitol. This sample condition is mentioned as "enzyme treated."

Finally, 100 µl of cell suspensions in each sample condition were placed in a glass-bottom dish (φ = 12 mm, Iwaki). All photoinjection experiments were done within 6 h after each treatment to maintain a healthy cell condition.

B. NIR fs laser photoinjection

The schematic illustration of the photoinjection setup is shown in Fig. 1. Amplified fs laser pulses from a regeneratively amplified fs Ti:sapphire laser system (Spectra-Physics, Solstice-Ref-MT5W, 800 nm, 150 fs) were directed into a laser-scanning confocal microscope (Olympus, IX71FVSF-2) and focused on a TBY-2 cell via a 100× oil-immersion objective lens (Olympus; Plan N, NA = 1.25). A single laser pulse was obtained using a mechanical shutter with a gate time of 1/125 s from 125 Hz pulse trains. The laser pulse energy was adjusted by using a half waveplate, polarizer, and neutral density filter. The laser beam was expanded to fill the back aperture of the objective lens, and the laser focal plane was set to the microscope image plane. The laser pulse energy through the objective lens was measured with a laser power meter (Ophir, Nova Display-Rohr). A sample was placed on a motorized microscope stage (Sigma Koki, E-65GR) placed on the microscope.

FIG. 1. Schematic illustration of the near-infrared (NIR)-femtosecond laser system coupled with a confocal laser scanning microscope. C. Fluorescence measurements

Ultrabright green fluorescent nanoparticles (G-FNPs) based on copolymer of styrene and fluorescent BDPMA [boron-dipyrromethene (BODIPY) methacrylate] were used to visualize their injection into TBY-2 cells sensitively. The synthesis of G-FNPs was described elsewhere.28,29 The prepared G-FNPs (7.3 ± 0.34 × 108 particles/ml) are 80 ± 2.0 nm in diameter. This particles' volume is roughly 20 times larger than conventional DNA plasmids (2 MDa) and 400 than average proteins (30 kDa) (Fig. 2).30-32 The maximum excitation and emission wavelengths were 529 nm and 547 nm, respectively.

FIG. 2. Comparison of hydrodynamic diameters among poly(styrene-co-BDPMA) nanoparticle, DNA plasmid with 2 MDa and an average protein (30 kDa).

The G-FNP injection was observed by confocal fluorescence microscopy. A diode-pumped solid-state (DPSS) laser (Spectra-Physics, PC14763) at 488 nm wavelength was used for G-FNP excitation. 100 µl of TBY-2 cell suspension in the glass-bottom dish with 1 µl of G-FNP solution were put on the microscope stage. Same acquisition parameters were used for capturing all fluorescence images.

The G-FNP injection level was evaluated as a differential fluorescence intensity (∆I) between before and after laser irradiation. A cytoplasmic region of a TBY-2 cell was selected using Image J software in the acquired fluorescence image. The mean intensity value before irradiation (Ibefore) was subtracted from that of 2 min after irradiation (Iafter). When laser irradiated cells were damaged, they were removed from the dataset and the ∆I histogram was made using data of 20 viable cells.

D. Cell viability assessment

Cell viability after the fs laser irradiations was assessed by the observation of cytoplasmic streaming in the transmission image and fluorescein diacetate (FDA) assay. Cytoplasmic streaming is driven from cytoskeletal myosin motion on actin filaments and transports nutrients inside the cytoplasm, thus regarding an indicator of plant cell viability. The FDA assay is a common cell viability test based on the enzymatic activity of plant cells.33 When plant cells are viable, fluorescent fluorescein (λex = 494 nm, λem = 521 nm) is yielded upon hydrolysis of non-fluorescent FDA. For the FDA assay, 2 µl of 0.36 mM FDA (Dojindo) aqueous solution was added to 100 µl of TBY-2 culture medium and its fluorescence signal was acquired after 20 min by the same confocal fluorescence imaging system as the G-FNPs measurement.

