Metallic molybdenum sulfide catalyses protometabolic carbon dioxide reaction networks under extreme conditions

Domain: chemistry
Published: 2026-02-05
Editorial outcome: major_revision

Abstract
The nonenzymatic synthesis of organics from H2–CO2 redox couple forms the foundational basis for prebiotic carbon metabolism. However, constructing comprehensive carbon cycling reaction networks with multiple interlinked subsystems preceding enzymatic catalysis remains challenging. Here, we demonstrate that metallic molybdenum sulfide mimicking Mo-S-pterin enzyme drives the construction of hydrothermal CO2 reaction networks across five carbon sequestration pathways including acetyl-CoA, reductive tricarboxylic acid, 3-hydroxypropionate-4-hydroxybutyrate, dicarboxylate-4-hydroxybutyrate, and ethylmalonyl-CoA pathways. A total of 32 intermediates and end-products are synthesized from CO2, encompassing five universal metabolic precursors. Spectroscopic and computational studies reveal that molybdenum sulfide containing Mo3+ with vacancy-induced distorted octahedral structure promotes the formation of radicals and enhances their adsorption and coupling in aqueous solutions, leading to CO2 conversion rate of 68.6% and selectivity of up to 70% for C2+ carboxylates. The metal sulfide-catalyzed multiple CO2 reaction networks under extreme conditions could function as a prebiotic precursor to ancient and core metabolic pathways.

Introduction
The origin and evolution of complex interplay within chemical reaction networks are fundamental to the diversity and stability of Earth’s biogeochemical cycles, which sustain life by transforming and circulating essential elements such as carbon, nitrogen, and phosphorus across various environmental and biological systems1–3. These networks underpin energy flow and material exchange within the biosphere, mediating interactions between geological and biological processes. At the earliest stages of life’s emergence, synthetic chemical pathways likely contributed to the construction of self-organized reaction networks from simpler material forms4. Among the biogeochemical cycles, the carbon cycling networks are particularly noteworthy due to their pivotal role in supporting life and influencing the global climate system. Understanding complex carbon cycling reaction networks originating from simple H2–CO2 redox couples is crucial for comprehending how life originated, evolved, and adapted to shifting environmental conditions5–7. Organisms use enzymatic pathways to construct and regulate carbon cycles, yet the origins of these pathways prior to life’s emergence remain elusive, highlighting the need to explore prebiotic chemistry that may have laid the groundwork for life’s carbon metabolism.

The acetyl-CoA pathway, also known as the Wood-Ljungdahl pathway, is distinguished by its ancient origins and serves as a key hub in carbon metabolism7–9, acting as a central intermediate in the reductive tricarboxylic acid (rTCA) cycle10,11, the 3-hydroxypropionate-4-hydroxybutyrate (3HP-4HB) cycle12, and the dicarboxylate-4-hydroxybutyrate (DC-4HB) pathway (Supplementary Fig. 1)13. Additionally, acetate in the acetyl-CoA pathway can be converted into cellular biomass, including C4 and larger carbon skeletons through the ethylmalonyl-CoA pathway (EMCP)14,15, offering an alternative to the glyoxylate cycle and producing intermediates for central metabolic pathways like the TCA cycle (Supplementary Fig. 2). Simulating and constructing these cycles using natural metals, metal oxides, metal sulfides, and other catalysts to produce non-enzymatic analogs is expected to link CO2 fixation chemistry to primitive metabolic pathways6,9,10,16. The prebiotic synthesis from CO2 under neutral or alkaline conditions produced intermediates and end-products of the acetyl-CoA pathway, such as formate, acetate, pyruvate, methanol, and methane on zero-valent transition metals (Fe, Ni, and Co)9,17, ferrous sulfide (FeS)18, greigite (Fe3S4)6, magnetite (Fe3O4)6, and awaruite (Ni3Fe)17,19. Using Zn2+, Cr3+, and zero-valent iron (Fe0) under specific conditions in an acidic aqueous solution promoted more than half of the individual reactions in the rTCA cycle10. Three consecutive steps of the rTCA from oxaloacetate into succinate can be driven by geologically produced H2 using metal (nickel or platinum group metals) or ground meteorite catalysis under mild conditions19. Moreover, the metabolic analogs of rTCA and glyoxylate cycles were constructed using active pyruvate and glyoxylate as a proxy for CO2, linking the components of the acetyl-CoA and rTCA pathway16,20. Despite these advances, constructing a complete and more diverse prebiotic network with multiple interlinked subsystems derived directly from CO2 remains challenging.

We previously demonstrated that the Fischer-Tropsch synthesis mechanism with carbon monoxide intermediate or free radical mechanism was responsible for carbon−carbon (C−C) coupling and carboxylation in simulated natural alkaline hydrothermal environments21,22. By leveraging this geo-inspired approach, here we showed that naturally-occurring 1T′ phase of molybdenum sulfide with a distorted octahedral structure akin to biological enzymes and transformed from 2H phase molybdenum sulfide under geological conditions such as high temperature and pressure or strong stress, can facilitate the construction of entire or partial CO2 reaction networks (CO2 RN) with an time-dependent oscillations, spanning five carbon sequestration pathways, including acetyl-CoA, rTCA, 3HP-4HB, DC-4HB, and EMCP. A total of 32 products identified using isotopic labeling were synthesized from CO2, including five universal metabolic precursors (acetate, pyruvate, oxaloacetate, succinate, and α-ketoglutarate). In situ spectroscopy and computational studies suggest that molybdenum sulfide with vacancy-induced distortions facilitates the formation of radicals and enhances their adsorption and coupling in aqueous solutions, leading to a CO2 conversion rate of 68.6% and a selectivity of 70% for C2+ products.

Results
The hydrothermal conversion of NaHCO3 into organics was conducted in a stainless steel autoclave with an inner volume of 7 mL at 250°C for 12 h, with 1 MPa H2 serving as the reducing agent. The resulting products were identified through GC−MS and NMR analyses following acidification or derivatization (Supplementary Fig. 3−63). Drawing inspiration from the transition metal-sulfur coordination structure of CO2-immobilized enzymes, we screened various sulfide minerals for their potential role in the prebiotic CO2 RN. Among the tested minerals (FeS2, Ni3S2, Cu2S, CuS, CoS, CoS2, MnS, 2H-MoS2, and 1Tʹ-MoS2), the twisted 1Tʹ-MoS2 polymorph, which mimics the distorted geometry of the carbon dioxide Mo-S-pterin enzyme23 and Mo-nitrogenase24 that produce hydrocarbons under certain conditions (Supplementary Fig. 64), enables the hydrothermal conversion of NaHCO3 into formate, acetate, glycolate, oxalate, propionate, lactate, 2-hydroxybutanoate, succinate, and methylsuccinate (Supplementary Figs. 3−61). Given the instability of pyruvate and other reaction products, the reaction temperature was reduced or the reaction time was shortened to identify additional intermediates. When the reaction time was shortened to 3 h, several additional compounds, including α-ketoglutarate, 3-hydroxybutanoate, crotonate, butyrate, fumarate, methylmalonate, 4-hydroxybutyrate, 3-hydroxypropionate, and hydroxyisobutyrate, were detected (Supplementary Fig. 65−96). At lower temperatures of 70°C and 100°C, further products such as pyruvate, acetoacetate, acetaldehyde, glyoxylate, methacrylate, ehtylmalonate, mesaconate, malonate, malate, itaconate and isobutyrate were identified (Supplementary Fig. 97−161).

Using intermediates and end-products of the ancient acetyl-CoA pathway (formate, acetate, and pyruvate) as substrates, all the aforementioned products of hydrothermal NaHCO3 reduction were reproduced, along with additional compounds β-methylmalate and citraconate at 70°C over 12 h during pyruvate conversion (Supplementary Figs. 162−207). This supports the central role of acetyl-CoA in protometabolic carbon fixation. A total of 32 products were synthesized from NaHCO3, encompassing 7 out of 11 products from the rTCA cycle, 9 out of 13 products from the 3HP-4HB pathway, 9 out of 11 products from the DC-4HB pathway, 6 out of the 9 intermediates of the glyoxylate cycle and all intermediates of the EMCP. The isotopic labeling studies using NaH13CO3, combined with GC–MS analysis validated by authentic standards and spiking experiments, confirmed that all intermediates originated from a single carbon source (Supplementary Fig. 208−244). Based on the product analysis, a fundamental chemical autocatalytic network for carbon fixation was constructed (Fig. 1), enabling a one-step conversion from CO2/NaHCO3 to multi-carbon carboxylic acids, specifically all five universal metabolic precursors (Table 1 and Supplementary Table 1). This observation of direct nonenzymatic CO2 conversion surpasses previous attempts to construct rTCA chemical reaction networks, which required both active pyruvate and glyoxylate as substrates16.

The variation of Gibbs free energy (ΔG) of the reactions under saturated vapor pressure at different temperatures was calculated based on the existing database (Supplementary Fig. 245; For detailed datasets, see Supplementary Table 2). Most reactions are thermodynamically favorable (ΔG < 0) under specific temperature conditions. For reactions generating malate, fumarate, succinate, α-ketoglutarate, oxalate, methylmalonate, and hydroxyisobutyrate (ΔG > 0), higher pressure from hydrogen replenishment and the consumption of these organics within reaction networks drive these reactions away from equilibrium.

To gain a deeper understanding of the transforming relationship in the prebiotic CO2 RN, we evaluated the effects of various parameters—including reaction temperature, time, NaHCO3 concentration, hydrogen pressure, and catalyst amount—on the production of formic acid in the acetyl-CoA pathway and five universal intermediates and their derivatives involved in C−C coupling (Fig. 2 and Supplementary Fig. 246). When the temperature is below 200°C, the primary products are C1−C3 compounds such as formate, acetate, propionate, and lactate (Fig. 2a). When the temperature exceeds 200°C, succinate and methylsuccinate begin to appear, indicating that higher temperatures promote the C−C coupling process. Pyruvate and α-ketoglutarate are detected only at lower temperatures, which aligns with existing literature9. Higher temperatures lead to the reduction of pyruvate to lactate, a phenomenon not observed in previous studies of prebiotic CO2 fixation6,9,26.

The acetate, which links acetyl-CoA, rTCA, 3HP-4HB, and DC-4HB, was primarily generated from formate (Fig. 2b, bottom panel), indicating that the acetyl-CoA pathway was the initial reaction to occur, consistent with the autocatalytic network described above. Another universal metabolic precursor, α-ketoglutarate, regenerated when the reaction time was extended to 24 h at 250°C, suggesting the presence of a dynamic reaction network (Supplementary Figs. 247−253). The selectivity of C2+ carboxylic acids in the time-dependent experiment exhibited an oscillatory distribution (Fig. 2b, upper panel), reflecting the characteristics of natural autocatalytic networks involved in carbon cycling27. The observation of oscillatory behavior not only enhances the diversity of reaction pathways but also increases the overall complexity of the reactions. Such properties may have significant implications for carbon cycling during the origin and evolution of early life, as they expand the array of possible chemical interactions and pathways that could have contributed to life’s emergence28.

The excessive use of substrates NaHCO3 (Fig. 2c) or H2 (Fig. 2d) decreased the selectivity for C2+ products, consistent with previous findings that high hydrogen pressure inhibited C−C coupling29, leading to formate as the dominant product. This observation indicated that formate/formyl was not responsible for C−C coupling, which differed from the previous study involving the formyl radical mechanism21. Additionally, optimizing the amount of catalyst (Supplementary Fig. 254) led to a CO2 conversion rate of 68.6% and a selectivity of 74.5% for C2+ products (Fig. 2e), which exceeded the performance of catalysts previously reported for CO2/NaHCO3 conversion (Supplementary Table 3). Notably, the catalyst demonstrated catalytic turnover over 21 consecutive cycles, with a cumulative turnover number (TON) of 1.02. When adjusted for the proportion of active Mo3+ sites (~58%), the effective TON reached 1.76, unequivocally confirming true catalytic behavior (Supplementary Fig. 255).

To elucidate the origin of the outstanding performance of CO2 RN, the atomic and electronic structure of 1Tʹ-MoS2 was investigated. The 1Tʹ-MoS2 was synthesized hydrothermally, as the geological conditions, such as high temperature and pressure or strong stress, can induce the structural transformation from 2H to 1T phase30. The XRD analysis revealed a peak corresponding to the typical 002 plane of the 1T phase, with a d-spacing of 9.4Å (Supplementary Fig. 256, Supplementary Table 4)31. High-resolution transmission electron microscopy (HRTEM) analysis confirmed the interlayer spacing and lattice fringes, showing structures with d-spacings of 0.64 nm (attributed to 2H phase) and 0.94 nm (attributed to 1T phase) in the 1T′-MoS2 sample (Supplementary Fig. 257)32. The enlarged HRTEM image clearly displayed the triangular atomic arrangement of the 1T phase, corroborating the XRD analysis33. The 1T phase in 1Tʹ-MoS2 was also confirmed by photoluminescence (PL), ultraviolet–visible (UV−Vis), and Raman spectroscopy. The PL spectra showed the absence of peaks attributed to interlayer coupling and spin-orbit coupling of the 2H phase in 1T′-MoS2 (Supplementary Fig. 258)34. The UV−vis spectrum of 1T′-MoS2 exhibited a monotonic change without absorption peaks at 635 nm, 458 nm, and 400 nm, which are characteristic of the 2H phase (Supplementary Fig. 259). The Raman peaks at 200 (J1), 225 (J2), 336 (J3), and 355 (J3) cm−1 were consistent with the formation of 1T phase containing Mo3+ (Fig. 3a)35.

