Cationic molybdenum oxo alkylidenes stabilized by N-heterocyclic carbenes: from molecular systems to efficient supported metathesis catalysts

Cationic d0 group 6 olefin metathesis catalysts have been recently shown to display in most instances superior activity in comparison to their neutral congeners. Furthermore, their catalytic performance is greatly improved upon immobilization on silica. In this context, we have developed the new family of molecular cationic molybdenum oxo alkylidene complexes stabilized by N-heterocyclic carbenes of the general formula [Mo(O)(CHCMe3)(IMes)(OR)[X−]] (IMes = 1,3-dimesitylimidazol-2-ylidene; R = 1,3-dimesityl-C6H3, C6F5; X− = B(3,5-(CF3)2C6H3)4−, B(ArF)4, tetrakis(perfluoro-t-butoxy)aluminate (PFTA)). Immobilization of [Mo(O)(CHCMe3)(IMes)(O-1,3-dimesityl-C6H3)+B(ArF)4−] on silica via surface organometallic chemistry yields an active alkene metathesis catalyst that shows the highest productivity towards terminal olefins amongst all existing molybdenum oxo alkylidene catalysts.

Introduction
Since the discovery of olen metathesis more than half a century ago, 1 the development of novel catalyst families has been at the core of extensive research efforts, with the goal to increase catalytic performance, from functional group tolerance to increased activities, selectivities and stabilities. 2 Apart from Ru-based "Grubbs-type" catalysts, 3 d 0 early-transition-metal metathesis catalysts mostly based on group 6 metals (Mo/W), i.e. Schrock-type catalysts of the general formula M(E)(CHR)(X)(Y) with E ¼ oxo or imido and X, Y ¼ anionic ligands of various types (alkyls, alkoxys, amidos), have emerged as a central class of olen metathesis catalysts. 4 Besides, the use of surface organometallic chemistry (SOMC) 5 has enabled to greatly improve their activity and stability through generation of the corresponding well-dened active sites dispersed at the surface of oxide supports like silica. 6 In parallel, DFT calculations have revealed that the right balance of the s-donation ability of the X, Y and E ligands is key to these improved catalytic performances. 7 The concurrent presence of both strong and weaker s-donating anionic X and Y ligands for instance avoids overstabilization of the metallacyclobutane intermediates while favouring [2 + 2] cycloaddition and cycloreversion processes as well as suppressing deactivation pathways. N-Heterocyclic carbenes (NHCs) have gained increasing importance as ancillary ligands in transition metal complexes since the 1990s. 8 In this regard, the introduction of NHCs in overall tetracoordinate, metathesis-active, cationic Mo and W species has illustrated how catalytic performance can be further improved. 9 Charge delocalization between the NHC and the cationic metal centre renders cationic complexes rather "so" according to the HSAB principle, which explains the high activity and functional group tolerance in cationic group 6 alkylidene complexes. 10 The combination of both conceptsintroduction of the strong s-donating NHC ligands and weak siloxy ligands provided by the silica surfacehas thus enabled the generation of some of the most active and stable olen metathesis catalysts to date. 9b,d,9g For the cationic tungstenbased catalysts, the oxo-bearing complexes outperformed their imido analogues by 1-2 orders of magnitude in activity; 9g this can be attributed to the combination of the smaller E ligand size, allowing for easier access of the incoming olen to the metal centre, and its strong s-donating ability, which simultaneously enhances catalyst stability. Previously published immobilized neutral Mo oxo alkylidene complexes proved to possess high activity for internal olens (Fig. 1a). 2j,11 Consequently, we wanted to investigate whether the activity of molybdenum oxo complexes can be boosted for terminal olens similar to what was shown for analogous tungsten oxo complexes. 9g

Synthesis of molecular catalysts
In this context, we developed and herein report the synthesis and characterization of the long-awaited family of molecular and well-dened silica-supported cationic Mo oxo species stabilized by NHC ligands (Fig. 1b) and investigated their activity towards internal and terminal olens. The synthesis of cationic molybdenum oxo alkylidene NHC complexes was rst attempted starting from the 4-methoxybenzylidene precursor Mo-1 (Scheme 1a). 12 Addition of 1 equiv. of 1,3-bis(2,4,6trimethylphenyl)imidazol-2-ylidene (IMes) to Mo-1 resulted in deprotonation of the alkylidene ligand. Through careful choice of a less basic chlorinated NHC, 13 this side-reaction could be vastly circumvented and the desired product was isolated in 34% yield. Single crystals of Mo-2 for X-ray diffraction were grown from a mixture of dichloroethane/n-heptane at À40 C (Fig. 2). Mo-2 crystallizes in the triclinic space group P 1 with a ¼ 1184.11(6) pm, b ¼ 1247.18(7) pm, c ¼ 1537.13(8) pm, a ¼ 80.252(2) , b ¼ 81.208(2) , g ¼ 66.112(3) , Z ¼ 2. In the solid state, the complex adopts a distorted square pyramidal geometry (s 5 ¼ 0.34) 14 with a syn alkylidene ( 1 J CH ¼ 134 Hz) in the apical position. The Mo-oxo bond (166.4 pm) is slightly shorter than in ve-coordinate W oxo alkylidene NHC complexes (169.0-176.0 pm) whereas the Mo-NHC bond distance of 226.3 pm is well in the expected range of comparable tungsten complexes.
Exchange of the uorinated alkoxides was achieved by protonation using N,N-dimethylanilinium chloride to furnish complex Mo-3. Due to their electron count of 16 (counting the free electron pair at the oxo ligand, too) and their binding site occupied by an NHC, both, Mo-2 and Mo-3 were not expected to exhibit high activity in olen metathesis. Therefore, we strived for the synthesis of highly active cationic species. 9k  Abstracting one of the X-type ligands using either N,Ndimethylanilinium B(Ar F ) 4 in the reaction with Mo-2 in the presence of pivalonitrile (pivCN) or Ag(pivCN) 3 B(Ar F ) 4 in the reaction with Mo-3 resulting in complexes of the formula [MoO(CH-4-(OMe)C 6 H 4 )(X)(IMesCl 2 )(pivCN)][B(Ar F ) 4 ] (X ¼ OC(CF 3 ) 3 , Cl) proved to be successful when conducted in CDCl 3 and monitored by 1 H NMR. However, the cationic species readily decomposed upon workup, which we attribute to the low steric demand of both, the 4-methoxybenzylidene moiety and the oxo ligand. And indeed, so far, all isolated cationic group 6 alkylidene complexes either bear a neophylidene or neopentylidene ligand 9c in line with the "almost magic properties" of the neopentylidene ligand contributing to the stability of group 6 complexes. 15 We consequently shied our attention to a lately published procedure leading to the Mo oxo neopentylidene complex MoO(CHCMe 3 )Cl(OHMT)(3-Brpy) (OHMT ¼ 2,6-dimesitylphenoxide; 3-Brpy ¼ 3-bromopyridine). 16 3-Bromopyridine as a relatively weak donor could then be readily replaced by strongly sdonating IMes (Scheme 1b). In this case, no deprotonation of the alkylidene ligand occurred. The resulting ve-coordinate NHC complex was not isolated on account of its poor crystallisation propensity. Nevertheless, the addition of NaB(Ar F ) 4 and lithium tetrakis(peruoro-t-butoxy)aluminate (LiPFTA), respectively, to the crude reaction mixture led to formation of the desired cationic complexes that could be puried by recrystallization and which were isolated in good yields of 72 and 80%, respectively, over two steps. The stable cationic species served as efficient and general precursors for immobilization but can also be further transformed by exchange of the X-ligand. Thus, upon addition of an excess of HOC 6 F 5 to Mo-7, quantitative protonation of the HMTO ligand was facilitated to form Mo-8 in a high isolated yield of 84%. The coordination of THF in complex Mo-8 that lacks the sterically demanding alkoxide is imperative for its purication by recrystallization. Single crystals of Mo-8 suitable for X-ray diffraction were grown from 1,2dichloroethane at À40 C (Fig. 3). As for Mo-2, the triclinic space group P 1 was found in Mo-8 with the unit cell dimensions being a ¼ 1296.54(8) pm, b ¼ 1388.76(9) pm, c ¼ 1930.09(12) pm, a ¼ 79.898(2) , b ¼ 78.994(2) , g ¼ 86.057(3) and two molecules in the unit cell. The distorted square pyramidal structure (s 5 ¼ 0.29) bears a syn-alkylidene ligand in the apical position and all other ligands in the equatorial plane. All bond lengths and angles are similar to those observed for ve-coordinate cationic W oxo alkylidene NHC complexes. 17 On a nal note regarding the synthesis of cationic Mo oxo alkylidene complexes, the synthesis of further species bearing uorinated alkoxides starting from aryloxide complexes Mo-6 and Mo-7 have been attempted using HOC(CF 3 ) 3 . Unfortunately, the reactions proved unsuccessful, and no conversion was observed. However, the aryloxide ligand in Mo-6 and Mo-7 was successfully protonated by the action of HCl in Et 2 O in the presence of pivCN to yield a mixture of complexes of the type [MoO(CHCMe 3 )Cl(IMes)(pivCN)][X] (X ¼ B(Ar F ) 4 or LiPFTA) and the protonated OHMT ligand as an inseparable oil. All attempts to isolate the complexes by crystallization or to further convert them in situ using MOC(CF 3 ) 3 (M ¼ Li, Ag) failed.

