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1.
Lizhu Zhang Cunbo Wei Jiawen Wu Dan Liu Yinchao Yao Zhuo Chen Jianxun Liu Chang-Jiang Yao Dinghua Li Rongjie Yang Zhonghua Xia 《Chemical science》2022,13(25):7475
Alkynes are widely used in chemistry, medicine and materials science. Here we demonstrate a transition-metal and photocatalyst-free inverse Sonogashira coupling reaction between iodoalkynes and (hetero)arenes or alkenes under visible-light irradiation. Mechanistic and computational studies suggest that iodoalkynes can be directly activated by visible light irradiation, and an excited state iodoalkyne acted as an “alkynyl radical synthetic equivalent”, reacting with a series of C(sp2)–H bonds for coupling products. This work should open new windows in radical chemistry and alkynylation method.A transition-metal and photocatalyst-free, photoinduced inverse Sonogashira coupling reaction was developed. Under visible-light irradiation, the excited state iodoalkyne acted as an “alkynyl radical synthetic equivalent”.Alkynes are among the most important class of compounds in organic chemistry. Because of their structural rigidity, special electronic properties and numerous methods available for the functionalization of the triple bond, alkynes are important tools and structural elements both in medicinal chemistry and materials sciences.1 Therefore, the development of a new methodology to introduce carbon–carbon triple bonds is of great importance in organic chemistry. The Sonogashira coupling reaction is typically used for the formation of C(sp)–C(sp2) bonds starting from hetero(aryl) halides and terminal alkynes.2 Recently, “inverse Sonogashira coupling” involving the direct alkynylation of unreactive C(sp2)–H bonds with readily available alkynyl halides has received growing interest in the development of a complementary strategy (Fig. 1a). Various main-group and transition metals have been developed to promote this transformation.3 In addition, a photomediated Sonogashira reaction without a photocatalyst was also developed by several groups (Fig. 1b).4Open in a separate windowFig. 1Models of alkynylation. (a) Conventional inverse Sonogashira reaction. (b) Photomediated Sonogashira reaction. (c) SOMOphilic alkynylation. (d) Photoinduced inverse Sonogashira reaction.In recent years, SOMOphilic alkylnylation (SOMO = singly occupied molecular orbital) has become an excellent method of introducing alkynyl groups (Fig. 1c).5 Based on photoredox and transition metal catalysis, numerous in situ generated radicals undergo α-addition and β-elimination to alkynyl reagents, like the broadly applicable ethynylbenziodoxolone (EBX) reagent. Various radical alkynylations were thus discovered by Li,6 Chen,7 Waser,8 and many other groups.9 However, extending the scope of radical precursors, more atom–economic reactions, and a deeper understanding of the mechanism in these transformations are still highly desirable.After the discovering of trityl radicals by Gomberg in 1900, the “rational” era of radical chemistry has since begun.10 Now, the development of radical reactions, especially those involving C(sp3) and C(sp2) radicals, enables rapid access to drug discovery, agrochemistry, materials science, and other disciplines.11 However, the C(sp) radical remains a baffling species. Due to their very high energy, short life time, and limited and harsh preparation methods, alkynyl radicals remain an elusive species, which just exists in some extreme environments, like outer-space and the petrochemical industry.12 Even though alkynyl radicals have been proposed as intermediates for some alkynylation methods, they were regarded as mysterious species and ignored by organic chemists for a long time.13 Recently, two approaches have been developed to aid the alkynyl radical generation step. In 2015, Hashmi and collaborators reported a [Au2(μ-dppm)2]2+ catalyzed free radical–radical C(sp)–C(sp3) bond coupling reaction between iodoalkynes and aliphatic amines.14 Under irradiation of sunlight, the dimeric gold complex was proposed to reduce the iodine acetylide to an alkynyl radical. In 2017, Li developed a transition-metal-free alkynylation reaction between iodoalkyne and 2-indolinone.15 Iodoalkynes could release alkynyl radicals under high temperature conditions. In 2019, we reported an Au(i) and Ir(iii) catalyzed alkynylative cyclization of o-alkylnylphenols with iodoalkynes, wherein the photosensitized energy transfer promoted the oxidative addition of a gold(i) complex with iodoalkynes.16 Based on our continuous interest in haloalkyne and photo-chemistry, we proposed that an iodoalkyne could be a potential “alkynyl radical precursor” under light irradiation. In this work, we uncovered a novel mode of transition-metal and photocatalyst-free, direct photoexcitation of iodoalkynes for the inverse Sonogashira coupling reaction with arenes, heteroarenes, and alkenes via an “alkynyl-radical type” transfer (Fig. 1d). 相似文献
2.
Long Yang Becky Bongsuiru Jei Alexej Scheremetjew Binbin Yuan A. Claudia Stückl Lutz Ackermann 《Chemical science》2021,12(39):12971
Copper-catalyzed electrochemical direct chalcogenations of o-carboranes was established at room temperature. Thereby, a series of cage C-sulfenylated and C-selenylated o-carboranes anchored with valuable functional groups was accessed with high levels of position- and chemo-selectivity control. The cupraelectrocatalysis provided efficient means to activate otherwise inert cage C–H bonds for the late-stage diversification of o-carboranes.Copper-catalyzed electrochemical cage C–H chalcogenation of o-carboranes has been realized to enable the synthesis of various cage C-sulfenylated and C-selenylated o-carboranes.Carboranes are polyhedral molecular boron–carbon clusters, which display unique properties, such as a boron enriched content, icosahedron geometry and three-dimensional electronic delocalization.1 These features render carboranes as valuable building blocks for applications to optoelectronics,2 as nanomaterials, in supramolecular design,3 organometallic coordination chemistry,4 and boron neutron capture therapy (BNCT) agents.5 As a consequence, considerable progress has been witnessed in transition metal-catalyzed regioselective cage B–H functionalization of o-carboranes6 and different functional motifs have been incorporated into the cage boron vertices.7–10 However, progress in this research arena continues to be considerably limited by the shortage of robust and efficient methods to access carborane-functionalized molecules. While C–S bonds are important structural motifs in various biologically active molecules and functional materials,11 strategies for the assembly of chalcogen-substituted carboranes continue to be scarce. A major challenge is hence represented by the strong coordination abilities of thiols to most transition metals, which often lead to catalyst deactivation.12 While copper-catalyzed B(4,5)–H disulfenylation of o-carboranes was achieved,7e elevated reaction temperature was required, and 8-aminoquinoline was necessary as bidentate directing group. The bidentate directing group13 needs to be installed and removed, which jeopardizes the overall efficacy. Likewise, an organometallic strategy was recently devised for cysteine borylation with a stoichiometric platinum(ii)-based carboranes.14 Meanwhile, oxidative cage B/C–H functionalizations largely call for noble transition metal catalysts15 and stoichiometric amounts of chemical oxidants, such as expensive silver(i) salts.16In recent years, electricity has been identified as an increasingly viable, sustainable redox equivalent for environmentally-benign molecular synthesis.17,18 While significant advances have been realized by the merger of electrocatalysis with organometallic bond activation,19 electrochemical carborane functionalizations continue unfortunately to be underdevelopment. In sharp contrast, we have now devised a strategy for unprecedented copper-catalyzed electrochemical cage C–H chalcogenations of o-carboranes in a dehydrogenative manner, assembling a variety of C-sulfenylated and C-selenylated o-carboranes (Fig. 1a). It is noteworthy that our electrochemical cage C–S/Se modification approach is devoid of chemical oxidants, and does not need any directing groups, operative at room temperature.Open in a separate windowFig. 1Electrochemical diversification of o-carboranes and optimization of reaction conditions. aReaction conditions: procedure A: 1a (0.10 mmol), 2a (0.3 mmol), CuOAc (15 mol%), 2-PhPy (15 mol%), LiOtBu (0.2 mmol), TBAI (2.0 equiv.), solvent (3 mL), platinum cathode (10 mm × 15 mm × 0.25 mm), graphite felt (GF) anode (10 mm × 15 mm × 6 mm), 2 mA, under air, r.t., 16 h. bYield was determined by 1H NMR with CH2Br2 as the internal standard. cIsolated yields in parenthesis. dKI (1.0 equiv.) as additive. eProcedure B: 2 (0.3 mmol), LiOtBu (0.2 mmol), TBAI (2.0 equiv.), solvent (3.0 mL), 2 mA, r.t., 3 h, then adding 1a (0.10 mmol), 2-PhPy (15 mol%), CuOAc (15 mol%), 2 mA, rt, 16 h. f2b (0.3 mmol), LiOtBu (0.2 mmol), KI (1.0 equiv.), TBAI (2.0 equiv.), solvent (3.0 mL), 2 mA, r.t., 3 h, then adding 1a (0.10 mmol), 2-PhPy (15 mol%), CuOAc (15 mol%), r.t., 16 h. TBAI = tetrabutylammonium iodide, TBAPF6 = tetrabutylammonium hexafluorophosphate. DCE = 1,2-dichloroethane, THF = tetrahydrofuran.We commenced our studies by probing various reaction conditions for the envisioned copper-catalyzed cage C–H thiolation of o-carborane in an operationally simple undivided cell setup equipped with a GF (graphite felt) anode and a Pt cathode (Fig. 1b and Table S1†). After extensive experimentation, we observed that the thiolation of substrate 1 proceeded efficiently with catalytic amounts of CuOAc and 2-phenylpyridine, albeit in the presence of 2 equivalents LiOtBu as the base, and 2 equivalents n-Bu4NI as the electrolyte at room temperature under a constant current of 2 mA (entry 1). The yield was reduced when other copper sources or additives were used (entries 2–5). Surprisingly, n-Bu4NPF6 as the electrolyte failed to facilitate the carborane modification, indicating that n-Bu4NI operates not only as electrolyte, but also as a redox mediator (entry 6). Altering the stoichiometry of the electrolyte or using KI did not improve the performance (entries 7–8). Product formation was not observed, when the reaction was conducted with DCE as the solvent, while CH3CN resulted in a drop of the catalytic performance (entries 9–10). Control experiments confirmed the essential role of the electricity and the catalyst (entries 11–12), while a sequential procedure was found to be beneficial (entries 13–15).With the optimized reaction conditions in hand, we explored the versatility of the cage C–H thiolation of o-carborane 1a with different thiols 2 (Scheme 1). Electron-rich as well as electron-deficient substituents on the arenes were found to be amenable to the electrocatalyzed C–H activation, providing the corresponding thiolation products 3aa–3ao in good to excellent yields. Thereby, a variety of synthetically useful functional groups, such as fluoro (3ae, 3am), chloro (3af, 3ak, 3an) and bromo (3ag, 3al), were fully tolerated, which should prove instrumental for further late-stage manipulations. Various disubstituted aromatic and heterocyclic thiols afforded the corresponding cage C–S modified products 3ap–3as. Notably, aliphatic thiols efficiently underwent the electrochemical transformation to provide the corresponding cage alkylthiolated products 3at–3au. Notably, the halogen-containing thiols (2e–2f, 2k–2n and 2q) reacted selectively with o-carboranes to deliver the desired products without halide coupling byproducts being observed. The connectivity of the products 3aa, 3am and 3ao was unambiguously verified by X-ray single crystal diffraction analysis.22Open in a separate windowScheme 1Electrochemical C–H thiolation of o-carborane 1a. (a) Procedure B. (b) KI (1 equiv.). (c) Cul as the catalyst.Encouraged by the efficiency of the cupraelectro-oxidative cage C–H thiolation, we became intrigued to explore the chalcogenantion of differently-decorated o-carboranes 1 (Scheme 2). Electronically diverse carboranes 1 served as competent coupling partners, giving the corresponding thiolation products 4bo–4do with high levels of efficacy in position-selective manner. The strategy was not restricted to phenyl-substituted o-carboranes. Indeed, substrates bearing benzyl and even alkyl groups also performed well to deliver the desired products 4eo–4ga. It is noteworthy that the C–H activation approach was also compatible with selenols to give the o-carboranes 4av–4fv. The molecular structures of the carborane 4br and 4av were unambiguously verified by single-crystal X-ray diffraction.22Open in a separate windowScheme 2Electrochemical cage C–H chalcogenation of o-carboranes. (a) Procedure B. (b) KI (1 equiv.).Scaffold functionalization of the thus obtained carborane 3ag provided the alkynylated derivative 5a and amine 5b (Scheme 3), giving access to carborane-based host materials of relevant to phosphorescent organic light-emitting diodes.20Open in a separate windowScheme 3Late-stage diversification.Next, we became attracted to delineating the mode of the cupraelectro-catalyzed cage C–H chalcogenation. To this end, control experiments were performed (Scheme 4a). First, electrocatalysis in the presence of TEMPO or Ph2C CH2 gave the desired product 3aa. EPR studies of thiol 2a, LiOtBu and THF under the electrochemical conditions showed a small radical signal, which might be attributed to a thiol radical.21 Second, the cupraelectrocatalysis occurred efficiently in the dark. Third, detailed cyclovoltammetric analysis of the thiol and iodide mediator (Scheme 4b and ESI†)21 revealed an irreversible oxidation of the thiol anion at Ep = −0.62 V vs. Ag/Ag+ and two oxidation events for the iodide, including an irreversible oxidation at Ep = 0.12 V vs. Ag/Ag+ and a reversible oxidation at Ep = 0.44 V vs. Ag/Ag+, which is in good agreement with the literature reported iodide oxidation potentials,18c,d and is suggestive of the preferential oxidation of the iodide as a redox mediator. In this context, the use of n-Bu4NI as a redox mediator to achieve copper-catalyzed electrochemical arene C–H aminations had been documented.18d Furthermore, we calculated the redox potential of complex C by means of DFT calculations at the PW6B95-D4/def2-TZVP + SMD(MeCN)//TPSS-D3BJ/def2-SVP level of theory.21 These studies revealed a calculated oxidation half-wave potential for complex C is Eo,calc1/2 = −0.08 V vs. SCE. Hence, iodide is a competent redox mediator to achieve the transformation from complex C to complex D. Analysis of non-covalent interactions21 in complex C (Fig. 2) show the presence of a weak stabilization interaction between the chalcogen''s anisole group and the 2-phenylpyridine. In contrast, in complex D these interactions were found more relevant between the o-carborane phenyl group and the chalcogen aromatic motif.Open in a separate windowFig. 2Non-covalent interaction plots for the complexes C and D. Strong attractive interactions are shown in blue, weak attractive interactions are given in green, while red corresponds to repulsive interactions. Ar = 4-MeOC6H4.Open in a separate windowScheme 4Control experiments and cyclic voltammograms.On the basis of the aforementioned findings,18 a plausible reaction mechanism is proposed in Scheme 5, which commences with an anodic single electron-transfer (SET) oxidation of the thiol anion E to form the sulfur-centered radical F. Subsequently, the copper(i) species A reacts with the sulfur radical F to deliver copper(ii) complex B, which next reacts with o-carborane 1 in the presence of LiOtBu to generate a copper(ii)-o-carborane complex C. Thereafter, the complex C is oxidized by the anodically generated redox mediator I2 to furnish the copper(iii) species D,18d which subsequently undergoes reductive elimination, affording the final product and regenerating the catalytically active complex A. Alternatively, the direct oxidation of copper(ii) complex C by electricity to generate copper(iii) species D can not be excluded at this stage.18a,bOpen in a separate windowScheme 5Proposed reaction mechanism.In conclusion, a sustainable electrocatalytic C–H chalcogenation of o-carboranes with thiols and selenols was realized at room temperature by earth abundant copper catalysis. The C–H activation was characterized by mild reaction conditions and high functional group tolerance, leading to the facile assembly of various o-carboranes. Thereby, a transformative platform for the design of cage C–S and C–Se o-carboranes was established that avoids chemical oxidants by environmentally-sound electricity in the absence of directing groups. A plausible mechanism of paired electrolysis was established by detailed mechanistic studies. 相似文献
3.