The X-ray photoelectron spectroscopy (XPS) analysis showed that the binding energy of the Mo 3d peaks in 1Tʹ-MoS2 was ∼1 eV lower than that in 2H-MoS2, indicating the formation of Mo3+ (Fig. 3b, Supplementary Fig. 260). Further deconvolution of the XPS Mo 3d spectra revealed that the 1T phase content was 58.0% ± 0.5% in 1Tʹ-MoS2, confirming Mo3+ as the predominant species (Supplementary Table 5)35. The formation of Mo3+ was also corroborated by electron paramagnetic resonance (EPR) and X-ray absorption fine structure (XAFS) analysis. The EPR spectra of 1Tʹ-MoS2 showed a signal centered at g=2.014, typical of Mo3+36, distinct from sulfide-related paramagnetic states (g=2.002) of 2H phase (Fig. 3c)37. The Mo K-edge X-ray absorption near edge structure (XANES) spectra showed that the absorption edge energy (defined as the energy where the normalized absorption is 0.5) for the 1Tʹ-MoS2 was lower than that of the 2H phase, supporting the presence of Mo3+ (Fig. 3d). The fitting of X-ray absorption fine structure data revealed the decreased coordination number of Mo−S in 1Tʹ-MoS2 compared to that of 2H-MoS2, suggesting the existence of sulfur vacancy (Supplementary Figs. 261−263, Supplementary Table 6). The presence of the sulfur vacancies in 1Tʹ-MoS2 was further confirmed by aberration-corrected transmission electron microscopy (AC-TEM) (Fig. 3e), where some sulfur atoms octahedrally coordinated to Mo were not observed (light green dashed circle). The electron energy loss spectroscopy (EELS) (Fig. 3f) showed that the electron energy loss of Mo-M5, Mo-M4, Mo-M3, Mo-M2, and S-L2,3 edge in layer 1Tʹ region (Area 2) shown in Fig. 3f, located at 228, 234, 396, 414, and 164 eV, respectively, were lower than those in the 2H region (Area 1), further confirming the formation of Mo3+ in 1Tʹ-MoS238.

Compared to that of 1T’-MoS2, 2H-MoS2 primarily catalyzed NaHCO3 to organics dominated by formate, and the selectivity of C2+ products decreased by 8.1-fold (Fig. 4a). To elucidate the mechanism behind phase-induced CO2 RN, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) was employed to detect key reaction intermediates (Fig. 4b, c and Supplementary Figs. 264−265). The C–H stretching region (3200–2850 cm−1) in FTIR spectra showed distinct characteristics between 1T′- and 2H-MoS2 (Fig. 4b). The 1T phase displayed a methane-derived C–H vibrational mode, νasym(CH3), νsym(CH3), νasym(CH2), and νsym(CH2) stretching modes at 3016, 2958, 2927, and 2862 cm−1, respectively, as a function of reaction temperature and time39,40. These observations suggest the preferential stabilization of CHx intermediates, facilitating the formation of long-chain organics41. In contrast, no peaks corresponding to adsorbed CHx species were detected in 2H-MoS2, which aligns with the low selectivity of C2+ products.

The carbon monoxide (CO), proposed as the precursor of CHx in hydrogenation, was observed in the FTIR spectra of both 1T′- and 2H-MoS2. The peaks located at 2171 and 2115 cm−1 were assigned to CO vibrations42, while the peak at 2188 cm−1 was associated with surface-bound CO coordinated to Mo sites (Fig. 4b)43. Quantitative analysis by deconvolution (Fig. 4c) revealed a higher content of surface-bound CO (69.74%) in the 1T′ phase compared to that of 2H-MoS2 (44.51%). The enhanced CO adsorption on 1T′-MoS2 can be attributed to the dynamic involvement of hydroxyl groups, as demonstrated by the time-dependent modulation of the terminal Mo–OH vibration at 3730 cm−1 (octahedral coordination) and the Mo–OH bending mode at 900 cm−144,45. Surface hydroxyl groups are believed to interact with carbonaceous species to enhance their adsorption affinity46. The CO2 and CO temperature-programmed desorption measurements confirmed that the 1T′-MoS2 facilitates the adsorption of carbonaceous species (Supplementary Figs. 266–267). Operando spectral analysis also revealed the simultaneous consumption and regeneration of bicarbonate-associated hydroxyl groups (3703−3600 cm−1) and the catalytically active Mo–OH species43, thereby supporting a self-sustaining catalytic cycle47. The increased concentration of surface-bound CO promotes the formation of CHx intermediates and subsequent C–C coupling reactions. In contrast, the absence of hydroxyl groups in 2H-MoS2 reduces the adsorption strength of carbonaceous species, resulting in the formation of formate via the reaction of free CO with hydroxide ions (CO + OH− → HCOO−). These findings are consistent with the phase-dependent performance illustrated in Fig. 4a.

EPR spectroscopy was performed to identify the forms of species observed in FTIR. Upon heating the solution, the formation of formyl radical (CH•), acetyl radical (CH3CO•), propionyl radical (CH3CH2CO•), and hydroxyl radical (OH•) was first observed at 150°C, and further increased as the reaction temperature was raised to 250°C in 1T′-MoS2-catalyzed CO2 RN (Fig. 4d and Supplementary Figs. 268−269). Without adding the NaHCO3 substrate, no EPR signals were detected, confirming that the radicals originated from NaHCO3 reduction (Supplementary Fig. 270). When the phenyl-tert-butylnitrone (PBN) radical scavenger (1 time the amount of NaHCO3) was added into the 1T′ MoS2-catalyzed CO2 RN system, the yield of organics decreased by 3.7-fold, and further increasing the concentration of PBN (4 times the amount of NaHCO3) led to complete suppressing of C–C coupling (Fig. 4e). Additionally, the reduced activity of radical formation (Fig. 4d) catalyzed by 2H-MoS2 correlated with the poor performance of CO2 RN. These observations suggested that the radicals served as active intermediates and radical-mediated C–C coupling mechanisms were involved in 1T′-MoS2-catalyzed CO2 RN system. Collectively, the 1T′-MoS2 with vacancy-induced distorted octahedral structure promotes the formation of radicals from adsorbed CO reduction and enhances their adsorption and coupling in aqueous solutions.

The first-principles density functional theory (DFT) calculations were performed to further elucidate the underlying mechanism difference of phase-dependent CO2 RN. CH3 and CO are commonly chosen as monomer species in C–C coupling reactions, and both were detected in our experimental studies. We first examined the adsorption energies of CH3 and CO on sulfur-deficient 1T′-MoS2 and pristine 2H-MoS2 (Fig. 5a and Supplementary Fig. 271). The results show that CH3 has a lower adsorption energy than CO on all MoS2 structures, suggesting a stronger affinity for CH3 to interact with MoS2. The introduction of sulfur defects decreases the adsorption energies of both CH3 and CO on MoS2, with the adsorption energy of CH3 reaching −4.44 eV on 1T′-MoS2 and −2.63 eV on 2H-MoS2, respectively. Experimental observations also demonstrated that increasing the sulfur vacancy concentration in 1T′-MoS2 enhances the selectivity for C2+ products (Supplementary Figs. 272−273). Specifically, as the S:Mo ratio decreases from 1.85 to 1.79, the selectivity for C2+ products increases from 67.59% to 74.77% (Supplementary Table 5). These findings suggest that the Mo sites in MoS2 serve as the primary active sites for the activation of carbon-containing species.

To investigate the impact of sulfur vacancies on product selectivity, we calculated the activation energy for CH3−CO coupling on both pristine MoS2 and 1T′-MoS2 with sulfur vacancies. Specifically, we examined the influence of different catalytic reaction mechanisms, the Langmuir–Hinshelwood (L−H) and Eley–Rideal (E−R) mechanisms, where the L−H mechanism represents high sulfur vacancy density, while the E−R mechanism represents low sulfur vacancy density in MoS2 (Fig. 5b and Supplementary Fig. 274). Fig. 5b shows that the presence of sulfur vacancies significantly enhances the activity for C−C coupling, both thermodynamically and kinetically, as evidenced by the reduction in both adsorption energy and activation energy. Compared to 2H-MoS2, we found that 1T′-MoS2 exhibits superior reactivity, with the lowest activation energy of 1.18 eV during C−C coupling via the L−H mechanism. Regarding the adsorption configuration, the C−C distance between the CH3 and CO monomers is 2.56 Å on 1T′-MoS2 and 3.12 Å on 2H-MoS2, which aligns with previous theoretical studies that a shorter C−C distance is expected to lower the activation energy for C−C coupling48.

We next analyzed the electronic structures of both sulfur-deficient 1T′-MoS2 and 2H-MoS2 to elucidate the origins of their catalytic activity for C−C coupling reactions. Fig. 5c presents a comparison of the partial density of states (PDOS) between 1T′- and 2H-MoS2. The Mo d-orbitals in 1T′-MoS2 display a more discrete structure than that in 2H-MoS2, with the d-band center shifting from −0.88 eV in 2H-MoS2 to −0.69 eV in 1T′-MoS2. The shift with the d-band center approaching the Fermi level in 1T′-MoS2 suggests an enhancement in catalytic activity. Moreover, the work function of 1T′-MoS2 is lower (4.65 eV) compared to that of 2H-MoS2 (5.41 eV), indicating that electrons at the Mo sites in 1T′-MoS2 are more readily transferred to carbon-based species, thereby facilitating their activation (Supplementary Fig. 275). Additionally, the orbital interactions between the CH3 group and the Mo site were examined, as the Mo site exhibited the lowest adsorption energy for CH3 (Fig. 5a). In 1T′-MoS2, there exists a greater overlap between the Mo d-orbitals and the CH3 p-orbitals below the Fermi level compared to that of 2H-MoS2, suggesting stronger d−p hybridization (Fig. 5d). This phenomenon is further corroborated by crystal orbital Hamiltonian populations (COHP) analysis (Fig. 5e), which reveals a more negative integrated COHP value for the Mo−C bond in 1T′-MoS2 (−2.06 eV) relative to 2H-MoS2 (−0.66 eV), implying stronger Mo−CH3 interactions in 1T′-MoS2. Charge density and electron localization function (ELF) (Fig. 5f−g) analyses also show significant differences between 1T′ and 2H-MoS2. Notably, the regions near the Mo sites in 1T′-MoS2 exhibit a lower charge density and smaller ELF values, indicating more delocalized electron distributions, which may facilitate charge carrier transfer. Moreover, Bader charge analysis (Fig. 5h) supports these findings, showing that CH3 accepts 0.43 |e| from 1T′-MoS2, whereas only 0.21 |e| is transferred from 2H-MoS2. In addition, CO accepts 0.32 |e| from 1T′-MoS2, in contrast to only 0.15 |e| transferred from 2H-MoS2. Based on integrated experimental and spectroscopic analyses of catalytically active intermediates, we propose a visual summary (Fig. 5i) illustrating the key steps in the C−C bond formation between CH3 and CO on the 1T-MoS2 catalyst surface.

Methods
Chemicals
Sodium molybdate (Na2MoO4, ≥99.0%), sodium pyruvate (CH3COCOONa, ≥99.5%), nickel sulfide (Ni3S2, ≥99.0%), ferrous disulfide (FeS2, ≥99.99%), copper(I) sulfide (Cu2S, ≥99.0%), copper(II) sulfide (CuS, ≥99.0%), molybdenum(IV) disulfide (2H-MoS2, ≥99.0%), tetrahydrofuran (C4H8O, ≥99.5%), 3-hydroxypropionate (C3H6O3, 28%–32% in water), mesaconate (C5H6O4, ≥98.0%), 2-hydroxybutanoate (C4H8O3, ≥98.0%), 3-hydroxybutanoate (C4H8O3, ≥95.0%), methacrylate (C4H6O2, ≥99.0%), itaconate (C5H6O4, ≥99.0%), malonate (C3H4O4, ≥99.0%), methylmalonate (C4H6O4, ≥97.0%), malate (C4H6O5, ≥98.0%), citraconate (C5H6O4, ≥98.0%) were purchased from Shanghai Titan Technology Co., LTD. L-cysteine (C3H7NO2S, ≥99.0%), nickel disulfide (NiS2, ≥99.0%), cobalt disulfide (CoS2, ≥99.5%), cobalt sulfide (CoS, ≥99.5%), 1,1,1,3,3,3-hexamethyldisilazane (C6H19NSi2, ≥99.0%), manganese sulfide (MnS, ≥99.5%), chlorotrimethylsilane (C3H9ClSi, ≥99.0%), crotonate (C4H6O2, ≥99.0%), hydroxyisobutyrate (C4H8O3, ≥98.0%), ethylmalonate (C5H8O4, ≥98.0%), and pyridine (C5H5N, ≥99.5%) were purchased from Beijing Enocai Technology Co., LTD. Ethanol (C2H5OH, ≥99.8%), methanol (CH3OH, ≥99.5%), propionate (C3H6O2, ≥99.5%), oxalate (C2H2O4, 1 mol/L), lactate (C3H6O3, 1 mol/L), succinate (C4H6O4, ≥99.5%), sodium bicarbonate (NaHCO3, ≥99%), sodium bicarbonate-13C (NaH13CO3, ≥99.0%), and sulfuric acid (H2SO4, 95.0–98.0%) were purchased from Sinopharm Chemical Reagent Co., Ltd. Sodium pyruvate-13C (13C3H3O3Na, ≥99.0%) were purchased from J&K scientific Technology Co., LTD. Formate (CH2O2, ≥98.0%), acetate (C2H4O2, ≥99.5%), glycolate (C2H4O3, ≥98.0%), fumarate (C4H4O4, ≥99.0%), glyoxylate (C2H2O3, 50% in water) were purchased Shanghai Macklin Biochemical Co., Ltd. Methylsuccinate (C5H8O4, ≥99.5%), α-Ketoglutarate (C5H5NaO5, ≥99.5%), butyrate (C4H8O2, ≥99.0%), isobutyrate (C4H8O2, ≥99.0%), acetaldehyde (C2H4O, 40% in water) were purchased Shanghai Aladdin Biochemical Technology Co., Ltd. Hydrogen (H2, 99.999%), nitrogen (N2, 99.999%), and helium (He, 99.999%) were purchased from Shanghai Weichuang Standard Gas Analysis Technology Co., LTD.