Immobilization of complexes
The reaction of Mo-7 with HOC 6 F 5 illustrates that protic functional groups can lead to the very selective exchange of the Xligand without notable side-reactions. Following the development of the molecular compounds, we also sought to exploit this reactivity for graing the complex on silica partially dehydroxylated at 700 C (SiO 2-700 ). For comparability with previously published complexes, 9b,d,9g the B(Ar F ) 4 -containing complex Mo-6 was selected for this purpose and yielded the corresponding supported species Mo-6@SiO 2 (Scheme 2).
Quantication by 19 F solution NMR spectroscopy of the remaining B(Ar F ) 4 of the complex indicates that ca. 29% of the surface silanols (0.32 mmol g À1 ) reacted with Mo-6, in line with a molybdenum loading of 0.79 wt% as determined by elemental analysis. IR spectroscopy conrms that a signicant amount of isolated silanols (ñ¼ 3747 cm À1 ) are consumed upon graing by protonolysis, although the large size of Mo-6 likely prevents full coverage.
Besides of the appearance of CH stretching bands between n1 3200 and 2800 cm À1 , a broad absorption band appeared at around ñ¼ 3700 cm À1 , typical of the non-covalent interactions of unreacted surface silanols with the aromatic ligands of the graed complex. 2h,9b,9d,11 Further characterization of Mo-6@SiO 2 by 1 H magic angle spinning (MAS) NMR showed the alkylidene proton resonance at 13.6 ppm, slightly upeld with respect to the molecular precursor signals in solution (13.9 ppm). The 13 C cross-polarization (CP) MAS NMR spectrum shows the presence of the different methyl moieties and aromatics, whereas the alkylidene carbon, unfortunately, was not observed (for further details see ESI Fig. S1 and S2 †).

Catalytic testing of molecular and immobilized complexes
Evaluation of the catalytic activity for Mo-6 was carried out for both molecular and immobilized complexes in the homo metathesis of the two benchmark substrates cis-4-nonene and 1nonene at 30 C (Table 1). While the homogeneous complex Mo-6 had to be tested in o-dichlorobenzene (DCB) due to solubility requirements, the immobilized analogue Mo-6@SiO 2 was tested in both DCB and toluene, prototypical metathesis solvents used in many studies. In all cases, the metathesis activity towards a-olens was higher than for internal ones. This behaviour was most pronounced for the molecular complex Mo-6, where no activity was observed for internal olens and only 86% conversion aer 8 hours was reached with 1-nonene. The lack of reactivity is likely associated with the presence of the large phenoxy (OHMT) ligand, whose steric encumbrance probably hinders the required distortion of the metal complex to a trigonal prism necessary to bind the incoming olen. 2j In fact, switching OHMT for the smaller surface siloxy ligand leads to an increase in activity towards both olens. 2g,h Notably, the initial turnover frequency recorded for the self-metathesis of cis-4-nonene was modest in comparison to other molybdenum oxo alkylidenes (TOF 3 min ¼ 26 min À1 vs. >500 min À1 ). 2j,11 This observation is in line with the reported activity difference for the cationic tungsten analogues 9b and established trends that show increased activity for internal olens in Schrock-type metathesis catalysts with decreasing s-donor strength of the X ligands (X ¼ NHC, pyrrolide, alkoxide). 9b, 11,18 Considering also the activity for terminal olens, the same correlation can only be observed in few cases. 11 The decisive factor for a high catalytic activity might be rather the right balance of strong and weak s-donor ligands as outlined above. 7 However, the activity of Mo-6@SiO 2 towards terminal olens exceeds those previously reported for this family of supported complexes with TOF 3 min ¼ 254 min À1 at 0.02 mol% catalyst loading compared to only 75 min À1 and 170 min À1 of their neutral congeners at their optimal loadings (Fig. 4). 2j,11 Indeed, this supports our working hypothesis that the catalytic activity of cationic Mo oxo alkylidenes is enhanced for terminal olens similarly as shown previously for the corresponding tungsten oxo complexes. 9g Furthermore, the catalyst remained active (94% aer 8 h), when increasing the substrate to catalyst ratio to 10 000 : 1.
The excellent performance of Mo-6@SiO 2 was additionally demonstrated by recycling tests, where maximum conversion was obtained within 8 h three times, albeit accompanied by a constant loss of activity (see ESI †).  In previous studies it was shown that the homometathesis of 1-nonene using neutral immobilized Mo alkylidene catalysts can lead to low selectivities through isomerization of the position of the double bond. This can result in a selectivity of the desired hexadec-8-ene of only 80-85%, especially in closed vessels and at prolonged reaction times. 2m,5b This type of isomerization can be attributed to Mo(IV) species resulting from methylidene complex decomposition that form p-complexes with olens in the reaction mixture. Subsequently, the pcomplexes undergo oxidative addition and reductive elimination processes and, thereby, lead to isomerization. 19 Monitoring the selectivity for the formation of the homocoupling product in the metathesis of 1-nonene using cationic Mo oxo alkylidene complexes, in contrast, demonstrated that neither the homogeneous nor the immobilized catalyst are prone to form structural isomers. Even aer long reaction times of 8 h, high selectivities of ca. 98% were obtained.

