Herein, we report that quantum-sized Au nanoparticles can photocatalytically reduce CO2 to CO with the help of H2O by electron-hole pairs mainly originating from interband transitions. Notably, the Au photocatalyst shows a CO production rate of 4.73 mmol g−1 h−1 (~100% selectivity), ~2.5 times the rate during CO2 reduction with H2 under the same experimental conditions, under low-intensity irradiation at 420 nm. Theoretical and experimental studies reveal that the increased activity is induced by surface Au–O species formed from H2O decomposition, which synchronously optimizes the rate-determining steps in the CO2 reduction and H2O oxidation reactions, lowers the energy barriers for the *CO desorption and *OOH formation, and facilitates CO and O2 production. Our findings provide an in-depth mechanistic understanding for designing active metal photocatalysts for efficient CO2 reduction with H2O.Achieving CO 2-to-CO conversion with H2O by charge carriers generated from interband transitions.While plasmonic metals show some activity for photocatalysis, they often must be paired with semiconductors or sacrificial reagents. Here, authors find quantum-sized Au nanoparticles can achieve photocatalytic CO In recent decades, photocatalytic studies have almost exclusively focused on semiconductors, which provide an opportunity for reducing CO2 with H2O at room temperature4. However, the reaction rates of semiconductor photocatalysts are usually proportional to the square root of light intensity, thereby limiting their efficiency in practical applications, in which relatively high light intensity is required5.Coupling CO12 or nonplasmonic (such as Ni and Ru)14 metal nanoparticles (NPs) have been recently reported to show photocatalytic activities for CO2 reduction. Under illumination, a collective oscillation of free electrons (conduction band electrons) is sometimes induced in metal NPs, a phenomenon defined as localized surface plasmon resonance (LSPR). Moreover, valence electrons in metal NPs can be excited to a much higher energy level through interband transitions15. Compared with the photoinduced electron-hole pairs in semiconductors, charge carriers produced in metal NPs usually possess sufficient energy for activating chemical bonds16. In addition, metal photocatalysts also have the ability to combine light and thermal energies to activate inert molecules in catalytic reactions15. Notably, a linear increase in reaction rates with increasing reaction temperature and light intensity was reported for metal photocatalysts, which sharply contrasts with semiconductor photocatalysts17. These intriguing properties make metal photocatalysts effective candidates for overcoming the severe challenges to photocatalytic CO2 reduction18.Other than semiconductor photocatalysts, some plasmonic 2 conversion on metal NPs, strong reductants  are generally required21. For application purposes, using inexpensive and abundant H2O as an electron donor should be the direction and goal of photocatalytic CO2 reduction22. In this reaction, a series of complex and consecutive multi charge transfer processes followed by simultaneous CO2 reduction and H2O oxidation reactions at two different sites should occur under illumination3. The hot carriers generated from the LSPR effect of metal photocatalysts always have a much shorter lifetime than those from semiconductors23, which leads to difficulty in accomplishing CO2 reduction with H2O. To solve this problem, combining plasmonic metal nanostructures with semiconductors (such as TiO2) has been a widely used method by which the lifetime of hot charge carriers is prolonged24. In this situation, the metal NPs work as photosensitizers via the LSPR effect, and their low interfacial charge transfer efficiency greatly limits the application of these composite photocatalysts25.However, for photocatalytic CO2O oxidation and CO2 reduction reactions26. However, to the best of our knowledge, photocatalytic CO2 reduction with H2O has been rarely realized on metal-based photocatalysts by interband transitions. Moreover, molecular-level insights into the fundamentals of photocatalytic reactions on metal photocatalysts are still challenging to obtain and lacking, greatly restricting the rational design of high-efficiency metal photocatalysts. Gold (Au) NPs have received increasing attention for the photocatalytic conversion of CO2 into C1 or C2 hydrocarbons by the LSPR effect, which is mainly performed in the presence of H2 or sacrificial agents27. Recently, it has been proven that the interband absorption of Au NPs can dominate plasmon absorption with decreasing particle size28. Therefore, quantum-sized Au NPs with unique interband excitations under visible light irradiation may provide unprecedented possibilities for driving the photocatalytic conversion of CO2 with H2O.In contrast to the LSPR effect, hot electron-hole pairs generated from the interband transitions of metal NPs always possess higher redox potentials and longer lifetimes, which are more conducive to driving the H2 to CO with H2O by interband transitions, are synthesized by a one-pot reduction approach. Molecular insight into the photocatalytic reaction mechanism reveals that the CO2 reduction and H2O oxidation reactions on Au followed the CO2→*COOH→*CO→CO(g) and H2O→*OH→*O−→*O→*OOH→O2 routes, respectively. Notably, the surface Au–O species formed from decomposing H2O are proven to be able to lower the activation energy of the CO2 reduction reaction (CO2RR) and improve the evolution of CO and O2 on the Au surface.In this work, quantum-sized Au NPs with a diameter of ~4 nm, which can be used as efficient photocatalysts for reducing CO4·xH2O with NaBH4 at room temperature. The representative transmission electron microscopy (TEM) image in Fig. 29.The ~4 nm Au NPs were fabricated by reducing HAuClsp-hybridized atomic orbitals of Au  in the 6sp band with a threshold at 2.4 eV , and the other is from the 5d band to unoccupied states in the 6sp band above EF with a tail at 1.8 eV . Considering that minor discrepancies between the theoretical prediction and experimental observation should be acceptable, the intraband and interband absorption regions of Au are marked with different colors in Fig. As revealed by the UV–Vis absorption spectrum in Fig.  Au Fig. . In addiNPs Fig. 26,30. Ac2 reduction with H2O was evaluated in a steel reactor with a quartz window on top, with temperature-controlled by a heating element. To evaluate the contributions of the LSPR and interband transitions to photocatalysis, monochromatic light-emitting diode (LED) light sources at wavelengths in the visible region (from 365 to 620 nm) were used. The intensities of the LED light sources are summarized in Supplementary Table −1 h−1 accompanied by 1.98 mmol g−1 h−1 O2 evolution (based on a 4 h reaction), which are much higher than most of the semiconductor-based photocatalysts, even with the low-intensity LED light used in our study 23, which is beneficial for the transfer of photogenerated charge carriers and thereby leads to improved photocatalytic activity on Au NPs. The TRPL result proves the superiority of the charge carriers induced by the interband transition of Au for photocatalysis.To gain insight into the dynamics of the photogenerated carriers, the time-resolved photoluminescence (TRPL) decay spectrum of Au NPs was obtained  was used in the present study. Under 420 nm LED light irradiation, the temperature of the sample can only increase from ambient temperature (24 °C) to 33 °C. Based on the above considerations, we can speculate that CO2-to-CO conversion on Au NPs should be reduced by the photogenerated electrons.As we know, thermal splitting of CO °C Fig. . Further2RR activity of Au. This phenomenon is possibly induced by the improved populations of the adsorbate in excited vibrational states at higher temperatures17. In the future, the influence of the reaction temperature on photocatalytic CO2 conversion on Au needs to be systematically studied. Moreover, a nearly linear increase in CO production with increasing light intensity is shown in Fig. 17. This result confirms that the CO2RR on Au is in fact a light-driven process. The results in Fig. 25.However, a linear dependence of the photocatalytic CO production rate on the reaction temperature at constant light intensity is shown in Fig. 2O in the photocatalytic CO2 reduction process over Au, desorption of absorbed H2O from the surface of Au was carried out by calcining the sample at 450 °C for 1 h in Ar gas, and the complete desorption of H2O was confirmed by FT-IR spectra  is –3.49 eV when the Au NPs are illuminated by 420 nm LED light, which is more positive than the reduction potential of CO/CO2 (–3.92 eV vs. vacuum level)37. The value of the corresponding hot hole energy  is determined to be –6.44 eV, which is more negative than the oxidation potential of O2/H2O (–5.67 eV vs. vacuum level)33. The details for calculating Ee,hot and Eh,hot are shown in the Method part. These results indicate that the Au NPs exhibit a suitable electronic band structure to achieve CO2 reduction and H2O oxidation simultaneously by interband transitions.To prove that the Au sample has a suitable electronic band structure for the CO2RR with H2O due to the competing H2 evolution reaction (HER). In the present study, no H2 production on Au was detected during photocatalytic CO2 reduction with H2O  was observed, which represents the chemical adsorption state of CO2 on Au40. The interaction between the CO2 and H2O (and/or surface oxygen atom) coadsorbed on the Au surface leads to the monodentate carbonate (m-CO32−) peaks at 1510 cm−1 and 1380 cm−1 and bidentate bicarbonate (b-HCO32−) peak at 1458 cm−141. Moreover, two new peaks at 1545 and 1180 cm−1 that can be ascribed to the *COOH group (the asterisk denotes the surface adsorption site)40 appear with increasing reaction time, demonstrating that *COOH is a key intermediate of the CO2-to-CO conversion process. Moreover, the changes in the peak intensities of the critical intermediates with reaction time are shown in Fig. 2O and CO2 adsorption process and the photocatalytic conversion process of the intermediate species during the reduction of CO2 on Au NPs.To elucidate the catalytic mechanism, in situ Fourier transform infrared (in situ FT-IR) spectroscopy was conducted