Fei Yu Tingting Huo Quanhua Deng Guoan Wang Yuguo Xia Haiping Li Wanguo Hou 《Chemical science》2022,13(3):754
Expediting the oxygen evolution reaction (OER) is the key to achieving efficient photocatalytic overall water splitting. Herein, single-atom Co–OH modified polymeric carbon nitride (Co-PCN) was synthesized with single-atom loading increased by ∼37 times with the assistance of ball milling that formed ultrathin nanosheets. The single-atom Co-N4OH structure was confirmed experimentally and theoretically and was verified to enhance optical absorption and charge separation and work as the active site for the OER. Co-PCN exhibits the highest OER rate of 37.3 μmol h−1 under visible light irradiation, ∼28-fold higher than that of common PCN/CoOx, with the highest apparent quantum yields reaching 4.69, 2.06, and 0.46% at 400, 420, and 500 nm, respectively, and is among the best OER photocatalysts reported so far. This work provides an effective way to synthesize efficient OER photocatalysts.Single-atom CoII-OH modified polymeric carbon nitride synthesized with increased single-atom loading under the assistance of ball milling exhibits high photocatalytic water oxidation activity with Co-N4OH as the highly active site.Massive fuel energy consumption induced environmental and ecological problems, especially the greenhouse effect, and the resultant extreme climates and rise in sea level are threatening human life.1 As a potential substitution for fuel energy, hydrogen energy conversion from solar energy via photocatalytic water splitting attracts great attention from scientists.2–5 However, the photocatalytic hydrogen evolution efficiency from overall water splitting is still restricted by the sluggish oxygen evolution reaction (OER) that involves energy absorption, four-electron transfer, breakage of O–H bonds, and formation of O–O bonds,6,7 and thus efficient OER photocatalysts become the key to achieving efficient overall water splitting. Though numerous hydrogen evolution photocatalysts have been reported, research on OER photocatalysts is mainly around a few semiconductors including BiVO4, WO3, Ag3PO4, α-Fe2O3, etc.8–11 and their activity is not high enough yet for practical applications. Therefore, exploring high-efficiency OER photocatalysts is still necessary.Polymeric carbon nitride (PCN) was first reported in 2009 (ref. 12) as a photocatalyst with a layered melon-type orthorhombic structure,13 and thereafter quickly became a “star” photocatalyst thanks to its advantages of being visible-light responsive and metal-free, non-toxic, and low cost, and its relatively high chemical stability.14 Because of several self-deficiencies including fast photogenerated charge recombination and a narrow optical absorption spectrum, PCN exhibits relatively low photocatalytic activity.15 Then, a series of strategies were put forward successively to enhance the photoactivity of PCN, such as enhancement of crystallinity,16 morphological control,17 structural modification18 (including extensively researched single atom modification in recent years19,20), exfoliation,21 construction of hetero-(homo-)junctions,22 and loading of noble metals.23 Though photocatalytic water splitting on PCN was extensively researched in the past, the research was mainly around the hydrogen evolution half-reaction used for exploring properties and the catalytic mechanism of photocatalysts, and little research was focused on the industrially useable overall water splitting process owing to the sluggish OER.15 Therefore, enhancing the photocatalytic OER activity of PCN becomes the key to practical applications.To increase OER rates of PCN, several kinds of methods were proposed, such as rational design of compound cocatalysts (e.g., CoOx, IrO2, CoP, CoPi, RhOx, RuOx, PtOx, MnOx, Co(OH)2, Ni(OH)2, and CoAl2O4 (ref. 24–30)), modification of carbon dots and carbon rings,31,32 fabrication of special architectures of PCN (e.g., PCN quantum dot stacked nanowires33), and single-atom (e.g., B, Co, and Mn34–36) modification. For instance, Zhao and coauthors prepared B and N-vacancy comodified PCN that exhibits the highest OER rate of ∼28 μmol h−1 (ref. 36) and recently their group further used these B doped PCN ultrathin nanosheets to fabricate a Z-scheme heterojunction for overall water splitting with a solar-to-hydrogen efficiency reaching ∼1.2%.37 Comparatively, PCN loaded with compound cocatalysts can only enhance OER activity to a limited degree and there are finite methods for carbon modification and special architecture fabrication. Single-atom modification shows a bright prospect, on account of metal atoms capable of being inserted into the framework of PCN and effectively increasing the OER activity. However, reported single metal atom modification routes are all based on direct ion adsorption on PCN or calcination of mixtures of metal salts and PCN feedstocks.34,35,38 New routes need be explored to increase effective loading of single atoms in PCN. Besides, the metal-OH structure is considered efficient for the OER,30,39,40 and a single metal atom-OH structure has never been reported for modification of PCN, though Mn–OH was thought to play a key role in the OER process.34Ball milling is an extensively used versatile and scalable way for preparation of heterogeneous catalysts and even single-atom catalysts,41,42 but was rarely used in synthesis of PCN-based single-atom photocatalysts. In this work, we synthesized single-atom Co–OH modified PCN (Co-PCN) with the single-atom content in PCN highly increased with the assistance of ball milling. The simple synthetic route is shown in Fig. 1a. PCN was ball-milled to obtain BM-PCN that then adsorbed Co2+ till saturation to form BM-PCN/Co which was calcined to obtain BM-PCN/Co-c (Co-PCN). For comparison, PCN was directly used to adsorb Co2+ till saturation to form PCN/Co which was calcined to obtain PCN/Co-c. PCN mainly comprises large blocks with the size of several micrometers (Fig. S1†), while BM-PCN contains massive irregular particles with the size reduced to several hundreds of nanometers (Fig. S2†), indicative of high efficacy of ball milling. BM-PCN/Co-c exhibits a similar morphology as BM-PCN (Fig. 1b and S3†) and PCN/Co-c exhibits a similar morphology to PCN (Fig. S4†), but the surface area and mesopore volume of BM-PCN and BM-PCN/Co-c are not higher than those of PCN and PCN/Co-c (Fig. S5†), manifesting that ball-milling and subsequent calcination did not form massive mesopores, which accords well with the particle size variation from several micrometers (before ball milling) to several hundreds of nanometers (after ball milling). However, the Co content in BM-PCN/Co-c, BM-PCN/Co, PCN/Co-c, and PCN/Co was measured to be 0.75, 0.50, 0.02, and ∼0.02 wt%, respectively, by inductively coupled plasma mass spectrometry (ICP-MS). The ∼37 times higher Co content in BM-PCN/Co-c than in PCN/Co-c suggests the ball-milling enhanced adsorption of Co2+ on surfaces of BM-PCN, which should arise mainly from the ball-milling induced increase of surface energy and adsorption sites.43Open in a separate windowFig. 1(a) Schematic illustration for synthesis of single-atom CoII-OH modified PCN (BM-PCN/Co-c); and (b) SEM, (inset in b) TEM, (c) AFM, (d) EDS elemental mapping, and (e) HAADF-STEM images of BM-PCN/Co-c.The TEM image shows the existence of small and ultrathin nanosheets in BM-PCN/Co-c (inset in Fig. 1b) which can also be observed in the atomic force microscopy (AFM) image with a thickness of ∼7–10 nm and lateral size of <70 nm (Fig. 1c), and formation of these ultrathin nanosheets results from the ball milling of PCN.44 It should be noted that most formed ultrathin nanosheets with high surface energy may stack into compact particles upon ball milling, leading to no increase of the total surface area. Energy dispersive X-ray spectroscopy (EDS) elemental mapping images of BM-PCN/Co-c indicate homogeneous distribution of C, N, O, and Co elements in the sample (Fig. 1d). The high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image of BM-PCN/Co-c shows massive white spots (marked by circles) with a mean size of <1 Å dispersed in the sample (Fig. 1e and S6†), which should correspond to single-atom Co.To further verify the single-atom Co structure in BM-PCN/Co-c, Co K-edge X-ray absorption near-edge structure spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS) analysis were performed. As shown in Fig. 2a, the absorption-edge position of BM-PCN/Co-c is quite close to that of CoO and their peak positions are similar and far from those of other reference samples, indicating that the valence of Co in BM-PCN/Co-c is about +2. The bonding structure around Co was determined by Fourier transformed (FT) k3-weighted EXAFS analysis. As shown in Fig. 2b, a distinct single Co-ligand peak at ∼1.6 Å for BM-PCN/Co-c is observed, which prominently differs from the Co–Co coordination peak at ∼2.2 Å for Co foil and the CoII–O coordination peak at ∼1.7 Å for CoO. The wavelet transform (WT) contour plot of BM-PCN/Co-c shows only one intensity maximum (Fig. S7†), and the Cl 2p core-level XPS spectrum of BM-PCN/Co-c reveals no residue of Cl (Fig. S8†). These further indicate the single-atom dispersion of Co species. Apparently, the Co-ligand peak is almost consistent with the CoII–N peak for Co porphyrin, suggesting that the single-atom Co in BM-PCN/Co-c mainly coordinates with N. Least-square EXAFS curve fitting was performed to confirm quantitative structural parameters of CoII in BM-PCN/Co-c, as shown in Fig. 2c, S9, and S10 and Table S1.† Simple Co–N single-shell fitting of BM-PCN/Co-c (Fig. S10†) gave a coordination number of 5.6 ± 0.4 (Table S1†), that is, CoII coordinates with five atoms. Considering that the PCN monolayer provides four appropriate N coordination sites at most,45 CoII likely coordinates with four N atoms and one OH atom. Thus, we further performed Co–N4/Co–O double-shell fitting (Fig. 2c) and the obtained R-factor (0.0011) remarkably reduces relative to that from Co–N single-shell fitting (0.0035), indicative of rationality of the proposed CoII–N4OH structure. Confirmed Co–N and Co–O bond lengths are 2.04 and 2.15 Å, respectively (Table S1†).Open in a separate windowFig. 2(a) Co K-edge XANES and (b) EXAFS spectra of Co foil, Co porphyrin (Copr), CoO, Co3O4, Co2O3, and BM-PCN/Co-c; EXAFS (c) R space-fitting and (inset in c) K space-fitting curves of BM-PCN/Co-c; (d) optimized structure of PCN and Co-doped PCN with different doping configurations and calculated formation energies (e) of Co doped PCN; and (e) Co 2p and (f) O 1s core-level XPS spectra of samples.To further confirm rationality of the Co–N4OH coordination structure, density functional theory (DFT) calculations were conducted. As shown in Fig. 2d, three possible CoII coordination structures in the PCN monolayer were explored. The Co–N4OH structure without removal of H from PCN exhibits a much lower formation energy (∼0.15 eV) than Co–N4 and Co–N3 structures with removal of two H atoms from PCN (∼2.51 and 3.55 eV), demonstrating a high probability of existence of the Co–N4OH structure in BM-PCN/Co-c. This structure can also be evidenced by X-ray photoelectron spectroscopy (XPS). As shown in Fig. 2e, the Co 2p core-level XPS spectrum of BM-PCN/Co-c shows two distinct peaks at binding energies of 796.8 and 781.4 eV beside satellite peaks, corresponding to Co 2p1/2 and 2p3/2 of CoII ions.46 The spectrum of BM-PCN/Co also shows two Co 2p peaks but at a binding energy ∼1.1 eV higher, suggesting variation of the Co coordination structure from BM-PCN/Co to BM-PCN/Co-c. PCN/Co-c exhibits no peaks because of its low Co content. Fig. 2f shows O 1s core-level spectra of PCN, BM-PCN, BM-PCN/Co-c, and PCN/Co-c. All the samples exhibit one peak at a binding energy of ∼532.0 eV, ascribed to surface hydroxyl species,47 but an additional peak could be obtained for BM-PCN or BM-PCN/Co-c after deconvolution. The peak at a binding energy of ∼531.3 eV for BM-PCN should be assigned to adsorbed H2O at new active adsorption sites generated by ball milling. This peak can also be observed in the spectrum of BM-PCN/Co, but with a ∼0.1 eV shift to a higher binding energy (Fig. S11†) owing to the influence of adsorbed CoII ions. The peak at ∼531.2 eV for BM-PCN/Co-c should be assigned to Co–OH,48 given that there is only one O 1s peak for BM-PCN-c (synthesized by direct calcination of BM-PCN) (Fig. S11†). The calculated Co/O(–Co) molar ratio, based on the XPS data, is ∼1.07 (Table S2†), close to 1, consistent with the Co–N4OH coordination structure.In C 1s and N 1s core-level XPS spectra, BM-PCN, BM-PCN/Co-c, PCN/Co-c, and BM-PCN/Co exhibit similar peaks to PCN (Fig. S12a–d†), indicative of their similar framework structure which can also be evidenced by their similar N/C molar ratios, 1.53 (Table S3†), but the N–H peak of BM-PCN shifts ∼0.2 eV to a lower binding energy relative to that of PCN, likely arising from the ball-milling induced destruction of intralayer hydrogen bonds (Fig. S13†). The Co content in BM-PCN/Co, BM-PCN/Co-c, and PCN/Co-c is too low to cause detectable variation of C 1s and N 1s peaks. Similar FT-IR absorption bands of the samples (Fig. S14a and b†) also indicate their basic frame structure, but in enlarged spectra (Fig. S14c†), ν(C–N) and ν(C N) absorption bands of BM-PCN shift 16 cm−1 to a higher wavenumber and 19 cm−1 to a lower wavenumber, respectively, relative to those of PCN at 1242 and 1640 cm−1,49 likely resulting from the ball-milling induced hydrogen bond destruction, and the shift of these two absorption bands turns smaller for BM-PCN/Co-c, suggesting calcination-induced reforming of the destroyed hydrogen bonds, which is consistent with the XPS results (Fig. S12c†). Besides, BM-PCN exhibits a wider and relatively stronger ν(N–H)/ν(O–H) absorption band than PCN (Fig. S14a†), probably owing to the hydrogen bond destruction and new adsorbed H2O, while this absorption band for BM-PCN/Co-c becomes much weaker, suggesting hydrogen bond reforming and loss of new adsorbed H2O (Fig. 2f). Zeta potentials of the samples dispersed in water reflect variation of surface adsorbed hydroxyl species. As shown in Fig. S15a,† all the samples exhibit negative zeta potentials because of dissociation of surface hydroxyl species. The zeta potentials, following the order PCN (−24 mV) > BM-PCN (−41 mV) < BM-PCN/Co-c (−30 mV) ≈ PCN/Co-c (−28 mV), suggest the ball milling-induced increase of surface hydroxyls in BM-PCN and calcination-induced decrease in BM-PCN/Co-c, consistent with the FT-IR results.Solid-state 13C magic-angle-spinning nuclear magnetic resonance (NMR) spectra of PCN, BM-PCN, BM-PCN/Co-c, and PCN/Co-c show two similar peaks at chemical shifts of ∼164 and 156 ppm (Fig. S15b†), ascribed to C−NHx and N C–N, respectively,50 indicating their similar molecular framework, but in enlarged spectra, BM-PCN exhibits ∼0.3° movement of the N C–N peak to a lower chemical shift compared with PCN, because of the ball-milling induced hydrogen bond destruction, and the C−NHx peak of BM-PCN/Co-c moves ∼0.2° to a lower chemical shift, likely owing to formation of the C–N–Co structure whose peak lies close to the C−NHx peak.51 The XRD patterns of the samples are shown in Fig. S15c.† PCN and PCN/Co-c exhibit typical diffraction peaks of melon-type carbon nitride with a layered orthorhombic structure and peaks at 13.1° and 27.6° correspond to (210) and (002) facets, respectively,13,52 but BM-PCN reveals remarkably decreased peak intensity and ∼0.2° shift of the (002) peak to a lower 2θ (indicative of the increased interlayer distance) relative to PCN, demonstrating the ball-milling induced hydrogen bond destruction and substantial decrease of crystallinity. The remarkable decrease of crystallinity and almost no change of the surface area of BM-PCN, compared with those of PCN, further suggest that ball milling may form massive thin nanosheets (Fig. 1c) most of which stack into compact particles (Fig. 1b) owing to their high surface energy. In comparison with BM-PCN, BM-PCN/Co-c exhibits a narrower (002) peak, suggesting enhanced crystallinity owing to the calcination-induced hydrogen bond reforming, consistent with the FT-IR results. On the whole, it is likely the ball-milling induced destruction of hydrogen bonds that contributes largely to the increase of surface energy and new active adsorption centers and thus Co2+ adsorption on BM-PCN.Optical absorption capability of samples was investigated by UV-vis diffuse reflectance spectroscopy (DRS). As shown in Fig. 3a, BM-PCN/Co-c, BM-PCN, and PCN/Co-c exhibit considerably higher, lower, and similar optical absorption than/to PCN, respectively. For BM-PCN/Co-c, the optical absorption enhancement at a wavelength of <400 nm may benefit from the electron-rich Co that enhances π–π* transitions in heptazine rings,53 and the Urbach tail absorption should arise from the Co–OH doping.54,55 Bandgaps (Eg) of PCN, BM-PCN, BM-PCN/Co-c, and PCN/Co-c were roughly confirmed as 2.70, 2.81, 2.56, and 2.73 eV, respectively, via the formula Eg/eV = 1240/(λed/nm)56 where λed is the absorption edge determined by solid lines in the spectra. The wider Eg of BM-PCN probably results from the quantum size effect of massive ultrathin crystal nanosheets (Fig. 1c) formed by ball milling, and the narrower Eg of BM-PCN/Co-c arises from the Co–OH doping that was then verified by DFT calculations. As shown in Fig. S16,† the calculated Eg of BM-PCN/Co-c, ∼1.90 eV, is much smaller than that of PCN (2.57 eV), in accordance with the experimental results. For PCN, the conduction band (CB) is contributed by C 2p and N 2p orbitals and the valence band (VB) mainly by N 2p orbitals, while for BM-PCN/Co-c, the CB is contributed by Co 3d, C 2p, and N 2p orbitals and the VB mainly by Co 3d and N 2p orbitals (Fig. S16c and d†), effectively manifesting that the narrowing of Eg of BM-PCN/Co-c results from the Co–OH doping. In addition, there are prominent doping levels (Ed) in the bandgap of BM-PCN/Co-c, mainly contributed by Co 3d and O 2p orbitals (Fig. S16d†), effectively proving the Co–OH doping effect in BM-PCN/Co-c. Similar calculation results have been reported for Pt–OH modified carbon nitride.57 Given that the experimental Co content (0.75 wt%) is much lower than the theoretical (6.71 wt%), practical doping levels in the bandgap may approach more to the VB. CB edges of the samples (ECB) could be roughly determined by using Mott-Schottky plots (Fig. S17†) and their Fermi levels (Ef) were subsequently confirmed based on VB-XPS spectra (Fig. S18†). Energy band levels of the samples are shown in Fig. 3b, and it seems that ball milling causes a slight downshift of the VB edge (EVB) of BM-PCN, favorable for photocatalytic water splitting, but the Co–OH doping causes a slight downshift of ECB and upshift of EVB of BM-PCN/Co-c. It is noteworthy that the Ed close to the VB edge (EVB) can capture photogenerated holes58 and thus the single-atom Co–OH works as the active site for the OER (Fig. 3b).Open in a separate windowFig. 3(a) UV-vis diffuse reflectance spectra of PCN, BM-PCN, BM-PCN/Co-c, and PCN/Co-c; (b) energy band levels of the samples and schematic illustration for water oxidation on BM-PCN/Co-c; (c) photoluminescence spectra, (d) time-resolved fluorescence spectra, and (e) anodic photocurrent (Ja) response of the samples; and (f) EPR spectra of the samples in the dark and under visible light irradiation. Data in (d) are the results of fitting decay curves to a tri-exponential model. Dark Ja in (e) was set as zero for distinct comparison.Spectroscopy and photoelectrochemical tests were conducted to evaluate photogenerated charge separation and transfer performance. As shown in Fig. 3c, photoluminescence (PL) spectra of all the samples show one emission peak, basically corresponding to their bandgap emission. BM-PCN exhibits weaker PL intensity than PCN, revealing a decreased photogenerated charge recombination efficiency, which originates from faster charge transfer from the inside to the surface of ultrathin nanosheets (Fig. S19†) and trapped by surface states.59 BM-PCN/Co-c exhibits the lowest PL intensity and the PL intensity of PCN/Co-c is lower than that of PCN, which arises from the Ed capturing photogenerated holes to reduce their direct recombination with electrons beside the ultrathin nanosheet effect in BM-PCN/Co-c. Fig. 3d shows time-resolved fluorescence spectra of the samples. Decay curves were well fitted to a tri-exponential model (S3) and the obtained results are shown in Fig. 3d. Three lifetimes (τ1–τ3) and their mean lifetime (τm, 89.2 ns) of BM-PCN are all much longer than those of PCN (τm = 17.9 ns), further suggesting the faster charge transfer from the inside to the surface of ultrathin nanosheets in BM-PCN, decreasing the direct charge recombination efficiency, but with subsequent surface radiative recombination.60 Interestingly, the τ1–τ3 and τm (10.8 ns) of BM-PCN/Co-c are much shorter than those of PCN, which should result from faster transfer of holes to Ed that effectively decreases the charge recombination efficiency, with subsequent nonradiative energy transformation.61 The Co–OH doping effect also makes PCN/Co-c exhibit shorter τ1–τ3 and τm (16.5 ns) than PCN. Fig. 3e shows the photocurrent response of the samples. Their anodic photocurrent density follows the order PCN < PCN/Co-c < BM-PCN < BM-PCN/Co-c, indicating gradually increased photogenerated charge separation efficiencies,62 basically consistent with the PL results. The relatively high photocurrent response of BM-PCN benefits from the applied bias that effectively inhibits surface recombination of photogenerated charge carriers.To assess charge mobility of the samples, their electrochemical impedance spectroscopy (EIS) spectra were tested with high-frequency data simply fitted to an equivalent circuit (Fig. S20†). The obtained charge transfer resistance (Rct) follows the order PCN (26 Ω) > BM-PCN (18 Ω) ≈ PCN/Co-c (19 Ω) > BM-PCN/Co-c (13 Ω). Apparently, BM-PCN/Co-c exhibits smaller Rct than BM-PCN and PCN/Co-c, and PCN/Co-c exhibits smaller Rct than PCN, indicating the highest charge transfer performance of BM-PCN/Co-c63 which originates from the single-atom Co modification64 that may increase the electron density to facilitate charge transport. The smaller Rct of BM-PCN than that of PCN indicates the additional favorable effect of ultrathin nanosheets.65Fig. 3f shows electron paramagnetic resonance (EPR) spectra of the samples. All reveal one single Lorentzian line centered at a g of 2.0039, attributed to unpaired electrons in heptazine rings.66 In the dark, the EPR signal intensity follows the order PCN < BM-PCN < PCN/Co-c < BM-PCN/Co-c, and the stronger signal of BM-PCN than that of PCN results from formation of ultrathin nanosheets that enhances delocalization of unpaired electrons, while the stronger signal of BM-PCN/Co-c and PCN/Co-c mainly benefits from the Co doping that increases the delocalized electron density.67 Under visible light irradiation, the samples exhibit remarkable signal enhancement, following the sequence PCN < BM-PCN < PCN/Co-c < BM-PCN/Co-c, similar to that of the signal intensity in the dark, suggesting that the increase in the delocalized electron density facilitates charge photoexcitation. The high delocalized electron density favors charge transport, consistent with the EIS results, and the high photoexcited charge density benefits enhancement of photocatalytic activity.Photocatalytic OER activity of various samples was well evaluated using Ag+ as the sacrificial agent (Fig. S21†). The Co content in BM-PCN/Co-c was optimized according to the photocatalytic OER rates and BM-PCN-c exhibits no detectable OER activity (Fig. S22†), indicating indispensability of the Co–OH structure for the OER. The influence of the calcination temperature (Tc °C) of BM-PCN/Co on OER rates of BM-PCN/Co-c (Tc = 460) and BM-PCN/Co-cTc was investigated and BM-PCN/Co-c exhibits the highest photoactivity (Fig. 4a), manifesting that the optimal calcination temperature is 460 °C. Under both simulated solar light and visible light irradiation (λ ≥ 420 nm), BM-PCN/Co-c exhibits substantially higher OER activity than PCN/Co-c (Fig. 4b), further suggesting the significance of the single-atom Co loading amount, and remarkably higher activity than common PCN/CoOx (with 0.75 wt% Co, obtained via photodeposition) and BM-PCN-c/Co(OH)2 (with 0.75 wt% Co), demonstrating the high efficacy of the single-atom distribution of Co–OH in BM-PCN/Co-c. Besides, urea was used as the feedstock to synthesize carbon nitride (marked as PCN-urea) with a larger surface area (76 m2 g−1 (ref. 68)) than PCN, and PCN-urea was further used to synthesize PCN-urea/Co-c similar to the synthesis of BM-PCN/Co-c. The OER activity of BM-PCN/Co-c is prominently higher than that of PCN-urea/Co-c (with the optimized Co content and Co single atom distribution, Fig. S23†), suggesting the significant role of ball milling in fabricating the single-atom Co–N4OH structure. To quantitively compare photoactivity of the samples, their mean OER rates under visible light illumination for 2 h are shown in Fig. 4c. The OER rate of BM-PCN/Co-c can reach ∼37.3 μmol h−1, about 13.8, 28.7, 2.6, and 2.0 times those of PCN/Co-c, PCN/CoOx, BM-PCN-c/Co(OH)2, and PCN-urea/Co-c, respectively. Comparatively, less N2 was generated for BM-PCN/Co-c (Fig. S24†), further demonstrating the significance of single-atom Co–OH modification.Open in a separate windowFig. 4(a) The influence of the calcination temperature (Tc °C) of BM-PCN/Co on photocatalytic OER activity of BM-PCN/Co-c (Tc = 460) and BM-PCN/Co-cTc, under Xe-lamp illumination, with AgNo3 as the sacrificial agent; (b) photocatalytic oxygen evolution on various samples under Xe-lamp illumination with or without using a 420-nm filter; (c) corresponding OER rates of the samples in 2 h; (d) photocatalytic OER rates of BM-PCN/Co-c under irradiation with various monochromatic light sources for 12 h; (e) apparent quantum yields (AQYs) of BM-PCN/Co-c at different wavelengths and reaction times and the highest AQY at every wavelength, along with the UV-DRS spectrum; and (f) proposed mechanism for photocatalytic water oxidation on the single-atom CoII-OH structure.Photocatalytic oxygen evolution on BM-PCN/Co-c was also tested under monochromatic light irradiation (Fig. S25†). Apparently, BM-PCN/Co-c can exhibit OER activity even at a wavelength of 500 nm. The mean OER rate in 12 h decreases from 1.85 to 0.54 μmol h−1 with increasing wavelengths from 400 to 500 nm (Fig. 4d), independent of light intensity of the Xe lamp and is mainly dependent on optical absorption capability of BM-PCN/Co-c at various wavelengths (Fig. 3a). Fig. 4e shows apparent quantum yields (AQYs) of BM-PCN/Co-c at different reaction times and wavelengths. Basically, there are maxima of AQYs with increasing reaction time at every wavelength, suggesting the adverse effect of excessive photodeposited Ag on surfaces of samples. These maxima are shown in Fig. 4e and accord well with the UV-vis DRS spectrum with increasing wavelengths. The maxima of AQYs at 400, 420, 450, and 500 nm can reach 4.69, 2.06, 1.07, and 0.46%, respectively. Compared with the reported photocatalytic OER results for PCN (Table S4†), BM-PCN/Co-c exhibits the top-class performance.To investigate chemical stability of BM-PCN/Co-c, the cyclic OER experiment was conducted. After five consecutive runs, OER rates of BM-PCN/Co-c decrease less (Fig. S26a†), with the morphology similar to the original (Fig. S26b†). Co single atoms in the sample could still be distinctly observed by HAADF-STEM (Fig. S26c and d†). In addition, N 1s core-level XPS spectra of BM-PCN/Co-c are almost similar before and after the cyclic experiment (Fig. S26e†). These indicate the high stability of the basic framework structure of the sample. However, Co 2p core-level spectra show remarkable differences before and after the experiment, not only the CoII peak shift, probably owing to ion (e.g., IO4−) adsorption, but also formation of a large amount of CoIII (Fig. S26f†). Coexistence of CoII/CoIII may suggest the photocatalytic OER mechanism.The proposed OER mechanism based on the Co–OH structure is shown in Fig. 4f, according to the reported results in Mn doped PCN.34 Four holes are needed to complete four oxidation steps and obtain one O2 molecule. The first step starting with one hole may involve formation of the CoIII O bond. The Co–N4OH structure should facilitate the water oxidation more compared with that of Co–N4 without OH coordination, by leaving out the initial adsorption process of H2O molecules.34 On the whole, the high photocatalytic OER activity of Co-PCN benefits from the Co–N4OH structure that not only effectively enhances optical absorption, and charge separation and transport, but also works as the highly active site for the OER. 相似文献
4.