Conclusions
In conclusion, catalysts belonging to the new family of cationic Mo oxo alkylidenes NHC complexes have been developed. The immobilization on silica yielded a catalyst with preferential activity towards a-olens over internal olens. This particular reactivity is attributed to the nature of the NHC ligand, whose steric demand and strong s-donor ability have an important inuence on the formation and reversion of the TBP metallacyclobutane. These results further contribute to the overall understanding on how structural and electronic properties of metal alkylidenes affect the catalytic performance and further highlight the role of the NHC ligand in providing highly active cationic d 0 -based olen metathesis catalysts.

General considerations
All experiments were carried out under an inert nitrogen or argon atmosphere using Schlenk techniques or an MBraun or GS glovebox equipped with a purier unit. Water and oxygen levels were kept below 0.1 ppm. Diethyl ether, methylene chloride, pentane, toluene and tetrahydrofuran were puried using double MBraun SPS alumina columns. Benzene and benzene-d 6 were distilled from Na/benzophenone. 1,2-Dichlorobenzene was distilled from CaH 2 . All solvents were degassed by three consecutive freeze-pump-thaw cycles.
Elemental analyses were performed at Mikroanalytisches Labor Pascher, Germany, and at the Institute of Inorganic Chemistry, University of Stuttgart, Germany. All infrared (IR) spectra were recorded using a Bruker FT-IR Alpha spectrometer placed inside a glovebox, equipped with the OPUS soware. The IR spectrometer had a total spectral range of 275-7500 cm À1 with a resolution <2 cm À1 and consisted of a RockSolid interferometer, a DTGS (triglycine sulfate) detector and a SiC globar source. Solid samples were investigated in a magnetic pellet holder. A typical experiment consisted of 32 consecutive transmission measurements in the region from 4000 to 400 cm À1 . Solution NMR spectra were recorded on a Bruker Avance III 400.
Chemical shis are reported in ppm relative to the solvent signal (CDCl 3 : 7.26 ppm, C 6 D 6 7.16 ppm, CD 2 Cl 2 5.13 ppm). 20 Data are reported as follows: chemical shi, multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, quint ¼ quintet, sept ¼ septet, br ¼ broad, m ¼ multiplet), coupling constants (Hz) and integration. The 1 H and 13 C solid-state NMR spectra were acquired on a Bruker AVANCE III spectrometer operating at 400 MHz 1 H frequency (9.4 T) and equipped with a 3.2 mm proton-heteronucleus magic angle spinning ( 1 H,X MAS) probe (Bruker). Samples were packed into a 3.2 mm MAS NMR zirconia rotor closed with a VESPEL drive cap in an argon-lled glovebox, transferred to the NMR spectrometer in a tightly sealed vial under argon, rapidly inserted into the NMR spectrometer and spun under dry nitrogen. The MAS frequency was set to 16 kHz for all experiments. For the 13 C CP MAS measurements, the Hartman-Hahn cross-polarization was performed by making use of a linear ramp from 70% to 100% with the contact time set to 3 ms. For decoupling, SPINAL64 was applied with 100 kHz irradiation on 1 H. The acquired solid-state NMR spectra are referenced externally with respect to the downeld signal of adamantane (38.5 ppm). All MAS NMR spectra were acquired at ambient temperature.
Liquid catalytic test aliquots were analysed using a GC/FID (Agilent Technologies 7890 A) equipped with a split-splitless injector heated to 250 C, injection volume 0.5 mL using hydrogen carrier gas. Chromatographic separations for 1-nonene and cis-4-nonene catalytic tests were performed using an HP-5 (Agilent Technologies) column (30 m, 0.32 mm, 0.25 mm stationary phase). Crystal data have been deposited with the Cambridge Crystallographic Data Centre (CCDC): Mo-2 CCDC 2151036, Mo-8 CCDC 2151037.
Silica. Silica (Aerosil Degussa, 200 m 2 g À1 ) was compacted with distilled water, sieved, calcined at 500 C under air for 12 h and treated under vacuum (10 À5 mbar) at 500 C for 8 h and then at 700 C for 14 h (referred to as SiO 2-700 ).

Author contributions
JVM and JDJS contributed equally.

Conflicts of interest
There are no conicts to declare.


s2fieldsofstudy: Chemistry Biology

text:
Structures and Anti-Inflammatory Evaluation of Phenylpropanoid Derivatives from the Aerial Parts of Dioscorea polystachya

Seven undescribed phenylpropanoid constituents, including three new bibenzyl derivatives (1–3) along with four new benzofuran stilbene derivatives (4–7), were isolated from the aerial parts of Dioscorea polystachya. The structures of these compounds were elucidated using a combination of spectroscopic analyses, including UV, IR, HRESIMS, 1D, and 2D NMR. Further, all the compounds were evaluated on the anti-inflammatory activity for their inhibition of nitric oxide (NO) production by RAW 264.7 macrophages cells, and some of them (1–3 and 6) displayed inhibitory activity with IC50 values in the range of 9.3–32.3 μM. Moreover, compound 3 decreased the expression of iNOS in Western blot analysis, suggesting compound 3 is mediated via the suppression of an LPS-induced NF-κB inflammasome pathway.

Introduction
The genus of Dioscorea is a perennial herbaceous and monocotyledon plant, and has large roots and/or rhizomes [1][2][3][4]. Dioscorea plants are widely distributed in China. The D. polystachya tuber has been edible with a history of more than 3000 years in China, and it is also an important traditional Chinese herbal plant. It is used for the treatment of atherosclerosis, coughing with lung heat, hypertension, pyretic stranguria, anthracia, hyperglycemia, coronary heart disease, swelling, gastric ulcers, and sprains [5][6][7][8][9]. Previous phytochemistry investigations focused on the genus of Dioscorea and resulted in the isolation of bibenzyls, phenanthrenes, biphenanthrenes, lignans, steroidal saponins, flavonoids, and polysaccharides [10][11][12].
However, there is still a deficiency of bioactive compounds from the aerial parts D. polystachya. With our ongoing research of chemical constituents and biological activity with herbal medicine [13][14][15], phytochemical studies of the aerial parts of D. polystachya were carried out in this research in consideration of explore anti-inflammatory ingredients. Here, seven undescribed phenylpropanoid constituents, including three new bibenzyl compounds (1)(2)(3) along with four new benzofuran stilbene compounds (4)(5)(6)(7), were isolated from the aerial parts of D. polystachya ( Figure 1) and their chemical structures were elucidated based on UV, IR, NMR, and HRESIMS spectroscopic data. Compound 3, a bibenzyl compound, exhibits strong anti-inflammatory effect, and it should inhibit the inflammatory signaling mechanism of NF-κB pathways. In this report, their isolation, structural characterization, and potential anti-inflammatory activities are described.