to detect the surface-adsorbed species on Au during the photocatalytic process. As shown in Fig. 2-to-CO conversion rate is greatly reduced after 3 h of photocatalytic reaction. To study the reasons for this phenomenon, FT-IR spectra over long time ranges were tested  spectra of the Au NPs before and after the photocatalytic reaction were obtained. As shown in Fig. 2O molecules with restricted mobility near the surface region43. The sharp peak at 3.43 ppm can be attributed to the weakly adsorbed H2O on the outer surface of the Au45. Furthermore, surface H2O species on Au can be also confirmed by thermogravimetric analysis (TGA). As shown in Fig. 2O (physisorbed water) from the surface of Au46. The second weight loss of ~1.2% in the temperature range of 258-450 °C can be attributed to the removal of highly immobilized water from the surface of Au47. This is well consistent with the 1H ssNMR results.To study the reaction pathway for H2RR for 3 h, the peak at 6.36 ppm disappeared  formed on the Au surface44. These results are very similar to the observations of the production of SiOH–H2O and isolated SiOH species on mesoporous silica NPs44.When the catalyst was subjected to photocatalytic COred Fig. , suggest1H ssNMR spectra, it can be concluded that molecular H2O was dissociated into *OH species under photocatalytic reaction conditions and that partial *OH species were not bound to H2O. This is consistent with the in situ FT-IR characterizations in Fig. −1) attributed to isolated *OH species on the Au surface was observed48. And a characteristic band at 1260 cm−1 that can be regarded as the vibration of the *OOH group appeared after the CO2RR 50, respectively. Upon irradiation at 200 °C, the peaks corresponding to the oxide state of Au (Au+) emerged at 84.8 eV and 88.4 eV50, which demonstrates that the Au–O species formed by H2O decomposition on Au during the reaction process. As shown in Fig. 2RR on Au.To study the species formed on Au during the photocatalytic process, quasi in situ XPS measurements were carried out. As shown in Fig. 51. In addition, Raman spectroscopy was also used to characterize the Au–O species formed on the Au surface. As shown in Fig. −1 appears during the photocatalytic CO2RR process, which can be attributed to the stretching vibrations of Au–O species52. Both the electrochemistry and Raman characterizations confirm that Au–O species formed on the surface of Au NPs during the photocatalytic CO2RR, which is consistent with the XPS result in Fig. To further confirm the formation of Au oxide species, electrochemical linear scan voltammetry (LSV) was performed on the Au samples before and after the photocatalytic reaction. As shown in Fig. s XPS spectra are conducted to distinguish the oxygen species on Au surface. Supplementary Fig. 2O molecules, respectively53. While the O 1 s spectrum of the Au sample after 3 h reaction shows two additional shoulder peaks at 530.0 eV and 535.0 eV, the former can be attributed to the Au–O species54, which is consistent with the XPS results of Au 4 f. The latter peak at 535.0 eV can be attributed to chemisorbed oxygen species from COOH or/and H2O55. As discussed above through 1H ssNMR, FT-IR, XPS, electrochemistry, and Raman analyses, we can confirm that Au–O species were formed on the Au surface in the photocatalytic CO2RR process by decomposing H2O. Thus, we speculate that the formed Au–O species might play a crucial role in CO2RR on Au. Meanwhile, the above results prove that the *COOH, *OH, and *OOH species are the main intermediates in photocatalytic CO2 reduction with H2O.Furthermore, the high-resolution O 1 2RR mechanisms occurring on the surface of Au. The experimental results above have proven that the photocatalytic CO2 reduction and H2O oxidation processes over Au NPs undergo pathways  calculations based on an electrochemical method to examine the possible COys Eqs. –9) that  that 2RR2O dissociation reaction on Au (111) surface without photoinduced charges. As shown in Fig.+ on the Au surface by direct H2O decomposition without the participation of photoinduced charges. Considering that CO2 reduction via the proton-coupled electron transfer process is favourable21, it was reasonable that the CO2RR on the Au surface was negligible in H2O without the presence of light since no available H+ and e− pairs were generated. However, according to the previous theoretical calculations, the energy barrier for H2O decomposition would be significantly reduced if the crossing of excited states is considered59. Therefore, both the presence of H2O and light illumination are critical for the CO2RR on Au, which is consistent with our experimental observations in Fig. 1H ssNMR, FT-IR, and XPS analyses, light irradiation is helpful for dissociating H2O into *OH and H*, then goes through the *O and *OOH intermediates and eventually forms O2. In this process, O bound to the Au surface inevitably formed under light irradiation. Therefore, the improved CO2RR activity on Au with H2O in Fig. 2RR with H2O.We first simulated the energetic barriers for the H2RR and oxygen evolution reaction (OER) activities, we calculated the free energy diagrams for CO and O2 evolution on Au with (O-preadsorbed Au) and without (clean Au) surface Au–O species. As shown in Fig. 2RR. Furthermore, the required free energy of the rate-determining step (the first protonation process of CO2 to form *COOH) on O-preadsorbed Au (0.59 eV) is lower than that (the last step of *CO desorption) on clean Au (0.68 eV), implying the higher activity of the O-preadsorbed Au surface in the CO2RR. Figure 2O is used for CO2 reduction. This is also helpful for enhancing the CO2-to-CO conversion activity on Au. As shown in Fig. 2O→*OOH + H+ + e−) is greatly reduced on Au in the presence of surface Au–O species (1.59 eV) compared with a clean Au surface (2.64 eV), demonstrating that the preadsorbed O can act as an effective structure to boost the OER activity by weakening the adsorption of the formed intermediates in the H2O oxidation process.To explore the effect of Au–O species on the CO2RR and OER performances, we further examined the electronic structure of the Au centers in the Au–O species using Bader charge analysis. As shown in Fig. d orbital of Au to O. As a result, the d electrons present on the Au atoms involved in adsorbing the intermediates (*COOH/*CO/*OH/O*/*OOH species) of the CO2RR and OER processes are reduced, verifying the superiority of Au–O for the enhancement of CO and O2 production. The relationship of catalytic activity, adsorption energy and d electrons obtained here is consistent with our previous studies60. Overall, based on the DFT calculation results, we can confirm that the generated Au–O species play a critical role in synchronously improving the CO2RR and OER activities on Au, which is consistent with experimental results in Fig. To understand the mechanism by which the Au–O species enhance the above CO2 with H2O is proposed as follows. Under illumination, hot electron-hole pairs are generated on the Au NPs through interband transitions  can possibly participate in photocatalytic CO2 reduction by the proton-coupled electron transfer process. In this process, negatively charged Au–O− species are left on the Au surface61. The formation of Au–O− species was confirmed by zeta potential measurements. Compared with the zeta potential of the fresh Au sample (–2.71 mV), the Au sample after 9 h of photocatalytic reaction possesses a much more negative potential (–14.3 mV), consistent with the formation of Au–O− species on the surface of Au in the photocatalytic process61. Moreover, the adsorbed CO2 on the Au NP surface reacts with photogenerated e− and H+ to yield CO based on the following equations:Unlike the H4  solution (0.1 M) was rapidly added into a 100 mL aqueous solution containing 0.25 mM HAuCl4·xH2O  under vigorous magnetic stirring. In this process, the color of the solution changed from yellow to wine red, which matched the formation of Au NPs. After stirring for 30 min, the obtained solution was aged for 24 h to completely hydrolyze the unreacted NaBH4. Then, the aged solution containing Au NPs was dialyzed in deionized water for a week to remove unnecessary ions, in which the water was exchanged for fresh water each day.Au NPs were routinely prepared by a one-step reduction method. In detail, 3 mL ice-cooled NaBHs peak at 284.6 eV can be regarded as the reference for the calibration binding energy. TEM and HRTEM images were acquired on an FEI Tecnai G2 F20 microscope working at 200 kV. The absorbance spectra of the samples were collected on a Shimadzu UV-2600 UV−Vis spectrophotometer, in which the dialyzed Au colloidal solution was dropped onto a sample plate with BaSO4 as the background, and the diffuse reflectance spectra were measured after drying. The absorption cross-section of 4 nm Au NPs was calculated using the method in a previous report33. Raman microscopy was performed using the emission of a 633 nm laser. The TRPL spectrum was obtained on an Edinburgh FS-5 spectrometer with an excitation wavelength of 340 nm. FT-IR was carried out on a Bruker VERTEX 70 V. TGA was measured on a TGA 5500 instrument with a heating rate of 10 °C min−1 under nitrogen flow. 1H ssNMR spectra were obtained on an Agilent 600 DD2 spectrometer at room temperature. The resonance frequency and the spinning rate used in this test were 600.19 MHz and 15 kHz, respectively. Zeta potential measurements were performed on a NAMO-ZS Zetasizer . Isotopic distributions of the gas products were analysed on a gas chromatograph-mass spectrometer .XPS and valence band XPS characterization was conducted with a ThermoFisher ESCALAB 250Xi instrument equipped with an excitation source of monochromatic aluminium, and the C12SO4 electrolyte at room temperature with an electrochemical workstation  with a standard three-electrode system. The prepared sample, Pt wire, and a saturated Ag/AgCl electrode were used as working, counter, and reference electrodes, respectively. The working electrode was prepared by attaching Au samples onto a 1 Electrochemical measurements were performed in 0.1 M H−1 and an accumulation time of 32 s. A photo of the instrument used for in situ FT-IR measurements is shown in Supplementary Fig. 2 and H2O vapour, as reaction gases, were introduced into the reaction chamber through