Chaohai Wang Hongyu Wang Jongbeom Na Yiyuan Yao Alowasheeir Azhar Xin Yan Junwen Qi Yusuke Yamauchi Jiansheng Li 《Chemical science》2021,12(46):15418
Heterogeneous Fenton-like processes are very promising methods of treating organic pollutants through the generation of reactive oxygen containing radicals. Herein, we report novel 0D–1D hybrid nanoarchitectonics (necklace-like structures) consisting of FeCo@N–C yolk–shell nanoreactors as advanced catalysts for Fenton-like reactions. Each FeCo@N–C unit possesses a yolk–shell structure like a nanoreactor, which can accelerate the diffusion of reactive oxygen species and guard the active sites of FeCo. Furthermore, all the nanoreactors are threaded along carbon fibers, providing a highway for electron transport. FeCo@N–C nano-necklaces thereby exhibit excellent performance for pollutant removal via activation of peroxymonosulfate, achieving 100% bisphenol A (k = 0.8308 min−1) degradation in 10 min with good cycling stability. The experiments and density-functional theory calculations reveal that FeCo dual sites are beneficial for activation of O–O, which is crucial for enhancing Fenton-like processes.Novel 0D–1D hybrid nanoarchitectonics consisting of FeCo@N–C yolk–shell nanoreactors are developed for Fenton-like reaction. With the multilevel advantages of this design, FeCo@N–C nano-necklaces exhibit excellent performance for BPA removal.Advanced oxidation processes (AOPs) are one of the most promising strategies to eliminate organic contaminants, sustainably generating reactive oxygen species (ROS) to ideally destroy all non-biodegradable, recalcitrant, toxic, or membrane-permeable organic impurities.1–4 Among these AOPs, sulfate radical (SO4˙−)-based Fenton-like processes have gained increasing attention as a water treatment strategy because of the strong oxidation potential of SO4˙− (3.1 V vs. normal hydrogen electrode) at wider pH ranges. SO4˙− is mainly produced by physical or chemical methods for activation of persulfate salts, such as peroxymonosulfate (PMS) and persulfate.5–9 Over the past two decades, heterogeneous catalysis has emerged as the most effective approach to water treatment, with much effort dedicated to developing better catalysts, including transition metal-based and carbonaceous materials.10,11 Unfortunately, most metal-based catalysts suffer from leaching of toxic metal ions, which can thwart their practical application,12,13 and although carbonaceous catalysts produce no secondary pollution, their cycle performance is always depressed.14 There is therefore an urgent need to find robust catalysts with adequate activity and stability for Fenton-like processes.To achieve superior performance, an ideal Fenton-like catalyst should contain oxidants with favorably reactive centers for cleavage of peroxyl bonds (O–O), have structure optimized for target pollutant attraction, and have chainmail to protect the vulnerable active sites for long periods.15–17 Recent studies have demonstrated Co–N–C active sites prefer to activate the O–O of PMS.18 Furthermore, introducing Fe-doping into the Co–N–C system not only suppresses Co2+ leaching, but also modulates the pyrrolic-N content, which is the adsorption site for capture of bisphenol A (BPA).19 We previously discovered that Co@C yolk–shell nanoreactors could enhance the catalytic activity because of the confinement effect in the nano-spaces between the core and shell, while the carbon shell acted like a chainmail protecting the Co active sites, keeping them highly reactive after five cycles.20,21Combining different kinds of materials to generate novel hybrid material interfaces can enable the creation of new kinds of chemical and physical functionalities that do not currently exist. However, one cannot simply mix these materials in an uncontrolled manner, because the ensemble of interfaces created by random mixing tends to favour thermodynamically stable interfaces that are functionally less active. Therefore, to prepare new materials with high functionality, it is necessary to carefully control the hybridization of components in interfacial regions with nanometric or atomic precision. By further hybridization of different components e.g., zero to one dimension (0D–1D) hybrid structures, we can prepare the structure to increase not only the specific surface area but also the interfacial region between different materials.In this work, we report novel 0D–1D hybrid nanoarchitectonics (necklace-like structures) consisting of FeCo@N–C yolk–shell nanoreactors as a PMS activator for Fenton-like processes. This catalyst has multilevel advantages: (i) each FeCo@N–C unit is a well-formed yolk–shell nanoreactor, which can guarantee sufficient contact of reactants and active sites, as well as defend them for good durability; (ii) all single nanoreactors are threaded along the carbon fibers, providing a highway for electron transport; and (iii) all the carbon fibers constructed into a thin film with macroscopic structure, which overcomes the complex recyclability of powder catalysts. Benefiting from favorable composition and unique structure, the FeCo@N–C catalyst delivers excellent performance for BPA removal via activation of PMS accompanied with good stability.The synthesis processes of necklace-like nanoarchitecture containing FeCo@N–C yolk–shell nanoreactors are illustrated in Fig. 1a. First, uniform Fe–Co Prussian blue analogue (Fe–Co PBA) nanocubes with an average size of 800–900 nm (Fig. 1b) are encapsulated in polyacrylonitrile (PAN) nanofibers by electrospinning. The obtained necklace-like FeCo PBA–PAN fibers (Fig. 1c) are then pyrolyzed at 800 °C in N2 atmosphere to produce FeCo@N–C nano-necklaces. The scanning electron microscopy (SEM) image (Fig. 1d) of the FeCo@N–C shows this necklace-like morphology with its large aspect ratio, with the FeCo@N–C particles strung along the PAN-derived carbon fibers. A broken particle (Fig. 1e) shows that the FeCo@N–C has a yolk–shell architecture, which is also identified by transmission electron microscopy (TEM). Fig. 1f and g show the well-defined space between the inner yolk and outer shell, which is attributed to the volume shrinkage of the original Fe–Co PBAs. During pyrolysis, Fe–Co PBA is reduced to FeCo (inner yolk) and PAN is carbonized (outer carbon shell), resulting in the unique necklace-like nanoarchitecture.22–24 The high-resolution TEM in Fig. 1h shows a lattice fringe of 0.20 nm, which matches well with the (110) plane of FeCo alloy.25 The scanning transmission electron microscopy (STEM) image (Fig. 1i) and corresponding elemental map (Fig. 1j) indicate that FeCo nanocrystals are well distributed in the inner core with some small FeCo nanocrystals located on external carbon shells. Furthermore, the control samples of Fe@N–C and Co@N–C nano-necklaces, prepared by only replacing the Fe–Co PBA nanocubes with Fe–Fe PB and Co–Co PBA (Fig. S1†), also demonstrate the versatility of this synthetic strategy. The formation of hierarchical porous structure, beneficial to the PMS transportation on the surface of catalysts, could be determined by N2 adsorption–desorption isotherms and corresponding pore volume analysis (Fig. S2 and Table S1†).Open in a separate windowFig. 1(a) Preparation of FeCo@N–C necklace-like nanoarchitecture. SEM images of (b) Fe–Co PBA cubic particles and (c) the electrospun FeCo PBA–PAN fibers. (d and e) SEM, (f and g) TEM, and (h) high-resolution TEM images of FeCo@N–C nano-necklaces. (i) STEM and (j) the corresponding elemental mappings of C, N, Fe, and Co.The X-ray diffraction patterns of the as-prepared products are depicted in Fig. S3,† with one prominent diffraction peak centered at 44.8° corresponding to the (110) lattice plane of FeCo alloy. All the products also have a characteristic signal at 26°, implying that graphite carbon is formed during pyrolysis. Raman spectroscopy further analyzed the crystal structures and defects of the FeCo@N–C nano-necklaces (Fig. S4†), where peaks found at 1349 cm−1 and 1585 cm−1 index the disordered (D band) and graphitic carbon (G band), respectively.26 X-ray photoelectron spectroscopy investigated the composition and valence band spectra of FeCo@N–C nano-necklaces. The survey spectrum (Fig. S5a†) reveals the presence of Fe (1.4%), Co (1.2%), C (86.4%), N (4.5%), and O (6.5%) in the composite. The high-resolution N 1s spectrum (Fig. S5b†) exhibits broad peaks at 398.1, 401.1, and 407.4 eV, corresponding to the pyridinic-N, graphitic-N, and σ* excitation of C–N, respectively.27 The high-resolution Fe 2p spectrum (Fig. S5c†) shows a broad peak at 707.4 eV, attributed to Fe0. Similarly, the 777.5 eV peak observed in the Co 2p spectrum (Fig. S5d†) corresponds to Co0, implying that FeCo dual sites have formed.28 The oxidation state of these sites was investigated by 57Fe Mössbauer spectroscopy, which found a sextet in the Mössbauer spectrum of the FeCo@N–C nano-necklaces attributed to FeCo dual sites (Fig. 2a and Table S2†).29 The coordination environment of the FeCo dual sites was also verified by X-ray absorption fine structure (XAFS) spectroscopy. Fig. 2b shows that the X-ray absorption near-edge structure (XANES) spectra of the Fe K-edge, which demonstrates a similar near-edge structure to that of Fe foil, illustrating that the main valence state of Fe in FeCo@N–C nano-necklaces is Fe0. Furthermore, the extended-XAFS (EXAFS) spectra (Fig. 2c) displays a peak at 1.7 Å, which is ascribed to the Fe–N bond, and a remarkable peak at approximately 2.25 Å corresponding to the metal–metal band.10,30 The Co K-edge and EXAFS spectra (Fig. S6†) also confirm the presence of Co–N and the metal–metal band. These results provide a potential structure of the FeCo dual sites in the FeCo@N–C nano-necklaces, as illustrated in Fig. 2d.Open in a separate windowFig. 2(a) 57Fe Mössbauer spectra of FeCo@N–C nano-necklaces at 298 K. (b) Fe K-edge XANES spectra of FeCo@N–C nano-necklaces and Fe foil. (c) Corresponding Fourier transformed k3-weighted of the EXAFS spectra for Fe K-edge. (d) Possible structure of the FeCo dual sites.This dual-metal center and necklace-like structure may be beneficial to enhance catalytic performance. Fig. 3a shows the Fenton-like performance for BPA degradation compared to Fe@N–C nano-necklaces, Co@N–C nano-necklaces, and FeCo@N–C particles (Fe–Co PBA directly carbonized without electrospinning). Here, the FeCo@N–C nano-necklaces display a higher catalytic performance, with BPA completely removed in 7 min. To clearly compare their catalytic behavior, the kinetics of BPA degradation was fitted by the first-order reaction. As shown in Fig. 3b, FeCo@N–C nano-necklaces exhibit the highest apparent rate constant (k = 0.83 min−1), which is approximately 6.4, 2.6, and 1.2 times that of FeCo@N–C particles, Fe@N–C nano-necklaces, and Co@N–C nano-necklaces, respectively. The significantly enhanced performance of FeCo@N–C nano-necklaces suggests that the FeCo dual sites and necklace-like nanoarchitecture are crucial. Furthermore, the concentration of BPA and PMS in the solution is higher than that in yolk–shell nanoreactor, resulting a concentration gradient which helps to accelerate the diffusion rates of reactants (Fig. 3c).31,32 For these nano-necklaces, the carbon shell acts like a chainmail protecting the FeCo active sites from attack by molecules and ions, and all the nanoreactors are threaded along the carbon fibers, providing a highway for electron transport, which is important for SO4˙− generation (SO4˙− production as eqn, HSO5− + e− → SO4˙− + OH−). Electrochemical impedance spectroscopy further confirms the good conductivity of the FeCo@N–C nano-necklaces (Fig. 3d). In addition, the concentration of metal-ion leaching and cycling performance (Fig. 3e and f) reveal the high reusability of FeCo@N–C nano-necklaces, with 95% BPA removal in 20 min after five cycles, which is also proved by the SEM and TEM characterization (Fig. S7†). The effect of other reaction parameters on the BPA degradation, such as pH, reaction temperature, PMS or catalysts dosage, and common anions, were investigated in detail (Fig. S8–S11†). All the results demonstrate that FeCo@N–C nano-necklaces deliver a better performance for PMS catalysis. In addition, the turnover frequency (TOF) value of FeCo@N–C nano-necklaces is 5.5 min−1 for BPA degradation, which is higher than many previously reported catalysts (detailed catalytic performance comparison as shown in Table S3†).Open in a separate windowFig. 3(a) BPA degradation efficiency in different reaction systems and (b) the corresponding reaction rate constants. (c) Schematic illustration of PMS activation in FeCo@N–C nano-necklaces. (d) Nyquist plots of the catalysts. (e) The metal leaching in different reaction systems. (f) Cycling performance of FeCo@N–C nano-necklaces for BPA removal. Reaction conditions: [catalyst] = 0.15 g L−1, [BPA] = 20 mg L−1, [PMS] = 0.5 g L−1, T = 298 K, and initial pH = 7.0.To examine the enhanced catalytic activity, radical quenching experiments were conducted. As shown in Fig. 4a, when NaN3 is added to the reaction solution as a scavenger for 1O2, there is no significant reduction of BPA decomposition, implying that non-radicals are not the dominant reactive species. By comparison, when tert-butanol (TBA) (radical scavenger for ˙OH) is added, there is a slight (2.8%) decrease in BPA removal. However, if methanol (radical scavenger for SO4˙− and ˙OH) is added, the efficiency of BPA degradation declines by up to 59.2%, indicating that the major radicals generated from the PMS activation are SO4˙−;33 the presence of these radicals is also verified by electron paramagnetic resonance (EPR) (Fig. 4b). Furthermore, the significant inhibition ratio can be observed when KI (quencher for the surface) is added, demonstrating that BPA degradation is mainly attributed to reactions with SO4˙−, which is produced by a surface catalytic process.34Open in a separate windowFig. 4(a) Effects of the radical scavengers on BPA degradation. (b) EPR spectra of SO4˙− and ˙OH. (c) The energy profiles of PMS on FeCo@N–C nano-necklaces surface. (d) Optimized configurations of PMS adsorbed on FeCo@N–C nano-necklaces.Density-functional theory was applied to calculate the surface energy of PMS activation at FeCo dual sites (Fig. 4c, d and S12†). The dissociation barrier of PMS into SO4˙− and OH− is −2.25 eV, which is much lower than that on an Fe or Co single site, suggesting that cleavage of O–O bonds of PMS occurs more easily on FeCo dual sites. This is because FeCo dual sites provide two anchoring sites for the dissociated O atoms, leading to more efficient activation of O–O. The FeCo@N–C nano-necklaces can reduce the energy barrier of O–O bond breaking, which results in high activity for PMS activation and thus high productivity of SO4˙−. 相似文献
5.
6.