Isolation of New Compounds 1-7 from the Aerial Parts of D. polystachya
The fresh aerial parts of D. polystachya (5 kg) were extracted three times with methanol to give a crude extract, then the extract was suspended in water and extracted with EtOAc, which were subjected to MCI, C 18 column chromatography, MPLC, preparative HPLC, and preparative TLC to obtain seven new phenylpropanoid compounds 1-7 ( Figure 1).
Compound 2 was obtained as a yellowish solid powder. Its molecular formula had the same HRESIMS data as compound 1, indicating they were isomers. The NMR data ( Figure S2) showed that compounds 1 and 2 had similar structures. Nevertheless, they have different polars in HPLC analysis. The 1D NMR spectra (Table 1)  In the HMBC spectrum (Figure 2), the correlations from H-2/6 to C-4 revealed that the methoxy group was linked to C-4. The HMBC correlations from H-2 to C-3 (δ C 151.2) indicated that a hydroxy group was linked to C-3. Therefore, compound 2 was determined as 4,5,2 -trimethoxy-3,3 -dihydroxy-bibenzyl and named diosbiben B.  Compound 3, a faint yellow solid, had the molecular formula of C 17 H 20 O 5 from HRESIMS data and an unsaturation of 8. The UV spectrum of 3 was similar to 1 and 2. Typical absorption peaks attributed to broad hydroxyl (3290 cm −1 ), aromatic ring (1612, 1518 cm −1 ), and methine (1474 cm −1 ) functionalities were observed in the IR spectrum. Its molecular formula and NMR data ( Figure S3) were similar to that of compounds 1 and 2, indicating that they were possible isomers and also were bibenzyl compounds. The HMBC correlations from H-2/6 to C-4 (δ C 133.1) indicated a hydroxy group was linked to C-4. One methoxy group was linked to C-3 , which was also demonstrated by the following correlations in the HMBC spectrum: H-5 to C-3 (δ C 154.0); 3 -OCH 3 to C-3 . Finally, the structure of 3 was identified as 5,2 ,3 -trimethoxy-3,4-dihydroxy-bibenzyl and named diosbiben C.

Inhibitory Effects of New Compounds 1-7 on NO Production of LPS-Activated RAW 264.7 Cells
All the isolates were tested for their effects on nitric oxide (NO) production inhibition in lipopolysaccharide (LPS)-activated RAW 264.7 cells. In order to exclude the inhibition of NO production caused by cytotoxicity, cell viability was evaluated by the MTT method. Results revealed that no obvious cytotoxicity (over 75% cell survival) for most of compounds at concentrations up to 50 µM was observed. The results shown in Table 4, most of the isolated compounds (1-3, 6) that displayed NO inhibitory activity (positive control: Aminoguanidine hydrochloride, IC 50 19.2 ± 0.78 µM). The NO inhibitory activity of compounds 1-3 (IC 50 9.3-32.3 µM) versus 6 (IC 50 24.1 µM) suggested the bibenzyl compounds were more active than benzofuran stilbene compounds. The position of substituent may play a significant role in mediating the activities.

Inhibitory Effects of New Compound 3 on LPS-Enhanced Inflammatory Mediators
Since compound 3 displayed the strongest inhibition on NO produce of all isolated compounds, so it was chosen for mechanistic research. Compound 3 displayed no cytotoxicity at concentrations up to 25 µM ( Figure 4A); thus, this concentration was used in subsequent experiments. As shown in Figure 4B, the significant NO production inhibitory effect of 3 with an IC 50 of 9.3 ± 1.03 µM was observed. As is well known, the production of NO is closely related to the key proteins iNOS and COX-2 [18]. Therefore, the expression levels on these proteins were detected by Western blotting. As shown in Figure 4C, a mild down-regulation of COX-2 expression was observed, but the levels of iNOS were inhibited in the presence of 3 at 10 µM. Based on these observations, it was inferred that 3 exhibited inhibitory effects on NO production may by means of suppression of iNOS in LPS-induced RAW 264.7 macrophages.

Discussion
The biological activities of D. polystachya, which have not been fully demonstrated. Moreover, few studies have been conducted with the metabolites of the aerial parts of D. polystachya. In the current study, the secondary metabolites of the aerial parts of D. polystachya were investigated by chromatographic purification and interpretation of spectroscopic data to generate seven new phenylpropanoid derivatives, and their antiinflammatory activities were explored on LPS-induced inflammatory molecules in RAW 264.7 cells.
The NO is a key signaling molecule and has been well known to regulate various physiological functions in many tissues of the human body [19]. However, an overproduction of NO is associated with many inflammatory diseases, so discovery of natural bioactive compounds plays a key role in research of new drugs for reducing the inflammatory molecules. Therefore, seven new phenylpropanoid compounds were isolated from the methanol extract of the aerial parts of D. polystachya by many kinds of chromatographs. The inhibitory rate on LPS-induced expression of nitric oxide in RAW 264.7 cells of all isolated compound were evaluated, and the results showed that compounds (1-3, 6) that displayed NO inhibitory activity. Notably, by comparing compound 1 with 3, we found when the methoxy group located at C-3 could cause a dramatic reduction in the inhibitory activity. The NO inhibitory activity of compounds 1-3 versus 4-7 suggested the bibenzyl compounds were more active than benzofuran stilbene compounds. The anti-inflammatory activities of bibenzyl derivatives may due to their special chemical structure, in which a carbon-carbon bond can have free rotation that produces multiple spatial conformations. These results demonstrated that structurally different phenylpropanoid compounds in D. polystachya were possible to play their anti-inflammatory function. Furthermore, proinflammatory molecules, including iNOS and COX-2, were involved in inflammation-associated diseases and act as inflammatory mediators or activators of inflammatory pathways, such as NF-κB [20]. Therefore, the expression levels on these proteins were detected by Western blotting. The result show that compound 3 obvious inhibited the expression of iNOS, which proposing compound 3 appears to be mediated NO release via the suppression of NF-κB inflammasome pathway.
In this study, a bibenzyl derivative, diosbiben C (3), isolated from the aerial parts of D. polystachya, significantly reduced the NO release of LPS-stimulated in RAW 264.7 cells, and the Western blot analysis resulting in a reduction in the expression inflammatory molecules of iNOS in NF-κB pathway. In addition to the NF-κB signaling pathway, our future research will investigate the anti-inflammatory mechanism of diosbiben C in more detail.

Plant Materials
The fresh aerial parts of D. polystachya were collected at Lu'an, Anhui province (China), in September 2019 and identified by associate Prof. Tao Xu.

Extraction and Isolation
The fresh aerial parts of D. polystachya (5 kg) were extracted three times with methanol to give a crude extract, then the extract was suspended in water and extracted with EtOAc, affording an EtOAc soluble extract (20 g). The extract was subjected to an MCI gel column CC (46-50 µm) using MeOH-H 2 O (v/v) in step gradient from 1:9 to 1:0 to obtain four fractions Fr.1-Fr.4. Fr. 2 (800 mg) was subjected to Sephadex LH-20 column (MeOH) and separated by semipreparative HPLC (MeCN-H

Cell Viability Assay
An MTT assay was used to evaluate RAW 264.7 cell viability as previously described. Briefly, cells were plated in 96-well plates (5 × 10 3 cells/well) for 18 h and then incubated with compounds 1-7 in various concentrations with or without LPS (1.0 µg/mL). Eighteen hours later, the prepared MTT solution (20 µL, 5 mg/mL) was added, and the cells were incubated for another 4 h. After the formazan that formed was fully dissolved in DMSO (150 µL/well), the absorbance was read at 570 nm on a microplate reader. The viability of RAW 264.7 cells for the control group (with DMSO only) is defined as 100%.