III. RESULTS

A. Effect of mannitol addition and enzyme treatment on cell structures

Morphological changes in the TBY-2 cell structure after mannitol and enzyme treatment were confirmed by transmission images before fs laser photoinjection (Fig. 3). After the mannitol addition to the culture medium, the cell vacuoles contracted in the hypertonic mannitol solution and the cell membrane was separated from the cell wall, namely, a plasmolyzed state [Fig. 3(b)]. The enzyme treatment was applied adjunctively with the mannitol addition to increase the cell wall permeability for larger molecules. In a general protocol for preparing protoplast, cell walls are completely digested after about 1 h of the enzyme treatment. In contrast, no noticeable cell morphological change was observed after 10 min of the enzyme treatment [Fig. 3(c)]. Instead of being removed, the cellulose fiber network of the cell wall would be degraded partially in this moderate process. Those results are similar to our previous report. 25 Then, the fs laser photoinjection was conducted under these three cell conditions: untreated, mannitol treated (plasmolyzed), and adjunctive enzyme treated conditions.

FIG. 3. Transmission images and their schematic structures of TBY-2 cells; (a) untreated, (b) mannitol treated, and (c) enzyme and mannitol treated states. In untreated TBY-2 cells, the cell membrane is pushed against the cell wall by turgor pressure. After osmotic pressure control by mannitol addition, the turgor pressure decreased and the cell membrane is separated from the cell wall (plasmolyzed).

The adjunctive enzyme treatment did not cause noticeable cell morphological change.

B. Nanoparticles injection in the hypertonic solution and with enzyme assistance

First, the photoinjection of G-FNPs into untreated and mannitol treated TBY-2 cells was examined. Fluorescence images of the cross section of target cells were taken before and 2 min after fs laser irradiation by confocal fluorescence microscopy. A fluorescence image of untreated TBY-2 cells before laser irradiation is shown in Fig. 4(a). There is no observable intracellular fluorescence ensuring that the nanoparticles did not diffuse into the cells spontaneously. Cell plasmolysis can be observed after the mannitol treatment from the transmission image in Fig. 4; meanwhile, the fluorescence signal could not be found within the apoplast, which is a region between the cell wall and membrane, as seen in Fig. 4(c). This observation proves that G-FNPs are too large to pass through the cell wall.

FIG. 4. Confocal fluorescence images of [(a) and (b)] untreated, [(c) and (d)] mannitol treated and [(e) and (f)] enzyme and mannitol treated TBY-2 cells before and after fs laser irradiation under the presence of G-FNPs. The top row shows corresponding transmission images before laser irradiation. The dashed line indicates the outline of a target single cell and the white arrowheads indicate the laser focal point. These images represent the change of fluorescence intensity: (b) and (d) have no differences from (a) and (c), and (f) has a clear intensity increase compared to (e).

A single 20 nJ fs laser pulse was then focused on the contact point of the cell wall and membrane in the medium containing the G-FNPs. The contact point was chosen as the laser focusing position for simultaneous pore formation on both the cell wall and membrane. The results for the different laser focusing positions are mentioned later. Figures 4(b)-4(d) shows representative fluorescence images of untreated and mannitol treated cells 2 min after irradiation. For untreated cells, the fluorescence images before and after fs laser irradiation were almost identical. For mannitol treated cells, the increase in fluorescence intensity within the cytoplasm was also quite small. Thus, the G-FNP injection into the cytoplasm of untreated and mannitol treated cells was not efficient. The pore in the cell wall and membrane formed by a 20 nJ laser pulse could not be large enough for the G-FNP injection.

Next, partial degradation of the cell wall with cellulase and pectolyase was performed in order to increase the cell wall permeability for large objects with minimal damage. Figure 4(e) depicts a representative fluorescence image of adjunctive enzyme treated TBY-2 cells before irradiation in the presence of G-FNPs in the culture medium. Unlike the image shown in Fig. 4(c), fluorescence was clearly seen in the apoplast. However, the cytoplasm was always darker, indicating that the cell membrane permeability was unchanged under this condition.

In Fig. 4(f), the fluorescence image of enzyme treated TBY-2 cells 2 min after laser irradiation is presented. The fluorescence intensity increase was obviously noticed within the cytoplasm. In the meantime, auto-fluorescence increase was not detected under the same photoinjection parameter without G-FNPs in the culture medium. Therefore, it was concluded that the G-FNPs were successfully injected into the cytoplasm.