The OH + HCl → H2O + Cl reaction releases Cl atoms, which can catalyze the ozone destruction reaction in the stratosphere. The measured rate coefficients for the reaction deviate substantially from the Arrhenius limit at low temperatures and become essentially independent of temperature when T < 250 K, apparently due to quantum tunneling; however, the nature of the quantum tunneling is unknown. Here, we report a time-dependent wave packet study of the reactions on two newly constructed potential energy surfaces. It is found that the OH + HCl reaction possesses many Feshbach resonances trapped in a bending/torsion excited vibrational adiabatic potential well in the entrance channel due to hydrogen bond interaction. These resonance states greatly induce quantum tunneling of a hydrogen atom through the reaction barrier, causing the reaction rates to deviate substantially from Arrhenius behavior at low temperature, as observed experimentally.The OH + HCl reaction possesses many Feshbach resonances trapped in the hydrogen bond well in the entrance channel, which substantially enhance the reaction rates at low temperatures.In the classical picture, a chemical reaction with an energetic barrier can only occur at collision energies higher than the barrier, which leads to the well-known Arrhenius formula for chemical reaction rates. However, chemical reactions can happen at energies below the reaction barrier through quantum tunneling,1–3 resulting in the deviation of the reaction rates from the Arrhenius behavior at low temperatures. The effect of quantum tunneling on the reaction rates increases with decreasing reaction temperature, and hence becomes especially important in low-temperature environments such as the interstellar medium and atmospheric processes.4,5 Reaction resonances are quasi-trapped quantum states in the transition state region with some lifetime, and can substantially promote quantum tunneling through the reaction barrier. Over the past decades, great efforts have been devoted to detecting resonances in chemical reactions and to studying their structures and dynamics.6–14 Theoretical studies on the O(3P) + HCl → OH + H reaction using the accurate 3A′′ potential energy surface revealed that the tunneling induced by the resonances trapped in the van der Waals well in the reactant channel can substantially enhance the thermal rate constants at low temperatures.15–17 A combined experimental and theoretical investigation discovered that resonance-induced quantum tunneling dramatically enhances the reactivity of the F + p-H2 → HF + H reaction,14 and is fully responsible for the unusually high chemical reactivity of the reaction in the low temperature interstellar medium. Recently, a quantum dynamics calculation showed that the presence of two resonance peaks strongly influence the rotational quenching of HF (j = 1, 2) with H, leading to an up to two-fold increase in the thermal rate coefficients at the low temperatures characteristic of the interstellar medium.18 Thus, understanding quantum tunneling, and in particular, resonance-induced quantum tunneling, in chemical reactions is of general interest and fundamental importance to low-temperature chemistry.The OH + HCl → H2O + Cl reaction is of great importance in atmospheric chemistry because it releases Cl atoms from one of the principal chlorine-containing species in the stratosphere, HCl. The Cl atoms generated from the reaction can catalyze the ozone destruction reaction in the stratosphere, which was responsible for the formation of the ozone hole over Antarctica.19 Since the chlorine-catalyzed ozone destruction is proportional to the steady-state Cl atom concentration, which is directly controlled by the rate of the reaction, extensive studies have been carried out to measure the rate coefficient for the reaction with high accuracy over a wide range of temperatures.20–29 The measured rate coefficients exhibit a small activation energy of a few hundred K, deviate substantially from the Arrhenius limit at low temperatures and become essentially independent of temperature when T < 250 K.25,28,29 In addition, a large H/D kinetic isotope effect has also been found.21,22,26,28 All these observations suggest the presence of an important quantum tunneling effect in the reaction.The dynamics of this reaction and its reverse have also attracted great attention in the past decades. In particular, the endothermic Cl + H2O → HCl + OH reaction with a late barrier has been extensively investigated as a benchmark system for mode specificity and bond selectivity chemistry.30–35 Recently, the construction of two high-quality potential energy surfaces (PESs) in its ground electronic state using the PIP-NN method have substantially advanced the theoretical study of the dynamics and kinetics of the system. The first PES was based on a large number of ab initio data points calculated at the multi-reference configuration interaction (MRCI) level of theory by Li, Dawes, and Guo (LDG),33 and the second one was fitted to ab initio energy points obtained using an explicitly correlated unrestricted coupled-cluster method with single, double, and perturbative triple excitations (UCCSD(T)-F12b) and the augmented correlation-consistent polarized valence triple-zeta (aug-cc-pVTZ, or AVTZ) basis set by Zuo, Zhao, Guo, and Xie (ZZGX) with a fitting error of 6.9 meV.36 The static barrier height is 2.86 and 2.23 kcal mol−1, respectively, for these two PESs. On both PESs, there exists a well of about 3.5 kcal mol−1 in the OH + HCl entrance channel due to the hydrogen bond (HB) interaction between OH and HCl. Extensive quantum dynamics studies on the PESs have revealed many interesting features of the reaction in both directions. In particular, time-dependent wave packet calculations for the title reaction with OH in the ground and vibrational excited states found one or two broad peaks in the total reaction probabilities, which are presumed to be the signature of the resonances supported by the reactant complex well.36,37 However, the impact of these peaks on the reaction rates has not been investigated. Ring-polymer molecular dynamics (RPMD) calculations38,39 were also carried out on both PESs to compute the thermal rate coefficients for the reaction.40–42 It was found that the RPMD rates on the LDG PES underestimate the experimental data, while the RPMD rates on the latest ZZGX PES agree with the experimental results much better, and do not decrease further when the temperature falls below 300 K, apparently due to quantum tunneling. Unfortunately, RPMD calculations cannot provide any clue regarding the nature of the quantum tunneling.Therefore, despite the significant progress that has been made in theoretical studies of the system, some key issues still remain to be addressed: Are the broad peaks in the total reaction probabilities obtained from the time-dependent wave packet calculations indeed the signature of the resonances in the reaction? If not, do there exist resonances in the reaction? How do resonances affect the reaction rates at low temperature? Here, we report a quantum dynamics study of the reaction on two new and more accurate PESs. Good agreement is achieved between the rate coefficients calculated on these two PESs and the experimental data. Our calculations reveal that the HB well in the entrance channel of OH + HCl supports many low energy resonance states. These resonance states substantially enhance the quantum tunneling effect and have an important impact on the reaction rates at low temperatures.In order to improve the fitting accuracy of the ZZGX PES, we constructed two new PESs using the fundamental invariant neural network (FI-NN) method, fitted to ∼70 000 ab initio energy points calculated at the UCCSD(T)-F12a and UCCSD(T)-F12b levels of theory, respectively, both with the AVTZ basis set. The fitting RMSE is 3.07 and 3.12 meV, respectively, for the F12a and F12b PES, which is about half that for the ZZGX PES.36 The spin–orbit coupling of the channels of both the reagent OH and the product Cl have been included using FI-NN fitting to about 38 000 points calculated at the MRCI/aug-cc-pVTZ level of theory with a fitting error of 0.19 meV. The CASSCF wave-function with an active space of (5e, 3o) was used as a reference for MRCI. Details of the new PESs are provided in the ESI†. The static barrier height for PESa is 0.088 eV (0.095 eV with SO correction included), and that for PESb is 0.097 eV (0.104 eV). As can be seen from Tables S1 and S2,† the geometries and energies of all the stationary points for the F12b PES without SO correction are in good agreement with the ZZGX PES. Table S2† also shows the corresponding complete basis set (CBS) energies for these stationary points based on AVTZ, AVQZ and AV5Z calculations. Because the F12b energies are slightly closer to the CBS results, we will present the dynamical results obtained on the F12b PES in the main text and provide those for the F12a PES in the SI.On the new PESs, we carried out potential-averaged five-dimensional (PA5D) time-dependent wave packet43,44 calculations to obtain the total reaction probabilities for the reaction by freezing the non-reacting OH bond in its ground vibrational state. Tests revealed that the PA5D treatment is capable of providing reaction probabilities for the ground rovibrational initial state that are essentially identical to those obtained using the full six-dimensional approach, as shown in Fig. S2.†Fig. 1(A) shows the total reaction probabilities for the HCl + OH reaction as a function of collision energy calculated on the F12b PES with both reagents in the ground rovibrational state at propagation times of 60 000, 120 000, 360 000, and 2 400 000 a.u. At high collision energies, the reaction probabilities converge quickly with respect to the propagation time, and one can barely see any difference among the four reaction probability curves, which exhibit smooth increases with collision energy. However, in the low collision energy region, large differences appear for different propagation times. At t = 60 000 a.u., the reaction probability presents a smooth curve with some small and broad oscillations, as was observed in the wave packet calculations of Guo and coworkers.36Open in a separate windowFig. 1(A) Total reaction probabilities for the ground initial state of the OH + HCl → Cl + H2O reaction on the F12b PES at wave packet propagation times of T = 60 000, 120 000, 360 000, and 2 400 000 a.u. (B) Same as (A), except showing the collision energy between 0.0 and 0.04 eV. The crosses mark the points for which the wavefunctions are shown in Fig. 2. (C) Total reaction probabilities for some partial waves J = 0, 30, 60, and 90 as a function of the collision energy. (D) Same as (C) except showing the collision energy between 0.0 and 0.04 eV.When the propagation time is increased to t = 120 000, the reaction probabilities at collision energies below 0.05 eV increase substantially. With further increasing the propagation time to 360 000 a.u., many oscillatory structures emerge at collision energies below the barrier height of 0.104 eV, in particular in the very low collision energy region as shown in Fig. 1(B). These sharp structures become fully converged after around 2 400 000 a. u. of wave packet propagation (∼58 ps). The reaction probability even at a collision energy close to zero reaches 3–4%. The convergence of the total reaction probabilities on the F12a PES is very similar to that on the F12b PES (Fig. S3†), except that the final converged reaction probabilities for these two PESs exhibit a small shift, apparently due to slightly different barrier heights. Therefore, it is clear that reaction resonances exist in the title reaction in the very low collision energy region, and that these resonance states substantially induce quantum tunneling and enhance the reactivity. The lifetimes for these resonance states are quite long, with many being longer than 6.5 ps and having corresponding widths smaller than 0.1 meV. Fig. 1(C) presents converged (t = 2 400 000 a.u. ≈ 58 ps) total reaction probabilities for the total angular momentum J = 0, 30, 60, and 90. With increasing J, the reaction probability curve shifts to higher energy. In the low collision energy region (<0.05 eV), the total reaction probabilities for J = 30 exhibit rich oscillatory structures as in the J = 0 case (Fig. 1(D)), which are expected to have a great influence on the rate constant at low temperature. With further increase of the total angular momentum, the influence of these resonances on the total reaction fades due to the centrifugal barrier, which prevents the low-energy wave function from entering the well. They only leave a small trace in the total reaction probabilities at low energies for J = 60, and do not have any effect for J = 90.To understand the nature of these resonances, we calculated scattering wave functions at two collision energies (1.08 and 4.26 meV) with the peak reaction probabilities indicated by x in Fig. 1(B). Fig. 2(A) shows the two dimensional (2D) contour at the collision energy of 1.08 meV in the Jacobi coordinates HCl bond length (rH–Cl) and center of mass distance between OH and HCl (RHCl–OH), with the other coordinates integrated. As can be seen, the wave function is localized in the HB well region in the entrance channel with a few nodes in the R coordinate and no node in the rH–Cl coordinate. Inspection of the scattering wave function for the bending and torsion coordinates reveals nodes exist in these coordinates (Fig. S4 and S5†). The 2D contour in the coordinates of RHCl–OH and rH–Cl at the collision energy of 4.26 meV shown in Fig. 2(B) looks similar to that shown in Fig. 2(A), except with more nodes in the R direction. Therefore, the observed resonance states in the reaction are Feshbach resonances trapped in a bending/torsion excited vibrationally adiabatic potential (VAP) well in the reactant complex region due to the HB interaction.Open in a separate windowFig. 2Reactive scattering wave functions for the OH + HCl → Cl + H2O reaction on the F12b PES in the two Jacobi coordinates R(HCl–OH) and r(H–Cl) with other coordinates integrated at the collision energies of 1.08 (A) and 4.26 meV (B). The contour lines are the corresponding 2D PESs along the two reactive bonds R(HCl–OH) and r(H–Cl) with other coordinates optimized. The geometries for the saddle point and the HB minimum are displayed in (A). The coordinate units in the figures are a0. Fig. 3(A) shows the accurate rate constants for the initial ground rovibrational state, kg, based on the probabilities for J = 0, 30, 60, 90 using the uniform J-shifting approach with a temperature-dependent shifting constant.45–47 A test shows that the J-shifting scheme based on these four individual J values only introduces a few percent error to the rate constants in the temperature region considered here (Fig. S6†). As can be seen from the figure, with decreasing temperature, kg first decreases rapidly from 1000 K to 700 K, then decreases slowly. It reaches a minimum at T ≈ 260 K, and increases slowly with further decrease of the temperature. At temperatures lower than 300 K, the rate constants for the ground rovibrational initial state are larger than the measured thermal rate coefficients, with kg being larger than kexp by ∼70% at T = 200 K.Open in a separate windowFig. 3(A) Accurate rate constants, kg, for the initial ground rovibrational state of the HCl + OH → H2O + Cl reaction calculated on the F12b PES, in comparison with kJSg (obtained using the J-shifting approximation) and kNRg (based on the background reaction probabilities for J = 0 with the resonance contribution removed shown below); (B) the background reaction probabilities up to E = 0.1 eV by connecting some valleys of the reaction probabilities marked by x.Also shown in Fig. 3(A) are the rate constants for the ground rovibrational initial state, kJSg, obtained from the J = 0 reaction probabilities using the J-shifting approximation (see ESI† for details). As can be seen, the J-shifting approximation works very well at high temperatures around 1000 K, but begins to overestimate the rates with decreasing temperature. At T = 500 K, kJSg is about 10% higher than the true rate. At temperatures below 300 K, the J-shifting approximation underestimates the reaction rate, with kJSg being smaller than kg by about 16% at T = 200 K. Overall, the J-shifting approximation works fairly well for the ground rovibrational initial state, although there are numerous resonance peaks in the reaction probabilities in the low collision energy region.Now we consider the issue of the effect of the resonance structures found in the reaction on the rate constant. For a reaction system with isolated resonances, it is rather straightforward to remove the resonance contributions from the reaction probabilities by fitting the resonance peaks to some Lorentzian functions and to obtain smooth background scattering probabilities, as demonstrated in the F + HD reaction and recently in the inelastic scattering of H + HF (ref. 6 and 18). However, the OH + HCl reaction possesses numerous highly overlapped resonances in the low energy region, as shown in Fig. 1(A), and it is impractical to fit these resonance peaks as accurately as the isolated resonances. Instead, we obtained an approximate background reaction probability curve, which is shown in Fig. 3(B), by smoothly connecting some resonance valleys as shown in the figure. The rate constants kNRg calculated using the background curve shown in Fig. 3(B) with the J-shifting approximation were compared with the original kg in Fig. 3(A). Given the fact that the J-shifting approximation works fairly well for kJSg, as shown in Fig. 3(A), it is very reasonable to expect that will work even better for the kNRg values obtained from the background reaction probabilities with the resonance contribution removed. As can be seen, kNRg exhibits rate behavior typical for systems with a low barrier with some quantum tunneling effects. At T = 1000 K, kNRg is essentially identical to kg, but decreases much faster than kg as the temperature drops. At T = 200 K, kg is larger than kNRg by a factor of about 5.6 (9.8 × 10−13vs. 1.74 × 10−13 cm3 s−1), indicating that the reaction probabilities in the low collision energy region due to resonances substantially enhance the rate constants for the reaction at low temperatures. It is worthwhile to note that for systems with overlapped resonances like that shown in Fig. 3(B), the reaction probabilities at the valleys must be considerably higher than the true background reaction probabilities; therefore, the background curve shown in Fig. 3(B) is the upper limit of the background reaction probabilities and the rate based on the curve shown in Fig. 3(A) is also the upper limit of the rate for the ground rovibrational initial state without the resonance contributions. Therefore, the true enhancement due to the resonances must be larger than that shown in Fig. 3(A).For reliable comparison with the measured thermal rate coefficients, we must take into account the contributions from all the thermally populated initial states of the reagents. Due to the very large number of thermally populated rotational states for this reaction even at 200 K, we opted to calculate the cumulative reaction probabilities, NE(E), (the sum of the reaction probabilities for all the initial states with a fixed total energy) for the reaction from which the thermal rate constants can be reliably evaluated.48–52 The transition state wave packet calculations were carried out to obtain NE(E) using the details given in the ESI†. Due to huge computational efforts required to obtain the cumulative reaction probabilities for J > 0, we only calculated NE(E) for J = 0 and employed the J–K-shifting approximation45,49,53 to obtain the thermal rate constant. NE(J = 0, E) as a function of total energy measured with respect to the ground rovibrational energy of OH and HCl is presented in Fig. S7.†In Fig. 4, we present thermal rate constants for the reaction calculated on both the F12a and F12b PES, together with the rate constants for the ground rovibrational state (kg) and the previous experimental measurements.21,22,24–29 As can be seen, the thermal rate coefficients are smaller than kg over the entire temperature region, indicating that reagent rotation excitations diminish the reaction rates. Overall, the thermal rate coefficients calculated on both PESs agree with the experimental results rather well, with the F12a PES slightly overestimating and the other PES slightly underestimating compared to the experimental measurements. As observed in the experiments, the thermal rate coefficients decrease quickly with decreasing temperature in the high-temperature region, but decrease much more slowly at low temperatures, in particular at T < 300 K, substantially deviating from Arrhenius behavior.Open in a separate windowFig. 4Thermal rate constants of the HCl + OH → H2O + Cl reaction calculated on both the F12a and F12b PES, compared with rate constants for the ground rovibrational state, the RPMD rates on ZZGX PES and the previous experimental measurements.21,22,24–29 Experimental data are taken from Husain et al. (in circles),21,22 Molina et al. (in diamonds),24 Ravishankara et al. (in upward-pointing triangles),25 Smith et al. (in squares),26 Sharkey et al. (in triangle left),27 Battin-Leclerc et al. (in downward-pointing triangles),28 and Bryukov et al. (in leftward-pointing triangles).29Also shown in Fig. 4 are the RPMD rates calculated (see ESI† for details) on the F12a, F12b and ZZGX PESs.42 As shown, the RPMD rates on the F12b PES agree with those on the ZZGX PES extremely well, although the barrier heights for the F12b PES are higher by 9 meV. The present RPMD rates agree with the J–K-shifting quantum rates rather well, except at 200 K, at which the RPMD rates are overestimated by about 40%. As a result, the RPMD rate is higher than the experimental value at T = 200 K, in particular for the F12a PES. Previous studies have shown that RPMD tends to underestimate reaction rates in the strong quantum tunneling region, even for systems with resonances such as the O(3P) + HCl → OH + H reaction;54 however, it overestimates the rates for the F + H2 reaction with pronounced post-barrier Feshbach resonances55 and some insertion reactions.56 The discrepancy between the RPMD and J–K-shifting quantum rate at T = 200 K could be caused by a possible underestimation of the rate by the J–K-shifting approximation, as in the J-shifting approximation of the ground rovibrational initial state shown in Fig. 3(A). On the other hand, the rapid increase in the rate with decreasing temperature (from 250 K to 200 K) could also be a feature of bimolecular reaction rates in the high-pressure limit for reactions with a pre-reactive minimum.57 RPMD rate theory in its original form employs a free energy calculation,38,39 which allows thermalization in the pre-reactive minimum, sampling tunneling pathways with energies not accessible in the low-pressure limit. Therefore, RPMD models a rate process that resembles rates in the high-pressure limit, which could lead to an overestimation of the rate at low temperature. A recent study also reported a similar issue with RPMD.58 The authors attributed it to spurious resonances in RPMD favoring energy transfer in the pre-reactive minimum, which is similar to our argument. Certainly, more quantum dynamics calculations to provide rigorous quantum rates at low temperatures to assess the accuracy of the J–K-shifting approximation and the RPMD method on this reaction system with strong HB interaction in the entrance channel would be highly desirable.Therefore, the OH + HCl reaction possesses many long-lifetime Feshbach resonances trapped in a bending excited VAP well in the entrance channel due to HB interaction. These resonance states substantially induce quantum tunneling of the hydrogen atom through the reaction barrier and enhance the reactivity in the low-collision region. Consequently, the reaction rate for the reaction becomes essentially independent of temperature in the low-temperature region and deviates substantially from Arrhenius behavior, as observed experimentally. The resonance states in the reaction are very different in location from those in the F + H2/H2O reactions,8,11,12,14,59–63 which are trapped in the VAP well in the product channel. Furthermore, they are trapped in the bending/torsion excited VAP well with HCl in the ground vibrational state, unlike those in the F + H2/H2O reactions with HF in vibrationally excited states.8,63,64 In nature, these resonance states in the reaction arise from HB interaction as in the F + H2O (v = 0) reaction,11,62,63 but are different from those in the F + H2/HD/HOD (v = 1) reactions8,10,12,14,64–66 due to chemical bond softening. Because the resonance states are trapped in the bending/torsion excited VAP well, their lifetimes are much longer than those observed in the F + H2/H2O reactions. As a result, they should have an important impact on the differential cross sections. More efforts, and in particular more joint efforts involving theory and experiment, should be devoted to studying these Feshbach resonances in this reaction of great atmospheric importance in detail. 相似文献
7.