Synthesis of metallic 1T′-MoS2
1T′-MoS2 in this work was synthesized using a hydrothermal method similar to previously described procedures37,49. In brief, 6 mmol of sodium molybdate and 6 mmol of L-cysteine were dissolved in 60 mL of ultrapure water (18.25 MΩ, Sichuan UPU Ultrapure Technology Co. Ltd). The mixture was stirred magnetically for 10 min and then transferred to a Teflon-lined 100 mL stainless steel autoclave reactor (Anhui Kemi Instrument Co., Ltd). The hydrothermal treatment was conducted at 200°C for 20 h, after which the reactor was allowed to cool naturally to room temperature. The resulting black precipitates were collected by filtration, thoroughly washed three times with ultrapure water and ethanol, and then lyophilized. Finally, the black solid was freeze-dried for 12 h.

NaHCO3 fixation catalyzed by 1T′-MoS2
The hydrothermal conversion of NaHCO3 to carboxylic acids was carried out in a stainless steel (SUS-316) autoclave with a 7 mL inner volume. The parameters such as temperature, reaction time, substrate concentration, water volume, and hydrogen pressure were varied according to the experimental design. The reactor was magnetically stirred at a speed of 800 rpm. After the reaction was completed, the gas, liquid, and solid products were cooled to room temperature by air cooling and then collected from the reactor. To verify the reaction pathways, NaH13CO3 and 13C3H3O3Na were also applied to the secondary experiments following the above steps. For comparison, 2H-MoS2 was used for the experiments following the above steps.

High-performance liquid chromatography (HPLC) analysis
The liquid products were obtained by filtering through a 0.22-μm membrane filter and then were quantified on an Agilent 1200 system high−performance liquid chromatography (HPLC) equipped with two KC-811 columns (SHODEX) and a tunable UV−vis absorbance detector, which was set at 210 nm. A 2 mmol/L HClO4 solution was applied as the mobile phase at a flow rate of 1.0 mL/min at 75°C. The HPLC external standard method was employed for quantitative analysis, which involved the preparation of a series of standard solutions with known concentrations of the analyte, followed by HPLC analysis to establish a calibration curve relating peak responses to concentrations. Subsequently, the same chromatographic conditions were applied to analyze the samples, and the concentration of the target analyte in the samples was determined by referencing the calibration curve.

Gas chromatography−mass spectrometry (GC−MS) analysis
After the reaction, the resulting liquid was adjusted to an acidic state (pH ~3) using H2SO4 and then esterified with either ethanol or methanol. The prepared sample was analyzed using an Agilent 7890A GC equipped with an Agilent 19091N-233 HP-Innowax Polyethylene Glycol column (30 m × 250 μm × 0.5 μm) and a model 5975C mass selective detector. The analysis was carried out in a splitless mode with a 1 μL injection volume, at an injection port temperature of 250°C. The column was maintained at 60°C for 1 min, then ramped up to 230°C at a rate of 7°C per minute, and was held at this final temperature for 40 min. Helium was used as the carrier gas. Formate, acetate, lactate, propionate, succinate, methyl succinate, glycolate, and oxalate were detected. To identify more compounds, some of the liquid products were acidified with HCl (pH ~3), frozen using liquid nitrogen, and further freeze-dried and concentrated. The esterified samples were then subjected to GC−MS analysis using the method described above. Lactate, pyruvate, butyrate, isobutyrate, fumarate, methylmalonate, crotonate, glyoxylate, acetaldehyde, malonate, methacrylate, 3-hydroxybutyrate, α-ketoglutarate, hydroxyisobutyrate, itaconate, methylmalate, and malate were detected. Additionally, some liquid products were silanized according to methods described in the literature and analyzed using GC−MS equipped with an Agilent 19091S-433 HP-5MSI Polyethylene Glycol column (30 m × 250 μm × 0.25 μm)50. The GC temperature program started at 35°C for 5 min, ramped to 320°C at 5°C min−1, and was held at this temperature for 10 min. Oxalate, malonate, itaconate, hydroxyisobutyrate, citraconate, lactate, glycolate, succinate, methyl succinate, ethylmalonate, mesaconate, α-ketoglutarate, 3-hydroxypropionate, and 4-hydroxybutyrate were detected. The corresponding isotope-labeled products were also qualitatively analyzed by GC−MS. The obtained mass spectra of the liquid products were analyzed using GC–MS post-run analysis software with commercial MS libraries. To reach the detection limit of GC−MS, we conducted sample freeze-drying and concentration, followed by derivatization for detection (a physical volume concentration factor of 3). These samples include: fumarate, methylmalonate, crotonate, glyoxylate, acetaldehyde, malonate, methacrylate, 3-hydroxybutyrate, hydroxyisobutyrate, itaconate, methylmalate, malate, itaconate, hydroxyisobutyrate, citraconate, ethylmalonate, mesaconate, α-ketoglutarate, 3-hydroxypropionate, and 4-hydroxybutyrate.

NMR analysis
1H NMR spectra were recorded at ambient temperature using a Bruker AVANCE III (600 MHz) spectrometer, employing a mixture (H2O/D2O: 6/1) as the solvent. The proton resonance frequency was 600.13 MHz. The main magnetic field was 14.09 Tesla (superconducting). The swept field range ranged from −1 to 13 ppm for 1H. Data were basically acquired 32 times with a recycle time of 7.6 s for a particle sample. A Diff50 RF double resonance diffusion probe was used with a gradient field strength of 28 T/m. Propionate and lactate were identified by adding an internal standard. Formate, acetate, succinate, methyl succinate, and ethanol were determined according to the literature51,52. Additionally, 13C-NMR data were obtained to determine the source of carbon according to the literatur53. The swept field range ranged from −12 to 220 ppm for 13C. Data were basically acquired 16 times. The rest of the conditions are the same as above.

Calculation of the product yield and selectivity
Product conversion was defined on the carbon-atom basis of NaHCO3:
Product yield = (nC in COOH / nNaHCO3) × 100% (1)
C2+ products selectivity was calculated based on the proportion of C2+ products among C2+ carboxylates:
C2+ selectivity = (Σ nC in COOH / Σ nC in COOH) × 100% (2)

X-ray diffraction analysis
X-ray diffraction (XRD) patterns were recorded using a Shimadzu XRD-6100PC, equipped with Cu Kα radiation (λ=1.54184 Å) operating at 40 kV and 40 mA. The scanning angle (2θ) was set from 10° to 80°, with a scan rate of 2° per minute and step intervals of 0.01°.

X-ray photoelectron spectroscopy
XPS spectra were obtained on an ESCALAB 250xi spectrometer equipped with a non-monochromatic Al Kα (photon energy 1486.6 eV) anode target. Conditions: vacuum, 1×10−8 bar; scanning range of binding energy, 0−1000 eV; pass energy, 40 eV; scanning step size, 0.1 eV/step.

Electron microscope measurements
HRTEM images were obtained using a JEOL JEM-2100F electron microscope at an accelerating voltage of 200 kV. The TEM samples were prepared by dropping the sample suspension onto a lacey carbon TEM grid, followed by air drying. HAADF-STEM (High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy) and EDS elemental mapping were conducted using an X-max Oxford electron microscope at an accelerating voltage of 200 kV.

Aberration-corrected transmission electron microscope (AC-TEM)
The structural and chemical characterization was performed using a spherical aberration-corrected scanning transmission electron microscope (STEM) operated at 200 kV. The microscope is equipped with an integrated differential phase contrast (iDPC-STEM) imaging system, which employs a four-segment detector to record electron beam deflection signals generated by the sample’s electrostatic potential gradients. The iDPC-STEM configuration enables atomic-resolution imaging with low electron dose (≤10 e/Å2), critical for minimizing beam-sensitive damage in materials such as MOFs and 2D layered compounds. For elemental and chemical state analysis, electron energy-loss spectroscopy (EELS) was conducted in STEM mode using a dual-EELS acquisition system with an energy resolution of 0.7 eV. The EELS spectra were collected over an energy range of 0−2000 eV, covering core-loss edges for light (e.g., C-K at 284 eV, O-K at 532 eV) and heavy elements. To enhance signal-to-noise ratio, multiple spectra (≥50 frames) were acquired with a dwell time of 0.1−0.5 ms/pixel and processed via background subtraction using power-law fitting. The EELS spatial mapping was synchronized with high-angle annular dark-field imaging to correlate atomic-scale structure with localized chemical information.

XAFS tests
XAFS data for the sample, as well as the metal and oxide references, were collected in raw transmission mode at Super Photon Ring-8 (SPring-8) in Japan. Pellet samples for the XAFS measurements were prepared by mixing granular samples with boron nitride (BN) from Kojundo Chemical Lab, Japan. The XANES spectra were derived by subtracting the post-edge background from the overall absorption signal and then normalizing the results with respect to the edge-jump step. The χ(k) data were subsequently Fourier transformed to real (R) space using Hanning windows (dk=1.0 Å−1) to isolate the EXAFS contributions from different coordination shells. To obtain quantitative structural parameters around the central atoms, least-squares curve fitting was performed using the ARTEMIS module of the IFEFFIT software package54,55.

Raman measurements
Raman spectra of MoS2 were obtained using a confocal laser micro-Raman spectrometer (Renishaw inVia Qontor) equipped with a 532 nm laser. The light source was a 1800 grooves per mm grating, and the objective was 50×. The laser intensity was adjusted to 2.5 mW. The co-additions of 10 spectra were recorded with an integration time of 5 s.

Electron paramagnetic resonance spectra
EPR characterization of molybdenum sulfide samples was performed on a Bruker A300 spectrometer equipped with an Oxford Instruments liquid helium cryostat. Spectra were acquired under the following conditions: microwave power 1−20 mW (optimized through power saturation studies), 100 kHz field modulation with 1 G amplitude, and constant temperature control at 40 K via a liquid helium-cooled quartz cryostat with integrated temperature and gas flow control.

Operando EPR analysis employed identical instrumentation with modified parameters: 9.85 GHz microwave frequency, 20 mW power, 2.0 G modulation amplitude, and 100 G sweep width centered at 3452 G. The detection protocol involved in situ thermal activation of 5 mL 0.1 M NaHCO3 solution (with/without 20 mg MoS2 catalyst) in a sealed Teflon-lined autoclave under 10°C/min heating ramp. At 20°C intervals during the temperature program, 200 μL aliquots were immediately mixed with 100 μL DMPO solution (100 mM) as a spin-trapping agent and transferred to quartz capillaries for analysis. Radical identification was achieved through comparative spectral analysis against established references for hydroxyl, formyl, acetyl, and propionyl.

Ultraviolet–visible spectra
A spectrometer (UV-3600, Shimadzu) equipped with a multifunctional large sample chamber (150 mm (length) × 260 mm (width) × 140 mm (height)) and a built-in integrating sphere (MPC-2200, Shimadzu) was used to collect the UV−Vis spectra of the powder samples in the diffuse mode. It is equipped with a three-detector system (PMT detector, InGaAs detector, and PbS detector), which can test wavelengths from 185 to 3300 nm.

Photoluminescence spectra
Photoluminescence (PL) spectra were obtained using a high-end steady-state/transient fluorescence spectrometer (FLS980, Edinburgh Instruments) equipped with a large sample chamber and an integrating sphere. A minority layer of MoS2 is excited at 532 nm on a Si/SiO2 wafer.

Temperature-programmed desorption (TPD) measurement
The CO/CO2 adsorption sites on the catalyst surface were characterized by using a high-precision programmed temperature desorption system (equipped with a mass flow controller, with a thermal drift of ≤ 0.08%). The experimental procedure is as follows: Firstly, the sample is pretreated at a temperature of 10°C/min to 400°C in a He atmosphere for 2 h to remove surface impurities; Subsequently, the temperature was reduced to 50°C, CO/CO2 gas was introduced for saturated adsorption, and the physical adsorbed species were removed through 30 min of Helium purging (at a flow rate of 40 cm3/min). During the desorption stage, the temperature rises at a rate of 10°C/min to 400°C, and the desorption signal is monitored in real time through a thermal conductivity detector (TCD). After baseline correction and Gaussian peak fitting of the experimental data, a CO/CO2 desorption peak was observed.

Operando DRIFTS spectra
Operando DRIFTS was utilized to detect the intermediates generated during the NaHCO3 conversion. Spectra were recorded with a Thermo Fisher iS10 Fourier transform infrared spectroscopy (FTIR) spectrometer equipped with a narrow-band mercury-cadmium-telluride detector. The catalyst, NaHCO3, H2, and water were sealed in a PC-50-300-DFIR high-temperature and high-pressure micro-cell equipped with a heating device (15°C/min) and an S5 liquid ethanol-cooling system. The reactor was integrated with the FTIR spectrometer for capturing operando signals.