Cell Culture and NO Production Measurements
The experimental procedures were followed by the literature [21]. Cells were seeded in 96-well plates at the density of 50,000 cells/well for 24 h, pretreated with the tested compounds for 30 min at 37 • C, and co-incubated with LPS (100 ng/mL) for 24 h. NO production was analyzed through Griess reaction. Momently, cell culture supernatant (50 µL) and Griess reagent (50 µL) were mixed for 10 min, and then monitored at 540 nm using a microplate reader. All the tested compounds were prepared as stock solutions with a concentration of 10 mM in DMSO. Aminoguanidine hydrochloride was used as the positive control.

Western Blot Analysis
Cells were pretreated with the test compound 3 for 30 min and stimulated with LPS (1 µg/mL) for 24 h. The total proteins were extracted and immunoblotted as previously described [22,23]. Briefly, the harvested cells were lysed by 1% RIPA (radioimmunoprecipitation assay) (Amresco, Solon, OH, USA) to achieve the cellular lysates. Cellular lysates were centrifuged, and the total protein concentration was measured by the BCA protein assay. Total proteins were electrophoresed on SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were washed with TBST buffer, blocking with 5% skim milk for 2 h at 25 • C, and then incubated with primary antibodies for 12 h at 4 • C. After being washed with TBST buffer, the membranes were treated with secondary antibody at room temperature.

Conclusions
In summary, seven undescribed phenylpropanoid derivatives (1-7) were isolated from the fresh aerial parts of D. polystachya. The new compounds were identified as bibenzyl and benzofuran stilbene derivatives. Those compounds will enrich the structural skeletons of natural occurring phenylpropanoids and the structural diversity of the Dioscoreaceae family. All new phenylpropanoid derivatives were screened for anti-inflammatory effects. The screened results revealed that compounds (1-3, 6) displayed strong NO inhibitory activity with the IC 50 range of 9.3-32.3 µM. Importantly, the potential compound 3 decreased iNOS levels, and indicated that 3 may mediated via the suppression of an LPS-induced NF-κB inflammasome pathway. These findings provide an insight into the potential therapeutic value of phenylpropanoid derivatives for inflammatory diseases. However, more studies are needed to determine whether these compounds can act on other inflammatory mechanisms.


s2fieldsofstudy: Chemistry Biology Engineering

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Engineering Fe/Mn-doped zinc oxide nanosonosensitizers for ultrasound-activated and multiple ferroptosis-augmented nanodynamic tumor suppression

As an effective tumor-therapeutic modality, ultrasound-triggered sonodynamic therapy (SDT) has been extensively explored to induce cancer cell death by activating sonosensitizers to generate reactive oxygen species (ROS). However, the traditional inorganic semiconductor-based sonosensitizers still suffer from inefficient ROS production because of the low separation efficiency of electrons and holes (e−/h+) and their fast recombination. Herein, the iron (Fe) and manganese (Mn) co-doped zinc oxide nanosonosensitizers have been rationally designed and engineered for augmenting the SDT efficiency against tumor by inducing both multiple ferroptosis and apoptosis of tumor cells. The Fe/Mn component was co-doped into the nannostructure of ZnO nanosonosensitizers, which not only catalyzed the Fenton reaction in the hydrogen peroxide-overexpressed tumor microenvironment to produce ROS, but also depleted intracellular glutathione to suppress the consumption of ROS. The doping nanostructure in the engineered nanosonosensitizers substantially augmented the SDT efficacy of ZnO nanosonosensitizers by promoting the separation and hindering the recombination of e−/h+ under ultrasound activation. The multiple ferroptosis and apoptosis in the enhanced SDT effect of Fe/Mn co-doped ZnO nanosonosensitizers were solidly demonstrated both in vitro and in vivo on tumor-bearing mice in accompany with the detailed mechanism assessment by RNA sequenching. This work provides a distinct strategy to augment the nanomedicine-enabled SDT efficency by engineering the inorganic semiconductor-based nanosonosensitizers with transitional metal doping and inducing multiple cell-death pathways including ferroptosis.

Introduction
Malignancy has threatened the human health for centuries with its high mortality [1][2][3]. Remarkble advances in the field of nanotherapy have provided tremendous opportunities for human to fight against tumor [4][5][6][7][8][9][10][11][12][13][14]. Among all possible efforts made by reserachers, sonodynamic therapy (SDT) presents a promising cancer treatment modality because of its noninvasiveness and deep tissue penetration [15][16][17][18][19]. SDT employs ultrasound (US) to activate sonosensitizers for generating toxic reactive oxygen species (ROS), which further evokes apoptosis of tumor cells. For inorganic semiconducotor-based SDT, when US irradiates on sonosensitizers, the electrons (e À ) transfer from the valence band (VB) to the conduction band (CB), and the holes (h þ ) will form in the VB. Then, the highly active e À and h þ can further react with oxygen (O 2 ) and water molecules (H 2 O) to generate different kinds of ROS. As the US features deep tissue penetration and high controllability, SDT is of great protential for tumor nanotherapy as compared to traditional photodynamic therapy, especially for tumor in deep site of body [20][21][22][23].
However, SDT-based tumor treatment is still at the preliminary stage of development and has not been extensively applied in clinic because of the unsatisfactory anti-tumor efficacy and unclear biological effects. Since the success of SDT highly denpends on the performance of sonosensitizers, both organic and inorganic sonosensitizers have been explored and utilized recently [24][25][26]. Organic sonosensitizers, including porphyrins and porphyrin derivatives, typically suffer from the critical issues of poor stability, fast clearance and high phototoxicity, resulting in unsatisfactory efficacy against tumor and potential biotoxicity in vivo [27]. In contrast, inorganic semiconductor-based sonosensitizers, such as zinc oxide (ZnO) nanoparticles, are more preferable for effective SDT-based tumor treatment with high stability and low phototoxicity [28][29][30][31][32]. As a typical semiconductor, the electrons in ZnO are transferred from VB to CB opon US irradiation, resulting in the separation of e À and h þ , which further react with O 2 and H 2 O to produce 1 O 2 and OH [33,34]. However, due to the rapid recombination rate of e À and h þ , the therapeutic limitation of ZnO sonosensitizers still exists, which would reduce the production of ROS. Therefore, it is highly necessary to explore more effective inorganic semiconductor-based sonosensitizers to reduce the recombination of e À and h þ in SDT under ultrasonic activation.
Inspired by the optical and electronic properties of ZnO, which has great potential in nanoelectronic applications, doping of metal component in ZnO is an effective method to improve the semiconducting properties [35][36][37]. The doping transition metals typically include iron (Fe) and manganese (Mn) elements. After doping into ZnO, Fe 3þ and Mn 4þ could attrack e À so as to form oxygen vacancy in ZnO, which leads to the decrease of e À /h þ recombination. Moreover, Fe 2þ and Mn 2þ can react with tumor-overexpressed H 2 O 2 in tumor site for triggering the typical Fenton reaction, which generates the highly toxic OH for inducing the enhanced SDT effect [29,[38][39][40][41].
Herein, the Fe/Mn-doping ZnO nanoparticle (D-ZnO NP) was designed and constructed by adding Fe and Mn source to the Zn precursor solution during the synthetic procedure. The as-synthesized D-ZnO NP could not only induce the ferroptosis effect in the presence of Fe and Mn, but also augment SDT effect by decreasing the recombination of e À /h þ pairs. In detail, the Fe 3þ and Mn 4þ doping could oxidize glutathione (GSH) to glutathione disulfide (GSSH), which decreases the consumption of ROS. In addition, during the GSH depletion process, the Fe 3þ and Mn 4þ were respectively reduced to Fe 2þ and Mn 2þ , which could trigger Fenton-like cataytic reaction in the presence of tumor-overexpressed H 2 O 2 in the tumor microenvironment (TME). Thus, the ferroptosis effect was induced. Furthermore, the D-ZnO also performed better SDT effect than pure ZnO. On one hand, the doping of Fe and Mn could effectively separate the e À /h þ pairs by reducing the band gap. On the other hand, the Fe 3þ and Mn 4þ could decrease the recombination of e À / h þ pairs by attracting e À . After polyethylene glycol (PEG) surface modification, the D-ZnO-PEG NPs could accumulate in the tumor site by enhanced permeation retention (EPR) effect after intravenous administration and subsequently achieve the ferroptosis-augmented SDT on suppressing tumors under US irradiation. Therefore, our work successfully designed and fabricated a high-performance nanosonosensitizer based on trasitional metal doping to achieve highly efficient tumortherapeutic outcome (Fig. 1).