Following immense growth and maturity of the field in the past decade, native mass spectrometry has garnered widespread adoption for the structural characterization of macromolecular complexes. Routine analysis of biotherapeutics by this technique has become commonplace to assist in the development and quality control of immunoglobulin antibodies. Concurrently, 193 nm ultraviolet photodissociation (UVPD) has been developed as a structurally sensitive ion activation technique capable of interrogating protein conformational changes. Here, UVPD was applied to probe the paratopes of nanobodies, a class of single-domain antibodies with an expansive set of applications spanning affinity reagents, molecular imaging, and biotherapeutics. Comparing UVPD sequence fragments for the free nanobodies versus nanobody·antigen complexes empowered assignment of nanobody paratopes and intermolecular salt-bridges, elevating the capabilities of UVPD as a new strategy for characterization of nanobodies.Ultraviolet photodissociation mass spectrometry is used to probe the paratopes of nanobodies, a class of single-domain antibodies, and to determine intersubunit salt-bridges and explore the nanobody·antigen interfaces.Antibodies must possess high specificity toward target antigens to enable recognition and activation of an immune response. Because of this specificity, antibodies or antibody fragments are being increasingly explored for deployment in diagnostic assays, vaccine design and other therapeutic applications.1 As such, unravelling the collection of noncovalent interactions and structural features that endow antibodies with specificity is a primary goal in biomedical research and critical for development of new biotherapeutics.2 Methods to decipher antibody–antigen interactions have advanced significantly in recent years, and well-established methods including X-ray crystallography, mutagenesis techniques, cryo-EM, and hydrogen–deuterium exchange mass spectrometry remain the most versatile methods.2–5 Another new strategy, native mass spectrometry (MS) has emerged as a powerful tool for structural biology, including analysis of macromolecular complexes and antibodies.6–10 In this technique, rapid but gentle ionization and transfer of protein complexes into the gas-phase via electrospray ionization (ESI) of aqueous solutions of near-physiological ionic strength preserves noncovalent interactions, allowing elucidation of ligand binding and stoichiometry of complexes.6–9 Coupled to advanced methodologies including ion mobility (IMS) or tandem mass spectrometry (MS/MS), native MS has proven an innovative strategy for interrogating protein structure, revealing topology, distinguishing conformations, identifying ligand binding sites, and determining folding thermodynamics.6–9,11,12 As one example of a premier application in biotherapeutic development, native MS has been utilized to rapidly measure stoichiometry, heterogeneity, and stability of antibody·antigen complexes.10,13–17Detailed structural analyses by native MS often rely on controlled dissociation or disassembly of protein complexes via ion activation in MS/MS workflows.7,8,18–21 Collision-based dissociation methods comprise the most ubiquitous ion activation techniques and are capable of dismantling protein assemblies into subunits, detaching ligands, and facilitating determination of stoichiometries.7,8,18 However, the slow-heating process of collision induced dissociation (CID) causes protein unfolding and less effective fragmentation of the peptide backbone, limiting characterization of native protein complexes.7,8,18 Significantly more structural information can be acquired by employing alternative ion activation techniques.7,8,18 For example, surface induced dissociation (SID) is a collision-based method that promotes disassembly of protein complexes into subunits through a single high energy collision that minimizes protein unfolding and enables robust characterization of native protein assemblies.20,21 SID has proven especially versatile for the analysis of quaternary structure, as protein complexes disassemble to produce subcomplexes and subunits that reflect the native architecture.20,21 Most impressive, the outcome of SID correlates with the magnitude of protein interfacial area (cleavage of the weakest protein:protein interfaces) and has been integrated into computational modelling workflows to enhance the accuracy of protein assembly predictions, emphasizing the value of native MS/MS for structural interrogation.22–24 Alternatively, electron-based dissociation methods result in cleavage of the protein backbone to produce sequence fragments that are enhanced at surface exposed or flexible protein regions, informing topology (through the preservation of noncovalent interactions that prevent separation of reaction products) and degree of protein disorder.7,8,19,25Also a method sensitive to protein structure, ultraviolet photodissociation (UVPD) is a photon-based ion activation method that has demonstrated exceptional use for structural biology. In particular, propensities of polypeptide backbone cleavages induced by 193 nm UVPD correlate with backbone flexibility and arrangement of non-covalent interactions, an outcome related to the likelihood of separation (and detection) of nascent product ions after a backbone cleavage event. Product ions enmeshed by stabilizing non-covalent interactions are less likely to separate (i.e. UVPnoD), causing an apparent suppression of backbone fragmentation. This correlation empowers characterization of conformational changes induced by point mutations, ligand binding, and protein complexation by UVPD.26–33 Additionally, the high sequence coverage and rapid timescale of photodissociation enable detailed analyses of protein gas-phase structure, even informing proton sequestration with single residue resolution.34 As native MS and UVPD increasingly gain broader utility for new protein applications, development and establishment of strategies to routinely study protein structure become imperative to cement these methodologies as cornerstones in the fields of structural biology and biotechnology that encompass development of new therapeutics, imaging agents, diagnostics and drug delivery agents.One fascinating new class of biotherapeutics are nanobodies, single domain antibodies derived from the variable domain of functional heavy chain antibodies found in camelids and certain shark species.35,36 In contrast to conventional ∼150 kDa heterotetrameric immunoglobulin (IgG) antibodies, nanobodies feature a single peptide chain and overall reduced size of ∼15 kDa. Nanobodies offer high stability, solubility, affinity, and specificity, features that have propelled these single domain antibodies as a valuable alternative biotechnology.37–42 Concurrently, these same features facilitate native MS analysis, for which decreased size, increased solubility and antigen affinity are favorable for rapid and routine analysis of nanobody complexes, circumventing analytical challenges and tedious sample preparations, such as proteolysis and deglycosylation steps,14,17,43 often required for native MS of typical IgG·antigen complexes. One recent native MS study mapped the location of the epitope of influenza A hemagglutinin (HA1) bound to an antibody based on a decrease in backbone cleavages of HA1 when bound in the Ab·2HA1 complex relative to the free HA1 antigen during UVPD-MS analysis.31 This innovative approach motivated our interest in exploring an intriguing inverse strategy to map paratopes of nanobodies. In the present work, native MS and 193 nm UVPD are showcased as a valuable combination for determination of intersubunit salt-bridges and nanobody·antigen interfaces, ultimately localizing nanobody paratopes.Three nanobodies44–46 with distinct proteinaceous antigens of green fluorescent protein (GFP), ribonuclease A (RNAseA), and porcine pancreatic amylase (PPA), referred to here as Gnb,44 Rnb,45 and Anb,46 respectively, were targeted to evaluate native MS and UVPD for characterization of nanobody·antigen complexes. Each of these nanobodies interact with the antigen via differing contributions of the complementarity-determining regions (CDR) 1–3 and framework residues to the protein interface, which vary in surface area from 554 Å2 to 683 Å2 to 1062 Å2 for Rnb, Gnb, and Anb, respectively. These three pairs of nanobodies and respective antigens were first ionized individually using native conditions (Fig. S1†), and as nanobody·antigen complexes (Fig. 1). In each case, native MS produced the expected 1 : 1 complex in the full mass spectrum (MS1), in accordance with known crystal structures, in a range of charge states. A single charge state of the antigen-bound (bound state) nanobody was isolated and subjected to 193 nm UVPD (Fig. 1B, D and F), resulting in disassembly to release the free nanobody and antigen as well as sequence fragments from backbone cleavage of each protein, the latter of which will be the focus of UVPD analysis. UVPD mass spectra of each free nanobody are shown in Fig. S2.† The corresponding backbone cleavage maps for the free nanobodies and corresponding nanobody·antigen complexes are shown in Fig. S3.†Open in a separate windowFig. 1Native MS and UVPD of nanobody·antigen complexes. (A) MS1 of Gnb·GFP complex and (B) UVPD of the 13+ charge state. (C) MS1 of Rnb·RNAseA complex and (D) UVPD of the 11+ charge state. (E) MS1 of Anb·PPA complex and (F) UVPD of the 15+ charge state. Insets display expanded views of m/z regions populated by sequence fragments of low relative abundance. For UVPD, 1 laser pulse at 3 mJ was applied.Backbone cleavages induced by UVPD have been shown to be favored at protein regions with higher flexibility, typically ones less stabilized by networks of non-covalent interactions which might prevent separation and release of fragment ions.26–31,47 Accordingly, comparing abundances of fragment ions produced by free and bound nanobodies should reveal regions in which backbone fragmentation is suppressed or enhanced, thus uncovering those residues involved in stabilizing interactions with the antigen and effectively localizing the paratope. Ten types of fragment ions (a, a + 1, b, x, x + 1, y, y − 1, y − 2, and z, where +1, −1, and +2 indicate the gain or loss of hydrogen atoms) commonly generated by UVPD were monitored across the three nanobodies in both the free and bound states. Among the collection of ions detected, a- and x-type ions are the most prevalent and dominant for the nanobodies and complexes, thus providing the greatest sequence coverage. Those fragment ions displaying statistically significant differences in abundances (p < 0.05, n = 5) upon complexation of the nanobody to the antigen are shown in Fig. S4–S6† and mapped onto the backbone position cleaved to generate the fragment ions. The high diversity of fragment types characteristic of UVPD originates from competing pathways: direct dissociation from excited electronic states yields a/a + 1/x/x + 1 ions, and internal conversion to the ground state following intramolecular vibrational energy redistribution (IVR) produces b/y fragments.32,33 IVR processes may preferentially sever weak non-covalent interactions, whereas direct dissociation from excited states occurs on a faster time-scale minimizing disruption of non-covalent interactions.32,33 Consequently, the latter dissociation pathways and respective products, a/a + 1/x/x + 1 ions, are best suited to evaluate antigen-induced changes in nanobody topology. Variations in abundances of these four fragment types tended to be greater at the interface and CDRs, and inspection of the color-coded maps in Fig. S4–S6† reveals that in most cases the abundances of these a/x-type ions decreased for the nanobody·antigen complexes relative to the free nanobodies, signifying suppression of backbone fragmentation.To underscore the impact of antigen binding on UVPD, significant changes in backbone fragmentation (ΔUVPD) based on differences in summed abundances of a/a + 1/x/x + 1 fragments for each free nanobody versus nanobody·antigen complex are shown in Fig. 2, along with color maps highlighting residues at the CDRs and protein interfaces. Generally, apparent suppression of backbone fragmentation (e.g., less efficient separation of nascent fragment ions owing to stabilizing non-covalent interactions) is the greatest at or adjacent to the interface residues, an outcome which is especially notable and consistent for patches of residues near CDR3 in Gnb (Fig. 2A), near CDR1 and CDR3 in Rnb (Fig. 2B), and near CDR2 and CDR3 in Anb (Fig. 2C). These patterns in suppression of backbone fragmentation upon antigen binding correlate with structural features of the respective crystal structures: main antigen contacts in Gnb are predominantly present on CDR3;44 only CDR1 and CDR3 participate in antigen binding for Rnb;45 CDR2 and CDR3 primarily mediate antigen binding for Anb.46 Although nanobodies characteristically feature a disulfide bond spanning C23 to a paired cysteine directly N-terminal to the CDR3, cleavage of the disulfide bond via UVPD was sufficient to unlock the nanobody and allow release of sequence ions from other concomitant backbone cleavages within this region, including CDR1 and CDR2. Moreover, any statistically significant enhancement of backbone cleavages induced by antigen binding was sparse and remote from interaction sites. These infrequent increases in UVPD fragmentation for nanobody·antigen complexes relative to the free nanobody possibly indicate disruption of non-covalent interactions and increased flexibility in those limited regions of the nanobody. However, this sporadic enhancement of backbone cleavages upon antigen binding occurs in stark contrast to more prevalent suppression of backbone cleavages spanning larger swaths of neighboring residues in the nanobody·antigen complexes. Overall, the observed suppression of fragmentation at the protein·protein interface serves as a strong basis for the development of native MS-UVPD strategies to discern nanobody paratopes.Open in a separate windowFig. 2Suppression and enhancement of backbone cleavage sites based on abundances of UVPD fragment ions induced for each nanobody by antigen binding for (A) Gnb, (B) Rnb, and (C) Anb. ΔUVPD heat plots display significant differences (p < 0.05, n = 5) for the abundances of a- and x-type fragment ions between the free and bound nanobody. Blue and red indicate suppression and enhancement of fragment abundances, respectively, for the nanobody upon complexation. Positions that display no significant change are shown in grey, while white indicates small significant changes. Color maps highlighting interface residues and CDRs are also included for each nanobody. ΔUVPD values correspond to the fragment abundance per residue for the bound state minus the fragment abundance per residue for the free state. All product ions had a signal-to-noise ratio > 3.Contrary to classical IgGs where paratopes are primarily localized to CDRs,48 nanobody paratopes feature greater diversity in terms of residue identity and position, and also include the involvement of framework residues as well as the potential absence of interactions from certain CDRs, obfuscating assignment of paratopes.48 By leveraging the trends in the reduction of fragmentation observed in Fig. 2 for the three nanobody·antigen complexes relative to the free nanobodies, a strategy for the approximation of surface patches contributing to the paratope was developed. Because the most structurally significant changes of fragmentation related to antigen binding are demarcated by apparent suppression of backbone cleavages for stretches of neighboring residues, the UVPD data was analyzed by averaging abundances of fragment ions originating from backbone cleavages across every five residues (non-overlapping box-car average) prior to comparing fragment abundances for the free and bound states using Welch''s t-test (n = 5). A stringent significance cutoff of p < 0.001 was subsequently applied to limit false assignments of protein sections involved at the interface. Protein sections (5 residues long) displaying significant suppression of backbone cleavages (i.e. reduction in abundances of fragment ions originating from backbone cleavages in each 5 residue segment) according to this method are plotted onto the crystal structures in Fig. 3. Impressively, only protein sections adjacent to the protein·protein interfaces displayed significant suppression for Gnb and Anb. Similarly, Rnb mostly featured suppression adjacent to the interface, while only two sections remote from the interface (spanning residues 15–24 and 45–49) were also suppressed. Furthermore, a significant enhancement of fragmentation was only observed for Rnb near the N-terminus and C-terminus (Fig. S7†), remote from the paratope. This Rnb complex featured the smallest interfacial area, which may lead to instability, compaction, or distortion during ion transmission, resulting in the unexpected suppression and enhancement at these regions. Regardless, UVPD suppression was predominant at the interfaces of each of the three nanobody complexes, presenting a new strategy for the approximation of nanobody residue patches that contribute to the paratope.Open in a separate windowFig. 3The a and x fragment ion abundances originating from backbone cleavages were averaged across every 5 residues for the free and bound forms of each nanobody. Sections displaying significant UVPD suppression upon complexation (p < 0.001, n = 5) were mapped onto the crystal structure as green spheres for (A) Gnb, (B) Rnb, and (C) Anb. Residue positions displaying significant suppression of backbone cleavages, interface residues, and CDR regions are shown for each nanobody as color maps. For comparative purposes, the CDR regions of each nanobody are demarcated on the crystal structures in Fig. S8.†Tracking charge states of specifically a- and x-type ions produced by 193 nm UVPD also informs proton sequestration along the protein sequence,34 a feature that is capitalized on here to identify the formation of inter-subunit salt-bridges. In this strategy, charge states for each detected a- or x-type ion are weighted based on intensity and plotted for each backbone cleavage position of the nanobody. For example, if both a42+ and a43+ have equal intensities, the weighted average charge state at backbone cleavage position 4 (between residues 4 and 5) would be 2.5. Because electrostatic interactions, such as salt-bridges, are highly stable in the gas-phase,49,50 applying this method to monitor changes in proton sequestration across free and bound states promises to reveal the locations of inter-subunit salt bridges introduced by complexation, if charge partitioning during subunit ejection is due to heterolytic scission of salt-bridges as previously proposed.26,51,52 Indeed, this is demonstrated in the analysis of the Rnb·RNAseA complex, for which one inter-chain salt bridge has been identified by X-ray crystallography between nanobody R107 and antigen E111. Charge site analysis of the a-ion series for free Rnb (6+ charge state) (Fig. 4A) displays discrete step-changes in charge states at residues R39, between residues 43–47 (suggesting protonation at R45), R68, Q111, and H128, indicating localized protons at these sites. Although coverage of the x-ion series is sparser, it nonetheless corroborates proton localization at Q111 as well as near the N-terminus in the span of residues Q2–L5. Protonation of side-chains is not unexpected for basic amino acids like R and K, but protonation of backbone heteroatoms is also possible for non-basic residues like Q at either the amide oxygen or the amide nitrogen of the peptide bond.53,54 The step analysis reported here localizes all 6 protons of the 6+ charge state of free Rnb.Open in a separate windowFig. 4Weighted average charge of a-type and x-type fragment ions attributed to Rnb produced by UVPD of (A) free Rnb (6+ charge state) and (B) Rnb·RNAseA (11+ charge state), delineated based on the backbone cleavage site along the sequence of the nanobody.Charge site analysis of Rnb in the Rnb·RNAseA complex was not as comprehensive, but nonetheless many charge sites were elucidated (Fig. 4B). Specifically, R29, R46, and R68 remained protonated, while a shift occurred from protonation at Q111 in free RNAseA to protonation of the span of residues 108–106 in the Rnb·RNAseA complex, indicating proton sequestration at R107, a residue engaged in a salt-bridge with the antigen in solution. Charge migration observed upon antigen binding based on this charge-site analysis thus evinces formation of salt-bridges between binding partners. Similarly for Gnb·GFP, charge site analysis enabled localization of multiple charges in both the free and bound states (Fig. S9†) including protonation at R36 on Gnb, which is involved in electrostatic interactions with E142 on the GFP antigen. Additionally, a charge was located at R58 for bound Gnb that is absent for free Gnb. Although slightly higher than canonical distance cutoff of 4 Å for salt bridges, Gnb R58 is within 4.5–5.5 Å of the E172 and D173 side-chains of GFP according to the crystal structure. Salt-bridge formation spanning this distance may be possible in the gas-phase, according to charge site analysis derived from the UVPD data. The companion residues, E45 and E104, of the nanobody are also engaged in putative interchain salt-bridges when bound to GFP based on the X-ray structure, however, localization of deprotonation sites of acidic residues is not possible in the positive ion mode.For Anb, coverage of a- and x-type fragments from the Anb·PPA complex was less comprehensive and precluded detailed charge site analyses (Fig. S10†). Regardless, data for Gnb and Rnb demonstrate that basic residues engaged in inter-subunit salt-bridges maintain a proton upon disassembly of the nanobody·antigen complexes, highlighting an exceptional use of MS/MS to localize gas-phase salt-bridges between protein subunits. However, it is also noted that these changes in protonation sites may be caused by reasons other than those postulated here. Specifically, it is possible that proton migration is caused by vibrational redistribution of deposited energy from photoabsorption, leading to proton mobilization,53,54 and is not due to heterolytic scission of salt bridges. These results are nonetheless intriguing and may guide future interpretations of charge partitioning during UVPD of protein complexes.Following incredible advances in native MS, routine and rapid analysis of biotherapeutics for quality control and drug development has become commonplace.13–17 At the same time, UVPD has been propelled over the past decade as a premier ion activation method capable of uncovering structural features of proteins that are not revealed by other MS/MS methods.47 As shown in this study, we extended the combination of native MS and UVPD to characterize nanobody·antigen complexes, particularly aiming to showcase UVPD for mapping the binding interface. The pattern of fragment ions generated by UVPD for nanobody complexes resulted in the discernment of inter-chain salt-bridges. Additionally, tracking trends in suppression of fragmentation enabled the localization of nanobody paratopes using only micromolar quantities of nanobodies and circumventing some of the limitations of traditional structural biology methods such as NMR and X-ray crystallography. We anticipate that further improvements in data analysis/informatics methods will further extend the level of structural detail gleaned from the very dense UVPD mass spectra generated for large macromolecular assemblies akin to nanobody·antigen complexes. 相似文献
8.