Computational details
Spin-polarized electronic structure calculations were performed using the plane-wave basis set approach as implemented in the Vienna ab initio simulation package (VASP 5.4.4)56. The projector augmented wave (PAW) method was used to represent the ion−core electron interactions57. The valence electrons were represented with a plane wave basis set with an energy cutoff of 450 eV. Electronic exchange and correlation were described with the Perdew−Burke−Ernzerhof (PBE) functional58. The DFT-D3 method was used to treat the van der Waals interaction59. 15 Å vacuum space was included to avoid interactions between surface slabs. A 3×3×1 Monkhorst−Pack scheme was used to generate the k-point grid for the modeled surfaces60. The convergence criteria for the self-consistent electronic structure and geometry were set to 10−5 eV and 0.05 eV/Å, respectively. Zero-point vibrational energy (ZPVE) corrections are calculated by assuming a quantum harmonic oscillator possessing the calculated vibrational frequency. Transition state calculations were performed using the climbing image nudged elastic band (CI-NEB) method, with the transition state identified to have an absolute tangent force below 0.05 eV/Å61. The adsorption energy ΔEads was defined as:
ΔEads = Etot − Esur − Emol (3)
where Etot, Esur, and Emol denote the total energy of the molecule on MoS2 surfaces, energy of bare MoS2 surfaces, and energy of the molecule in vacuum, respectively.
Activation barriers were defined as follows:
ΔEa = ETS − Eini (4)
where ΔEa is the activation energy barrier, Eini is the initial state energy, and ETS is the energy of the transition state.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This exciting paper presents the most complete set of biologically relevant carbon oxides synthesized from H2 and CO2 reported to date. It is almost too good to be true from some standpoints, because it provides perhaps the most comprehensive set of observations indicating that the core of central carbon metabolism unfolds spontaneously from H2 and CO2, given suitable conditions and catalysts. What they show is that MoS2 converts H2 and CO2 into 32 (!) biologically relevant compounds, mostly organic acids of CO2 fixation pathways, many of which have been reported from previous, well-cited work of others, but many of which are new. Two compounds, acetaldehyde and glyoxylate, caught my attention because aldehydes are reactive and might be short lived under these conditions. This is not to say that metabolism and life arose directly from MoS2 catalysts, but it does say that MoS2 under hydrothermal conditions certainly could have helped, in a surprisingly big way, which is welcome news.\n\nI am sure it is in the supplemental, but many readers will not find the energy to look through Supplemental figures 83–147, so it would really be helpful if the authors could indicate in a main text Table how much they found (M, mM, µM, nM concentrations) of these 32 compounds under some selected conditions. For specific values of interest, readers can then go to the Supplemental. The information is provided for some compounds in other figures, but they should show an overview of the concentrations for the compounds in Figure 1 for some important conditions in a main text table, is my suggestion. We see in the text that some compounds preferentially accumulate at low temperature, which is more biocompatible than 250°C.\n\nFigure 1 direly needs to cite [Fuchs, G. Alternative pathways of carbon dioxide fixation: Insights into the early evolution of life? Annual Review of Microbiology 65, 631–658 (2011).] as he elucidated several of the pathways shown in the figure and drew them in that paper in a manner similar to what we see here.\n\nMolybdenum is particularly interesting here because it is only involved in CO2 to formate conversion in primary metabolism. Its efficient synthesis of C5–C6 backbones here is surprising and interesting. I am reminded of the side activities of Mo-nitrogenase, which can produce hydrocarbons and other compounds under some conditions (work by M. Ribbe). Ribbe’s papers might deserve some brief mention, as they provide precedent for longer backbones being synthesized by a Mo-containing (MoS2-containing) enzyme. This connection makes the present work even more compelling and interesting.\n\nLine 96f. The use of H2 as the reductant should be mentioned early in the results discussion section. I find first mention of H2, 10 bar or 1 MPa, in the legend to Figure 2. Figure 1 does not make sense without naming the reductant. Assuming that H2 was the reductant for those experiments summarized in Figure 1, it is an impressive set of findings and an intuitive way to present the results. Please make the reductant clear.\n\nThe x-axis label of Figure 2d needs to indicate H2 pressure, not just pressure. Same with panel 2c, NaHCO3 concentration on axis label\n\nFor dummies like me, Figure 4 should have a panel sketching out the mechanism in structures and their inferred interactions with Mo orbitals. Not with a guarantee, but a suggestion for non-chemists to work with conceptually. This might be in one of the 277+ supplemental figures, which are too much to review. This referee is familiar with the topic at hand, but cannot clearly interpret Figure 2h for example, so a simplified, biochemistry-text-book like version, maybe with electon pair arrows, would aid understanding for some of us.\n\nLine 379, Mo in enzymes is usually as molybdenum cofactor (MoCo), Mo-nitrogenase is an exception. Perhaps a simplified structure comparison of the Mo environment here and in the MoS2 containing cofactor or Mo-nitrogenase would be instructive in the main text.\n\nFigure 6 is useful, and the legend indicates H2 being produced by serpentinization, it should therefore show H2 emerging from the crust (not swimming in the ocean). H2 that diffused into the primordial oceans might not have maintained sufficient partial pressure for CO2 reduction. Perhaps a citation to a recent paper on the serpentinization process as a source of H2 for CO2 reduction at biochemical origin (there are many such papers) would be useful.\n\nIn summary, this is a very interesting and surprising set of findings in a scholarly researched paper that strengthen the case for H2-dependent CO2 reduction over transition metal catalysts at the origin of metabolism. The findings can appeal to even a broader audience with attention to some of the comments here.", "reviewer_2": "The authors have claimed the formation of a large variety of metabolites from central carbon fixation pathways using the twisted 1T’-MoS2 polymorph, which mimics the distorted geometry found in certain molybdenum metalloenzymes. I focus my comments on the characterization and quantification of the small organic compounds and metabolites reported in this study, where several issues preclude the proper analysis of the data.\n\nMost of the compounds have been identified only by GC-MS using comparison with MS libraries. This identification process typically affords several scores to evaluate the reliability of the identification. These scores are not provided by the authors, which does not leave the reader with any confidence to justify the characterization. The authors only provide a visual comparison of the experimental/library MS traces. This is especially an issue when significant additional peaks with higher m/z are detected and/or the relative intensity of peaks differ significantly (the latter could indicate different fragmentation patterns, Fig. S81 is an example of both). Accordingly, the identification of several compounds reported in this manuscript appears doubtful.\n\nConcerning the products only identified by GC-MS, another issue is that the authors have not evaluated their derivatization methods on authentic standards: some important intermediates arise from dehydration and have been only observed after concentration of the reaction mixture. Although it’s not likely that freeze-drying will produce those, it is still an important control. The authors should treat authentic samples of the compounds they claim are detected with their derivatization methods and show the resulting GC-MS traces in a way that allows for easy comparison with the results reported in the present study.\n\nI would additionally recommend to spike the reaction mixture with some authentic sample of the supposed products to confirm their retention time and fragmentation pattern in the GC-MS (in case of co-elution, for example, complicate the analysis), either because they are essential to the authors’ argumentation, or because their MS spectra does not match unambiguously.\n\nFinally, the use of authentic standards would also enable the authors to provide some evaluation concerning the quantification: in the present state of the work, we do not know on which order of magnitude the products are detected after concentration of the mixture. Is it 10x lower than the products detected by NMR before concentration? 100x lower? 100,000x lower?\n\nThe authors should also have a deeper look if an experimental catalytic activity can indeed be demonstrated: they have used 0.1 mol/L NaHCO3 with a 0.5 mol/L 1-T’MoS2 loading: even if there is DFT calc. and IR analysis showing the potential for catalysis, it is not the same as experimentally demonstrating catalysis: deactivation/inhibition (and other processes) can result in a lack of catalytic turnover. This would not be catalysis. The authors must show experimental catalytic turnover (i.e. that more products are formed than the amount of “catalyst” used) to claim that the 1-T’MoS2 is a catalyst. If this cannot be demonstrated, the authors cannot claim “catalysis”. Rather, they should refer to it as a promoter or a reagent.\n\nBased on the above points, further work is required such that the authors can properly justify their claims that certain compounds have been produced and well quantified. As these claims are central to the manuscript, I must recommend major revisions."}, "author_response": "Explanations of our opinions and revisions to Reviewer #1\nManuscript ID: NCOMMS-25-64186\nNotice: All revisions based on this reviewer’s comments are highlighted in yellow in\nthe manuscript and supporting information.\nReviewer comment 1\nThis exciting paper presents the most complete set of biologically relevant carbon\noxides synthesized from H and CO reported to date. It is almost too good to be true\n2 2\nfrom some standpoints, because it provides perhaps the most comprehensive set of\nobservations indicating that the core of central carbon metabolism unfolds\nspontaneously from H and CO , given suitable conditions and catalysts. What they\n2 2\nshow is that MoS converts H and CO into 32 (!) biologically relevant compounds,\n2 2 2\nmostly organic acids of CO fixation pathways, many of which have been reported from\n2\nprevious, well-cited work of others, but many of which are new. Two compounds,\nacetaldehyde and glyoxylate, caught my attention because aldehydes are reactive and\nmight be short lived under these conditions. This is not to say that metabolism and life\narose directly from MoS catalysts, but it does say that MoS under hydrothermal\n2 2\nconditions certainly could have helped, in a surprisingly big way, which is welcome\nnews.\nAuthor reply 1\nThank you for your enthusiastic and insightful evaluation of our work. We are grateful\nto your recognition of the breadth and significance of the chemical syntheses\ndemonstrated in this work. We agree your point about acetaldehyde and glyoxylate, and\nthe identification of these reactive intermediates in catalytic systems such as MoS are\n2\nkey to understand the dynamic and potentially transient nature of abiotic metabolic-like\nnetworks. Thus, we share your view that while our results do not imply that life\noriginated directly from MoS , they strongly suggest that this catalyst could have played\n2\na pivotal and largely underappreciated role in the emergence of prebiotic organic\nchemistry.