the gas valve. The temperature of the reaction cell was raised to 200 °C with a heating rate of 5 °C min−1. After that, the background spectrum in the presence of the sample was collected. Next, the FT-IR spectra were recorded as a function of time, in which 420 nm LED light was used to illuminate the sample by a top quartz window.In situ FT-IR measurements were carried out on a Bruker Vertex 70 v spectrometer equipped with a liquid nitrogen-cooled HgCdTe detector. All spectra were obtained with a resolution of 2 cm−10 mbar. The Au catalyst used for the XPS measurement was pressed onto stainless steel mesh. The CO2 photoreduction process was conducted in a load-lock chamber. After loading the catalyst, the reaction chamber was first treated with 5 mbar CO2 and H2O vapour and then heated to 200 °C for the photocatalytic reaction. In this process, 420 nm LED light was used to illuminate the sample. The distance between the light source and the sample was approximately 13.5 cm. After the photocatalytic reactions proceeded for different reaction times , the load-lock chamber was pumped down to high vacuum (<10–9 mbar). Then, the sample was transferred to the analysis chamber through the sample transfer axis for XPS measurements without exposing the sample to air.Quasi in situ XPS spectra were acquired with a customized SPECS UHV system equipped with a SPECS PHOIBOS 150 analyser, a SPECS XR50 X-ray source, and a SPECS 1D DLD detector. The basic pressure of the analysis chamber was less than 3 × 102 conversion with H2O was carried out in a custom-built stainless-steel reaction chamber (450 mL) with a quartz window on top for light irradiation. The temperature of the reaction system was controlled by a heating element. Before the photocatalytic reaction test, 3 mL of the prepared Au colloidal solution (~100 mg L−1) was transferred into a quartz container and then dried in a vacuum freeze dryer overnight. The obtained Au photocatalyst was evenly distributed in the stainless-steel reaction chamber. After that, the gas-tight reactor was purged with Ar (99.999%) gas for 30 min to ensure air removal. Subsequently, 50 mL CO2 (99.999%) and 0.2 mL H2O were injected into the reactor. Monochromatic LED light sources at 365 nm, 420 nm, 450 nm, 520 nm, 590 nm and 620 nm  were used to drive photocatalytic CO2 conversion. The power of the light was measured with a PLS-LED100B photoradiometer . The gas products were analysed by a gas chromatograph (Agilent 7890B) equipped with two thermal conductivity detectors using He and Ar as the carrier gases.Photocatalytic CO2.The AQEs at different wavelengths were calculated by the following equation. The measured intensities of LED light at 365 nm, 420 nm, 450 nm, 520 nm, 590 nm, and 620 nm are shown in Supplementary Table Ee,hot and Eh,hot generated under light excitation by the interband transitions of Au NPs can be calculated from the following two equations62:EF is the Fermi level of Au sample (–5.1 eV vs. vacuum level)33, and hv represents photon energy, which can be given by hv = 1240/λ. Meanwhile, the relationship between the standard hydrogen electrode (ESHE) and the vacuum energy level (Evac) is33:The 63. The projector augmented wave method was adopted to describe the electron-ion interactions64. The exchange-correlation potentials were expressed by the Perdew–Burke–Ernzerhof functional with generalized gradient approximation65. The wave functions at each k-point were expanded with a plane wave basis set and a 400 eV cutoff energy was set. A 1 × 1 × 1 Monkhorst-Pack grid was selected to conduct the integration of the Brillouin zone66. The convergence threshold was set to 1.0 × 10−4 eV for electronic relaxation and 0.05 eV/Å for force. The Van der Waals (vdW) with vdW-DF approximation and spin polarization was involved in our current study67. The transition states were obtained by the climbing image nudged elastic band method68.Theoretical calculations were carried out using DFT with Vienna ab-initio simulation packageThe Au(111) surface was constructed by cutting the bulk Au along (111) direction and 3 × 3 supercell with three layers was selected as the computational model. During the optimization, the atoms in the last layer were fixed to maintain the bulk structure and the other atoms were allowed to fully relax. A vacuum layer of 15 Å was used along the c direction normal to the surface to avoid periodic interactions.2RR and OER were estimated (15)The free energy diagrams of CO69:ΔE is the energy difference of reactants and products obtained by DFT calculations; ΔZPE and ΔS are the energy difference of zero-point energy and entropy, which are evaluated from the vibrational frequencies; T is the temperature, here 298.15 K is considered; The free energy of H2O in bulk water was calculated in the gas phase under a pressure of 0.035 bar (the equilibrium vapor pressure of H2O at 298 K). The free energy of O2 was obtained from the free energy change of the reaction O2 + 2H2→2H2O under the standard condition and the value of −4.92 eV was chosen. The free energy of (H++e−), according to a computational hydrogen electrode model proposed by Nørskov et al, was estimated as the energy of 1/2 H2 under standard conditions. The entropies and vibrational frequencies of the molecules in the gas phase were extracted from the NIST Chemistry WebBook .according to the method presented by Nørskov et al.Supplementary InformationPeer Review File


Aspergillus calidoustus yielded spirocyclic calidoustene, which exhibits a novel skeleton, besides five known sesterterpenes. The complex cyclisation mechanism to all six compounds was investigated by isotopic labelling experiments in combination with DFT calculations. Chemically synthesised 8‐hydroxyfarnesyl diphosphate was converted with isopentenyl diphosphate and AcAS into four oxygenated sesterterpenoids that structurally resemble cytochrome P450 oxidation products of the sesterterpene hydrocarbons. Protein engineering of AcAS broadened the substrate scope and gave significantly improved enzyme yields.The multiproduct chimeric sesterterpene synthase AcAS from  Aspergillus calidoustus was characterised that produces calidoustene, a compound with a new skeleton, besides five known compounds. A deep mechanistic investigation through isotopic labelling experiments, site‐directed mutagenesis and DFT calculations is presented. The substrate analog 8‐hydroxy‐FPP was converted into several oxygenated terpenoids.A sesterterpene synthase from  A genetically clustered cytochrome P450 monooxygenase (CYP450) then converts the four aspergildienes into the alcohol derivatives aspergilols A–D (6–9), following the natural cyclisation‐oxidation sequence of terpene biosynthesis  with a novel skeleton, besides the five known compounds 1–5. Isotopic labelling experiments in conjunction with DFT calculations revealed the cyclisation mechanism for all six compounds showing complex rearrangements for 10 and a mechanism for 5 that is different to a previous proposal.Class I fungal di‐ and sesterterpene synthases are composed of an N‐terminal prenyltransferase (PT) domain for the generation of geranylgeranyl (GGPP) or geranylfarnesyl diphosphate (GFPP) and a C‐terminal terpene cyclase (TC) domain for further conversion into di‐ or sesterterpenes. Their phylogenetic analysis has revealed that these chimeric enzymes can be divided into two clades that reflect their mechanisms of cyclisation.1, 2 IA. calidoustus, which is a homologue of AuAS from A. ustus , was cloned into the fungal expression vector pArgB‐TAAAspergillus oryzae NSAR1 using the protoplast‐polyethylene glycol method,1–5 by a comparison of mass spectra to published data,10  into the expression vector pYE‐Express, expression in Escherichia coli BL21 (DE3), and purification by Ni2+‐NTA affinity chromatography  yielded a protein that efficiently converted GFPP into the same six sesterterpenes [The gene encoding the bifunctional terpene synthase AcAS from  Figure S, was clo Figure S, was close (PaFS)16, 17 Figure S, was clo1 to 5  and allowed to elucidate the structure of 10  that exhibits a new skeleton and for which we suggest the name calidoustene. The enzyme characterised here is designated Aspergillus calidoustusAspergildiene Synthase (AcAS). The absolute configurations of the AcAS products were investigated through stereoselective deuteration (a summary of labelling experiments is given in Table S8). For this purpose, (E)‐ and (Z)‐IPP13C‐labels at the deuterated carbons allow for a highly sensitive analysis through HSQC spectroscopy, showing for each experiment one strongly enhanced and one vanished crosspeak for the diastereotopic hydrogens . Together with the NOESY based assignment of their relative orientation with respect to the naturally present stereogenic centres, these data allowed to conclude on the absolute configurations of all six compounds as shown in Figure R)‐ or (S)‐IPP,R)‐ or (S)‐FPP1 that was previously determined through comparison of experimental and calculated ECD spectra,2–5 and 10 are established here.A large‐scale incubation 800×1 mL) of FPP (400 mg) and IPP (400 mg) with AcAS yielded the mixture of all six compounds that were isolated by preparative HPLC. One‐ and two‐dimensional NMR spectroscopy confirmed the structures of  mL of FPA, followed by a 1,5‐hydride shift to B and 6,10‐cyclisation to C, the precursor of 1 by deprotonation  to achieve the trans substitution at the newly closed 6‐membered ring in F1, while for the ring closure from F2 to J Me25 must point down to install the correct stereochemistry at C3 in J. Related enzyme mechanisms were previously reported for other cyclopentane forming di‐ and sesterterpene synthases and are summarised in a recent review.A mechanistic hypothesis for the conversion of GFPP to the AcAS products starts with a 1,15‐14,18‐cyclisation to 13C)GFPP, enzymatically generated by the PT domain of AcAS from their correspondingly labelled biosynthetic precursors that were synthesised as reported previously,[1–5 and 10 . In particular, these data confirmed the skeletal rearrangements from J to K and from K to L along the pathway to 10.To obtain evidence for this mechanistic proposal, a series of isotopic labelling experiments were carried out (Table S8). Conversion of all 25 isotopomers of  indicate the specific migration of 4HE incorporated