C. Injection efficiencies under three conditions

The G-FNP injection levels under untreated, mannitol treated, and adjunctive enzyme treated conditions were estimated by differential fluorescence intensities between before and after laser irradiation (∆I). Even without laser irradiation, a small background fluorescence signal from cell's autofluorescence and/or scattered fluorescence by cells was found inside the cytoplasm. The changes in background fluorescence intensity during 2 min without laser irradiation are presented as a histogram in Fig. 5(a). The temporal fluctuations of background fluorescence for 20 cells varied from -2.25 to 1.25. The negative values were due to fluorescence bleaching throughout the laser scanning. The particle injection level is defined as ∆I, and the ∆I value of 2 is defined as injection threshold. The summary of ∆I values for 20 cells for each condition is shown as histograms in Fig. 5(b). The observations in Fig. 3 were well represented in the histograms as mentioned below.

FIG. 5. The histograms of (a) intensity changes of the background signal inside the cytoplasm in 2 min without laser irradiation, and (b) differential fluorescence intensities (∆I) between before and after laser irradiations. Untreated (UT, blue), mannitol treated (MA, yellow) and adjunctive enzyme treated (EM, green) cells (N = 20).

All ∆I values were less than the injection threshold for untreated and mannitol treated cells, suggesting that the G-FNPs were not able to be injected under these conditions. The adjunctive enzyme treatment shifted the ∆I distribution to a higher level than the injection threshold. Although outflow of cell contents was observed for 30% of untreated cells, as seen in transmission images (Fig. A1, supplementary material), such leakage was not observed for any mannitol and adjunctive enzyme treated cells. In addition, cytoplasmic streaming was noticed, and thus, they could be regarded as viable. Presumably, reduction of turgor pressure minimizes the cell damage by preventing cytosol leakage. The photoinjection efficiencies under the enzyme treatment with 10 nJ, 15 nJ, 20 nJ, 25 nJ, and 30 nJ pulse energies were also investigated [Figs. A2(a Long term cell viability after laser irradiation was evaluated by the FDA assay. All enzyme treated TBY-2 cells in a cell culture dish were exposed by the 20 nJ laser pulse and kept in an incubator at 27 °C for 12 h. After adding FDA in the cell culture medium, 95.7% of cells showed the fluorescence signal from fluorescein, as shown in Fig. 6(a), and the difference of the staining rate between laser irradiated cells and those without laser irradiation was very close, indicating that cells did not lose their physiological activity after the laser irradiation. Thus, photoinjection with a 20 nJ fs laser pulse under enzyme treatment was used as a standard condition hereafter.

FIG. 6. (a) Confocal fluorescence and transmission images of TBY-2 cells stained by FDA 12 h after irradiation of a single 20 nJ laser pulse. (b) The percentages of FDA-stained TBY-2 cells without laser irradiation (N = 104) and with single 20 nJ pulse laser irradiation (N = 116).

It should be noted here that the injection of G-FNPs into the cytoplasm was also able to be observed for the plasmolyzed cells when over 30 nJ laser pulse was exerted. Therefore, the enzyme treatment is not critical for this technique. However, the injection efficiency was still quite low compared to that with enzyme treatment, and further increase in the pulse energy caused a damage for cells [Figs. A2(a) and A2(b), supplementary material]. Moreover, the additional enzyme treatment is not a big drawback because such enzyme treatment is very common in plant biotechnology.

The temporal changes in ∆I with a 20 nJ fs laser pulse under enzyme treatment were also displayed in Fig. 7. The ∆I started rising about 15 s after laser irradiation. In Fig. 4(f), G-FNPs are accumulated near the laser focal point. To understand where G-FNPs accumulate, the fs laser was focused only on the cell membrane in the presence of G-FNPs in the apoplast [Figs. A3(c) and A3(d), supplementary material]. In this case, the accumulation also occurred around the laser focal point. Therefore, it is considered that G-FNPs are trapped not in the cell wall but in the cytoplasm, indicating that this delayed ∆I rising is due to the slower diffusion of G-FNPs in the cytoplasm than in the apoplast. The ∆I reached the maximum value in about 240 s in average, indicating that the formed pore was resealed roughly in this timescale.