\nIn response to your comments and suggestions below, we have thoroughly revised the\nmanuscript. We hope our revised manuscript adequately addresses the scientific merits\nand broader implications of the work as you have emphasized, and we sincerely\nappreciate your positive and thoughtful feedback.\nReviewer comment 2\nI am sure it is in the supplemental, but many readers will not find the energy to look\nthrough Supplemental figures 83–147, so it would really be helpful if the authors could\nindicate in a main text Table how much they found (M, mM, µM, nM concentrations)\nof these 32 compounds under some selected conditions. For specific values of interest,\nreaders can then go to the Supplemental. The information is provided for some\ncompounds in other figures, but they should show an overview of the concentrations\nfor the compounds in Figure 1 for some important conditions in a main text table, is my\nsuggestion. We see in the text that some compounds preferentially accumulate at low\ntemperature, which is more biocompatible than 250 °C.\nAuthor reply 2\nThank you very much for your insightful suggestions. We agree that presenting a\nsummarized overview of the main concentrations of the 32 compounds in Figure 1,\nunder selected important experimental conditions, would greatly improve the\naccessibility and usability of the data for the readers. In the revised manuscript, we have\nprepared a new Table in the main text (Table 1, first mentioned on Page 8, Lines 156-\n158) to summarize the concentration ranges of the key compounds under the selected\nconditions, and provided the corresponding calibration curves as Supplementary table\n1.\nTable 1 Summary of compound concentrations under key experimental conditions from\nFigure 1.\nTemperature\nEntry Compounds Concentrations\n(°C)\n7 α-Ketoglutarate 6 µM 250\nGlycolate, Oxolate, 3-Hydroxybutyrate, Crotonate,\nHydroxyisobutyrate, 2-Hydroxybutyrate, Ethanol\nAcetoacetate, Acetaldehyd, Glyoxylate, 4-\nHydroxybutyrate, Hydroxypropionate, Methacrylate,\nEhtylmalonate, Mesaconate, Malonate, Malate,\nItaconate, Isobutyrate, β-Methylmalate, Citraconate\nMD: mass detected.\nTable added (Page 301-303)\nSupplementary Table 1. Calibration curves for the quantitative analysis of metabolites\nby HPLC. The curves were constructed using the peak areas of authentic standards at\ndifferent concentrations. Each standard solution was analyzed in triplicate. The linear\nregression equation is expressed as y = ax + b, where y is the peak area and x is the\nconcentration (mmol/L). The correlation coefficient (R²) and linear range are listed for\neach compound.\nConcentration Area\nProducts Calibration curves\n(mM) (a.u.)\n50 85590.6\n25 42898.6\nFormate 12.5 21770.6\n6.25 10864.2\n3.125 5412.2\n20 51056.5\n10 25480.6\n5 12571.7\nAcetate\n2.5 6462.2\n1.25 3496\n0.625 1740\n20 78625\n10 39388.6\n5 19678.4\nPropionate\n2.5 9898.2\n1.25 4926.6\n0.25 975.1\n5 17419.5\nLactate\n2.5 8783\n1.25 4400\n).u.a(\naerA\n1 0\n8\n6\n4\n2\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\ny = 1 7 0 8 .7 5 x + 1 9 2 .1 0\n1 0 2 0 3 0\nC o n c e n tra tio n (m M\n4 0\n)\n5 0\n60000\ny = 2543.12 x + 111.94\n50000 R2 = 1.00\n40000\n).u\n.a30000 (\na e\nrA20000\n10000\n0\n0 5 10 15 20\nConcentration (mM)\n).u.a(\naerA\n8\n6\n4\n2\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\n0\ny = 3 ,9 3\n10\n5\n.0 2\nC\nx + 3 0 .3 7\n1 0\no n c e n tra tio n\n1\n(m\n2 0\n0.625 2280.2\n0.3125 1235.8\n0.25 865\n1 9007\n0.5 4544\nSuccinate\n0.25 2303\n0.125 1311\n0.0625 632\n5 46386.2\n2.5 21329.6\n1.25 12324.9\nMethylsuccinate\n0.625 5972.5\n0.3125 3905.9\n0.03125 389.2\n1 934.259\n0.5 465\n0.1 94.4\nα-Ketoglutarate\n0.05 48.9\n0.01 8.7\n0.005 4.3\nPyruvate\n2.5 2037.8\n).u.a(\naerA\n).u.a(\naerA\n).u.a(\naerA\n1 8 0 0 0 y = 3 4 6 9 .0 1 x + 8 5 .0 4\nR = 1 .0 0 1 6 0 0 0 2\n1 4 0 0 0\n1 2 0 0 0\n1 0 0 0 0\n8 0 0 0\n6 0 0 0\n4 0 0 0\n2 0 0 0\n0\n0 1 2 3 4\nC o n c e n tra tio n (m M )\n1 0 0 0 0\ny = 8 ,8 8 0 .4 9 x + 1 1 8 .2 1\n8 0 0 0\n6 0 0 0\n4 0 0 0\n2 0 0 0\n00\n.0 0 .2 0 .4 0 .6 0 .8\nC o n c e n tra tio n (m M )\n5 0 0 0 0 y = 9 ,0 7 4 .6 8 x + 3 5 2 .2 9\n4 0 0 0 0\n3 0 0 0 0\n2 0 0 0 0\n1 0 0 0 0\n0\n0 1 2 3 4\nC o n c e n tra tio n (m M )\n1\n5\n.0\n5\n1000 y = 933.17x + 0.3049\nR2= 1\n800\n).u\n.a 600\n(\na e\nrA 400\n200\n0\n0.0 0.2 0.4 0.6 0.8 1.0\nConcentration (mM)\n1.25 1017\n0.625 511.3\n0.3125 254.2\n0.15625 128.7\n0.015625 12.42\n0.0078125 6.3\nReviewer comment 3\nFigure 1 direly needs to cite [Fuchs, G. Alternative pathways of carbon dioxide fixation:\nInsights into the early evolution of life? Annual Review of Microbiology 65, 631–658\n(2011).] as he elucidated several of the pathways shown in the figure and drew them in\nthat paper in a manner similar to what we see here.\nAuthor reply 3\nThank you for your valuable feedback regarding Figure 1. We appreciate your\nsuggestion to cite the work by Fuchs (2011), as it offers a comprehensive overview of\nalternative CO₂ fixation pathways, including the acetyl-CoA and rTCA cycles, which\nare central to our Figure 1. Citing this work enhances the scholarly context of our figure\nby acknowledging prior elucidations of similar pathways. In the revised manuscript, we\nhave added this citation in the figure legend of Figure 1.\nCitation added (Page 7, Line 154)\nThese cycles are interconnected via shared key intermediate hubs (acetate, succinate,\npropionate, crotonate), forming a coherent and autocatalytic network architecture26.\nRef. 26. Fuchs, G. Alternative pathways of carbon dioxide fixation: insights into the\n).u.a(\naerA\n2\n1\n1\n0\n5\n0\n5\n0\n0\n0\n0\n0\n0\n0\n0\n00\ny = 8 1 4 .7\n.0 0 .5\n7 x + 0 .3 5 8 5\n1 .0\nC o n c e n tra\n1 .5\ntio n (m\n2 .0\nM )\n2 .5\nearly evolution of life? Annu. Rev. Microbiol. 65, 631-658 (2011).\nReviewer comment 4\nMolybdenum is particularly interesting here because it is only involved in CO to\n2\nformate conversion in primary metabolism. Its efficient synthesis of C5–C6 backbones\nhere is surprising and interesting. I am reminded of the side activities of Mo-nitrogenase,\nwhich can produce hydrocarbons and other compounds under some conditions (work\nby M. Ribbe). Ribbe’s papers might deserve some brief mention, as they provide\nprecedent for longer backbones being synthesized by a Mo-containing (MoS -\n2\ncontaining) enzyme. This connection makes the present work even more compelling\nand interesting.\nAuthor reply 4\nThank you for your insightful comment regarding the role of molybdenum (Mo) in our\nstudy. We appreciate your observation that Mo is typically involved only in CO -to-\n2\nformate conversion in primary metabolism, and you find its efficient synthesis of C5–\nC6 backbones here surprising and interesting. Your point enhances the biological\nrelevance of our work by linking the catalytic behavior of 1T'-MoS to known\n2\nenzymatic activities such as Mo-nitrogenase. In response to your comment, we have\nrevised the following sentence to briefly mention Ribbe's work related with the side\nactivities of Mo-nitrogenase for producing longer backbones, which not only\nstrengthens the significance of the current findings but also places them in a broader\nbiochemical context.\nOriginal (Page5, Lines 104-108)\nAmong the tested minerals (FeS , Ni S ,Cu S, CuS, CoS , CoS, MnS, 2H-MoS , and\n2 3 2 2 2 2\n1Tʹ-MoS ), the twisted 1Tʹ-MoS polymorph, which mimics the distorted geometry of\n2 2\nthe carbon dioxide Mo-S -pterin enzyme, enables the hydrothermal conversion of\n3\nhydroxybutanoate, succinate, methylsuccinate (Supplementary Fig. 3−61).\nRevised (Page 5, Lines 104-110)\nAmong the tested minerals (FeS , Ni S ,Cu S, CuS, CoS , CoS, MnS, 2H-MoS , and\n2 3 2 2 2 2\n1Tʹ-MoS ), the twisted 1Tʹ-MoS polymorph, which mimics the distorted geometry of\n2 2\nthe carbon dioxide Mo-S -pterin enzyme24 and Mo-nitrogenase25 that produce\n2\nhydrocarbons under certain conditions, enables the hydrothermal conversion of\nNaHCO into formate, acetate, glycolate, oxolate, propionate, lactate, 2-\n3\nhydroxybutanoate, succinate, methylsuccinate (Supplementary Fig. 3-61).\nCitations added (Page 22, Lines 467-470)\nRef. 24. Maia, L.B., Moura, I. & Moura, J.J. Molybdenum and tungsten-containing\nformate dehydrogenases: Aiming to inspire a catalyst for carbon dioxide\nutilization. Inorg. Chim. Acta. 455, 350-363 (2017).\nRef. 25. Rebelein, J.G., Lee, C.C., Hu, Y. & Ribbe, M.W. The in vivo hydrocarbon\nformation by vanadium nitrogenase follows a secondary metabolic pathway.\nNat. Commun. 7, 13641 (2016).\nReviewer comment 5\nLine 96f. The use of H as the reductant should be mentioned early in the results\n2\ndiscussion section. I find first mention of H , 10 bar or 1 MPa, in the legend to Figure\n2\n2. Figure 1 does not make sense without naming the reductant. Assuming that H was\n2\nthe reductant for those experiments summarized in Figure 1, it is an impressive set of\nfindings and an intuitive way to present the results. Please make the reductant clear.\nAuthor reply 5\nThank you for your valuable comment. We agree that the use of H as the reductant\n2\nshould be clearly stated earlier in the Results and Discussion section and Figure 1. As\nsuggested, we have revised the following sentence at the beginning of the Results and\nDiscussion section.\nOriginal (Page 5, Lines 99-100)\nThe hydrothermal conversion of NaHCO into organics was conducted in a stainless\n3\nsteel autoclave with an inner volume of 7 mL at 250 °C for 12 h.\nRevised (Page 5, Lines 99-100)\nThe hydrothermal conversion of NaHCO into organics was conducted in a stainless\n3\nsteel autoclave with an inner volume of 7 mL at 250 °C for 12 h with 1 MPa H serving\n2\nas the reducing agent.\nWe have also revised the following sentence in the figure legend of Figure 1 to improve\nthe clarity.\nOriginal (Page 7, Lines 143-145)\nNon-enzymatic self-organized metabolic networks are constructed through CO\n2\nreduction catalyzed by 1T′-MoS .\nNon-enzymatic self-organized metabolic networks are constructed through CO\n2\nreduction catalyzed by 1T′-MoS using H as reductant.\n2 2\nReviewer comment 6\nThe x-axis label of Figure 2d needs to indicate H pressure, not just pressure. Same with\n2\npanel 2c, NaHCO concentration on axis label.\nThank you for pointing out this important detail. We agree that the x-axis labels in\nFigure 2c and 2d should be more specific to clearly indicate what the values represent.\nIn response to your comment, the x-axis labels of Figure 2c and 2d have been updated\nto NaHCO concentration (mM) and H pressure (MPa), respectively, as illustrated in\n3 2\nthe revised figure below.\n90\n75\n60\n45 43\nKA M--D M--D M--D M--D M-D- M--DMD M-D- M-D- MD-- M-D-\nMSA 1.8 3.1 7.7 8.4 6.5 4.9 4.4 5.1 5.8 5.3 34\nSA 0.3 0.7 0.8 0.8 0.7 0.6 0.7 0.8 0.7 0.7 26Y LA 2.4 2.1 1.9 0.9 M--D 1.0 1.1 1.0 1.2 0.9\n17\nield\n(% PA 1.5 5.3 14.3 17.4 17.0 12.6 13.7 16.4 16.7 14.7 )\nAA 1.2 3.8 16.5 17.7 16.2 11.4 17.9 25.3 26.8 42.6 9\nFA 4.3 11.3 23.3 21.7 21.0 19.7 21.0 32.6 39.9 25.9\n0\n3 6 9 12 15 18 21 24 27 30\nTime (h)\nReviewer comment 7\nFor dummies like me, Figure 4 should have a panel sketching out the mechanism in\nstructures and their inferred interactions with Mo orbitals. Not with a guarantee, but a\nsuggestion for non-chemists to work with conceptually. This might be in one of the\n277+ supplemental figures, which are too much to review. This referee is familiar with\nthe topic at hand, but cannot clearly interpret Figure 2h for example, so a simplified,\nbiochemistry-text-book like version, maybe with electon pair arrows, would aid\nunderstanding for some of us.