Fabrication and characterization of D-ZnO-PEG NPs
The D-ZnO NPs were synthesized by a simple nonaqueous sol-gel method [35]. Briefly, the pure ZnO NPs were prepared by adding 1.89 mmol Zn acetylacetonate hydrate to 10 ml anhydrous benzyl alcohol, then the mixture was heated in a Teflon autoclave at 200 C for 3 days. The white ZnO nanorods (NRs) were obtained after washing several times by ethanol. During the synthesis procedure, 10 mol% of Fe acetylacetonate and 10 mol% of Mn acetylacetonate were added to the reaction mixture, by which the doping was achieved (Fig. 2a). Transmission electron microscopy (TEM) image showed that the pure ZnO NRs featured the average size of 70 nm (Fig. 2b). The as synthesized D-ZnO presented globular morphology compared with the rod-like pure ZnO according to TEM image (Fig. 2c). The high-angle annular dark-field scanning TEM (HAADF-STEM) images exhibited that the engineered D-ZnO NPs remained uniform and discrete with the average size of~70 nm ( Fig. 2d and e).
To confirm the success of Fe/Mn doping, the elemental mapping, energy dispersion spectrum (EDS) and the high-resolution TEM (HRTEM) were applied. For the elemental mapping and EDS, the distribution of O, Zn, Fe and Mn was present in D-ZnO NPs (Fig. 2f, S1). The HRTEM images provided a closer observation of the crystal structure in D-ZnO, and the lattice space was measured to be 0.26 nm (Fig. 2g and h). To discuss the element-valance status of Fe and Mn in D-ZnO NPs, X-ray photoelectron spectroscopy (XPS) was applied (Fig. S2). The Mn 2þ /Mn 4þ and Fe 2þ /Fe 3þ all existed in the structure of D-ZnO NPs ( Fig. 2i and j).
In order to improve the biocompatibility and stability of D-ZnO NPs in physiological solution and enable their further biomedical application, mPEG-NH 2 was employed for surface modification. To confirm the successful PEGylation of D-ZnO-PEG, Fourier transform infrared (FT-IR) was applied to analyze the linkage of mPEG-NH 2 . The absorption bands at 1210 cm À1 and 1156 cm À1 were ascribed to C-O stretching vibration of PEG, which demonstrated the successful PEGylation ( Fig. S3) [42,43]. After PEGylation, the morphology of D-ZnO-PEG showed barely variation, which indicated the stability of the nanoparticle structure (Fig. S4). Afterwards, the diameters of D-ZnO-PEG NPs in different solutions were measured by dynamic light scattering (DLS). The results displayed around 95 nm in water, 138 nm in phosphate buffered saline (PBS) and 146 nm in fetal bovine serum (FBS), respectively (Fig. S5), which indicated the high dispersity of the as-prepared D-ZnO-PEG NPs for guaranteeing both in vitro and in vivo applications.

In vitro ferroptosis-augmented SDT performance
To assess the SDT performance of D-ZnO-PEG NPs, we employed 1,3diphenylisobenzofuran (DPBF) as the probe of 1 O 2 to monitor the ROS production. DPBF was added into different groups of solutions (Control, US, D-ZnO-PEG and D-ZnO-PEG þ US). Each group was added with 0.1 mM H 2 O 2 , and the absorbance intensity of DPBF at 410 nm was recorded and analyzed by UV-vis spectrometer. The DPBF in D-ZnO-PEG þ US group exhibited more decrease rate of intensity than that in the Control, US, ZnO-PEG þ US group, indicating better SDT effect of D-ZnO than pure ZnO by generating 1 O 2 ( Fig. 3a and b). Moreover, different groups of solutions based on doping content (0%, 3%, 5% and 10% D-ZnO-PEG NPs) were also analyzed. The DPBF in 10% D-ZnO-PEG þ US group exhibited more decrease rate than that in other groups (0%, 3% and 5%), indicating the distinct effect of enhanced SDT by promoting e À /h þ sepration and hindering their recombination (Fig. S6). Electron spin resonance (ESR) was employed to further detect the 1 O 2 generation quantitatively. Compared with the Control, US, and ZnO-PEG þ US group, the D-ZnO-PEG þ US group exhibited the highest singlet oxygen intensities, which further confirmed the enhanced SDT effect of D-ZnO-PEG NPs (Fig. 3c).
Furthermore, we employed 3,3,5,5-tetramethylbenzidine (TMB) as the probe of OH. Briefly, TMB and 0.1 mM H 2 O 2 were mixed with different groups of solution (Control, US, D-ZnO-PEG, D-ZnO-PEG þ US), and the characteristic peak at 650 nm was recorded afterwards. Noteworthly, the D-ZnO-PEG group showed nonnegligible increase of intensity, indicating the Fenton reaction of the doped Fe/Mn in ZnO NPs. Furthermore, when US was applied, the characteristic peak of D-ZnO-PEG þ US group inceased most significantly, confirming the enhanced SDT effect of D-ZnO-PEG triggered by US irradiation (Fig. 3d and e). ESR was also employed to quantitatively detect the OH production. As predicted, the D-ZnO-PEG þ US group exhibited the highest increase of intensity, corresponding to the results of TMB OH detection (Fig. 3f).
To investigate the underlying mechanism of the ferroptosisaugmented SDT effect, we first took GSH into consideration. As reducing substances, GSH can react with intracellular oxidizing agents and provent them from damaging cell membrane and mitochondrion, which could protect tumor cells from being killed by ROS [44]. Therefore, it is important to consume GSH during the procedure of ferroptosis-augmented SDT. To confirm the GSH consumption capability of D-ZnO-PEG NPs, we used UV-vis spectrometer to detect the GSH depletion. As shown in Fig. 3g, the GSH consumption was enhanced obviously as the concentration of D-ZnO-PEG increased. The D-ZnO-PEG þ US group exhibited more GSH consumption than ZnO-PEG þ US group because of the existense of Fe/Mn in these doping NPs. Notebly, the D-ZnO-PEG þ US group also induced greater degree of GSH depletion than D-ZnO-PEG group. When US was applied, GSH was further oxidized by US-triggered ROS during the SDT procedure (Fig. 3h). Furthermore, the underlying mechanism of enhanced SDT effect by such doping nanostructure was also studied. It has been demonstarted that ZnO is a semiconductor with the band gap of about 3.37 eV, from VB to CB [45]. Herein, we plotted the Tauc plot of the Kubelka-Munk function to calculate the band gap of D-ZnO after the solid ultraviolet spectrum was recorded [46]. The band gap was determinded to be 2.02 eV, which was significantly lower than that of pure ZnO (3.37 eV), indicating the capability of easier triggering of e À stimulation from VB to CB by US irradiation (Fig. 3i). Moreover, the Fe 3þ and Mn 4þ in such doping structure could attract the activated e À , so as to hinder e À /h þ from recombination. In this way, ROS production was improved and SDT effect was enhanced.