Directed evolution is a powerful approach to engineer enzymes via iterative creation and screening of variant libraries. However, assay development for high-throughput mutant screening remains challenging, particularly for new catalytic activities. Mass spectrometry (MS) analysis is label-free and well suited for untargeted discovery of new enzyme products but is traditionally limited by slow speed. Here we report an automated workflow for directed evolution of new enzymatic activities via high-throughput library creation and label-free MS screening. For a proof of concept, we chose to engineer a cyclodipeptide synthase (CDPS) that synthesizes diketopiperazine (DKP) compounds with therapeutic potential. In recombinant Escherichia coli, site-saturation mutagenesis (SSM) and error-prone PCR (epPCR) libraries expressing CDPS mutants were automatically created and cultivated on an integrated work cell. Culture supernatants were then robotically processed for matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS analysis at a rate of 5 s per sample. The resulting mass spectral data were processed via custom computational algorithms, which performed a multivariant analysis of 108 theoretical mass-to-charge (m/z) values of 190 possible DKP molecules within a mass window of 115–373 Da. An F186L CDPS mutant was isolated to produce cyclo(l-Phe–l-Val), which is undetectable in the product profile of the wild-type enzyme. This robotic, label-free MS screening approach may be generally applicable to engineering other enzymes with new activities in high throughput.A robotic workflow for directed evolution of new enzymatic activities via high-throughput library creation and label-free MS screening.Mimicking random mutagenesis and natural selection in the laboratory,1–3 directed enzyme evolution is valuable in improving a variety of properties of biocatalysts (i.e., catalytic activity, stability, substrate specificity, and enantioselectivity).4–6 While it is straightforward to generate thousands of enzyme variants using established methods such as error-prone PCR (epPCR), site-saturation mutagenesis (SSM), and DNA shuffling, phenotypic screening remains a major bottleneck due to the substrate and product versatility of biocatalysts.7,8 Optical assays are widely applied in high-throughput screening (HTS) during directed evolution, often requiring a surrogate substrate or a coupling assay with a colorimetric or fluorogenic readout.9,10 However, it is very challenging to apply such approaches to discover new catalytic activities. On the other hand, mass spectrometry (MS) is label-free and suitable for untargeted molecular profiling.11 The high sensitivity and chemical resolution of MS further highlight its potential in discovering new products, because new catalytic activities are often weak when first emerging from enzyme promiscuity.12We and others have recently developed a range of high-throughput MS screening methods for directed protein evolution.13–16 However, the label-free advantage of MS has not been fully demonstrated in engineering new enzymatic activities, possibly due to the difficulties in untargeted MS screening. Particularly, it requires careful standardization and optimization of sample preparation, MS acquisition, and data processing, which are necessary to minimize experimental noise and spot the weak signals of a new product. On the other hand, biofoundries provide an emerging infrastructure to assist the design–build–test–learn (DBTL) cycles in biological engineering via robotic standardization and parallelization.17–19 Using an integrated biofoundry, here we report a workflow for unlabeled MS screening of recombinant libraries to rapidly isolate enzyme mutants that catalyze the formation of new products. This new workflow extends our previous matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS-based screening approach from agar colonies16 to liquid cultures in industry-standard microplates for better uniformity. Unlike colony biomass, liquid culture media often contain high concentrations of nonvolatile components, which interfere with MALDI matrix crystallization and cause severe ion suppression during MS analysis. Therefore, sample preparation steps, such as liquid–liquid extraction and solid-phase extraction, need to be incorporated and automated.Cyclodipeptide synthases (CDPSs) are a family of enzymes that catalyze the formation of a diketopiperazine (DKP) from two aminoacyl-tRNA substrates (aa-tRNAs), which is an important pharmacophore for modern drug development20–22 (Fig. 1A and S1†). To produce new DKP derivatives for therapeutic and industrial applications, the catalytic mechanism of CDPSs has been studied23,24 to guide engineering. AlbC (239 aa) is the first identified CDPS protein, taking phenylalanyl-tRNAPhe (Phe-tRNAPhe) and leucyl-tRNALeu (Leu-tRNALeu) as substrates to synthesize cyclo(l-Phe–l-Leu) (cFL) in its native host Streptomyces noursei.25,26 Recombinant E. coli with AlbC overexpression is able to produce more cyclodipeptide derivatives in addition to cFL.27 To date, eight CDPSs have been structurally characterized,26,28–32 and mutagenesis studies reveal that the residues within the P1 and P2 catalytic pockets are the key determinants of substrate specificity. However, the detailed mechanism of substrate recognition and catalysis is still elusive, and CDPSs are recognized as recalcitrant targets for rational engineering.33,34 To our knowledge, large-scale screening of CDPS variants has not been reported, possibly due to the lack of applicable HTS assays.Open in a separate windowFig. 1(A) Scheme of diketopiperazine (DKP) biosynthesis catalyzed by cyclodipeptide synthase (CDPS). (B) Workflow of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS)-based high-throughput screening method for directed evolution of AlbC protein in E. coli.Here we develop and apply untargeted MS screening on a biofoundry for directed evolution of AlbC mutants that form new CDP products. The workflow consists of strain library creation, colony picking, microtiter cultivation, organic solvent extraction, transfer of the sample to a MALDI target and acquisition of mass spectra, followed by data processing and visualization (Fig. 1B). For library creation, AlbC variants were generated by either SSM or epPCR approaches and expressed from a pET28a plasmid under control of a T7 promoter. Upon genetic transformation of E. coli Rosetta(DE3), individual clones were transferred by a colony picker into microtiter plates. Subsequent cultivation, inducible production by addition of isopropyl β-d-1-thiogalactopyranoside (IPTG), ethyl acetate extraction, and MS sample preparation were performed using an integrated robotic workcell in the Shenzhen Synthetic Biology Infrastructure. This workcell integrates common instruments for synthetic biology, including a liquid-handling station, shaking incubators, centrifuge, plate reader, and so on. Organic extracts of the liquid cultures were spotted onto a MALDI target and overlaid with 4-CHCA matrix solution. MS targets were then manually transferred to a stand-alone MALDI mass spectrometer in this study, although a robotic configuration has been previously reported to automate this step.35We first applied the workflow to analyze the wild-type (WT) AlbC-expressing strain, Rosetta(DE3)/pET28-AlbC(WT). After cell cultivation, inducible production, and sample preparation, MS analysis was performed using an Autoflex MALDI-ToF mass spectrometer in the reflector positive ion mode (Fig. 1). In the resulting MALDI mass spectra, we observed [M + H]+ ion peaks with m/z values corresponding to cFL (m/z 261.17), cFY (m/z 311.15), cFM (m/z 279.12), cYL (m/z 277.16), cFF(cYM) (m/z 295.15), cLL (m/z 227.16) and cMM (m/z 263.12), all of which were absent from the control strain Rosetta(DE3)/pET28 (Fig. S2†). Production of these CDP molecules was further confirmed by examining tandem MS results using LC-MS in the multiple reaction monitoring (MRM) mode (Table S3†), where ion fragmentation patterns were consistent with the literature.26,27 Moreover, the observed production profile was also consistent with previous studies.26,27 These results validated our workflow to detect CDP products from microplate cultures of AlbC-expressing E. coli using MALDI-ToF MS.To engineer AlbC mutants that synthesize new products, we first focused on the key residues in the substrate-binding pockets. AlbC employs a “ping-pong” catalytic mechanism: the initial step transfers the aminoacyl moiety of the first aa-tRNA onto a conserved serine, leading to the formation of an aminoacyl enzyme intermediate; then, the aminoacyl enzyme reacts with the aminoacyl moiety of the second aa-tRNA to form a dipeptidyl enzyme.32 Previous biochemical experiments demonstrated that two binding pockets P1 and P2 of AlbC accommodate the aminoacyl moieties of the two aa-tRNA during the biosynthesis24 (Fig. 2). Only a limited number of mutations were examined on P1 and P2 residues,25,26 possibly due to the lack of HTS assays for large-scale analysis. Instead, we applied our workflow to create and screen the SSM libraries of select residues, including 10 residues in the binding pockets (L33, V65, L119, L185, L200, M152, M159, I204, T206 and P207) and 4 residues (R99, R101, R102 and D205) outside the pockets which have interactions of the tRNA moiety reported in the literature (Fig. 2A).Open in a separate windowFig. 2(A) Structural analysis of the AlbC (PDB ID, 3OQV). Enlarged view of the catalytically active pocket. The possible catalytic residues are shown in green (pocket-1) and orange (pocket-2) and basic residues on helix α4 are labelled in cyan. (B) Heatmap of the relative activity change in the cFL production levels of mutation of 14 amino acid residues in AlbC. WT residues are labeled with a black dashed rectangle. Dark blue boxes indicate that a specific mutant was not covered in randomly picked clones (activity assigned as −1).For SSM libraries, we adopted the “22c-trick” strategy to design degenerative primers, and 94 library clones were randomly picked for each residue library to reach a >98.6% probability of full library coverage of the 20 canonical amino acids.36 In addition to library variants, the WT and control strains were also included in the same 96-well plate. Overall, the abovementioned workflow (Fig. 1B) takes approximately 5 s for sample preparation and MS analysis for each mutant culture. Tentative ion peaks of cyclodipeptides were assigned based on theoretical m/z values (ESI Section 1†) using the MetaboAnalyst webserver.37 For each ion peak, one-way analysis of variance (ANOVA) was used to evaluate statistical differences between the mean peak areas of a library variant and those of the control group. AlbC gene mutations were revealed by Sanger sequencing for all 14 SSM library members.To visualize the MALDI-ToF MS screening results, a heatmap was generated based on the cFL production of library members relative to that of WT (Fig. 2B), and we observed a general consistency between our results and literature data (Fig. 3A), respectively, which was similar to a previous study.26 Also, mutagenesis of basic residues (R99, R101 and R102) outside the pockets led to the decline of cFL production in the mutants, although R101 was more tolerant to mutations than R99 and R102. Furthermore, the substitution of D205 in the β6-α8 loop to alanine enhanced AlbC catalytic activity as observed previously.25 On the other hand, some previously unnoticed phenomena were observed. For example, in the D205 SSM library, relative ion intensities of cFL (m/z 261.17) in many mutants are higher than that of WT (Fig. S3†). The top 3 mutants, D205M, D205K and D205R, were subsequently analyzed by LC-MS/MS. The results not only confirmed augmented synthesis of cFL, but also revealed increased production of other leucine-containing products including cYL, cLL and cML (Fig. 3B). Also interestingly, the discovery that most mutations of the T206 residue in the P2 pocket abolished the biosynthesis of the main product cFL was unprecedented (Fig. S3 and ESI Section 2†). When examining MALDI mass spectra in detail, we noticed that although the T206F mutation greatly reduced cFL production, cFF and cFY products were largely not affected. Replacing the small threonine side chain with a bulky aromatic phenylalanine side chain could affect the activity of WT, because the phenyl ring structure increased the steric hindrance of T206F, thus impairing binding with its substrate and causing loss of its enzyme activity for cFL (Fig. 5A). These results suggested that T206 is also a key residue for the selectivity of the second Leu-tRNA substrate binding in the pocket. Unfortunately, we did not observe any new CDP molecules produced from the 14 SSM library mutants.Impact of AlbC mutations on the main product cFL
Open in a separate windowaCalculated from MALDI-TOF MS data.bCalculated from LC-MS/MS data.Open in a separate windowFig. 3Cyclodipeptide product analysis of AlbC mutants. (A) LC-MS characterization of the L200N mutant; (B) LC-MS characterization of select mutants in the D205 saturation mutagenesis library. Normalized production levels relative to WT (set as 1) are reported. Error bars represent the standard deviations of three biological replicates.Open in a separate windowFig. 5Structural illustration of the substrate binding pocket of the wildtype AlbC and the mutants F186L and T206. Note: Panel (A) Superimposition of the WT with T206F. Panels (B and C) Schematic representation of the hydrogen-bond and hydrophobic interaction between cFL·WT (B) and cFV·F186L (C). Dashed lines indicate hydrogen bond (yellow) interactions. The key amino acid residues of WT, F186L, and T206F are drawn as sticks in green, yellow and orange, respectively.The failure of isolating AlbC mutants with new substrate specificities from the SSM libraries highlights the limitation of semi-rational approaches that target manually selected residues. Therefore, we further turned to profile epPCR libraries that contain random mutations throughout the whole protein sequence. Under optimized conditions using the Agilent GeneMorph II Random Mutagenesis Kit, on average 2 nucleotides were introduced per gene variant so that most epPCR library members contain no more than one amino acid change. In total around 4500 independent clones were screened using the above workflow. The peak intensities at the theoretical m/z values of predicted cyclodipeptide ions in each MALDI mass spectrum were analyzed by the one-way ANOVA with Tukey''s multiple comparison test. A new peak (m/z 247) absent from WT that corresponds to the [M + H]+ ion of cFV was observed with three clones (Fig. 4A), all of which harbored the F186L mutation as revealed by Sanger sequencing results. Product identification was performed using LC-MS/MS and high-resolution (HR)-MS, and the retention time (5.7 min, Fig. 4B), exact mass (C14H19N2O2 [M + H]+m/z: 247.1441, Table S3†), and MS/MS daughter ions23 (Fig. S4†) of the new product were consistent with that of the chemically synthesized cFV standard. When comparing LC-MS/MS traces, we also noticed substantial reduction of the native main product cFL in the F186L variant relative to WT, and a slight enhancement in production of other products including cFM, cFF, and cFY, indicating a shift towards more bulky substrates with the F186L mutant (Fig. 4B). Another round of epPCR library screening was performed to further extend the AlbC substrate scope using the F186L variant as a parent, but unfortunately, no mutants were isolated to produce new cyclodipeptides.Open in a separate windowFig. 4(A) Box–whisker plots of 1152 samples at m/z 247.18 based on ANOVA analysis. Group 1, epRCR variants; 2, Rosetta(DE3)/pET28a-AlbC (WT); 3, Rosetta(DE3)/pET28a (control). Three clones containing F186L were labeled in red. ANOVA, analysis of variance. (B) LC-MS traces of WT and F186L mutant.Then, we investigated possible mechanisms underlying new substrate specificity of the F186L variant from structural aspects using computational modeling. In the substrate binding pocket, the cFL substrate formed three hydrogen bonds with two amino acid residues (N40 and E182) in the WT AlbC. The phenyl side chain of cFL forms a π–π stacking interaction with F186 (Fig. 5B and S5†). However, when the residue at position 186 was mutated to leucine, this π–π stacking interaction was abolished. This leads to a change in the conformation of the Phe1 ring and thereby the hydrogen bond between Phe1 and N40 is abolished (Fig. 5C and S5†), which ultimately results in outward movement of loop G33-S44 (Fig. S6†). Therefore, the volume of the substrate binding cavity, which was measured to be 197 Å3, increased up to 288 Å3 when the residue at position 186 of WT was mutated to leucine (Table S4†). Overall, it is possible that an increase in the production of cFV occurs precisely due to the larger volume of the substrate-binding cavity in F186L. Moreover, cFL and the new derivative cFV were separately docked into the binding pockets of the WT and F186L variant for 100 ns MD simulations (Fig. S5†). The results showed that the binding energy of F186L was significantly increased when docking with the substrate cFV (l-lysine complexes (Fig. S5†). Both WT and F186L reached the equilibrium state from an early stage.Binding energy of ligand for AlbC WT and F186L
Open in a separate windowaWT-cFL.bF186L-cFV.cF186L-cFL.dvan der Waals energy.eElectrostatic energy.fPolar-solvation energy.gNonpolar solvation energy.hΔGbinding = ΔGVDW + ΔGEt + ΔGpolar + ΔGapolar.In conclusion, we developed a robotic assay for directed evolution of AlbC, a model CDPS, using MALDI-ToF MS for label-free screening. Compared with conventional LC-MS (typically more than 5 min per sample), our HTS workflow represents a two-magnitude reduction of analytical time (5 s per sample). Contrary to previous reports that only study limited mutations of select residues, 14 SSM libraries were created and profiled for sequence-activity profiling, which not only confirmed the impact of known mutations (38,39 may also serve as label-free HTS assays, but further development is needed to address ion suppression issues caused by direct infusion of complex culture media into a mass spectrometer. On the other hand, the scarcity of positive hits in the single-residue SSM and epPCR libraries in this study confirmed AlbC as a difficult target for evolving new activities. Therefore, it is desirable to create and screen new AlbC mutant libraries that are comprehensive (i.e., deep mutational scanning40), combinatorial (i.e., combinatorial active site saturation test/iterative saturation mutagenesis, CAST/ISM41), or data-driven (i.e., machine learning-assisted directed evolution, MLDE42,43) in the future. Together, we envision that the label-free MS screening method should be generally applicable to engineering other enzymes with new activities. 相似文献
Target residue | Mutants and production levels relative to WT (literature) | Mutants and production levels relative to WT (this study) |
---|---|---|
WT: cFL (set as 100%) | WT: cFL (set as 100%) | |
Within binding pockets | ||
L33/L185D | L33Y/L185D: cFL (0)26 | L33Y: cFL (n.d.)a |
L200 | L200N: cFL (<10%)26 | L200N: cFL (n.d.,a 5.6%)b |
N159 | N159A: cFL (45%)25 | N159A: cFL (60%)a |
Outside the pockets | ||
R98 | R98A: cFL (20%)26 | |
R99 | R99A: cFL (<10%)26 | R99A: cFL (n.d.)a |
R98A/R99A: cFL (0) | ||
R101/R102 | R101A/R102A: cFL (20%)26 | R101A: cFL (>50%)a |
R102A: cFL (23%)a | ||
D205 | D205A: cFL (>100%)25 | D205A: cFL (>100%)a |
Binding energya (kcal mol−1) of WT | Binding energyb (kcal mol−1) of F186L | Binding energyc (kcal mol−1) of F186L | |
---|---|---|---|
ΔGVDWd | −35.01 | −23.67 | −34.27 |
ΔGEte | −31.95 | −8.70 | −29.37 |
ΔGpolarf | 48.71 | 31.08 | 47.60 |
ΔGapolarg | −5.60 | −4.08 | −5.50 |
ΔGbindingh | −23.85 | −5.37 | −21.54 |
9.