\n)%(\n+2C\nfo\nytiviticeleS\nM ACTr\nffo P /ni\netaidemretnI\n9 0\n7 5\n6 0\n4 5\nK A\nS A\nS A Y A\nLA P A\nA A\nF A\n0.1\n--MD\n--MD 0.0\n0.7 1.3\n1.2\n0.6\n100\n0.0\nMD--MD\n--MD -- MD\n0.8 3.2\n2.2\n1.3\n-- M D\n--MDMD\n--MD -- MD\n2.1 4.8\n4.2\n3.0\nMD\n-- M DMD\n-- MD\n-- MD --MD\n2.6 4.5\n7.5\n5.4\nM D -- -- M D\n3.0 4.4\n0.3 0.5 -- -- MD MD\n4.0 3.7 6.1 7.7\n5.4 7.1\n15.5 17.6\n200 225\no perature ( C )\n--MD\n8.4\n0.8 --MD\n0.9 17.4\n17.7\n21.7\n250\n--MD\n6.0\n1.0 -- MD\n--MD 13.8\n15.3\n21.9\n275\n--MD\n5.9\n1.0 -- MD\n-- MD 12.1\n15.6\n16.7\n300\n22\n18\n13\n9\n4\n(%)\na b\nc )% 90 d 90 e100\nC fo\nyti\n(\nv itice\n+2 7\n6\n4\n3 1\n5\n0\n5\n0 5 30\n7\n6\n4\n3 1\n5\n0\n5\n0 5 34 )%\nA C T r\nffo/n\nl\ni\ne\ne\nS\ntaidem\nM\nS\nP\nS\nLA\nA\nA\nA\n1\n0\n.\n.\n. -D\n7\n5\n5\n4\n0\n.\n.\n. -\n1\n7\n8\n0\n0 7\n.\n.\n.\n4\n8\n9 .4\n3\n0\n1 6\n.\n.\n. .\n5\n3\n3 4\n1\n0\n1 2\n.\n.\n. .\n5\n2\n4 7\n2\n1 1\n4\n8 2 Y ield (%\n)\nM\nS\nP L\nS\nA\nA\nA\nA\n1\n7\n.\n.\n-D\n3\n8\n.3 1\n7\n0\n1 7\n.\n.\n.\n7\n9\n3 .3 1\n8\n0\n0 7\n.\n.\n.\n4\n8\n9 .4 1\n5\n.\n.\n-D\n6\n6\n.7\n1\nM\n-\n-\n.\n.\n-\n-\nD\nD\n8\n6\n2\n2 1\n7\n0 4 Y ie ld (%\n)\n(\nC fo ytivitce\nle S\n+2 6\n4\n2\n0\n0\nM M M\ni e\ne e e\nm t\nt t t\na\na a a\ne l\nl l l\nt\n/\na\na s M\nl\nu ll e o lf t y i a d l e oxide\nretnI AA 11.9 16.4 17.7 8.6 4.4\n0 0 0\n0.025 0.05 0.1 0.2 0.4 0.25 0.5 1 2 3 0 20 40 60 80 100\nNaHCO3 concentration (mM) H2 pressure (MPa) Conversion of CO2 / NaHCO3 (%)\nAuthor reply 7\nWe sincerely appreciate your excellent suggestion and agree that providing a clearer\nconceptual illustration would greatly enhance accessibility to readers with a non-\nchemical background. In response to this suggestion, we have created a new schematic\n(Figure 5i and the corresponding figure legend), which provides a conceptual overview\nof the key reaction intermediates and their proposed interactions with the MoS catalyst\n2\norbitals. We hope this addition addresses the reviewer’s concern and improves the\nclarity of the mechanism for non-specialist readers.\nFigure 5i, Schematic illustration of the C−C coupling between CH and CO on 1T-MoS .\n3 2\nWe also have added the following sentence in the main text of revised manuscript.\nSentence added (Page 18, Lines 380-383)\nBased on integrated experimental and spectroscopic analyses of catalytically active\nintermediates, we propose a visual summary (Fig. 5i) illustrating the key steps in the\nC–C bond formation between CH and CO on the 1T-MoS catalyst surface.\n3 2\nInspired by your suggestion, we have conducted a new Bader charge analysis for the\nCO adsorption systems. The results reveal that CO accepts 0.32 |e| from 1T′-MoS ,\n2\nin contrast to only 0.15 |e| transferred from 2H-MoS . We have updated Figure 5h\n2\naccordingly and incorporated the charge differential density value of the 1T′-MoS −CO\n2\nstructure into the schematic representation in Figure 5i.\n+ 3 e\n+─ e ─\n0\no\n.4\nS\n3\n-S v\n2\nle l\n/D f: C -C c o u p lin g\nM o S C O H\nOriginal Figure 5h\nRevised Figure 5h\nFurthermore, we have added the following sentence in the main text of revised\nmanuscript to clarify the Bader charge analysis.\nSentence added (Pages 18, Lines 379-380)\nIn addition, CO accepts 0.32 |e| from 1T′-MoS , in contrast to only 0.15 |e| transferred\n2\nfrom 2H-MoS .\nLine 379, Mo in enzymes is usually as molybdenum cofactor (MoCo), Mo-nitrogenase\nis an exception. Perhaps a simplified structure comparison of the Mo environment here\nand in the MoS containing cofactor or Mo-nitrogenase would be instructive in the main\n2\ntext.\nAuthor reply 8\nThank you for this insightful suggestion regarding the comparison of the molybdenum\n2\n2\nH\n3\n3\n0 .4 3 |e\n0 .2\n0 .4 3 |e |\n0 .2 1 |e |\n|\n1 |e\n1\n2\n|\nT\nH\n-M\n-M\no\no\nS\nS\n-C 2\n-C\nO\nT o p v ie w\nT o p v ie w\n0 .3 2 |e |\n0 .1 5 |e |\nenvironment in our catalyst with biological Mo-containing systems. As shown in\nAuthor reply 4, we have revised the main text to clarify that the twisted 1Tʹ-MoS\n2\npolymorph mimics the distorted geometry of the carbon dioxide Mo-S -pterin enzyme\n2\nand Mo-nitrogenase that produce hydrocarbons under certain conditions (Page 5, Lines\n105-107). In response to this suggestion, we have created a new Supplementary Fig. 64\nto highlights the structural differences between our MoS -based system and biological\nFigure added (Page 76)\nSupplementary Fig. 64\n1T\n1T’-MoS\nVS\n（SeCys）\nCO Mo-S -pterin Mo-nitrogenase\n2 2\nFe Se C Mo O S\nSupplementary Fig. 64: Comparative structural illustration of molybdenum\nenvironments in 1T′-MoS and representative molybdenum-containing enzymes. The\n2\ndiagram highlights the distorted coordination geometries around molybdenum (blue) in\nthree systems: the inorganic 1T′-MoS catalyst (top), the CO -converting Mo-S -pterin\n2 2 2\nenzyme with selenocysteine (SeCys; left), and the FeMo-cofactor of Mo-nitrogenase\n(right). Key elemental colors are indicated in the legend: Fe (purple), C (grey), Mo\n(blue), Se (pink), O (yellow), and S (green). This comparison underscores the structural\nmimicry between the abiotic 1T′-MoS surface and native Mo-active sites in\n2\nbiocatalysts, supporting the hypothesis that similar Mo–S motifs could drive CO\n2\nfixation in both geochemical and biological contexts.\nReviewer comment 9\nFigure 6 is useful, and the legend indicates H being produced by serpentinization, it\n2\nshould therefore show H emerging from the crust (not swimming in the ocean). H\n2 2\nthat diffused into the primordial oceans might not have maintained sufficient partial\npressure for CO reduction. Perhaps a citation to a recent paper on the serpentinization\n2\nprocess as a source of H for CO reduction at biochemical origin (there are many\n2 2\nsuch papers) would be useful.\nAuthor reply 9\nThank you for your valuable feedback regarding Figure 6 and the suggestion to clarify\nthe depiction of H production via serpentinization. We appreciate your insight that\n2\nshowing H emerging from the crust, rather than diffusing into the ocean, more\n2\naccurately represents the geological process and emphasizes the potential for\nmaintaining sufficient partial pressure for CO reduction. This comment has helped us\n2\nenhance the figure's scientific accuracy. In response to your suggestion, we have revised\nFigure 6 to explicitly illustrate H emanating from crustal fissures, as opposed to being\n2\ndissolved in the primordial ocean. This modification aligns with the geological\nunderstanding of serpentinization as a deep crustal/Hadean process. Additionally, we\nhave added a citation (Nat. Ecol. Evol., 2020, 4(4): 534-42), which provides robust\nsupport for serpentinization as a key source of H in prebiotic CO reduction scenarios,\n2 2\nto the corresponding figure legend (Page 20, Lines 418-419).\nReviewer comment 10\nIn summary, this is a very interesting and surprising set of findings in a scholarly\nresearched paper that strengthen the case for H -dependent CO reduction over\n2 2\ntransition metal catalysts at the origin of metabolism. The findings can appeal to even\na broader audience with attention to some of the comments here.\nAuthor reply 10\nThanks for your positive assessment of our work. We are grateful for your constructive\ncomments and suggestions that enhance the clarity, accessibility, and interdisciplinary\nimpact of our paper.\nS\nC\nM\ne a b e\nr u s t\na n te\nd\nl\n1 5 0 o C\nMNC oiSo S\n2\n2F\nF\n2\ne S N iS\n2\ne S\nH y\no 0 - 3 0 0\nH e a t\nu\nd\nC\nS\nr\nC O\no th e\n+\nr m\n2\n7\n2\na l V e\no 0 0 C\n0\nn\nM\n-\nt\nSx\n2 5\n7 0\ny\no 0 C\nC O\nN a H\nC a C\no C\n2CO\nO\n3\n2\n3\n- 4\nA c C\no C\no A\nExplanations of our opinions and revisions to Reviewer #2\nManuscript ID: NCOMMS-25-64186\nNotice: All revisions based on this reviewer’s comments are highlighted in cyan in the\nmanuscript and supporting information.\nReviewer comment 1\nThe authors have claimed the formation of a large variety of metabolites from central\ncarbon fixation pathways using the twisted 1T’-MoS polymorph, which mimics the\n2\ndistorted geometry found in certain molybdenum metalloenzymes. I focus my\ncomments on the characterization and quantification of the small organic compounds\nand metabolites reported in this study, where several issues preclude the proper analysis\nof the data.\nAuthor reply 1\nWe sincerely appreciate your valuable and constructive comments regarding the\ncharacterization and quantification of small organic compounds and metabolites in our\nstudy. We agree that the reliability of our data analysis is crucial for enhancing the rigor\nand reproducibility of our work. We have made a comprehensive revision of the\nmanuscript based on your suggestions, and have provided detailed explanations of the\nmeasures we have taken as well as the relevant evidence in the document.\nReviewer comment 2\nMost of the compounds have been identified only by GC-MS using comparison with\nMS libraries. This identification process typically affords several scores to evaluate the\nreliability of the identification. These scores are not provided by the authors, which\ndoes not leave the reader with any confidence to justify the characterization. The\nauthors only provide a visual comparison of the experimental/library MS traces. This\nis especially an issue when significant additional peaks with higher m/z are detected\nand/or the relative intensity of peaks differ significantly (the latter could indicate\ndifferent fragmentation patterns, Fig. S81 is an example of both). Accordingly, the\nidentification of several compounds reported in this manuscript appears doubtful.\nAuthor reply 2\nThanks for your important feedback regarding the reliability of compound\nidentification based on GC−MS library matching. We agree that providing the matching\nscores is essential to support the interpretation of the MS data and to allow the reader\nto judge the confidence level of each identification. In response, we would like to clarify\nthat we have already included the matching scores in our analysis. However, these\nscores were originally reported in the Chinese version of the software, using the term\n“匹配度” to denote the matching confidence. For example, the matching scores (匹配\n度) in original Fig. S81 is 94. In the revised manuscript, we have translated these scores\ninto the standard English terminology “matching score” and re-presented all\nsupplementary data in English to ensure clarity and international accessibility. We\nbelieve these revisions significantly improve the transparency and validity of our\ncompound identification, and we are grateful to the reviewer for prompting this\nimportant clarification.\nReviewer comment 3\nConcerning the products only identified by GC-MS, another issue is that the authors\nhave not evaluated their derivatization methods on authentic standards: some important\nintermediates arise from dehydration and have been only observed after concentration\nof the reaction mixture. Although it’s not likely that freeze-drying will produce those,\nit is still an important control. The authors should treat authentic samples of the\ncompounds they claim are detected with their derivatization methods and show the\nresulting GC-MS traces in a way that allows for easy comparison with the results\nreported in the present study.\nI would additionally recommend to spike the reaction mixture with some authentic\nsample of the supposed products to confirm their retention time and fragmentation\npattern in the GC-MS (in case of co-elution, for example, complicate the analysis),\neither because they are essential to the authors’ argumentation, or because their MS\nspectra does not match unambiguously.\nAuthor reply 3\nThank you for your valuable feedback regarding the evaluation of our derivatization\nmethods and the recommendation for spiking experiments. We sincerely appreciate\nyour suggestions, as they are crucial for ensuring the reliability and accuracy of our\nGC−MS-based compound identifications. We have carefully addressed your concerns\nby conducting additional experiments and revising the manuscript accordingly.