from IPP (blue H in Scheme 13C)GPPE)‐ or (Z)‐IPP with AcldOS‐PT and AcAS‐TC revealed its 1,5‐migration from A to B for the compounds 2–5 . For the minor compound 10 very weak signals could be observed in the 13C‐NMR spectra, and no corresponding triplet for 1 was detected, but GC/MS analysis showed migration of the same hydrogen by loss of deuterium for the fragment ion arising by cleavage of the iPr group, if HE is substituted by 2H .The hydride shifts in the biosynthesis of 2–4 and 10 the 1,2‐hydride shift from C to D (dark green H) was evident from an incubation of FPP and DMAPPSerratia plymuthica5 the broad peak for C7 in the 13C‐NMR prevented the observation of a triplet, while for 1 the hydrogen from C6 was lost in the deprotonation to 1 . The same experiment indicated the 1,2‐hydride transfers from F1 to G and from L to M in the biosynthesis of 4 and 10, respectively . Since the experiment with FPP and DMAPP does not rule out another mechanism involving hydride migrations from C2 to C7 and from C6 to C2 or C3 , another labelling experiment with conversion of GGPP and DMAPP by IDI and AcAS was performed. The results confirmed the 1,2‐hydride shifts towards 4 and 10 , while the slightly upfield shifted signals for C3 of 1–3 (between Δδ=−0.03 and −0.10 ppm) indicated a deuterium bound to a neighbouring carbon .For compounds 5 the migration of H2 to C10 in the reaction of H to I was more difficult to follow, because the 13C‐NMR peak for the target carbon was again broad. However, the signal for C11 of 5 is sharp, and therefore the neighbouring effect was used to follow this step. The incubation of (3‐13C)FPP2H)DMAPP5 (Δδ=−0.03 ppm), in agreement with deuterium bound to C10 . This deuterium must originate from C2, as H6 was shown to migrate to C7 . Furthermore, this finding ruled out a mechanistic alternative for 5 that was proposed earlier (dashed box in Scheme E to N), followed by cyclisation to O, 1,3‐hydride migration to P and deprotonation from C2.For compound D to E (pink H) is relevant for 2–5 and 10, and was investigated through conversion of (2‐2H)FPP13C)IPP5 peak broadening was observed, like reported for other terpenes before,[5 this phenomenon prevented the detection of the expected triplet signal. The 1,3‐hydride shift from F1 to H in the biosynthesis of 5 (red H) turned out to be the most intricate problem in the mechanistic investigation of AcAS, because not only the target carbon C3, but also all three neigbouring carbons showed peak broadening. In consequence, neither the strategy through detection of a triplet signal (migration of deuterium from C12 to 13C‐labelled C3) nor through a neighbouring effect  could be applied successfully. However, the incubation of FPP, synthesised from (2H6)acetone , and (5‐13C)IPP5 demonstrated retainment of four deuterium atoms, with loss of one deuterium from Me23 in the final deprotonation step . As C12 of 5 is quarternary, both hydrogens must migrate away from this carbon. In the above‐described labelling experiments, the 12‐pro‐S hydrogen (blue) shifts to C19 , H6 (dark green) moves to C7 , H2 (purple) to C10 , and H10 (pink) to C6 . The stereoselective deuteration experiments revealed that all hydrogens at C1, C4, C5, C8, C9 and C16 stay at their places , and assuming that the carbons C13, C14, C17, C18 and C20–C22 in the left portion of 5 do not participate in hydrogen migrations, the only remaining target position for the 12‐pro‐R hydrogen (red) is C3. Finally, the stereoselective labelling experiments also revealed the specific loss of the 4‐pro‐R hydrogen (orange) in the deprotonation to 2  and of the 1‐pro‐R hydrogen (cyan) in the deprotonations to 4 and 10 . These hydrogens are located in the top hemispheres of F, G and M, similar to the situation for the abstracted H6 in C to 1, which is the side to which also the diphosphate in the initial cyclisation step to A must leave, suggesting that diphosphate may act as the base in these deprotonations.The 1,2‐hydride shift from s before,26, 27A to the central intermediate F1 low transition state (TS) barriers were found (Scheme Neosartorya fischeri Sesterfisherol Synthase (NfSS),A to B could be realised with specific migration of the 12‐pro‐S hydrogen. The transition state barrier to G is with +11.41 kcal mol−1 slightly higher. The reaction from F1 to H proceeds smoothly, while the subsequent 1,4‐hydride shift to I is associated with the highest barrier in the profile , but still achievable at room temperature. The conformational change from F1 to F2 results in the direct barrierless cyclisation to J. Also the subsequent rearrangement to K is directly followed by a barrierless transition to L and a smooth 1,2‐hydride shift to M. Overall, these computational data further support the experimental findings for the AcAS enzyme mechanism.The cyclisation mechanism of AcAS was also investigated through DFT calculations . For all reactions from 11) synthase (PbSS) from Penicillium brasilianum . This enzyme is with ca. 20 mg L−1 under heterologous expression in A. oryzae high yielding. In order to investigate the mechanistic divergence between multiproduct AcAS and the single product PbSS and to increase the activity of AcAS, site‐directed mutagenesis experiments were carried out targeting active site residues of AcAS. A sequence comparison between AcAS and PbSS resulted in the identification of several key residues near the pyrophosphate sensor and the NSE triad . Exchange of the AcAS residues against those found in the analogous positions of PbSS yielded the enzyme variants V197S, G199V, E202R, G232L, G235A, V236A, and A238M. In addition, the Gly residues were exchanged against Ala, giving the variants G199A and G232A. All enzyme variants were expressed, purified and tested for their product profile and relative activity in comparison to the wildtype by incubation with FPP and IPP . Several enzyme variants showed a reduced  or completely lost activity (G199V). Two enzyme variants resulted in an increased production of all six compounds . While the single mutations of E202R and G235A showed increased and moderately reduced activity, respectively, the double exchange E202R‐G235A was inactive. A combination of other successful mutations in the E202R‐V236A and E202R‐A238M variants was also less productive than the single exchanges. Taken together, the mutational studies resulted in the identification of the more productive enzyme variants E202R and A238M, but in no case a new product or simplified product profile was observed, although according to a protein homology model based on the structure of the TC domain of Fusarium graminearum GJ1012 synthase (FgGS)[m) which was most pronounced for the substrate combination of FPP and IPP with the A238M variant .The closest characterised homolog of AcAS and AuAS is the sesterbrasiliatriene (se (FgGS)29, 302–5 are oxidised by a CYP450 that is genetically clustered with AuAS.12–15 (Scheme E)‐ and (Z)‐IPP as well as (R)‐ and (S)‐IPP  revealed the absolute configurations as shown in Scheme 12, 13 and 15 are derived from (S)‐8‐hydroxy‐FPP, while 14 arises from (R)‐8‐hydroxy‐FPP, but the configurations of the stereogenic centres within the carbon skeleton are the same as for the sesterterpene hydrocarbons produced by AcAS. Their formation can be understood by elongation of both enantiomers of 8‐OH‐FPP with IPP to 16‐OH‐GFPP by the AcAS‐PT domain. Subsequent cyclisation of (S)‐16‐OH‐GFPP by the AcAS‐TC domain leads to (16S)‐A* that yields calidoustatriene ether (12) by intramolecular attack of the hydroxy function and 16‐hydroxycalidoustatetraene (15) by deprotonation. Alternatively, (16S)‐A* can react in a 1,5‐hydride shift to (16S)‐B*, and subsequent analogous steps as for 4 in Scheme epi‐aspergilol C (13), the 16‐epimer of natural product 8. Starting from (R)‐16‐OH‐GFPP, analogous reactions lead to (16R)‐A* and (16R)‐B* that can be further converted into 16‐hydroxycalidoustene (14) by similar reactions as for 10 in Scheme 14 represents the putative natural CYP450 oxidation product of 10.Naturally, the sesterterpene hydrocarbons 10) was deeply studied. The absolute configurations of all six sesterterpenes were determined through a stereoselective deuteration approach, and the cyclisation mechanism towards each of products was elucidated by isotopic labelling experiments and computational chemistry in great detail, revealing an interesting sequence of hydride transfers for 5 and a remarkable skeletal rearrangement for 10. Protein engineering through domain swapping yielded an enzyme with broadened substrate scope which was critical for the acceptance of DMAPP and GPP in conjunction with IPP for labelling experiments targeting the first two terpene units, while site‐directed mutagenesis and improvement of incubation conditions by the addition of β‐cyclodextrin resulted in strongly enhanced enzyme activity. Naturally, the sesterterpene hydrocarbons become oxidised at C16 by a tailoring CYP450, but neither the sesterterpene hydrocarbons nor their CYP450 oxidation products could be observed in culture extracts of A. calidoustus, suggesting that the corresponding genes are not expressed under laboratory culture conditions. In a reversal of biosynthetic steps, the oxygen functionality was introduced first through chemical synthesis of 8‐hydroxy‐FPP, followed by late‐stage enzymatic elongation with IPP and cyclisation into four new oxygenated sesterterpenes by AcAS whose absolute configurations were likewise addressed through use of stereoselectively deuterated precursors. This work demonstrated for the first time that chimeric bifunctional terpene synthases with their two domains can efficiently convert non‐natural substrate analogues into functionalised sesterterpenes. This approach is particularly interesting, because cytochromes P450 are difficult to handle in heterologous expressions and especially in in vitro reactions. As a chiral catalyst AcAS provides asymmetric active site pockets in its PT and TC domains, and consequently showed the potential to discriminate between the enantiomers of 8‐OH‐FPP, with more efficient conversion of the S enantiomer.In summary, the bifunctional terpene synthase AcAS producing five known and the unusual spirocyclic compound calidoustene  should be addressed to the authors.Supporting InformationClick here for additional data file.