FIG. 7. Temporal evolution of fluorescence intensity in the cytoplasm for enzyme treated cells. The graph is averaged for ten cells and normalized by the ∆I at 240 s. The error bar indicates standard deviation.

Interestingly, even when the fs laser pulse was focused beside a cell in culture medium, injection of G-FNPs was still observed. Figure 8(a) shows fluorescence images of the TBY-2 cell before and after irradiation of the fs laser pulse at 10 µm distance from the cell wall. Fluorescence intensity increase is confirmed in the image, although the injection efficiency was lower compared to the direct fs laser irradiation, as seen in Fig. 8(b). For other laser focusing positions, we also examined at 10 µm distance from the cell membrane in the apoplast [Figs. A3(a) and A3(b), supplementary material]. In this case, G-FNPs were injected into the cytoplasm with a similar injection efficiency to the case when the laser was focused 10 µm outside the cell wall. G-FNP accumulations were observed on the nearest part of the cell membrane from the laser focal point. We even examined the laser irradiation in the cytoplasm although cells would have damages and not suitable as an injection method. G-FNPs were injected very efficiently and accumulated at a broad area on the cell membrane [Figs. A3(e

D. Transient morphological change induced by the fs laser irradiation

When the intense fs laser pulse was focused on the cell wall and membrane, laser ablation would involve the pore formation at the laser focal point and induce a tensile stress wave that propagates to the periphery.18,23 In our previous report, we suggested that such mechanical stress might be a reason why efficient injection of large molecules occurred by the photoinjection using the fs laser amplifier. Besides, we demonstrated the injection of G-FNPs by the fs laser focusing nearby TBY-2 cells without direct irradiation on the cell wall and membrane. In this case, a rapid expansion of a cavitation bubble accompanied by an impulsive hydrodynamic force spreading to the periphery can deliver mechanical force to the TBY-2 cells. Therefore, the effects of the cavitation bubble on morphological changes of TBY-2 cells were verified by high-speed imaging.

The representative transient morphological changes of TBY-2 cells by the 20 nJ fs laser pulse are shown in Fig. 9. When the fs laser pulse was focused on the cell wall [Figs. 9(a)-9(c)], the cavitation bubble generation was seen and instantaneous deformation of the cells around the laser focal point was confirmed in their differential images as we reported elsewhere.25 Similarly, when the fs laser pulse was focused on the cell culture medium at 10 µm distance from the cell wall [Figs. 9(d)-9(f)], the cavitation bubble expanded and hit the cells, causing instantaneous deformation. This observation proved that the cells received a mechanical force from the cavitation bubble.

FIG. 9. [(a)-(f)] A series of high-speed images at 4 µs intervals. White arrowheads indicate the laser focal points (a) on the contact point of the cell wall and membrane and (d) at 10 µm distance from the cell. The differential images were acquired by image calculation

IV. DISCUSSION

The injection of G-FNPs into TBY-2 cells was achieved by employing a single intense fs laser pulse under the hypertonic condition by mannitol addition. From the transmission (Fig. 3) and fluorescence images (Fig. 4), the enzyme treatment led to the partial degradation of the network structure of the cell wall and enhanced the particle diffusion rate through the cell wall without making a protoplast. The high G-FNP concentration in the apoplast allows the efficient G-FNP injection into the cytoplasm. However, the enzyme treatment alone did not introduce G-FNPs into the cytoplasm, and the pore formation on the cell membrane by the fs laser irradiation was essential for the G-FNP injection.

Under the hypertonic condition, G-FNPs were injected when the fs laser pulse was focused on the contact point of the cell wall and membrane. With the enzyme treatment, G-FNP injection was confirmed even when the fs laser pulse was focused in the cell culture medium beside TBY-2 cells. Based on these two injection schemes, we propose mainly two mechanisms that involve the pore formation on the cell membrane.