\n1. Evaluation of derivatization methods using authentic standards\nWe have now performed comprehensive derivatization experiments using authentic\nstandards for all compounds reported in our study (acetoacetate, 4-hydroxybutyrate,\nand methylmalateare not available due to chemical safety issues). These standards were\nsubjected to the same derivatization protocol (e.g., esterification with ethanol or\nsilylation) as described in our Methods section. The resulting GC−MS traces for the\nauthentic standards are now included in the Supplementary Figs. 217−244, allowing for\ndirect visual comparison with the traces obtained from our reaction mixtures.\nThe results show that our derivatization methods do not artificially generate or\nalter compounds, particularly those prone to dehydration. By comparing retention times\nbetween authentic standards and samples, we demonstrate the specificity of our\napproach. For instance, the derivatized authentic standards show consistent peaks\nwithout unexpected by-products, confirming that freeze-drying and concentration steps\ndid not introduce artifacts.\n2. Spiking experiments to confirm retention times and fragmentation patterns\nWe have conducted spiking experiments by adding authentic standards directly to\nthe reaction mixtures prior to derivatization and GC−MS analysis. The resulting\nchromatograms and spectra are now presented in the Supplementary Figs. 217-244,\nshowcasing side-by-side comparisons of spiked vs. unspiked samples.\nThese experiments confirm that the retention times and fragmentation patterns of\ntarget compounds align perfectly with the authentic standards. For example, in cases\nwhere co-elution was suspected, the spiked samples showed increased peak intensities\nwithout shifting retention times, eliminating ambiguity. This approach also validates\ncompounds whose MS spectra initially appeared unclear, providing unambiguous\nidentification and supporting the conclusions drawn in our manuscript.\nFigures added\nSupplementary Figs. 217-244\nSupplementary Fig. 217. Validation of formate identification by GC−MS analysis. (a)\nGC−MS chromatogram of the authentic formate peak. (b) Overlay of chromatograms\nfor the real sample (NaHCO , 250 °C, 3 h) and the same sample spiked with authentic\n3\nformate standard. (c) Mass spectrum of the authentic formate. (d) Mass spectrum\nextracted at the formate peak position in the real sample. (e) Mass spectrum at the same\nretention time in the formate-spiked sample. The consistent retention time confirmed\nthe identity of the formate peak of the products. The match in fragmentation patterns\nbetween (d) and (e) verified the authenticity of formate detection.\na\nb\n).u.a(\nytisnetnI\n).u.a(\nytisnetnI\n8 ×\n7 ×\n6 ×\n5 ×\n4 ×\n3 ×\n2 ×\n1 ×\n1 ×\n9 ×\n8 ×\n7 ×\n6 ×\n5 ×\n4 ×\n3 ×\n2 ×\n1 ×\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n0\n1 2\n7 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n0\n1 2\nF\nN\nN\no rm\na H\na H\na te\n1 4\nC O -2\n3\n1 4\n5\n5\n1 6\nC o u n ts (%\n0 °C -3 h\n0 °C -3 h + F\n1 6\nC o u n ts (%\n1 8\n) v s A c q u\no rm a te\n1 8\n) v s A c q u\nis itio\nis itio\nn\nn\nT\nT\n2 0\nim e\n2 0\nim e\n(m\n(m\nin\nin\n)\n)\n2 2\n2 2\n2 4\n2 4\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5\n5\n5\n0\n0\na H C\nF o rm a te\n1 0 0 1 5 0 2 0 0\nm /z\no N a H C O -2 5 0 C -3 H\n3\n1 0 0 1 5 0 2 0 0\nm /z\no O -2 5 0 C -3 H + F o rm a te\n3\n1 0 0 1 5 0 2 0 0\nm /z\nSupplementary Fig. 218. Validation of acetate identification by GC−MS analysis. (a)\nGC−MS chromatogram of the authentic acetate peak. (b) Overlay of chromatograms\nfor the real sample (NaHCO , 250 °C, 3 h) and the same sample spiked with authentic\n3\nacetate standard. (c) Mass spectrum of the authentic acetate. (d) Mass spectrum\nextracted at the acetate peak position in the real sample. (e) Mass spectrum at the same\nretention time in the acetate-spiked sample. The consistent retention time confirmed the\nidentity of the acetate peak of the products. The match in fragmentation patterns\nbetween (d) and (e) verified the authenticity of acetate detection.\na\n).u.a(\nytisnetnI\nb\n2\n1\n1\n8\n4\n).u.a(\nytisnetnI\n7 .0 × 1 0\n7 .6 × 1 0\n7 .2 × 1 0\n6 .0 × 1 0\n6 .0 × 1 0\n0 .0\n1 6\n7 1 × 1 0\n6 9 × 1 0\n6 8 × 1 0\n6 7 × 1 0\n6 6 × 1 0\n6 5 × 1 0\n6 4 × 1 0\n6 3 × 1 0\n6 2 × 1 0\n6 1 × 1 0\n0\n1 6\nA\nN\nN\nc e ta\na H C\na H C\nte\nO\nO\n-2\n3-2\n3\n5\n0 °C -3 h\n0 °C -3 h + A\nC o u n ts (%\n) v s A\nc e ta te\n) v s A\n1 7\nc q u is itio n T\n1 7\nc q u is itio n T\nim\nim\ne (m\ne (m\nin )\nin )\n1 8\n1 8\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5 0\n5 0\nN\n5 0\na H\n1 0 0\nm /z\nN\n1 0 0\nm /z\nC O -2 5\n3\n1 0 0\nm /z\na\n1 5 0 2 0 0\no H C O -2 5 0 C -3 H\n3\n1 5 0 2 0 0\no C -3 H + A ce ta te\n1 5 0 2 0 0\nSupplementary Fig. 219. Validation of propionate identification by GC−MS analysis.\n(a) GC−MS chromatogram of the authentic propionate peak. (b) Overlay of\nchromatograms for the real sample (NaHCO , 250 °C, 3 h) and the same sample spiked\n3\nwith authentic propionate standard. (c) Mass spectrum of the authentic propionate. (d)\nMass spectrum extracted at the propionate peak position in the real sample. (e) Mass\nspectrum at the same retention time in the propionate-spiked sample. The consistent\nretention time confirmed the identity of the propionate peak of the products. The match\nin fragmentation patterns between (d) and (e) verified the authenticity of propionate\ndetection.\na\nb\n).u.a(\nytisnetnI\n).u.a(\nytisnetnI\n8\n7\n6\n5\n4\n3\n2\n1\n1\n9\n8\n7\n6\n5\n4\n3\n2\n1\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n6 0\n6 0\n6 0\n6 0\n6 0\n6 0\n6 0\n6 0\n0\n1\n7 0\n6 0\n6 0\n6 0\n6 0\n6 0\n6 0\n6 0\n6 0\n6 0\n0\n1\n7\nN\nN\nro p io n\na H C O\na H C O\na te\n-2 5\n3-2\n5\n0 °C -3\n0 °C -3\nC o u n\nts\nh\nh\nts\n1\n1\n8\n8\n(% \n+ P\n(% \n) v\nro p\n) v\ns A c q\nio n a te\ns A c q\nu\nu\nis\nis\nitio\nitio\nn\nn\nT\nT\n1\nim\n1\nim\n9\ne\n9\ne\n(m\n(m\nin\nin\n)\n)\n2\n2\n0\n0\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5 0\n5 0\nN a H\n5 0\nC O\n1 0 0\nm /z\nN a\n1 0 0\nm /z\no -2 5 0 C\n3\n1 0 0\nm /z\nP ro p io n a te\n1 5 0 2 0 0\no H C O -2 5 0 C -3 H\n3\n1 5 0 2 0 0\n-3 H + P ro p io n a te\n1 5 0 2 0 0\nSupplementary Fig. 220. Validation of butyrate identification by GC−MS analysis. (a)\nGC−MS chromatogram of the authentic butyrate peak. (b) Overlay of chromatograms\nfor the real sample (NaHCO , 250 °C, 3 h) and the same sample spiked with authentic\n3\nbutyrate standard. (c) Mass spectrum of the authentic butyrate. (d) Mass spectrum\nextracted at the butyrate peak position in the real sample. (e) Mass spectrum at the same\nretention time in the butyrate-spiked sample. The consistent retention time confirmed\nthe identity of the butyrate peak of the products. The match in fragmentation patterns\nbetween (d) and (e) verified the authenticity of butyrate detection.\na\n).u.a(\nytisnetnI\nb\n).u.a(\nytisnetnI\n3\n3\n2\n2\n1\n1\n5\n2\n1\n1\n1\n1\n1\n8\n6\n4\n2\n.5\n.0\n.5\n.0\n.5\n.0\n.0\n.0\n.8\n.6\n.4\n.2\n.0\n.0\n.0\n.0\n.0\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n6 1 0\n0 .0\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n0 .0\nB\nN\nN\nu\na\na\nty ra te\n1 9\nH C O -2\n3\n1 9\n5\n0 °C -3\n0 °C -3\nC o u n\nts\nh\nh\nts\n(% \n+ B\n(% \n) v s A\nu ty ra te\n) v s A\nc\nc\n2\nq\n2\nq\n0\nu\n0\nu\nis\nis\nitio\nitio\nn\nn\nT\nT\nim\nim\ne\ne\n(m\n(m\nin\nin\n)\n)\n2\n2\n1\n1\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5\n5\n5\n0\n0\na H C\n1\nm\n1\nm\nO\n1\nm\n0 0\n/z\nN\n0 0\n/z\n-2 5\n3\n0 0\n/z\nB\n1 5 0\na H C O -2 5 0 3\n1 5 0\no 0 C -3 H + B\n1 5 0\nu tyra\n2 0 0\no C -3\n2 0 0\nu tyra\n2 0 0\nte\nH\nte\nSupplementary Fig. 221. Validation of isobutyrate identification by GC−MS analysis.\n(a) GC−MS chromatogram of the authentic isobutyrate peak. (b) Overlay of\nchromatograms for the real sample (NaHCO , 250 °C, 3 h) and the same sample spiked\n3\nwith authentic isobutyrate standard. (c) Mass spectrum of the authentic isobutyrate. (d)\nMass spectrum extracted at the isobutyrate peak position in the real sample. (e) Mass\nspectrum at the same retention time in the isobutyrate-spiked sample. The consistent\nretention time confirmed the identity of the isobutyrate peak of the products. The match\nin fragmentation patterns between (d) and (e) verified the authenticity of isobutyrate\ndetection.\na\n).u.a(\nytisnetnI\nb\n).u.a(\nytisnetnI\n3\n3\n2\n2\n1\n1\n5\n4\n3\n3\n2\n2\n2\n1\n1\n8\n4\n.5\n.0\n.5\n.0\n.5\n.0\n.0\n.0\n.6\n.2\n.8\n.4\n.0\n.6\n.2\n.0\n.0\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n6 1 0\n0 .0\n1\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n6 1 0\n6 1 0\n0 .0\n1\n8\nN a H\nN a H\nu\nC\nC\nty\nO\nO\nra te\n-2 5\n3-2\n5\n0 °C -3\n0 °C -3\nC o u n\nts\nh\nh\nts\n(% ) v\n+ Is o b\n(% ) v\ns\nu\ns\nA\nty\nA\nc q u\nra te\nc q u\nis\nis\nitio\nitio\nn\nn\nT\nT\n1\nim\n1\nim\n9\ne\n9\ne\n(m\n(m\nin\nin\n)\n)\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5 0\n5 0\nN a H\n5 0\nC O\n1\nm\n1\nm\n-2\n3\n1\nm\n0 0\n/z\nN a\n0 0\n/z\no 5 0 C\n0 0\n/z\nIso b u tyra\n1 5 0 2 0 0\no H C O -2 5 0 C -3\n3\n1 5 0 2 0 0\n-3 H + Iso b u tyra\n1 5 0 2 0 0\nte\nH\nte\nSupplementary Fig. 222. Validation of lactate identification by GC−MS analysis. (a)\nGC−MS chromatogram of the authentic lactate peak after concentration and\nsilanization. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h)\n3\nand the same sample spiked with authentic lactate standard after concentration and\nsilanization. (c) Mass spectrum of the authentic lactate. (d) Mass spectrum extracted at\nthe lactate peak position in the real sample. (e) Mass spectrum at the same retention\ntime in the lactate-spiked sample. The consistent retention time confirmed the identity\nof the lactate peak of the products. The match in fragmentation patterns between (d)\nand (e) verified the authenticity of lactate detection.\na\nb\n).u.a(\nytisnetnI\n).u.a(\nytisnetnI\n5\n4\n3\n2\n1\n1\n8\n6\n4\n2\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n7 0\n7 0\n7 0\n7 0\n7 0\n01\n8 0\n7 0\n7 0\n7 0\n7 0\n01\n0\n0\n.0\n.0\nL\nN\nN\na\na\na\nc ta\nH C\nH C\nte\nO\nO\n-2\n3-2\n3\n1 0 .5\nC o u n\n5 0 °C -3\n5 0 °C -3\n1 0 .5\nC o u n\nts\nh\nh\nts\n(% \n+ L\n(% \n) v s A\na c ta te\n) v s A\n1 1 .0\nc q u is\n1 1 .0\nc q u is\nitio\nitio\nn\nn\nT\nT\nim\nim\ne\ne\n1 1 .5\n(m in )\n1 1 .5\n(m in )\n1\n1\n2\n2\n.0\n.0\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5 0\n5 0\nN\n5 0\na H\nL a cta te\n1 0 0 1 5 0 2 0 0\nm /z\no N a H C O -2 5 0 C -3 H\n3\n1 0 0 1 5 0 2 0 0\nm /z\no C O -2 5 0 C -3 H + L a cta te\n3\n1 0 0 1 5 0 2 0 0\nm /z\nSupplementary Fig. 223. Validation of glycolate identification by GC−MS analysis. (a)\nGC−MS chromatogram of the authentic glycolate peak after concentration and\nsilanization. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h)\n3\nand the same sample spiked with authentic glycolate standard after concentration and\nsilanization. (c) Mass spectrum of the authentic glycolate. (d) Mass spectrum extracted\nat the glycolate peak position in the real sample. (e) Mass spectrum at the same retention\ntime in the glycolate-spiked sample. The consistent retention time confirmed the\nidentity of the glycolate peak of the products. The match in fragmentation patterns\nbetween (d) and (e) verified the authenticity of glycolate detection.