These, together with the apparent benefit of cyclizing the peptides, improved the detoxification of the two lead peptides by approximately two folds compared to GSH and the benchmark chelating agents against Pb poisoning. Moreover, the two peptides did not show any toxicity and, therefore, were thoroughly investigated to determine their potential as next‐generation remedies for Pb poisoning.A rationally‐designed scaffold of cyclic octapeptides composed of two units of the natural tripeptide glutathione (GSH) was optimized to strongly and selectively capture toxic lead ions (Pb(II)). Using state‐of‐the‐art computational tools, a list of eleven plausible peptides was shortened to five analogs based on their calculated affinity to Pb(II) ions. We then synthesized and investigated them for their abilities to recover Pb‐poisoned human cells. A clear pattern was observed from the  Glutathione was incorporated into rationally‐designed cyclic octapeptides to enhance its binding affinity towards lead (Pb) and detoxify it. We computationally screened different variations of the scaffold and identified promising binders, five of which were subjected to intensive investigation. This included in vitro testing, which revealed their enhanced Pb‐detoxification capability due to high Pb‐affinity and selectivity, together with lack of toxicity. Lead (Pb) is a non‐essential, toxic metal widely distributed in the environment. More than one million deaths are attributed to Pb poisoning annually.Pb(II) ion, which is the most common Pb oxidation state,apo and the holo compounds, low toxicity, ability to penetrate cellular membranes, formation of a non‐toxic complex, and effective clearance of the complex from the body.⋅n{H2O} complex , as reflected in its computed free energy of complexation, ΔGcomp.1 a poor ligand for Pb(II) ions, we repeated the calculation with an octapeptide analog that contains two additional alanine (Ala) residues spacing the two GSH units (peptide 2). Ala was chosen as the simplest yet chiral proteinogenic amino acid. The extension of the macrocycle was proved effective as the ΔGcomp was shifted to a more favorable value, which is comparable to GSH and yet offers more potential for tuning the scaffold.We aimed to investigate whether a head‐to‐tail‐cyclized peptide scaffold containing two units of GSH would increase the low affinity of GSH to Pb(II) ions  inverting cysteine (Cys) stereochemistry; (b) varying the macrocycle size by contracting or expanding the backbone carbon skeleton of γ‐glutamic acid (γGlu); (c) replacing Ala with other amino acids (AAs) to tune properties, such as polarity and coordinating moieties; and (d) modifying functionalization of the scaffold to increase binding sites by coupling additional entities to γGlu free carboxylic acid.We then varied four structural and functional parameters of this basic octameric scaffold to further optimize Pb(II) binding 2⋅(H2O)m]⋅n{H2O} complex , both Cys participate in binding the ion m]⋅n{H2O} complex  upon inversion of one Cys to its D isomer indicates that the preorganization of the Cys residues in the all‐L scaffold is more favorable and was therefore adopted as the default configuration for subsequent structures.As expected, in the most stable isomer of the [Pb‐4), β‐aspartic acid , or δ‐homoglutamic acid , asparagine , and the noncanonical AA (ncAA) cysteic acid , that harbors a sulfonate group on its side‐chain, which aside of its high polarity can serve as an additional binder of Pb(II) ions.Since cyclization of the peptides neutralizes the charges at the N‐ and C‐termini, the aqueous solubility should be optimized, as it is a crucial parameter for achieving therapeutic efficacy.comp for 7 and a substantial stabilization for 8 and 9  ion has an affinity for thiols and imidazole in ligands with mixed donors,10 and 11, respectively. Congruent with the calculations for 4, the enlarged side‐chains resulted in an increase in ΔGcomp, which was about 30 and 40 kcal mol−1 for 10 and 11, respectively. These changes likely result from unfavorable steric interactions between the relatively large opposing chains. Both modifications were therefore excluded for further investigation. We then synthesized peptides 2, 3, 5, 8, and 9 and tested their ability to recover Pb‐poisoned human cells in vitro.The last parameter investigated was the further functionalization of the free carboxylic acid of γGlu. This modification would be synthetically feasible, as it allows for introducing new functional groups into the scaffold by coupling functionalized amines 2, 3, 5, 8, and 9 were synthesized by standard Fmoc‐based solid‐phase peptide synthesis (SPPS) on 2‐chlorotrityl chloride resin, where the synthesis was initiated with Gly at the C‐terminus. The octapeptides were then cleaved off resin while being side‐chain protected,Linear analogs of peptides 2 (lin2) was also synthesized by cleaving it off the resin fully unprotected and purified by HPLC.In addition, the linear analog of 3)2 (200 μM) and 1 h later treated with various concentrations of peptides3)2, respectively. Each dataset was then compared to the negative control to report the peptide recovery abilities , Cu(II), etc.comp values for Zn(II)‐5 and Cu(II)‐5 were calculated (Table S2).5 with Zn(II) and Cu(II) were more favorable  than for Pb(II)‐5, indicating a dramatically higher stability of complexation with physiologically occurring metal ions compared to Pb(II)‐5. The calculations thus corroborated the lack of efficacy identified in vitro.Furthermore, treatment of the poisoned cells with all five cyclic peptides improved viability with increasing concentrations Figures A, S2. No8 and 9, reaching approximately 2.5 times the viability of the negative control , in the presence or absence of Pb(II) ions 2; Figure 2DCF‐DA) as a fluorescent probe for reactive oxygen species (ROS).[We then tested the effect of  Figures C, S3. WeDCF assay34, 358 with or without Pb(II) decreased the oxidative stress compared to untreated cells, corroborating the antioxidative nature of 8.Comparing the fluorescence of the various conditions to untreated cells as a positive control reveals that 1 h after adding peptides under all conditions, ROS production is slightly below the baseline of 100 % Figure D. After 8 and 9, their complexation with Pb(II) ions was experimentally probed. UV titrations of 8 and 9 with Pb(NO3)2 were conducted to investigate the complex formation and determine their stoichiometries 2 (0–400 μM) to peptide 8 (100 μM) gave rise to a shoulder at 260 nm and a peak at λmax=335 nm. The shoulder and the peak were gradually blue‐shifted to 255 and 317 nm,[8 and Pb(NO3)2 was reached .[3)2 indicates a mixture of various Pb(II)‐8 complexes with varied stoichiometries as 1 : 1 and 1 : 2. It also shows the participation of at least one of the thiols in Pb(II) coordination, as concluded in the computed lowest‐energy complex. HR‐ESI‐MS analysis of an equimolar mixture of 8 and Pb(NO3)2 corroborates the concluded stoichiometries by which the most dominant species is a homo‐binuclear complex, and the apo species is peripheral .Titrations indicated the formation of Pb(II)‐peptide complexes at various ratios for both peptides. Adding Pb2 0–00 μM to 9 with Pb(NO3)2 indicated the complexation at the same wavelengths as with 8 but without any maxima shift  ions to 9, the absorption started to decay. This could be due to the formation of Pb(II)‐9 complexes that no longer have a similar print in the UV or due to aggregation.The titration of t Figure B–C. Unli8 and 9 to Pb(II) ions by isothermal titration calorimetry  are 4.4±0.5 and 2.6±0.3 μM for 8 and 9, respectively. With an excellent alignment to the reported ΔGcomp values  and Ca(II) ions as essential metals are frequently reported during chelation treatment for Pb‐poisoning and represent a significant drawback of the current CAs.9, 42 2  or CaCl2 , and the stability was assessed by following the LMCT bands at 317–335 nm. Titrating CaCl2 to Pb(II)‐8 shifted the LMCT band from 317 nm to 330 nm but did not lower its intensity, indicating the stability of the complex in the presence of Ca(II) ions ‐8 complex. Noteworthy, no direct evidence of a Zn(II)‐8 complex was observed, as the absorption spectrum of a mixture containing ZnCl2 and 8 (10 : 1) did not show any additional features compared to the spectrum of apo8 .To assess complex stability, equimolar mixtures of Pb(II) and peptides (100 μM) were back‐titrated with either ZnCl8 with both Pb(NO3)2 and ZnCl2, all with an equimolar ratio of peptide and Pb(II) ions and differed in the concentrations of Zn(II) ions; 1, 3, and 5 equivalents . When adding 1 equivalent of Zn(II) ions, the most dominant species were the homo‐binuclear complex with Pb(II) ions only and the hetero‐binuclear complex, where only minuscule amounts of Zn(II)‐8 were detected . Adding 3 or 5 equivalents of Zn(II) ions increased the ratio between the hetero‐binuclear Pb(II)‐Zn(II)‐8 and the Pb(II)2‐8 complexes, indicating that Zn(II) could displace one of the Pb(II) ions from the binuclear Pb(II)2‐8 species . This hints that the decrease in the LMCT as a response of Zn(II) ions addition is not a result of a complete ion replacement but the formation of a different complex with Pb(II) ion.To further understand these observations, we examined by HR‐ESI‐MS three mixtures of 9, the titration with both CaCl2 and ZnCl2 resulted in a steady decrease of the absorption at 317 nm to 29 % and 48 % of holo9, respectively, at the highest concentrations of the essential metals 2 compared to the peptide to mixtures of peptides and CaCl2 or ZnCl2 in molar ratios of 1 : 100 and 1 : 10, respectively. 