One is a pore formation by photochemical ablation of the cell membrane. To be precise, there is no direct proof for this mechanism. However, under the laser irradiation condition, which induces breakdown of water along with the cavitation bubble as seen in Fig. 9, it is hard to consider only that the breakdown occurs without the dissociation of the cell membrane through multiphoton absorption because the absorbance of cell membrane components (e.g., proteins, lipids) at 800 nm is normally higher than that of water. Meanwhile, the pore formation using low energy and high repetition rate fs laser pulses was capable of injecting only a few nm sized molecules into plant protoplasts in the previous reports.24 Therefore, we assumed the that the photochemically induced pore was not sufficient for the injection of nanoparticles.

The second mechanism is a rupture of the cell membrane driven by photo-mechanically induced tensile stress.18 This photomechanical effect is particularly prominent with amplified intense laser pulses as higher laser energy density at the focal point results in a higher stress elevation and its rapid relaxation to the periphery.18 As seen in Fig. 9, instantaneous cell deformations were induced in both injection schemes at around the laser focal point and impact area of the cavitation bubble. In general, the plasma membrane of plant cells has less flexibility compared to that of animal cells. Therefore, a tensile stress caused by the deformation would rupture the cell membrane. This injection behavior is more clearly shown for the laser focusing on the cytoplasm. The cavitation bubble expands almost isotropically in the cytoplasm from inside cell, and the pressure would stretch the cell membrane and cause several breaks in various directions.

At last, the G-FNP injection efficiency looks lower than expected when the 40 nJ fs laser pulse forms pore on the cell wall and membrane under hypertonic condition without the enzyme treatment even though that can lead to a leakage from the cytoplasm. We consider that the softened cell wall by the enzyme treatment would have a role to intensify the mechanically induced deformation that accelerates G-FNP injection from a small pore. Without the enzyme treatment, propagation of the tensile stress at the laser focal point and deformation of the cell membrane might be restricted by the rigid cell wall.

V. CONCLUSION

The photoinjection of fluorescent nanoparticles into the cytoplasm of single plant cells with cell walls was successfully demonstrated by applying the fs laser amplifier. The injection of nanoparticles shall have a great impact since they can have specific surface properties or functional molecule delivery ability. The combination of the precise targeting capability of the fs laser photoinjection and smart nanoparticles would be a significant breakthrough that opens wide opportunity for the study of plant biology and genetic engineering. Moreover, contribution of the mechanical effect of the intense fs laser pulse on the cell membrane to the nanoparticle injection was confirmed. However, precise states of the cell membrane or cell wall after the laser irradiation or the impact of cavitation bubble (e.g., formed pore size and shape) are still under discussion. Understanding of the exact photomechanical action of amplified fs laser pulses on biological membrane would offer new perspectives in its application in biophotonics.

SUPPLEMENTARY MATERIAL

See the supplementary material for the transmission image of a damaged cell and G-FNPs injection depending on the laser pulse energies and focusing positions.

  ) and A2(b), supplementary material]. The injection efficiencies with 10 nJ and 15 nJ were very low, and they increased along with the pulse energy from 20 nJ/pulse to 30 nJ/pulse. Meanwhile, enzyme treated cells started causing cytosol leakage with 25 nJ/pulse and 30 nJ/pulse.

  ) and A3(f), supplementary material].

FIG. 8 .

 8 FIG. 8. (a) Transmission and confocal fluorescence images of the enzyme treated TBY-2 cell before and after the fs laser irradiation at 10 µm distance from the cell under the presence of G-FNPs. The 20 nJ laser pulse energy was used. The dashed line indicates the outline of target single cell and the white arrowhead indicates the laser focal point. The same conditions as those in Fig. 4 were used for acquiring the fluorescence images. (b) The histogram of ∆I between before and after laser irradiations in medium beside enzyme treated cells (purple, N = 20). The case of direct irradiation is also shown as a reference (green).

ACKNOWLEDGMENTS

This work was supported, in part, by the Grant-in-Aid for Scientific Research (C) (Grant No. JP18042069), Scientific Research on Innovative Areas (Grant No. JP18H05493) from the Japan Society for the Promotion of Science (JSPS). The collaboration between Japan and France was supported by JSPS and MEAE-MESRI under the Japan-France Integrated Action Program (PHC Sakura 40951TG).