Natural systems produce various γ-dicarbonyl-bearing compounds that can covalently modify lysine in protein targets via the classic Paal–Knorr reaction. Among them is a unique class of lipid-derived electrophiles – isoketals that exhibit high chemical reactivity and critical biological functions. However, their target selectivity and profiles in complex proteomes remain unknown. Here we report a Paal–Knorr agent, 4-oxonon-8-ynal (herein termed ONAyne), for surveying the reactivity and selectivity of the γ-dicarbonyl warhead in biological systems. Using an unbiased open-search strategy, we demonstrated the lysine specificity of ONAyne on a proteome-wide scale and characterized six probe-derived modifications, including the initial pyrrole adduct and its oxidative products (i.e., lactam and hydroxylactam adducts), an enlactam adduct from dehydration of hydroxylactam, and two chemotypes formed in the presence of endogenous formaldehyde (i.e., fulvene and aldehyde adducts). Furthermore, combined with quantitative chemoproteomics in a competitive format, ONAyne permitted global, in situ, and site-specific profiling of targeted lysine residues of two specific isomers of isoketals, levuglandin (LG) D2 and E2. The functional analyses reveal that LG-derived adduction drives inhibition of malate dehydrogenase MDH2 and exhibits a crosstalk with two epigenetic marks on histone H2B in macrophages. Our approach should be broadly useful for target profiling of bioactive γ-dicarbonyls in diverse biological contexts.Natural systems produce various γ-dicarbonyl-bearing compounds that can covalently modify lysine in protein targets via the classic Paal–Knorr reaction.Synthetic chemistry methods have been increasingly underscored by their potential to be repurposed as biocompatible methods for both chemical biology and drug discovery. The most-known examples of such a repurposing approach include the Staudinger ligation1 and the Huisgen-based click chemistry.2 Moreover, bioconjugation of cysteine and lysine can be built upon facile chemical processes,3 while chemoselective labelling of other polar residues (e.g., histidine,4 methionine,5 tyrosine,6 aspartic and glutamic acids7,8) requires more elaborate chemistry, thereby offering a powerful means to study the structure and function of proteins, even at a proteome-wide scale.The classical Paal–Knorr reaction has been reported for a single-step pyrrole synthesis in 1884.9,10 The reaction involves the condensation of γ-dicarbonyl with a primary amine under mild conditions (e.g., room temperature, mild acid) to give pyrrole through the intermediary hemiaminals followed by rapid dehydration of highly unstable pyrrolidine adducts (Fig. S1†).Interestingly, we and others have recently demonstrated that the Paal–Knorr reaction can also readily take place in native biological systems.11–13 More importantly, the Paal–Knorr precursor γ-dicarbonyl resides on many endogenous metabolites and bioactive natural products.14 Among them of particular interest are isoketals15 (IsoKs, also known as γ-ketoaldehydes) which are a unique class of lipid derived electrophiles (LDEs) formed from lipid peroxidation (Fig. S2†)16 that has emerged as an important mechanism for cells to regulate redox signalling and inflammatory responses,17 and drive ferroptosis,18 and this field has exponentially grown over the past few years. It has been well documented that the γ-dicarbonyl group of IsoKs can rapidly and predominantly react with lysine via the Paal–Knorr reaction to form a pyrrole adduct in vitro (Fig. 1).15 Further, the pyrrole formed by IsoKs can be easily oxidized to yield lactam and hydroxylactam products in the presence of molecular oxygen (Fig. 1). These rapid reactions are essentially irreversible. Hence, IsoKs react with protein approximately two orders of magnitude faster than the most-studied LDE 4-hydoxynonenal (4-HNE) that contains α,β-unsaturated carbonyl to generally adduct protein cysteines by Michael addition (Fig. S3†).15 Due to this unique adduction chemistry and rapid reactivity, IsoKs exhibit intriguing biological activities, including inhibition of the nucleosome complex formation,19 high-density lipoprotein function,20 mitochondrial respiration and calcium homeostasis,21 as well as activation of hepatic stellate cells.22 Furthermore, increases in IsoK-protein adducts have been identified in many major diseases,23 such as atherosclerosis, Alzheimer''s disease, hypertension and so on.Open in a separate windowFig. 1The Paal–Knorr precursor γ-dicarbonyl reacts with the lysine residue on proteins to form diverse chemotypes via two pathways. The red arrow shows the oxidation pathway, while the blue one shows the formaldehyde pathway.Despite the chemical uniqueness, biological significance, and pathophysiological relevance of IsoKs, their residue selectivity and target profiles in complex proteomes remain unknown, hampering the studies of their mechanisms of action (MoAs). Pioneered by the Cravatt group, the competitive ABPP (activity-based protein profiling) has been the method of choice to analyse the molecular interactions between electrophiles (e.g., LDEs,24 oncometabolites,25 natural products,26,27 covalent ligands and drugs28–30) and nucleophilic amino acids across complex proteomes. In this regard, many residue-specific chemistry methods and probes have been developed for such studies. For example, several lysine-specific probes based on the activated ester warheads (e.g., sulfotetrafluorophenyl, STP;31N-hydroxysuccinimide, NHS32) have recently been developed to analyse electrophile–lysine interactions at a proteome-wide scale in human tumour cells, which provides rich resources of ligandable sites for covalent probes and potential therapeutics. Although these approaches can also be presumably leveraged to globally and site-specifically profile lysine-specific targets IsoKs, the reaction kinetics and target preference of activated ester-based probes likely differ from those of γ-dicarbonyls, possibly resulting in misinterpretation of ABPP competition results. Ideally, a lysine profiling probe used for a competitive ABPP analysis of IsoKs should therefore possess the same, or at least a similar, warhead moiety. Furthermore, due to the lack of reactive carbonyl groups on IsoK-derived protein adducts, several recently developed carbonyl-directed ligation probes for studying LDE-adductions are also not suitable for target profiling of IsoKs.33,34Towards this end, we sought to design a “clickable” γ-dicarbonyl probe for profiling lysine residues and, in combination with the competitive ABPP strategy, for analysing IsoK adductions in native proteomes. Considering that the diversity of various regio- and stereo- IsoK isomers15 (a total of 64, Fig. S2†) in chemical reactivity and bioactivities is likely attributed to the substitution of γ-dicarbonyls at positions 2 and 3, the “clickable” alkyne handle needs to be rationally implemented onto the 4-methyl group in order to minimize the biases when competing with IsoKs in target engagement. Interestingly, we reasoned that 4-oxonon-8-ynal, a previously reported Paal–Knorr agent used as an intermediate for synthesizing fatty acid probes35 or oxa-tricyclic compounds,36 could be repurposed for the γ-dicarbonyl-directed ABPP application. With this chemical in hand (herein termed ONAyne, Fig. 2A), we first used western blotting to detect its utility in labelling proteins, allowing visualization of a dose-dependent labelling of the proteome in situ (Fig. S4†). Next, we set up to incorporate this probe into a well-established chemoproteomic workflow for site-specific lysine profiling in situ (Fig. 2A). Specifically, intact cells were labelled with ONAyne in situ (200 μM, 2 h, 37 °C, a condition showing little cytotoxicity, Fig. S5†), and the probe-labelled proteome was harvested and processed into tryptic peptides. The resulting probe-labelled peptides were conjugated with both light and heavy azido-UV-cleavable-biotin reagents (1 : 1) via CuI-catalyzed azide–alkyne cycloaddition reaction (CuAAC, also known as click chemistry). The biotinylated peptides were enriched with streptavidin beads and photoreleased for LC-MS/MS-based proteomics. The ONAyne-labelled peptides covalently conjugated with light and heavy tags would yield an isotopic signature. We considered only those modified peptide assignments whose MS1 data reflected a light/heavy ratio close to 1.0, thereby increasing the accuracy of these peptide identifications. Using this criterium, we applied a targeted database search to profile three expected probe-derived modifications (PDMs), including 13 pyrrole peptide adducts (Δ273.15), 77 lactam peptide adducts (Δ289.14), and 557 hydroxylactam peptide adducts (Δ305.14), comprising 585 lysine residues on 299 proteins (Fig. S6 and S7†). Among them, the hydroxylactam adducts were present predominately, since the pyrrole formed by this probe, the same as IsoKs, can be easily oxidized when being exposed to O2. This finding was in accordance with a previous report where the pyrrole adducts formed by the reaction between IsoK and free lysine could not be detected, but rather their oxidized forms.37 Regardless, all three types of adducts were found in one lysine site of EF1A1 (K387, Fig. S8†), further confirming the intrinsic relationship among those adductions in situ.Open in a separate windowFig. 2Adduct profile and proteome-wide selectivity of the γ-dicarbonyl probe ONAyne. (A) Chemical structure of ONAyne and schematic workflow for identifying ONAyne-adducted sites across the proteome. (B) Bar chart showing the distribution of six types of ONAyne-derived modifications formed in situ and in vitro (note: before probe labelling, small molecules in cell lysates were filtered out through desalting columns).State-of-the-art blind search can offer an opportunity to explore unexpected chemotypes (i.e., modifications) derived from a chemical probe and to unbiasedly assess its proteome-wide residue selectivity.38,39 We therefore sought to use one of such tools termed pChem38 to re-analyse the MS data (see Methods, ESI†). Surprisingly, the pChem search identified three new and abundant PDMs (Fig. 1 and Table S1†), which dramatically expand the ONAyne-profiled lysinome (2305 sites versus 585 sites). Overall, these newly identified PDMs accounted for 74.6% of all identifications (Fig. 2B and Table S2†). Among them, the PDM of Δ287.13 (Fig. 1 and S7†) might be an enlactam product via dehydration of the probe-derived hydroxylactam adduct. The other two might be explained by the plausible mechanism as follows (Fig. 1). The endogenous formaldehyde (FA, produced in substantial quantities in biological systems) reacts with the probe-derived pyrrole adduct via nucleophilic addition to form a carbinol intermediate, followed by rapid dehydration to a fulvene (Δ285.15, Fig. S7†) and immediate oxidation to an aldehyde (Δ301.14, Fig. S7†). In line with this mechanism, the amount of FA-derived PDMs was largely eliminated when the in vitro ONAyne labelling was performed in the FA-less cell lysates (Fig. 2B and Table S3†). Undoubtedly, the detailed mechanisms underlying the formation of these unexpected PDMs require further investigation, and so does the reaction kinetics. Regardless, all main PDMs from ONAyne predominantly target the lysine residue with an average localization probability of 0.77, demonstrating their proteome-wide selectivity (Fig. S9†).Next, we adapted an ABPP approach to globally and site-specifically quantify the reactivity of lysine towards the γ-dicarbonyl warhead through a dose-dependent labelling strategy (Fig. 3A) that has been proved to be successful for other lysine-specific probes (e.g., STP alkyne).31 Specifically, MDA-MB-231 cell lysates were treated with low versus high concentrations of ONAyne (1 mM versus 0.1 mM) for 1 h. Probe-labelled proteomes were digested into tryptic peptides that were then conjugated to isotopically labelled biotin tags via CuAAC for enrichment, identification and quantification. In principle, hyperreactive lysine would saturate labelling at the low probe concentration, whereas less reactive ones would show concentration-dependent increases in labelling. For fair comparison, the STP alkyne-based lysine profiling data were generated by using the same chemoproteomic workflow. Although 77.5% (3207) ONAyne-adducted lysine sites can also be profiled by STP alkyne-based analysis, the former indeed has its distinct target-profile with 930 lysine sites newly identified (Fig. S10 and Table S4†). Interestingly, sequence motif analysis with pLogo40 revealed a significant difference in consensus motifs between ONAyne- and STP alkyne-targeting lysines (Fig. S11†).Open in a separate windowFig. 3ONAyne-based quantitative reactivity profiling of proteomic lysines. (A) Schematic workflow for quantitative profiling of ONAyne–lysine reactions using the dose-dependent ABPP strategy (B) Box plots showing the distribution of R10:1 values quantified in ONAyne- and STP alkyne-based ABPP analyses, respectively. Red lines showing the median values. ***p ≤ 0.001 two-tailed Student''s t-test. (C) Representative extracted ion chromatograms (XICs) showing changes in the EF1A1 peptide bearing K273 that is adducted as indicated, with the profiles for light and heavy-labelled peptides in blue and red, respectively.Moreover, we quantified the ratio (R1 mM:0.1 mM) for a total of 2439 ONAyne-tagged lysines (on 922 proteins) and 17904 STP alkyne-tagged lysines (on 4447 proteins) across three biological replicates (Fig. S12 and Table S5†). Strikingly, only 26.7% (651) of quantified sites exhibited nearly dose-dependent increases (R1 mM:0.1 mM > 5.0) in reactivity with ONAyne, an indicative of dose saturation (Fig. 3B and C). In contrast, such dose-dependent labelling events accounted for >69.1% of all quantified lysine sites in the STP alkyne-based ABPP analysis.31 This finding is in accordance with the extremely fast kinetics of reaction between lysine and γ-dicarbonyls (prone to saturation). Nonetheless, by applying 10-fold lower probe concentrations, overall 1628 (80.2%) detected lysines could be labelled in a fully concentration-dependent manner with the median R10:1 value of 8.1 (Fig. 3B, C, S12 and Table S5†). Next, we asked whether the dose-depending quantitation data (100 μM versus 10 μM) can be harnessed to predict functionality. By retrieving the functional information for all quantified lysines from the UniProt Knowledgebase, we found that those hyper-reactive lysines could not be significantly over-represented with annotation (Fig. S12†). Nonetheless, among all quantified lysines, 509 (25.1%) possess functional annotations, while merely 2.5% of the human lysinome can be annotated. Moreover, 381 (74.8%) ONAyne-labelled sites are known targets of various enzymatic post-translational modifications (PTMs), such as acetylation, succinylation, methylation and so on (Fig. S13†). In contrast, all known PTM sites accounted for only 59.6% of the annotated human lysinome. These findings therefore highlight the intrinsic reactivity of ONAyne towards the ‘hot spots’ of endogenous lysine PTMs.The aforementioned results validate ONAyne as a fit-for-purpose lysine-specific chemoproteomic probe for competitive isoTOP-ABPP application of γ-dicarbonyl target profiling. Inspired by this, we next applied ONAyne-based chemoproteomics in an in situ competitive format (Fig. 4A) to globally profile lysine sites targeted by a mixture of levuglandin (LG) D2 and E2, two specific isomers of IsoKs that can be synthesized conveniently from prostaglandin H2 (ref. 41) (Fig. S2†). Specifically, mouse macrophage RAW264.7 cells (a well-established model cell line to study LDE-induced inflammatory effects) were treated with 2 μM LGs or vehicle (DMSO) for 2 h, followed by ONAyne labelling for an additional 2 h. The probe-labelled proteomes were processed as mentioned above. For each lysine detected in this analysis, we calculated a control/treatment ratio (RC/T). Adduction of a lysine site by LGs would reduce its accessibility to the ONAyne probe, and thus a higher RC/T indicates increased adduction. In total, we quantified 2000 lysine sites on 834 proteins across five biological replicates. Among them, 102 (5.1%) sites exhibited decreases of reactivity towards LGs treatment (P < 0.05, Table S6†), thereby being considered as potential targets of LGs. Notably, we found that different lysines on the same proteins showed varying sensitivity towards LGs (e.g., LGs targeted K3 of thioredoxin but not K8, K85 and K94, Table S6†), an indicative of changes in reactivity, though we could not formally exclude the effects of changes in protein expression on the quantified competition ratios. Regardless, to the best of our knowledge, the proteome-wide identification of potential protein targets by IsoKs/LGs has not been possible until this work.Open in a separate windowFig. 4ONAyne-based in situ competitive ABPP uncovers functional targets of LGs in macrophages. (A) Schematic workflow for profiling LGs–lysine interactions using ONAyne-based in situ competitive ABPP. (B) Volcano plot showing the log2 values of the ratio between the control (heavy) and LGs-treated (light) channels and the −log10(P) of the statistical significance in a two-sample t-test for all quantified lysines. Potential targets of LGs are shown in blue (RC/T>1.2, P < 0.05), with the validated ones in red. (C) Bar chart showing the inhibitory effect of 2 μM LGs on the cellular enzymatic activity of MDH2. Data represent means ± standard deviation (n = 3). Statistical significance was calculated with two-tailed Student''s t-tests. (D) Pretreatment of LGs dose-dependently blocked ONAyne-labelling of MDH2 in RAW264.7 cells, as measured by western blotting-based ABPP. (E and F) LGs dose-dependently decreased the H2BK5 acetylation level in RAW 264.7 cells, as measured either by western blotting (E) or by immunofluorescence imaging (F). n = 3. For G, nuclei were visualized using DAPI (blue).We initially evaluated MDH2 (malate dehydrogenase, mitochondrial, also known as MDHM), an important metabolic enzyme that possesses four previously uncharacterized liganded lysine sites (K157, K239, K301 and K329, Fig. 4B) that are far from the active site (Fig. S14†). We found that LGs dramatically reduced the catalytic activity of MDH2 in RAW264.7 cells (Fig. 4C), suggesting a potentially allosteric effect. We next turned our attention to the targeted sites residing on histone proteins, which happen to be modified by functionally important acetylation, including H2BK5ac (Fig. 4B) that can regulate both stemness and epithelial–mesenchymal transition of trophoblast stem cells.42 We therefore hypothesized that rapid adduction by LGs competes with the enzymatic formation of this epigenetic mark. Immunoblotting-based competitive ABPP confirmed that LGs dose-dependently blocked probe labelling of H2B (Fig. 4D). Further, both western blots and immunofluorescence assays revealed that LG treatment decreased the level of acetylation of H2BK5 (average RC/T = 1.3, P = 0.007) in a concentration-dependent manner (Fig. 4E and F). Likewise, a similar competitive crosstalk was observed between acetylation and LG-adduction on H2BK20 (average RC/T = 1.2, P = 0.01) that is required for chromatin assembly43 and/or gene regulation44 (Fig. 4B and S15†). Notably, these findings, together with several previous reports by us and others about histone lysine ketoamide adduction by another important LDE, 4-oxo-2-noenal,11,45,46 highlight again the potentially important link between lipid peroxidation and epigenetic regulation. In addition to the targets validated as above, many other leads also merit functional studies considering diverse biological or physiologic effects of LGs in macrophages. 相似文献
10.