8 showed the same behavior when the CaCl2‐containing mixture was titrated as in the titration without Ca(II) ions  addition  ions, Pb(II) binds to both thiols of 8, as detected in the UV spectrum and with an agreement with calculations. Once Zn(II) is added, 8 binds Pb(II) via the two carboxylates, which is the second lowest‐energy complex according to calculations, with ΔGcomp=−5.3 kcal ⋅mol−1, only 0.8 units weaker than the thiol‐based binding mode. With such coordination, 8 could bind Zn(II) ion via the thiolates without losing the toxic ion. Additionally, mixed coordination environments would also be possible. In fact, HR‐ESI‐MS spectra of 8 with Pb(II) and excess of Zn(II)  show that the most dominant species is, indeed, the hetero‐binuclear complex. Such a complex can explain the disappearance of the clear LMCT at 317–335 nm.To learn more about the selectivity of these peptides, we performed reversed back‐titrations Figure F–J, addis Figure F, 5J. Ho9 with CaCl2 indicated a complete complexation with Pb(II) ions, even when the former was in a 100 fold excess and Pb(II) in an only equimolar ratio  ions, but the overall intensity reached only 42 % of the holo absorption.The reversed back titrations of o Figure H, 5J. Wh8 and 9 could form complexes with Pb(II) ions, even in the presence of significant excess of essential metal ions. Peptide 8 formed a complex with Pb(II) that was superior in stability to Pb(II)‐9 in the presence of Ca(II) and Zn(II) ions. We were, therefore, encouraged to continue further with our investigation with 8.The analytical investigations showed that both peptides 8, by which it enables to achieve a high recovery of Pb‐poisoned HT‐29 cells, inductively coupled plasma mass spectrometry (ICP‐MS) was employed to determine the Pb content of the Pb‐poisoned cells and their respective supernatant. First, HT‐29 cells were poisoned with Pb(NO3)2 (0.2 mM) and the Pb content in the cells and in the medium was determined after 1 and 24 h.via an extracellular mechanism such as membrane disruption. Next, we added 8 to the cells before and after poisoning them with Pb(II) ions to assess whether 8 prevents Pb(II) ions from entering the cells, expels them from the cells or transforms Pb(II) into a non‐toxic complex locally. The results clearly show that when 8 is added to cells pre‐ or post‐poisoning, the supernatant‐to‐cells ratio of detected Pb is reduced by approximately half compared to cells that were not treated with the peptide. Since treating Pb‐poisoned cells with 8 enables higher viability, despite the fact that their relative intracellular Pb content is elevated, we hypothesize that Pb crosses the cellular membrane from outside as a membrane permeable, non‐toxic Pb(II)‐8 complex.In order to determine the mode of action of 8 as a next‐generation chelating agent against Pb‐poisoning, we determined the oxidation rate of the peptide at which it forms a disulfide bond. Since all peptides within this scaffold contain two Cys residues that are oriented towards each other, intramolecular oxidation is plausible to occur spontaneously since no significant entropic penalty is apparent.To further examine the potential of 8 was incubated in H2O, and the Ellman's reagent was added at given time points .8 was started to be slightly oxidized within the first 2 h and was steadily forming disulfide bonds, after 24 h, roughly 40 % of the thiols were still reduced, enabling metal coordination within the experimental time frame of our in vitro study. Furthermore, 8 was found to be stable in human blood serum and at 37 °C for at least 48 h , revealing, once again, the advantages of cyclization.To examine this, we used the Ellman's test,in vitro evaluations of five members, the two lead peptides revealed an outstanding capability to recover Pb‐poisoned human cells of up to 2.5 folds compared with untreated cells outcompeting the standards of care as well as GSH. The peptides were also found to be non‐toxic, one of which showed antioxidant activity as it also enhanced cell viability when administered alone. Noteworthy, the utilized computational tools were capable, in general, of predicting complex stability, which is correlated to the bioactivity of the examined peptides. The calculations proved efficient in prioritizing plausible candidates within the scaffold.With the aid of state‐of‐the‐art computational tools, we rationally designed and investigated a peptidic scaffold that contains two units of GSH and a total of eight AAs. Upon sequence optimization based on computational scoring of eleven peptides and in vitro.The lead peptides were found to be highly selective for Pb(II) ions over Ca(II), and even though the presence of Zn(II) ions changed the complexation with Pb(II) of one of the peptides, it still captures the toxic ion. The increase in peptide efficacy compared to GSH is beyond an additive improvement, indicating the contribution of three parameters; cyclization, cavity size, and polarity. All of these enhance metal affinity and selectivity that are then translated into the detoxification of Pb(II) ions The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


In the Abstract and Results, there was a typographical error in the percentage of Hispanic patients in the prepandemic period. It should have read 17.1%. Additionally, the title of panel A in Figure 1 should have read “Fetal death or stillbirth,” and the title of panel B should have read “Maternal death during delivery hospitalization.” This article has been corrected.1In the Original Investigation titled “Comparison of Pregnancy and Birth Outcomes Before vs During the COVID-19 Pandemic,”


Their high Mn‐content allows reducing both costs and the environmental footprint. In this work, Co‐free Li1.2Mn0.6Ni0.2O2 was investigated. A simple water and acid treatment step followed by a thermal treatment was applied to the LRLO to reduce surface impurities and to establish an artificial cathode electrolyte interface. Samples treated at 300 °C show an improved cycling behavior with specific first cycle capacities of up to 272 mAh g−1, whereas powders treated at 900 °C were electrochemically deactivated due to major structural changes of the active compounds. Surface sensitive analytical methods were used to characterize the structural and chemical changes compared to the bulk material. Online DEMS measurements were conducted to get a deeper understanding of the effect of the treatment strategy on O2 and CO2 evolution during electrochemical cycling.Li‐rich layered oxides (LRLO) with specific energies beyond 900 Wh kg Surface treatment: Co‐free Li1.2Mn0.6Ni0.2O2 cathode material was treated with acid or water to modify the surface. A subsequent calcination step at lower temperature provided materials with high capacity (up to 270 mAh g‐1) and cycling stability, whereas high temperatures lead to deactivation. Simultaneously, the amount of critical raw materials like Co or in the future Ni can be reduced to lower both raw material costs and the environmental footprint.Driven by the climate change, the importance of energy storage increased in the last years due to the expansion of renewable energies and the need for balancing their power fluctuations.x Li2MnO3 ⋅ (1−x) LiMny−z1−NiyCozO2 have to be taken into consideration as next generation cathode materials. Their high discharge capacity (over 250 mAh g−1) with an average discharge potential of >3.5 V vs. Li/Li+ is attributed to the combination of cationic and anionic redox reactions.O2 material,2 originates mainly from the active electrode material rather than from the organic carbonate based electrolyte.More detailed information on the potential dependence of the gas evolution is obtained from Figure aterials,68, 712 evolution curves of LRLO, W300 and A300 are predominantly caused by differences in their potential curves and the corresponding potential variation rates. As evident from the potential curves  impurities[18O labeled layered Li1.2Ni0.2Mn0.6O2 material with Li2CO3 impurities, showed that the CO2 evolution observed for this material includes significant amounts of labelled C18O16O. They assumed that this originated from the oxidation of electrolyte with released lattice oxygen species, over the entire range of CO2 evolution, from 4.2 to 4.8 V. However, it should be noted that the 18O exchange treatment used by these authors results in partial 18O exchange both in the oxide and in the Li2CO3 impurity.2CO3, according to Equation Moving on to the COn Figure d, is foun Figure f. A simin Figure e. It canmpurities75, 76The protons are likely formed by oxidation of trace impurities in the electrolyte or of the electrolyte itself.2 peak, which appears at potentials above E≈4.6 V and essentially corresponds to the O2 evolution peak, is attributed to oxidation of the electrolyte by reaction with reactive (singlet) oxygen, which is released from the electrode upon activation of the Li2MnO3 domains.[The high‐potential CO domains.72, 772 evolution rate with a limited quantity of possible reactants (impurities) for the reaction, being responsible for the first peak.2 evolving side reaction is related to the surface potential, the reaction has to reach a chemical equilibrium, which leads to a decrease of the gas evolution during the occurring phase transformation of the material at the plateau.Plotting the MS signal against the potential highlights the strength of this DEMS setup. Here, the decrease of the signal at 4.5 V after the first maximum is clearly visible for W300 and A300, compared to the time dependent plot Figure  and data2 signal in the second cycle can be mainly attributed to different Li species, like Li2O2 or Li2CO3, which can be formed at the end of the discharge and to the reaction of further released oxygen at potentials >4.6 V with the electrolyte.2 signal. Consequently, the higher CO2 signal for A300 compared to W300 supports the observation of a faster capacity decrease of A300 due to a higher oxygen release.The remaining CO2CO3 in the powder. There is the pronounced shoulder at ∼4.6–4.7 V, which is clearly visible in the plots vs. time . A competing electrochemical oxidation of Na2CO3, which cannot be fully excluded from the present data, would not affect the signal shape in Figure 2CO3 cover layer can increase the reaction efficiency between the electrolyte and reactive oxygen species due to the slower off‐transport of the oxygen species and the related reaction products, like protons. This mechanism explains the much