Intracellular ferroptosis-augmented SDT performance
The in vitro anti-tumor effect of D-ZnO-PEG NPs was further evaluated on 4T1 cancer cell (a mouse breast cancer cell model). First of all, the effective cellular uptake is a vital ensurance for therapeutic effects. Therefore, we employed confocal laser scanning microscopy (CLSM) to observe the cellular uptake of D-ZnO-PEG NPs by 4T1 cells after labeling D-ZnO-PEG with fluorescein isothiocyanate (FITC) and 4T1 cells with 4 0 ,6-diamidino-2-phenylindole (DAPI). After co-incubating 4T1 cells with D-ZnO-PEG NPs, the intensity of green fluorescence increased with time lasting and matched well with blue fluorescence of nucleus at 8 h, which indicated large amount of cellular uptake of D-ZnO-PEG NPs by endocytosis (Fig. S7). The typical cell-counting kit 8 (CCK-8) assay was further applied to evaluate the intracellular anti-tumor effect of D-ZnO-PEG NPs. The cell viability was calculated after different groups of treatments. The cytotoxicity of D-ZnO-PEG NPs with different concentrations was initially calculated after different durations of co-incubation to evaluate their biocompatibility. It was shown that the cell viability  (Fig. 4a). When 0.1 mM H 2 O 2 was added to simulate tumor microenvironment and US was applied, cell viability at different concentrations was differently decreased. With the increase of D-ZnO-PEG concentration and the elevation of US energy, the cell viability stepped down to 20% at 200 ppm of concentration and 1.5 W/ cm 2 of power density (Fig. 4b). In addition, we compared the therapuetic efficiency of D-ZnO-PEG þ US group with other different groups (Control, D-ZnO-PEG, US, ZnO-PEG þ US). Each group was added with 0.1 mM H 2 O 2 , the concentrations of D-ZnO-PEG and ZnO-PEG were set to be 200 ppm based on Zn content and the US energy was set to be 1.5 W/cm 2 . The results showed that the ZnO-PEG þ US group exhibited nonnegligible decrease of cell viability (69.4%). Notebly, the cell viability of D-ZnO-PEG þ US group significantly dropped to 20%, suggesting the ferroptosis-augmented SDT effect by the doping nanostructure and composition of D-ZnO-PEG nanosonosensitizers (Fig. 4c).
To obtain more accurate anti-tumor efficiency of D-ZnO-PEG NPs, the results carried out by CCK-8 were also evaluated by CLSM and flow cytometry. For CLSM, the 4T1 cancer cells were first co-stained with propidium iodide (PI, dead cells) and calcein AM (AM, live cells), then images of different groups (Control, D-ZnO-PEG, US, ZnO-PEG þ US and D-ZnO-PEG þ US, each group with 0.1 mM H 2 O 2 ) were observed and the intensities of red fluorescence and green fluorescence were recorded. The D-ZnO-PEG þ US group exihited the highest intensity of red fluorescence, which was much higher than ZnO-PEG þ US and D-ZnO-PEG group (Fig. 4d). Then, the flow cytometry quantitively determined the apoptosis rates of the above different groups. The apoptosis rate of the D-ZnO-PEG þ US group reached 28.8% (early apoptosis) þ 23.5% (late apoptosis), which further confirmed the distinct in vitro anti-tumor outcome of the enhanced SDT effect by D-ZnO-PEG NPs under US irradiation in TME (Fig. 4e).
To offer accurate mechanism of in vitro anti-tumor efficiency, we further employed CLSM and flow cytometry to evaluate the ROS production during the SDT procedure. For the CLSM observation, 2,7dichlorofluorescein diacetate (DCFH-DA) was used as the probe to detect ROS and DAPI was used as the probe to detect nucleus after different groups of treatment (Control, US, D-ZnO-PEG, ZnO-PEG þ US and D-ZnO-PEG þ US, each group was added with 0.1 mM H 2 O 2 ). Among all groups, the D-ZnO-PEG and the ZnO-PEG þ US group displayed nonnegligible intensity of green fluorescence, confirming the Fenton reaction effect and SDT effect. Most importantly, the D-ZnO-PEG þ US group induced the highest intensity of green fluorescence, which indicated the synergistic effect of TME-triggered Fenton reaction and UStriggered SDT enabled by these D-ZnO-PEG nanosonosensitizers (Fig. 5a). Flow cytometry was also used to quantitively evaluate the ROS production among the above all groups. The ROS staining rates were determined to be 46.1% in D-ZnO-PEG, 56.8% in ZnO-PEG þ US and 71.5% in D-ZnO-PEG þ US, correspondingly (Fig. 5b).
In the ferroptosis program, ROS produced by Fenton reaction damaged the cell membrane and mitochondria, and Fe 3þ prevented GSH from protecting cell membrane and mitochondria [41,47,48]. Herein, the Fe 2þ and Mn 2þ in the doping nanostructure triggered Fenton reaction in the presence of H 2 O 2 , then the produced Fe 3þ and Mn 4þ enhanced GSH consumption effect. Therefore, we observed cell membrane and mitochondria damage by bio-TEM and GSH consumption by western blot analysis. The bio-TEM images showed that the membranes of 4T1 cancer cells were cracked obviously, and the mitochondria were also damaged to varying degrees (Fig. 5c). The key protein expression of ferroptosis glutathione peroxidase 4 (GPX4) was analyzed by western blot to evaluate the GSH consumption in different groups (Control, US, D-ZnO-PEG, ZnO-PEG þ US, D-ZnO-PEG þ US) [49,50]. The results showed that GPX4 expression level of D-ZnO-PEG þ US group decreased to 0.32 (GPX4/GAPDH) compared with the Control group (1.30). In contrast, the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), which is a biomarker and contributor of ferroptosis increased to 0.52 (ACSL4/GAPDH) in D-ZnO-PEG þ US group compared with the Control group (0.28), confirming the ferroptosis-induced cell membrane damage (Fig. 5d).
Therefore, the underlying mechanism of ferroptosis-augmented SDT against tumor was described as follows. On one hand, the doping of Fe/ Mn in the ZnO nanosonosensitizer could augment the ROS production by US-triggered SDT. On the other hand, the Fenton reaction and GSH depletion in ferroptosis process could enhance the tumor cell-killing effect. The synergistic effect by both enhanced SDT and ferroptosis induced by D-ZnO-PEG NPs guarantees the high antitumor efficiency.