Jos García-Calvo Javier Lpez-Andarias Jimmy Maillard Vincent Mercier Chlo Roffay Aurlien Roux Alexandre Fürstenberg Naomi Sakai Stefan Matile 《Chemical science》2022,13(7):2086
HydroFlippers are introduced as the first fluorescent membrane tension probes that report simultaneously on membrane compression and hydration. The probe design is centered around a sensing cycle that couples the mechanical planarization of twisted push–pull fluorophores with the dynamic covalent hydration of their exocyclic acceptor. In FLIM images of living cells, tension-induced deplanarization is reported as a decrease in fluorescence lifetime of the dehydrated mechanophore. Membrane hydration is reported as the ratio of the photon counts associated to the hydrated and dehydrated mechanophores in reconvoluted lifetime frequency histograms. Trends for tension-induced decompression and hydration of cellular membranes of interest (MOIs) covering plasma membrane, lysosomes, mitochondria, ER, and Golgi are found not to be the same. Tension-induced changes in mechanical compression are rather independent of the nature of the MOI, while the responsiveness to changes in hydration are highly dependent on the intrinsic order of the MOI. These results confirm the mechanical planarization of push–pull probes in the ground state as most robust mechanism to routinely image membrane tension in living cells, while the availability of simultaneous information on membrane hydration will open new perspectives in mechanobiology.HydroFlippers respond to membrane compression and hydration in the same fluorescence lifetime imaging microscopy histogram: the responses do not correlate.The detection and study of membrane mechanics in living cells is a topic of current concern.1–14 To enable this research, appropriate chemistry tools, that is small-molecule fluorescent probes that allow imaging of membrane tension, are needed.15 With the direct imaging of physical forces being intrinsically impossible, design strategies toward such probes have to focus on the suprastructural changes caused by changes in membrane tension.15 These suprastructural changes are divers, often interconnected, and vary with the composition of the membrane.15–25 Beyond the fundamental lipid compression and decompression, they include changes in membrane curvature, from rippling, buckling and budding to tubules extending from the membrane and excess lipid being ejected. Of similar importance are changes in membrane organization, particularly tension-induced phase separation and mixing, i.e. assembly and disassembly of microdomains. Consequences of these suprastructural changes include microdomain strengthening and softening and changes in membrane hydration and viscosity.16–25The currently most developed fluorescent flipper probes have been introduced26,27 to image membrane tension by responding to a combination of mechanical compression and microdomain assembly in equilibrium in the ground state.15 Extensive studies, including computational simulations,28 have shown that flipper probes align non-invasively along the lipid tails of one leaflet and report changes in membrane order and tension as changes in fluorescent lifetimes and shifts of excitation maxima.15 Among other candidates, solvatochromic probes respond off-equilibrium in the excited state to changes in membrane hydration and have very recently been considered for the imaging of membrane tension in living cells.29–36 So far not considered to image tension, ESIPT probes also report off equilibrium in the excited state on membrane hydration, but for different reasons.37,38 Mechanosensitive molecular rotors respond off equilibrium in the excited state to changes in microviscosity.17,30,32,39–53 The same principle holds for the planarization of bent, papillon or flapping fluorophores.54–57 The response of all possible probes to tension can further include less desired changes in positioning and partitioning between different domains, not to speak of more catastrophic probe aggregation, precipitation, disturbance of the surrounding membrane structure, and so on. Although the imaging of membrane tension is conceivable in principle with most of above approaches, the complex combination of parameters that has to be in place can thus far only be identified empirically, followed by much optimization.15The force-induced suprastructural changes are accompanied by the alteration in several unrelated physical properties of membranes. It is, for instance, well documented that membrane hydration increases with membrane disorder, from solid-ordered (So) to liquid-disordered (Ld) phases.29,58 Increasing cholesterol content decreases membrane hydration in solid- and liquid-ordered membranes.59 However, studies in model membranes also indicate that membrane hydration and membrane fluidity do not necessarily correlate.59 The dissection of the individual parameters contributing to the response of fluorescent membrane tension probes would be important for probe design and understanding of their responses, but it remains a daunting challenge. In this study, we introduce fluorescent flipper probes that simultaneously report on mechanical membrane compression and membrane hydration at equilibrium in the ground state. Changes of both in response to changes in membrane tension and membrane composition are determined in various organelles in living cells.The dual hydration and membrane tension probes are referred to as HydroFlippers to highlight the newly added responsiveness to membrane hydration. The mechanosensing of lipid compression in bilayer membranes by flipper probes has been explored extensively.15 Fluorescent flippers27 like 1 are designed as bioinspired60 planarizable push–pull probes26 (Fig. 1). They are constructed from two dithienothiophene fluorophores that are twisted out of co-planarity by repulsion of methyls and σ holes on sulfurs61,62 next to the twistable bond. The push–pull system is constructed first from formal sulfide and sulfone redox bridges in the two twisted dithienothiophenes. These endocyclic donors and acceptors are supported by exocyclic ones, here a trifluoroketone acceptor and a triazole donor.63 To assure stability, these endo- and exocyclic donors are turned off in the twisted ground state because of chalcogen bonding and repulsion, respectively.62Open in a separate windowFig. 1The dual sensing cycle of HydroFlippers 1–5, made to target the indicated MOIs in living cells and responding to membrane compression by planarization and to membrane hydration by dynamic covalent ketone hydration. With indication of excitation maxima (ref. 63) and fluorescence lifetimes (this study).Mechanical planarization of the flipper probe establishes conjugation along the push–pull systems, electrons flow from endocyclic donors to acceptors, which turns on the exocyclic donors and acceptors to finalize the push–pull system.62 This elaborate, chalcogen-bonding cascade switch has been described elsewhere in detail, including high-level computational simulations.62 The planar high-energy conformer 1dp excels with red shifted excitation and increased quantum yield and lifetime compared to the twisted conformer 1dt because the less twisted Franck-Condon state favors emission through planar intramolecular charge transfer (PICT) over non-radiative decay through twisted ICT, or conical intersections.15Flipper probe 1 was considered for dual responsiveness to membrane tension and hydration because of the trifluoroketone acceptor.63 Dynamic covalent hydration of 1dt yields hydrate 1ht.64–76 Blue-shifted excitation and short lifetime of 1ht are not expected to improve much upon planarization because the hydrate is a poor acceptor and thus, the push–pull system in 1hp is weak. The dynamic covalent chemistry of the trifluoroketone acceptor has been characterized in detail in solution and in lipid bilayer membranes.63To explore dual responsiveness to membrane tension in any membrane of interest (MOI) in living cells, HydroFlippers 2–5 were synthesized. While HydroFlipper 1 targets the plasma membrane (PM), HydroFlippers 2–4 were equipped with empirical targeting motifs.77 HydroFlipper 5 terminates with a chloroalkane to react with the self-labeling HaloTag protein, which can be expressed in essentially any MOI.78 Their substantial multistep synthesis was realized by adapting reported procedures (Schemes S1–S4†).The MOIs labeling selectivity of HydroFlippers was determined in HeLa Kyoto (HK) cells by confocal laser scanning microscopy. Co-localization experiments of flippers 1–4 with the corresponding trackers gave Pearson correlation coefficients (PCCs) >0.80 for the targeting of mitochondria, lysosomes and the endoplasmic reticulum (ER, Fig. S4–S6†). HydroFlipper 5 was first tested with stable HGM cells, which express both HaloTag and GFP on mitochondria (referred to as 5M).78,79 The well-established chloroalkane penetration assay demonstrated the efficient labeling of HaloTag protein by 5 as previously reported HaloFlippers (Fig. S3†).78 By transient transfection, HydroFlippers 5 were also directed to lysosomes (5L), Golgi apparatus (GA, 5G)80 and peroxisomes (5P) with HaloTag and GFP expressed on their surface.78 PCCs >0.80 for co-localization of flipper and GFP emission confirmed that MOI labeling with genetically engineered cells was as efficient as with empirical trackers (Fig. S7–S11†).Dual imaging of membrane compression and hydration was envisioned by analysis of fluorescence lifetime imaging microscopy (FLIM) images using a triexponential model (Fig. 2).81 FLIM images of ER HydroFlipper 4 in iso-osmotic HK cells were selected to illustrate the concept (Fig. 3a). Contrary to classical flipper probes, the fluorescence decay curve of the total FLIM image (Fig. 2a, grey) showed a poor fit to a biexponential model (Fig. 2a, cyan, b). Consistent with their expected dual sensing mode, a triexponential fit was excellent (Fig. 2a, dark blue, c). Lifetimes τ1i = 4.3 ns (†) were obtained besides background. This three-component model was then applied to every pixel of FLIM images (Fig. 3c). The resulting reconvoluted FLIM histogram revealed three clearly separated populations for τ1 (red), τ2 (green), and background (τ3, blue, Fig. 2d). Maxima of these three clear peaks were at the lifetimes estimated by triexponential fit of the global decay curve, thus demonstrating the validity of the methodology at necessarily small photon counts. Irreproducible fitting would give randomly scattered data without separated peaks.Open in a separate windowFig. 2(a) Fluorescence decay curve (grey, corresponding to the total image, not to a single pixel) with biexponential (cyan) and triexponential fit (dark blue). (b, c) Residual plots for bi- (b) and triexponential fit (c). (d) Histogram with the intensities associated with the τ1 (red), τ2 (green), and τ3 (blue, background) components obtained by triexponential fit of the fluorescence decay curve of each pixel of the FLIM image, fit to Gaussian function (black solid curves).Open in a separate windowFig. 3FLIM images of HK cells labelled with ER flipper 4 before (a, c) and after (b, d) hyper-osmotic shock, showing average lifetimes τav (a, b) and τ1 (c, d) from triexponential reconvolution; scale bars = 10 μm. (e) Distribution of the photon counts associated with the τ1 component of 4 in HK cells after triexponential reconvolution of FLIM images before (c, τ1i) and after (d, τ1h) hyper-osmotic shock, showing decreasing lifetimes for τ1 (4d). (f) The dehydration factor dhi defined as total integrated photon counts for τ1 (Στ1) divided by Στ2 (i.e., dhi = area Στ1i/area Στ2i) for 4 in strongly hydrated ER (dhi < 2, turquoise) and 1 in weakly hydrated plasma membrane (dhi > 6, purple) of HK Kyoto cells under iso-osmotic conditions.Dual response of HydroFlippers to changes in membrane tensiona
Open in a separate windowaFrom triexponential fit of FLIM images in HK cells (errors, see ESI).bFlipper (target MOI).cdhi = area Στ1i/area Στ2i in FLIM histogram under iso-osmotic (i) conditions (e.g.Fig. 3f).ddhh = area Στ1h/area Στ2h in FLIM histogram under hyper-osmotic (h) conditions.eFlipper hydration change in response to membrane tension: Δdh = (1 – dhh/dhi) × 100%.fFluorescence lifetime value of the slowest component from the fitted fluorescence decay under iso-osmotic (i) conditions (e.g.Fig. 2d).gSame as f, under hyper-osmotic (h) conditions.hFlipper planarization in response to membrane tension: Δτ1 = (1 – τ1h/τ1i) × 100%.iMeasured after cholesterol (C) removal from cells with MβCD.jCompared to dhi of 1 (6.6) in untreated cells measured on the same day.kCompared to τih of 1 (5.0) in untreated cells measured on the same day.lAs j using 4 and compared to dhi = 1.8.mAs k using 4 compared to τih = 4.5.nMeasured in transiently transfected HK cells with ST-HaloTag-HA expressed inside GA.80oMeasured in transiently transfected HK cells with HaloTag-Sec61B expressed inside ER.78pMeasured in SM/C GUVs.qMeasured in DOPC GUVs.Extensive lifetime data for monofunctional flipper probes supported that the intensities associated to τ1i (i for iso-osmotic, see below) originate from at least partially planarized flippers 4d in the ER (Fig. 2d, red, 3c, 1). The population of the τ2i component in the reconvoluted FLIM histogram was attributed to the presence of hydrated 4h in the ER (Fig. 2d, green, 1). This assignment was consistent with lifetime differences in solution between τ = 2.7 ns for the dehydrated and τ = 0.7 ns for the hydrated form of a hydrophobic flipper analog in dioxane-water mixtures (Fig. S2†), and model studies in GUVs (see below).63The ratio between the τ1i (red) and τ2i (green) populations in the reconvoluted FLIM histogram was used to extract a quantitative measure for hydration of the MOI (Fig. 2d, ,3f).3f). A dehydration factor dh was defined by dividing the total integrated counts for τ1 (Στ1) by Στ2. For 4 in iso-osmotic ER, dhi = 1.8 ± 0.1 was obtained (Fig. 3f, †63 Thus, these results implied that the dehydration factor dh obtained from reconvoluted triexponential FLIM images reports quantitatively on membrane hydration, that is the local water concentration around HydroFlippers in their MOI.In uniform model membranes composed of only one lipid, flipper probes like 1 respond to increasing membrane tension with decreasing lifetimes.15,18 This response can be explained by flipper deplanarization upon lipid decompression. In the mixed membranes composed of different lipids, flipper probes reliably respond to increasing membrane tension with increasing lifetimes, and lifetime changes can be calibrated quantitatively to the applied physical force.18,77 This indicates that in these biologically relevant membranes, the response is dominated by factors other than lipid decompression. Tension-induced microdomain formation is confirmed to account for, or at least contribute to, increasing lifetimes with increasing tension, or membrane decompression.15,18 Not only microdomain disassembly but also changes in membrane curvature from rippling, budding and microdomain softening to tube formation and lipid ejection combine to afford decreasing lifetimes with membrane compression, or decreasing tension.17,18Membrane tension was applied to the ER by extracellular hyper-osmotic stress. This causes membrane tension to decrease, i.e., membrane compression to increase.18,77 Consistent with tension-induced deplanarization from 4p to 4t (Fig. 1), lifetimes of 4 visibly decreased in response to decreasing membrane tension (Fig. 3b). The reconvoluted FLIM histogram clearly shows that compression caused the decrease of τ1 of 4 in the ER from τ1i = 4.3 ns to τ1h = 3.7 ns, whereas τ2i = 1.5 ns was less mechanosensitive (τ2h = 1.4 ns, Fig. 3e, 4a–c). These different mechanosensitivities were meaningful considering that in three-component histograms, τ1 originates from dehydrated HydroFlipper 4d that loses a strong push–pull dipole and thus shortens lifetime upon tension-induced deplanarization from 4dp to 4dt (Fig. 1). In contrast, hydrated HydroFlipper 4h accounting for τ2 lacks a strong dipole and thus features short lifetimes with poor sensitivity for tension-induced deplanarization from 4hp and 4ht. This result was consistent with the central importance of turn-on push–pull systems for flipper probes to function as mechanosensitive planarizable push–pull probes.81Open in a separate windowFig. 4(a) Reconvoluted FLIM histograms for 1–5 obtained by fitting each pixel of the FLIM image to a three-exponential model under iso-osmotic (top) and hyper-osmotic (bottom) conditions in HK cells; *dhi analysis in Fig. 3f; **Δτ1 analysis in Fig. 3e. (b–e) Trend plots for membrane compression (τ1) and hydration (dh) for 1–5 in HK cells without (b, e) and in response to hyper-osmotic membrane tension (c–e). (b) τ1i (iso-osmotic compression) vs. dhi (iso-osmotic hydration). (c) τ1i–τ1hvs. τ2i–τ2h (compression response in ns). (d) Δτ1 (compression response, %) vs. Δdh (hydration response, %), (e) Δτ1 and Δdh upon compression (σ) and cholesterol depletion (C). #Discontinuous, see 17,18The uniform response of HydroFlipper planarization and hydration thus provided corroborative support that membrane deformation and reorganization dominate the fluorescence imaging of membrane tension under the condition that the probe partitions equally between different phases.63 However, the dual response HydroFlipper dissects the consequences of these tension-induced suprastructural changes. HydroFlipper planarization 4t/4p detected by τ1 reports on lipid compression in the local environment in the MOI. HydroFlipper hydration 4d/4h detected by the dehydration factor dh reports on local membrane hydration. Pertinent reports from model membranes in the literature indicate that the two do not have to be the same.59To elaborate on these implications, FLIM images were recorded for all HydroFlippers 1–5 in their respective MOIs before and after the application of hyper-osmotic stress and then analyzed using the three-component model (Fig. 4a, Fig. 4a) and estimated by global triexponential fit (Fig. 3f, ,4a).4a). However, these changes do not affect dhi, which compares areas rather than maxima in the histograms.Trends for membrane hydration and compression reported by dhi and τ1i, respectively, should reflect the overall composition and thus nature of the different membranes. For PM 1, Lyso 2, GA 5G and ER 5E, coinciding trends were found for hydration (dhi, blue) and compression (τ1i, red, Fig. 4b). Hydration and deplanarization increased in parallel, consistent with increasingly disordered membranes. With Mito 3 and ER 4, increasing hydration (blue) was not reflected in increasing deplanarization (red, Fig. 4b).For the comprehensive analysis of the changes caused by hyper-osmotic stress, the differences in lifetimes for τ1 and τ2 were clarified first. Whereas τ1i–τ1h values (red) around 0.3 ns were large and significant in all MOIs, τ2i–τ2h values (pink) were negligible (Fig. 4c). The mechano-insensitive τ2, corresponding to hydrate 4h, were thus not further considered as a valid measure of membrane compression.To facilitate direct comparability, membrane compression Δτ1 and membrane dehydration Δdh in response to hyper-osmotic stress were converted in percentage of decrease (positive) or increase (negative) from the value under iso-osmotic conditions (Fig. 4d, Fig. 4d, red). In clear contrast, dehydration Δdh varied from 3% increase to 29% decrease (Fig. 4d, blue). The most extreme deviations concerned ER probes with maximal Δτ1 responsiveness for tracker 4 and minimal Δτ1 responsiveness for Halo flipper 5E. For dehydration Δdh, both probes showed high responsiveness. These extremes could reflect the diverse membrane properties of the ER, with τ = 4.1, 3.5 and 3.4 ns reported previously for different flipper mechanophores in tubular, sheet, and nuclear membranes of COS7 cells, respectively.15,77 Although less resolvable in HK cells, this heterogeneity of ER membranes is also visible in the FLIM images with 4 (Fig. 3). Tracker 4 and Halo flipper 5E both react covalently with membrane proteins and report on the respective surrounding ER membrane, which differs significantly according to the two HydroFlipper probes. The extreme values for Halo flipper 5E suggested that other factors like fractions of mispositioned flipper in more hydrophilic environment could also contribute to the global outcome (Fig. 4b, Fig. 4d, blue) increased with membranes disorder characterized by shorter τ1i and low dhi (Fig. 4b), while Δτ1 remained more constant until the possible onset of decreases at very high hydration (5E, Fig. 4d, red). Both observations - independence of mechanical flipper planarization and dependence of dynamic covalent hydrate formation on the water concentration in the surrounding membrane - were chemically meaningful.The validity of these conclusions was tested by removing cholesterol with methyl-β-cyclodextrin (MβCD). As expected for the increased hydration level and decreased order of cholesterol depleted membranes, Δdh and Δτ1 of 1 and 4 increased by MβCD treatment compared to those obtained on the same day without the treatment (Fig. 4e, C). Stronger response of ER HydroFlipper 4 to the cholesterol removal can be attributed to the poorer cholesterol content in ER membranes than in PM.82 Consistent with the overall trend, Δdh was more significantly affected by changes of the MOI by MβCD treatment than by tension change (Fig. 4e, blue, C vs. σ), while Δτ1 responded better to membrane tension than MOI change (Fig. 4e, red, C vs. σ).Taken together, these results reveal HydroFlippers as first dual mode fluorescent membrane tension probe, reporting on membrane hydration and membrane compression at the same time. Mechanical compression is reported as shift in τ, while tension-induced hydration is reported as change in relative photon counts for hydrated and dehydrated probes in the reconvoluted FLIM histograms. The response of flipper deplanarization to membrane tension is robust and less dependent on the nature of the MOI, including plasma membrane, ER, mitochondria, lysosomes and Golgi. In contrast, the responsiveness of flipper hydration to membrane tension depends strongly on the nature of the MOI, generally increasing with increasing intrinsic disorder, that is hydration, already under iso-osmotic conditions. These results validate the flipper probes as most reliable to routinely image membrane tension in cells, while the simultaneous information provided on membrane dehydration provides attractive possibilities for biological applications. 相似文献
Probeb | dhic | dhhd | Δdhe (%) | τ 1i f (ns) | τ 1h g (ns) | Δτ1h (%) | |
---|---|---|---|---|---|---|---|
1 | 1 (PM) | 6.3 | 6.5 | -3 | 4.8 | 4.4 | 8 |
2 | 1 (-C)i | 6.1 | — | 8j | 4.8 | — | 3k |
3 | 2 (Lyso) | 2.9 | 2.8 | 4 | 4.4 | 4.0 | 10 |
4 | 3 (Mito) | 2.3 | 1.9 | 17 | 4.4 | 4.0 | 8 |
5 | 4 (ER) | 1.8 | 1.5 | 17 | 4.3 | 3.7 | 15 |
6 | 4 (–C)i | 1.1 | — | 39l | 4.1 | — | 10m |
7 | 5G (GA)n | 2.5 | 2.3 | 8 | 4.2 | 3.8 | 10 |
8 | 5E (ER)o | 1.7 | 1.2 | 29 | 3.8 | 3.7 | 5 |
9 | 1 (Lo)p | 11 | — | — | 5.2 | — | — |
10 | 1 (Ld)q | 1.2 | — | — | 3.4 | — | — |
11.
Multisubstituted pyrroles are important fragments that appear in many bioactive small molecule scaffolds. Efficient synthesis of multisubstituted pyrroles with different substituents from easily accessible starting materials is challenging. Herein, we describe a metal-free method for the preparation of pentasubstituted pyrroles and hexasubstituted pyrrolines with different substituents and a free amino group by a base-promoted cascade addition–cyclization of propargylamides or allenamides with trimethylsilyl cyanide. This method would complement previous methods and support expansion of the toolbox for the synthesis of valuable, but previously inaccessible, highly substituted pyrroles and pyrrolines. Mechanistic studies to elucidate the reaction pathway have been conducted.This method is a toolbox for the synthesis of valuable, but previously inaccessible, highly substituted pyrroles and pyrrolines.Pyrroles are molecules of great interest in a variety of compounds including pharmaceuticals, natural products and other materials. Pyrrole fragments for example are key motifs in bioactive natural molecules, forming the subunit of heme, chlorophyll and bile pigments, and are also found in many clinical drugs, including those in Fig. 1a.1 Although many classical methods of pyrrole synthesis, including the Paal–Knorr condensation,2 the Knorr reaction,3 the Hantzsch reaction,4 transition metal-catalyzed reactions,5 and multicomponent coupling reactions,6 have been developed over many years, the efficient synthesis of multisubstituted pyrroles is still challenging. In condensation syntheses of pyrroles, the major problems lie in the extended syntheses of complex precursors and limited substitution patterns that are allowed. Multicomponent reactions are superior when building pyrrole core structures with more substituents. Among these, the [2+2+1] cycloaddition reaction of alkynes and primary amines is attractive because of the readily available alkyne and amine substrates and the ability to construct fully substituted pyrroles.7 However, with the exception of some rare examples,8 most [2+2+1] cycloaddition reactions afford pyrroles with two or more identical substituents. The synthesis of multisubstituted pyrroles with all different substituents from simple starting materials therefore remains a major challenge.Open in a separate windowFig. 1Previous reports and this work on propargylamides transformation.Easily accessible propargylamides are classical, privileged building blocks broadly utilized for the synthesis of a large variety of heterocyclic molecules such as pyrroles, pyridines, thiazoles, oxazoles and other relevant organic frameworks.9 For example, Looper10et al. reported the synthesis of 2-aminoimidazoles from propargyl cyanamides and Eycken11 reported a method starting from propargyl guanidines which undergo a 5-exo-dig heterocyclization as shown in Fig. 1b. Subsequently, Wan12et al. revealed the cyclization of N-alkenyl propargyl sulfonamides into pyrroles via sulfonyl migration. Inspired by these transformations and multi-substituted pyrrole synthesis, we report herein a direct synthesis of pentasubstituted pyrroles and hexasubstituted pyrrolines with all different substituents from propargyl sulfonylamides and allenamides.Previously, Zhu,13 Ji14 and Qiu13b,15 reported efficient syntheses of 2-aminopyrroles from isocyanides. Ye16 and Huang17 independently developed gold-catalyzed syntheses of 2-amino-pentasubstituted pyrroles with ynamides. Despite the many advantages of these methods, they all afford protected amines, rather than free amines. The deprotection of these amines may cause problems in further transformations of the products. Our method delivers pyrroles with an unprotected free amino group and are often complementary to the previously well-developed classical methods.Initially, the cyclization reaction of N-(1,3-diphenylprop-2-yn-1-yl)-N-ethylbenzenesulfonamide (1a) with trimethylsilyl cyanide (TMSCN) was carried out with Ni(PPh3)2Cl2 as a catalyst, a base (Cs2CO3) and DMF as a solvent. Different metal catalysts, such as Ni(PPh3)2Cl2, Pd(OAc)2, Cu(OAc)2, and Co(OAc)2 provided the desired product with similar yields ( Entry Cat. Base Solvent Yield 1 Ni(PPh3)2Cl2 Cs2CO3 DMF 67% 2 Pd(OAc)2 Cs2CO3 DMF 65% 3 Cu(OAc)2 Cs2CO3 DMF 65% 4 Co(OAc)2 Cs2CO3 DMF 63% 5 Cs2CO3 DMF 65% 6 KF DMF Trace 7 K3PO4 DMF Trace 8 K2CO3 DMF 48% 9 KOH DMF 52% 10 KOtBu DMF 46% 11 Et3N DMF Trace 12 Cs2CO3 CH3CN 18% 13 Cs2CO3 DME 23% 14 Cs2CO3 Toluene Trace 15 Cs2CO3 DCE Trace 16 Cs2CO3 Dioxane Trace