smaller O2 peak (first cycle) observed for LRLO compared to the treated W300 and A300 electrodes.For LRLO, the situation differs significantly from the W300 and A300 materials by the high amounts of Nae Figure a, but hae Figure d. This d2 signal of LRLO in the second cycle is lower compared to the first cycle, but still high relatively to the other electrodes. Most likely, the Na2CO3 impurities are not fully removed in the first cycle, further decomposition can occur in the second cycle and presumably also in the following ones. Additionally, it is possible that the amount of reactive oxygen evolved on the LRLO electrode in the second cycle is also significantly higher than on the other samples. This can be understood when considering that part of the surface was not accessible in the first cycle due to the Na2CO3 impurities, resulting in a successive activation of the material, which was already suggested during the electrochemical cycling.The CO2 formation for the treated samples and the opposite trend for the CO2 formation clearly indicate that the Na2CO3 impurities were removed during the treatment and that the chemical activation of the material was successful. Additionally, the high CO2 formation of LRLO supports the assumption that during subsequent cycling the active material becomes more accessible and that the Na2CO3 impurities are not fully decomposed even after the first two cycles. Finally, the high O2 signal in the first and the increased CO2 signal in the second cycle of A300 underlines in combination with the decreasing discharge capacity during electrochemical cycling the hypothesis that the acidic treatment was too aggressive and therefore A300 undergoes a faster surface densification into a structure similar to A900.Summarizing the DEMS results, the more pronounced O1.2Mn0.6Ni0.2O2 was synthesized using a scalable process. Post‐treatment of the as‐synthesized material with water or diluted HNO3, followed by calcination at 300 °C and 900 °C, respectively, had a strong effect on the electrochemical performance. Chemical impurities from the synthesis process were eliminated independently from the applied washing procedure. The effect of the post‐treatment steps could be classified into two groups. While the 300 °C treated samples were electrochemically activated (up to 270 mAh g−1), heat treatment at 900 °C lead to deactivation of the materials. The effect of the post‐treatment increased with higher acidity of the washing media, leaching more metal ions out of the materials, and thus leading to local Mn‐ and Ni‐enrichments. The activation could be explained by the formation of a thin spinel phase on top of the surface of the primary crystallites. This was indicated by the increasing fraction of MnIII ions detected by XPS analysis, and supported by XRD and Raman spectroscopy. DEMS measurements showed an increased O2 release during the first delithiation with a simultaneously reduced CO2 formation, which underlined the complete removal of Na2CO3. It was clearly shown that the deactivation of the materials calcinated at 900 °C was caused by the phase separation of the powders into Mn‐enriched LRLO particles and Ni‐rich rock‐salt structures, such as Ni6MnO8. The latter was located predominantly at the grain boundaries and the surface regions of the crystallites and it was assumed to block Li+ diffusion pathways. Overall, this work showed that simple post‐treatment steps could have significant positive impact on the electrochemical performance of Co‐free LRLO material. Critical parameters, like the pH of the applied solution or the temperature of the subsequent heat treatment, have a strong influence on the electrochemical behavior and need to be adjusted carefully.Spherical, Co‐free Li1.2Mn0.6Ni0.2O2 (LRLO) was synthesized by coprecipitation route. In a continuous stirred tank reactor  a solution of manganese and nickel nitrate (Roth) was mixed under vigorous stirring with a NH4OH (Roth), a Na2CO3 (Roth) and a small amount of NaOH  solution. The excess suspension was filtrated continuously. The resulting spherical precursor material (Mn/Ni=3 : 1) was washed extensively with deionized water, dried and a small amount was mixed with stoichiometric amount of LiOH x H2O . The final calcination step was conducted at 900 °C under air.The pristine powder Li2O (σ <0.55 μS cm−1). For the water treatment, pristine LRLO (5.5 g) was mixed with hot H2O (20 mL), stirred for 20 min and finally the water was decanted. This procedure was repeated three times and after the last residence time the suspension was filtrated and the powder was dried . For acidic treatment HNO3  was used. LRLO (5.5 g) was mixed with H2O , acid was carefully added until the pH of the solution reached a value of pH=7, the suspension was stirred for 20 min and afterwards the solution was decanted, too. In the second and third repetition, only H2O without additional acid was used. As a last step, the mixture was filtrated and the solid residue was dried . Both water and acid treated powders were divided in two equal portions. They were calcinated at 300 °C (W300 and A300) as well as at 900 °C (W900 and A900).The different post treatment steps were done in a similar way, using ultrapure H3 , diluted to a defined volume and analyzed by ICP‐OES (Spectro Arcos SOP). For further understanding of the heat treatment step after washing, the freshly washed powders were dried overnight under vacuum (80 °C) and subsequently TGA‐DSC‐MS measurements (heating rate: 10 K min‐1) were conducted in Al2O3 crucibles under air, using a TGA‐DSC device (Netzsch STA 449 C) coupled with a mass spectrometer (Netzsch QMS 403). DSC measurements were corrected with a blank measurement under the same conditions. The same powders were used without further heat treatment for the quantification of surface carbonates and hydroxides through acid‐base titration measurements. 0.20 g of powder was stirred in degassed ultrapure H2O  under a N2 gas flow to avoid further carbonate contamination. The suspension was filtrated and the filtrate was instantaneously titrated with hydrochloric acid  under N2 gas flow. The equivalence points (EPs) were determined using potentiometric endpoint detection with a pH electrode. The amount of carbonate and hydroxide ions were calculated from the EPs based on the method of Warder.Both types of washing solutions were collected, filtrated, acidified with HNOII solution, buffering the pH with NH4+/NH3 buffer. In a second titration step the powder was digested in an acidic (NH4)2Fe(SO4)2 solution under nitrogen and the remaining FeII was back titrated with CeIV solution. All titration experiments were conducted with an OMNIS titrator (Metrohm). The oxygen stoichiometry was calculated by the combination of the ICP and cerimetry results. XRD measurements were performed using a D8 Advance diffractometer . TOPAS V6 was used for Rietveld refinement of the diffraction data, using a Li1.2Mn0.6Ni0.2O2 structural model of Fell et al.6MnO8 structural model was applied.+ ion beam . The Raman investigations of the powders and the cross section were carried out with an alpha300 R (WITec) confocal Raman microscope over a spectral range between −70 and 1108 rel. cm−1. The system consists of a spectrometer with a grating of 1800 l mm−1 and a CCD‐camera (1600×200 pixels) as well as a Nd:YAG laser with an excitation wavelength of 532 nm. The laser power was adjusted to 1.5 mW after microscope transit. The powders were measured using a 10x objective and for the cross sections a 100x objective was used (both Zeiss).The chemical characterization of the powders was done by ICP‐OES measurements of a diluted aqua regia digestion (Spectro Arcos SOP) and cerimetric redox titration. For the latter, the amount of transition metal in the powders was determined through complexometric back titration of Na‐EDTA with a CuElectron transparent TEM samples were prepared using a focused Ga‐ion beam (FIB) system . The surface of a typical sample particle of about 15 μm in diameter was protected using carbon deposition and applying the in situ gas injection (GIS) system integrated into the FIB. A slab of material of about 10 μm by 4 μm by 10 μm  was etched out of the particle using a 30 kV Ga‐ion beam. This slab was lifted out and welded to a TEM lift out grid (Pelco company) using a piezo micromanipulator. The sample was subsequently thinned down to a final thickness of about 50 to 80 nm. The Ga‐ion beam current was reduced and the voltage was lowered to 5 kV to minimize amorphization damage.The TEM investigations were carried out using a Talos 200X STEM (Thermofisher) operated at 200 kV. The system was equipped with an energy dispersive X‐ray (EDX) detector (type Thermofisher SuperX). TEM images were acquired using a CMOS camera . HAADF images together with EDX mappings were acquired to determine the local elemental composition. HRTEM images and diffraction pattern were used to determine the crystal structure.