Biological mechanism of ferroptosis-augmented SDT by RNA sequencing
To further determine the biological mechanism of ferroptosisaugmented SDT by D-ZnO-PEG nanosonosensitizers, we employed RNA expression sequencing (RNAseq) for mechanism analysis. Briefly, gene expression profiles of different groups (Control and D-ZnO-PEG þ US) were analyzed by htseq-count and cufflinks after 4T1 cancer cells were treated [51,52]. The heat map demonstrated 15,776 co-expressed genes between the D-ZnO-PEG þ US group and the Control group (Fig. 6a). There were 1998 significantly differentially expressed genes involved in the D-ZnO-PEG þ US group according to the volcano map, including 781 up-regulated and 1127 down-regulated mRNAs (Fig. 6b and c). Moreover, the interaction relationship between key genes was illustrated by the protein-protein interactions network analysis (Fig. 6d). For further investigation of the biological functions, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed, in which ferroptosis, apoptosis, the TNF signaling pathway, the MARK signaling pathway and the IL-17 signaling pathway were highly relative to the ferroptosis-augmented SDT effect. Notably, the MARK signaling pathway was related to ROS-induced cell apoptosis, which stressed the effect of US-triggered SDT (Fig. 6e and f) [53].

In vivo ferroptosis-augmented SDT against tumor
The in vivo ferroptosis-augmented SDT was further assessed on the mouse model bearing 4T1 tumor cells based on the demonstrated in vitro therapeutic performance. It is important to ensure efficient accumulation of D-ZnO-PEG NPs in tumor site for guaranteeing high therapeutic effect. Therefore, we employed in vivo fluorescence imaging to monitor the enrichment of D-ZnO-PEG NPs in tumor site directly after intravenous injection of indocyanine green (ICG)-labelled D-ZnO-PEG NPs. With prolonged time, the fluorescence signals in tumor site gradually Briefly, the Kunming mice were divided randomly into four groups (Control, ZnO-PEG 20 mg/kg, D-ZnO-PEG 10 mg/kg, D-ZnO-PEG 20 mg/ kg), then body weight was recorded every 2 days till the 28th day. All groups of injected mice showed no significant difference in biosafety evaluation indexes compared with the control group (Fig. S10). The haematology and biochemistry indexes in each group were all within normal range after feeding for 4 weeks (Figs. S11 and S12). Moreover, the H&E staining of main organs obtained on the 28th day exhibited no obvious differences among the 4 groups, which further confirmed the desirable biocompatibility and biosafety of D-ZnO-PEG NPs in vivo (Fig. S13).
Encouraged by the efficient accumulation and desirable biocompatibility of D-ZnO-PEG NPs, we further conducted in vivo antitumor evaluations on nude female 4T1 tumor-bearing balb/c mice, which were divided randomly into 5 groups (Control, US, D-ZnO-PEG, ZnO-PEG þ US and D-ZnO-PEG þ US, n ¼ 5 in each group). US irradiation was applied at tumor site every other day for 3 times after 6 h post intravenous administration of D-ZnO-PEG NPs (Fig. 7a). Tumor size and body weight of each mouse were measured every 2 days till the last day of 14 days' feeding. Notably, the D-ZnO-PEG and ZnO-PEG þ US group showed some sort of tumor suppression (48.7% and 46.8% correspondingly), indicating the effect of both ferroptosis and SDT. Meanwhile, the D-ZnO-PEG þ US group induced the most significant degree of tumor suppression (92.8%), confirming the effect of ferroptosis-augmented SDT, which corresponded to the tumor photograph observed on the 14th day after mice sacrifice (Fig. 7b, c, d, e). The H&E staining of main organs and body weight monitoring exhibited no significant differences among all 5 groups during the 14 days' procedure, which further confirmed the high therapeutic biosafety of the as-fabricated D-ZnO-PEG NPs (Figs. S14 and S15). The therapeutic effect of D-ZnO-PEG NPs was further evaluated by DCFH-DA staining, H&E, TUNEL and Ki67 staining to analyze the underlying mechanism of in vivo ferroptosis-augmented SDT. For the DCFH-DA staining, the D-ZnO-PEG þ US group presented the highest intensity of green fluorescein compared with the other 4 groups, indicating the highest level of ROS production (Fig. 7f). The H&E and TUNEL staining was further employed to evaluate the necrosis and apoptosis of the tumor tissues, where the D-ZnO-PEG þ US group (ferroptosis-augmented SDT group) displayed the most degree of necrosis and apoptosis compared to Control, US, D-ZnO-PEG, ZnO-PEG þ US groups. Furthermore, the D-ZnO-PEG þ US group exhibited the lowest level of Ki67 protein, which is a tumor proliferation marker highly related to cell mitosis, confirming the efficient ferroptosis-augmented SDT induced by such a doping nanostructure under US irradiation (Fig. 7g).

Conclusions
In summary, we successfully designed and engineered a distinct Fe/ Mn-doped ZnO nanosonosensitizer for simultaneously triggering multiple ferroptosis effect in the specific tumor microenvironment and augmenting the therapeutic efficacy of SDT against tumor. The engineered (d) Tumor-growth curves of mice dependent on time in 5 groups after different treatments. (n ¼ 5 in each group, *p < 0.05, **p < 0.01, ***p < 0.001). (e) Weight of tumor from each mouse after sacrifice. (f) DCFH-DA staining of different treatment groups to detect ROS production. (g) H&E, TUNEL and Ki67 staining in tumor tissues after different treatments. The applied US irradiation parameters were set to be 1 MHz, 50% duty cycle, 1.5 W/cm 2 , 2 min. D-ZnO-PEG NPs not only tringgered the catalytic Fenton reaction for efficient ROS production and depleted GSH in the presence of Fe and Mn for inducing multiple ferroptosis effect, but also augmented the SDT effect by decreasing the recombination of e À /h þ pairs under US irradiation during the SDT procedure. The simultaneously endowed multiple ferroptosis and synergistically enhanced SDT achieved high in vivo tumorsuppression efficiency as demonstrated on tumor-bearing mice model. The underlying high therapeutic mechanism was revealed by multiple characterization techniques including the specific RNA sequencing. This work provides the unique paradigm of enhancing the SDT performance of nanosonosensitizers by trasitional metal doping, which is highly informative to further explore other ferroptosis and SDT-based therapeutic modality for efficient tumor nanotherapy with simultaneous desirable therapeutic biosafety, precision, efficency and outcome.