α X‐ray source (1486.6 eV) and a flood gun to avoid charging of the sample was used. Survey spectra were recorded using a pass energy of 93.9 eV, detail spectra with 29.35 eV. Both angles (angle of photon incidence on the sample and angle of emitted photoelectrons) are 45° with respect to the surface normal . The binding energies (BEs) of all spectra were calibrated with respect to the C 1s peak of ubiquitous carbon, which was fixed at a binding energy (BE) of 284.8 eV. The data were evaluated (deconvolution of spectra) by using the commercial software package CasaXPS . The fitting of the Mn 2p spectra was performed according to data by Biesinger et al.For the XPS measurements, a commercial XPS machine from Physical Electronics (PHI 5800 ESCA) equipped with a hemispherical electron analyzer, a monochromatic Al KN‐methyl‐2‐pyrrolidinone  was coated on an aluminum foil using the doctor blade technique. After drying, disk electrodes with a diameter of 12 mm were punched out, pressed with a hydraulic press (8 t cm‐2 for 60 s), dried overnight under vacuum (120 °C) and transferred directly without contact to air into an argon‐filled glovebox . Subsequently, the CR2032 coin cells (Hohsen) were assembled with one disk electrode, a lithium metal foil , two glass fiber coins  as a separator and electrolyte . The typical active material loading of the electrodes was between 3 and 4 mg. The cells were cycled between 2.5 and 4.8 V using a BaSyTec galvanostat at room temperature. The applied currents are presented in the corresponding figures (1 C=250 mA g−1). The presented capacities of the electrochemical measurements are mean values of at least three cells.A homogeneous slurry of each active material, polyvinylidene difluoride  and conductive carbon Super C45  (dry ratio of 85 : 5 : 10) dispersed in an adequate amount of m LiPF6 in EC/DMC=1 : 1 by wt, Solvionic, purity >99.9 %, water content <20 ppm) was filled into the DEMS cell (0.7 mL). The assembly of the cell and all measurements were performed in an Ar filled glovebox . Prior to the measurements the electrodes were resting in the electrolyte‐filled cell for at least two hours, to achieve stable OCP values  and stable background signals of the selected ion currents. A Princeton Research Instruments (PAR) 263 A potentiostat was used to conduct the galvanostatic experiments with a current of 0.1 mA, which corresponds to the C/10 rate at the applied loading of the active material, at cut‐off potentials 2.5 and 4.8 V (vs. Li+/Li), simultaneously measuring the intensity of selected ion currents.The design of the DEMS cell used was similar to that already reported.The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


Biological α‐keto acids were found to show pronounced nonenzymatic reactivity differences for the formation of amino acids . Accordingly, the flow of ammonia passes through the least reactive α‐keto acid of the Krebs cycle. One possible explanation for this choice is the position of the corresponding amino acid, glutamate, at the top of the thermodynamic landscape for subsequent transamination reactions.Amino acid biosynthesis initiates with the reductive amination of α‐ketoglutarate with ammonia to produce glutamate. However, the other α‐keto acids derived from the glyoxylate and Krebs cycles are converted into amino acids by transamination, rather than by reductive amination. Why is only one amino acid synthesized by reductive amination and not the others? To explore this question, we quantified the inherent reactivities of keto acids in nonenzymatic reduction and reductive amination by using BH Why does biochemical amino acid synthesis proceed the way it does? Kinetic and mechanistic experiments with a model hydride donor were used to determine the intrinsic electrophilic reactivities of keto acids in reduction and reductive amination reactions. Comparing the nonenzymatic reactivity trends with those found in biology provides new insight into the structure of amino acid metabolism. Next, we studied the underlying equilibria of imine and iminium ion formation from keto acids and quantified their reactivity within reductive amination. Lastly, we compared the relative reactivities of all investigated species to test whether fundamental differences in reactivity can explain the biochemical preference for the reductive amination of α‐ketoglutarate.Using BH3CN were studied. NMR spectroscopic analysis of the reaction of pyruvate (K1) with stoichiometric NaBH3CN in aqueous phosphate solution  showed the formation of 71 % lactate, hydrogen gas, boric acid, 25 % of cyanohydrin and 4 % unreacted K1 after a reaction time of 18 h . Table k2,0 for the reaction of BH3CN− with the different keto acids K1–K4 at pH 4. To identify the reasons for the observed reactivity differences of K1–K4 and to verify how they hold under different pH and buffer concentrations, initially the pH‐rate profile for the reaction of K1 with BH3CN− was studied  dominates at lower pH and is characterized by a slope of −1, while a “water‐catalyzed” background reaction (via kw) takes over at neutral pH. Lastly, we correlated the rate constants kHA for catalysis by the protonated component of the buffer with the corresponding pKa values  was found to be only slightly more electrophilic than K1 and the reduction reaction is analogously subject to general catalysis . However, the reactivity trends change drastically with decreasing acidity and at neutral pH the reduction of K2 is two orders of magnitude faster than that of K1  and α‐ketoglutarate (K4) react approximately 85 and 21 times faster than K1  for both competing electrophiles allows to calculate the competition constants κ which corresponds to the ratio of the second order rate constants [Figure K]t was derived from the concentration of the hydroxy acid [H]t  with the rate law for disappearance of K [Eq. (3)] results in Equation (5). On one hand, 3CN− and NH4+ and, accordingly, the concentrations of BH3CN− and NH4+ will not change largely during the reaction. With the definition of the pseudo‐first order rate constants k2ψ [Eq. (6)] and k4ψ [Eq. (7)], Equation (5) thus simplifies to Equation (8).Equation (5) can be simplified under pseudo‐first order conditions when there is a large excess of BHK [Eq. (9)] as well as for the formation of the products H and A.kobs for the disappearance of K1 in Figure k2ψ+k4ψ and the product ratios of H and A can be used to differentiate the terms k2ψ and k4ψ.A rate law like Equation (8) can be solved analytically for the disappearance of 4+ but with variation of the excess of BH3CN−, a correlation of kobs (=k2ψ+k4ψ) vs [BH3CN−] should have a slope of k2+k4KI‐H+[NH4+]. These two terms relate to the sum in Equation (5): The first term describes the contribution of the general acid catalyzed and pH‐dependent keto acid reduction. The second term accounts for the reductive amination, the rate of which should not change largely between pH 4 and 8 due to the constant concentration of the iminium ion  showed the expected linear correlation  into k2 and k4KI‐H+[NH4+]. At pH 7, the product ratio of hydroxy to amino acid was found to be independent of [BH3CN−] in line with the mechanism in Figure 3CN−] was observed and the lactate/alanine ratio at [BH3CN−]=100 mM was 27 % higher than at [BH3CN−]=33 mM  that becomes slower due to the decreased amount of free NH3 present at pH 4. To ascertain our analysis, we extrapolated the product ratio of hydroxy and amino acid at [BH3CN−]=0 from the linear correlation of the product ratios and [BH3CN−] . A more detailed explanation of our approach and a verification with numerical simulations is shown in the Supporting Information on page S48–50.In a series of measurements with different excesses of BHk2 and k4KI‐H+[NH4+] as well as the associated errors determined in this way for the reductive amination of pyruvate (K1). For the other keto acids K2‐K4, the measurements were performed in an analogous way as described for K1, using the same concentration of NH4Cl. However, the reductive aminations of K3 and K4 were studied at pH 5, as, at pH 4, the kinetics were found to result almost exclusively from keto acid reduction. The product k4KI‐H+ allows us to rank the relative reactivities of keto acids K1–4 in the reductive amination under an identical NH4+ excess. The formation of aspartate and alanine is faster by factors of 1.7 and 1.9, respectively, than the formation of glutamate. The rate for reductive amination of K2 is 11 times larger than for K1.Table H and amino acids A under these conditions  and glutamate (A4) while the ratio stays almost constant in the case of glycine (A2). However, even for A1 and A4, the ratio of amino vs. hydroxy acids is surprisingly low despite the large excess of NH4+, even at neutral pH. This contrasts with the high selectivity typically observed in reductive amination reactions with classical organic substrates.A4) compared to alanine (A1) also observed in the direct kinetic experiments (Table K2) compared with other keto acids at neutral pH to yield either glycine (A2) or glycolate (H1) is also found in competition experiments.Figure glycine A. Howeverutamate A while thK1 and K4 . When performing the reaction in 50 mM and 500 mM buffer, the observed changes were rather small and neither the ratio of glutamate (A4) to alanine (A1) nor the rates changed significantly.Lastly, competition experiments were used to study the buffer dependency of the reductive amination reaction in a mixture of + with glutamate or alanine.3CN−: The fastest reductive amination is observed for the most exergonic substrate (glyoxylate/glycine), while the reaction is slowest (and least exergonic) for glutamate synthesis. However, the reductive amination of oxaloacetate proceeds slower (based on k4KI‐H+) than expected from thermochemistry.Experimental thermochemistry for reductive amination has previously been derived from kinetic measurements of the enzyme catalyzed reaction of NAD3CN− to C=O and C=N functionalities of α‐keto acids and related iminium ions were used to investigate the reaction mechanism and to study the relative electrophilic reactivities of these biologically relevant functionalities.Kinetic studies of the hydride transfer of BHK3) and α‐ketoglutarate (K4), while only intermolecular catalysis can enhance the reaction rates of pyruvate (K1) or glyoxylate (K2) reductions.The relative electrophilic reactivities of keto acids in hydride transfer reactions are highly dependent on the pH as well as the presence or absence of buffer species that can act as general acid catalysts Figure  and 5. I3 system relevant for the understanding of the subsequent reduction reactions , synthesizing glutamate by reductive amination renders the subsequent transamination reactions to other α‐keto acids exergonic and might, therefore, establish glutamate as a high‐energy amine donor within amino acid synthesis.Why then did evolution select for amino acid biosynthesis to initiate with the kinetically and thermodynamically least favorable reductive amination? One possible answer lies within the thermodynamics of the subsequent transamination reactions.6, 37 The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.