\na\nb\n).u.a(\nytisnetnI\n).u.a(\nytisnetnI\n5\n4\n3\n2\n1\n1\n8\n6\n4\n2\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n7 0\n7 0\n7 0\n7 0\n7 0\n01\n8 0\n7 0\n7 0\n7 0\n7 0\n01\n1\n1\n.0\n.0\nG\nN\nN\nly c o\na H C\na H C\nla\nO\nO\nte\n-2\n3-2\n3\n1 1 .5\nC o u n\n5 0 °C -3\n5 0 °C -3\n1 1 .5\nC o u n\nts\nh\nh\nts\n(% \n+ G\n(% \n1 2 .0\n) v s A c q u is\nly c o la te\n1 2 .0\n) v s A c q u is\nitio\nitio\nn\nn\nT\nT\nim\nim\ne\ne\n1 2 .5\n(m in )\n1 2 .5\n(m in )\n1\n1\n3\n3\n.0\n.0\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5\n5\n5\n0\n0\n1\n1\n0\n0\n0\n0\n1 5 0\nm /z\nN a H\n1 5 0\nm /z\no -2 5 0 C\n3\n1 5 0\nm /z\nC O\n-3 H\nG\n2 0\n-2 5\n3\n2 0\n+ G\n2 0\nlyco la te\n0 2 5\no 0 C -3 H\n0 2 5\nlyco la te\n0 2 5\n0\n0\nanalysis. (a) GC−MS chromatogram of the authentic 3-hydroxypropionate peak after\nconcentration and silanization. (b) Overlay of chromatograms for the real sample\n(NaHCO , 250 °C, 3 h) and the same sample spiked with authentic 3-\n3\nhydroxypropionate standard after concentration and silanization. (c) Mass spectrum of\nthe authentic 3-hydroxypropionate. (d) Mass spectrum extracted at the 3-\nhydroxypropionate peak position in the real sample. (e) Mass spectrum at the same\nretention time in the 3-hydroxypropionate-spiked sample. The consistent retention time\nconfirmed the identity of the 3-hydroxypropionate peak of the products. The match in\nfragmentation patterns between (d) and (e) verified the authenticity of 3-\nhydroxypropionate detection.\na\n).u.a(\nytisnetnI\nb\n).u.a(\nytisnetnI\n2\n1\n1\n8\n4\n1\n7\n5\n2\n.0\n.6\n.2\n.0\n.0\n.0\n.5\n.0\n.5\n×\n×\n×\n×\n×\n×\n×\n×\n×\n8 1 0\n8 1 0\n8 1 0\n7 1 0\n7 1 0\n0 .01\n8 1 0\n7 1 0\n7 1 0\n7 1 0\n0 .01\n3\n3\n.0\n.0\nN\n-H y d ro x y p ro p io n a te\n1 3 .5 1 4 .0\nC o u n ts (%\na H C O -2 5 0 °C -3 h\n3\na H C O -2 5 0 °C -3 h + 3\n3\n1 3 .5 1 4 .0\nC o u n ts (%\n) v\n-H y\n) v\ns\nd\ns\nA\nro\nA\n1 4 .5 1 5 .0\nc q u is itio n T im e (m\nx y p ro p io n a te\n1 4 .5 1 5 .0\nc q u is itio n T im e (m\nin\nin\n)\n)\n1\n1\n5\n5\n.5\n.5\n1\n1\n6\n6\n.0\n.0\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n5 0\n5 0\nC O\n5 0\n-2\n3\n5 0\n1 0 0\nm\n1 0 0\nm\no C -3 H\n1 0 0\nm\n3 -H yd ro xyp ro p io n a te\n1 5 0 2 0 0 2 5 0\n/z\no N a H C O -2 5 0 C -3 H\n3\n1 5 0 2 0 0 2 5 0\n/z\n+ 3 -H yd ro xyp ro p io n a te\n1 5 0 2 0 0 2 5 0\n/z\nSupplementary Fig. 225. Validation of succinate identification by GC−MS analysis. (a)\nGC−MS chromatogram of the authentic succiante peak after concentration and\nsilanization. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h)\n3\nand the same sample spiked with authentic succiante standard after concentration and\nsilanization. (c) Mass spectrum of the authentic succiante. (d) Mass spectrum extracted\nat the succiante peak position in the real sample. (e) Mass spectrum at the same retention\ntime in the succiante-spiked sample. The consistent retention time confirmed the\nidentity of the succiante peak of the products. The match in fragmentation patterns\nbetween (d) and (e) verified the authenticity of succiante detection.\na\n).u.a(\nytisnetnI\nb\n).u.a(\nytisnetnI\n1\n1\n8\n4\n1\n7\n5\n2\n.6\n.2\n.0\n.0\n.0\n.5\n.0\n.5\n×\n×\n×\n×\n×\n×\n×\n×\n8 1 0\n8 1 0\n7 1 0\n7 1 0\n0 .01\n8\n8 1 0\n7 1 0\n7 1 0\n7 1 0\n0 .01\n8\n.0\n.0\n0\nN\nN\nu\na\na\nc\nH\nH\nc ia n te\nC O -2\n3\n5\n0 °C -3\n0 °C -3\nC o u n\nts\nh\nh\nts\n(% \n+ S\n(% \n) v s A\nu c c in a\n) v s A\n1 8 .2\nc q u\nte\n1 8 .2\nc q u\n5\nis\n5\nis\nitio\nitio\nn\nn\nT\nT\nim\nim\ne\ne\n(m\n(m\nin\nin\n)\n)\n1\n1\n8\n8\n.5\n.5\n0\n0\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5 0\n5 0\n5 0\n1 0 0\n1 0 0\nN a H C\n1 0 0\nO\n1 5 0 2 0 0 m /z\nN a H C O\n1 5 0 2 0 0\nm /z\no -2 5 0 C -3 H 3\n1 5 0 2 0 0\nm /z\nS u ccin a te\n2 5 0 3 0 0\no -2 5 0 C -3 H 3\n2 5 0 3 0 0\n+ S u ccin a te\n2 5 0 3 0 0\na\n1×108 Methylsucciante\n).u8×107\n.a\n( y\ntis\nn\ne\ntn4×107 I\n).u\n.a NaHCO 3 -250 oC-3H\n1\n0\n8.00 18.25 18.50 18.75 19.00\n(\ne cn\na\nd\nb Counts (%) vs Acquisition Time (min) n u\nb\n1.0×108\nNaHCO 3 -250 °C-3 h\na\ne vita\nNaHCO\n3\n-250 °C-3 h+Methylsucciante le\nR\n7.5×107 0 50 100 150 200 250 300\n).u m/z\n.a ( y tis\nn\ne tn\nI\n5\n2\n.\n.\n0\n5\n×\n×\n1\n1\n0\n0\n7\n7\n).u .a ( e\ncn\na d\nn u b\nNaHCO 3 -250 oC-3H + Methylsuccinate\na\ne\nvita\n0.0\nle\nR\n18.00 18.25 18.50 18.75 19.00 0 50 100 150 200 250 300\nCounts (%) vs Acquisition Time (min) m/z\nSupplementary Fig. 226. Validation of methylsuccinate identification by GC−MS\nanalysis. (a) GC−MS chromatogram of the authentic methylsucciante peak after\nconcentration and silanization. (b) Overlay of chromatograms for the real sample\n(NaHCO , 250 °C, 3 h) and the same sample spiked with authentic methylsucciante\n3\nstandard after concentration and silanization. (c) Mass spectrum of the authentic\nmethylsucciante. (d) Mass spectrum extracted at the methylsucciante peak position in\nthe real sample. (e) Mass spectrum at the same retention time in the methylsucciante-\nspiked sample. The consistent retention time confirmed the identity of the\nmethylsucciante peak of the products. The match in fragmentation patterns between (d)\nand (e) verified the authenticity of methylsucciante detection.\n).u.a(\necnadnuba\nevitaleR\n5 0\nH C O -2\n3\n1 0 0\n5 0 o C -3\n1 5 0\nm /z\nH + M e th\n2 0 0\nylsu ccin\n2 5 0\na te\n3 0 0\nc\nd\ne\nSupplementary Fig. 227. Validation of ethylmalonate identification by GC−MS\nanalysis. (a) GC−MS chromatogram of the authentic ethylmalonate peak after\nconcentration and silanization. (b) Overlay of chromatograms for the real sample\n(NaHCO , 250 °C, 3 h) and the same sample spiked with authentic ethylmalonate\n3\nstandard after concentration and silanization. (c) Mass spectrum of the authentic\nethylmalonate. (d) Mass spectrum extracted at the ethylmalonate peak position in the\nreal sample. (e) Mass spectrum at the same retention time in the ethylmalonate-spiked\nsample. The consistent retention time confirmed the identity of the ethylmalonate peak\nof the products. The match in fragmentation patterns between (d) and (e) verified the\nauthenticity of ethylmalonate detection.\na\nb\n).u.a(\nytisnetnI\n).u.a(\nytisnetnI\n1\n7\n5\n2\n1\n8\n4\n.0\n.5\n.0\n.5\n×\n×\n×\n×\n×\n×\n×\n8 1 0\n7 1 0\n7 1 0\n01\n7\n8 1 0\n7 1 0\n7 1 0\n7 1 0\n0 .01\n7\n.0\n.0\n0\nN\nN\nth y lm a lo n a te\n1 7 .2 5\nC o u n\na H C O -7 0 °C -2 4\n3\na H C O -7 0 °C -2 4\n3\n1 7 .2 5\nC o u n\nts\nh\nh\nts\n(% \n+ E\n(% \n) v s A\nth y lm a\n) v s A\n1 7 .5\nc q u\nlo n a\n1 7 .5\nc q u\n0\nis itio\nte\n0\nis itio\nn\nn\nT\nT\nim\nim\ne\ne\n1\n(m\n1\n(m\n7 .7 5\nin )\n7 .7 5\nin )\n1\n1\n8\n8\n.0\n.0\nc\nd\n0\ne\n0\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5\n5\n5\n0\n0\na H\n1\n1\n0\n0\n0\n0\n-2\n3\n0\n1 5 0 2 0 0\nm /z\nN a H C O\n1 5 0 2 0 0\nm /z\no 0 C -3 H + E\n1 5 0 2 0 0\nm /z\nth ylm a lo n a te\n2 5 0 3 0\no -2 5 0 C -3 H\n3\n2 5 0 3 0\nth ylm a lo n a te\n2 5 0 3 0\n0\n0\n(a) GC−MS chromatogram of the authentic mesaconate peak after concentration and\nsilanization. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h)\n3\nand the same sample spiked with authentic mesaconate standard after concentration and\nsilanization. (c) Mass spectrum of the authentic mesaconate. (d) Mass spectrum\nextracted at the mesaconate peak position in the real sample. (e) Mass spectrum at the\nsame retention time in the mesaconate-spiked sample. The consistent retention time\nconfirmed the identity of the mesaconate peak of the products. The match in\nfragmentation patterns between (d) and (e) verified the authenticity of mesaconate\ndetection.\na\nb\n).u.a(\nytisnetnI\n).u.a(\nytisnetnI\n1\n7\n5\n2\n1\n8\n4\n.0\n.5\n.0\n.5\n×\n×\n×\n×\n×\n×\n×\n8 1 0\n7 1 0\n7 1 0\n01\n9\n8 1 0\n7 1 0\n7 1 0\n7 1 0\n0 .01\n9\n.0\n.0\n0\nN\nN\ne s\na H\na H\na c o\nC O\nC O\nn a te\n1 9 .2 5\nC o u n ts (%\n-7 0 °C -2 4 h\n3-7\n0 °C -2 4 h + M\n3\n1 9 .2 5\nC o u n ts (%\n1 9 .5 0\n) v s A c q u is itio\ne s a c o n a te\n1 9 .5 0\n) v s A c q u is itio\nn\nn\nT\nT\nim\nim\ne\ne\n1\n(m\n1\n(m\n9 .7 5\nin )\n9 .7 5\nin )\n2\n2\n0\n0\n.0\n.0\nc\nd\n0\ne\n0\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5 0\n5 0\n5 0\nN\n1 0 0\n1 0 0\na H C O\n1 0 0\n1 5 0 2 0 0\nm /z\nN a H C O\n1 5 0 2 0 0\nm /z\no -2 5 0 C -3 H +\n3\n1 5 0 2 0 0\nm /z\nM e sa co n a te\n2 5 0 3 0 0\no -2 5 0 C -3 H\n3\n2 5 0 3 0 0\nM e sa co n a te\n2 5 0 3 0 0\nSupplementary Fig. 229. Validation of citraconate identification by GC−MS analysis.\n(a) GC−MS chromatogram of the authentic citraconate peak after concentration and\nsilanization. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h)\n3\nand the same sample spiked with authentic citraconate standard after concentration and\nsilanization. (c) Mass spectrum of the authentic citraconate. (d) Mass spectrum\nextracted at the citraconate peak position in the real sample. (e) Mass spectrum at the\nsame retention time in the citraconate-spiked sample. The consistent retention time\nconfirmed the identity of the citraconate peak of the products. The match in\nfragmentation patterns between (d) and (e) verified the authenticity of citraconate\ndetection.\na\n).u.a(\nytisnetnI\nb\n).u.a(\nytisnetnI\n8 1 × 1 0\n7 8 × 1 0\n7 4 × 1 0\n01\n8 .0 0\n7 7 × 1 0\n7 6 × 1 0\n7 5 × 1 0\n7 4 × 1 0\n7 3 × 1 0\n7 2 × 1 0\n7 1 × 1 0\n01\n8 .0 0\nC\nC\nC\nitra c o n a\n1 8 .2 5\nH N a O\n3 3\nH N a O\n3 3\n1 8 .2 5\nte\n1 8 .5 0 1 8 .7 5 1 9 .0 0 1 9 .2 5\nC o u n ts (% ) v s A c q u is itio n T im\n-7 0 °C -1 2 h\n3-7\n0 °C -1 2 h + C itra c o n a te\n3\n1 8 .5 0 1 8 .7 5 1 9 .0 0 1 9 .2 5\nC o u n ts (% ) v s A c q u is itio n T im\n1 9 .5 0\ne (m in )\n1 9 .5 0\ne (m in )\n1 9 .7 5\n1 9 .7 5\nc\nd\n2 0 .0 0\ne\n2 0 .0 0\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5 0\n5 0\n5 0\nN a\n1 0 0\n1 0 0\nH C O\n1 0 0\n1 5 0\nm /z\nN\n1 5 0\nm /zo\n-2 5 0 C\n3\n1 5 0\nm /z\nC itra co n a te\n2 0 0 2 5 0 3 0 0\no a H C O -2 5 0 C -3 H\n3\n2 0 0 2 5 0 3 0 0\n-3 H + C itra co n a te\n2 0 0 2 5 0 3 0 0\nSupplementary Fig. 230. Validation of itaconate identification by GC−MS analysis. (a)\nGC−MS chromatogram of the authentic itaconate peak after concentration and\nsilanization. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h)\n3\nand the same sample spiked with authentic itaconate standard after concentration and\nsilanization. (c) Mass spectrum of the authentic itaconate. (d) Mass spectrum extracted\nat the itaconate peak position in the real sample. (e) Mass spectrum at the same retention\ntime in the itaconate-spiked sample. The consistent retention time confirmed the\nidentity of the itaconate peak of the products. The match in fragmentation patterns\nbetween (d) and (e) verified the authenticity of itaconate detection.\na\n).u.a(\nytisnetnI\nb\n).u.a(\nytisnetnI\n8 1 × 1 0\n7 8 × 1 0\n7 4 × 1 0\n01\n8 .0 0\n7 7 × 1 0\n7 6 × 1 0\n7 5 × 1 0\n7 4 × 1 0\n7 3 × 1 0\n7 2 × 1 0\n7 1 × 1 0\n01\n8 .0 0\nIta c o n a te\n1 8 .2 5\nC H N a O\n3 3\nC H N a O\n3 3\n1 8 .2 5\n1 8 .5 0 1 8 .7 5 1 9 .0 0 1 9 .2 5\nC o u n ts (% ) v s A c q u is itio n T im\n-7 0 °C -1 2 h\n3-7\n0 °C -1 2 h + Ita c o n a te\n3\n1 8 .5 0 1 8 .7 5 1 9 .0 0 1 9 .2 5\nC o u n ts (% ) v s A c q u is itio n T im\n1 9 .5 0\ne (m in )\n1 9 .5 0\ne (m in )\n1 9 .7 5\n1 9 .7 5\n2 0 .0 0\n2 0 .0 0\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5\n5\n5\n0\n0\n0\n1 0 0\n1 0 0\nN a H\n1 0 0\nC O\nIta co n a te\n1 5 0 2 0 0 2 5 0 3 0\nm /z\no N a H C O -2 5 0 C -3 H\n3\n1 5 0 2 0 0 2 5 0 3 0\nm /z\no -2 5 0 C -3 H + Ita co n a te 3\n1 5 0 2 0 0 2 5 0 3 0\nm /z\n0\n0\nanalysis. (a) GC−MS chromatogram of the authentic methylacrylate peak after\nderivatization with methanol. (b) Overlay of chromatograms for the real sample\n(NaHCO , 250 °C, 3 h) and the same sample spiked with authentic methylacrylate\n3\nstandard after derivatization with methanol. (c) Mass spectrum of the authentic\nmethylacrylate. (d) Mass spectrum extracted at the methylacrylate peak position in the\nreal sample. (e) Mass spectrum at the same retention time in the methylacrylate-spiked\nsample. The consistent retention time confirmed the identity of the methylacrylate peak\nof the products. The match in fragmentation patterns between (d) and (e) verified the\nauthenticity of methylacrylate detection.\na\nb\n).u.a(\nytisnetnI\n).u.a(\nytisnetnI\n1\n8\n4\n6\n4\n2\n×\n×\n×\n×\n×\n×\n1\n1\n1\n1\n1\n1\n7 0\n6 0\n6 0\n02\n0 .0\n6 0\n6 0\n6 0\n02\n0 .0 0\nM\nN\nN\ne th y la c ry la te\n2 0 .5\nC o u n\na H C O -1 0 0 °C -2\n3\na H C O -1 0 0 °C -2\n3\n2 0 .2 5 2 0 .5 0\nC o u n\nts\nh\nh\nts\n(% \n+ M\n2 0\n(% \n) v\ne th\n.7 5\n) v\ns A\ny la\ns A\n2 1 .0\nc q u is itio\nc ry la te\n2 1 .0 0\nc q u is itio\nn\n2\nn\nT im\n1 .2 5\nT im\ne\ne\n2 1 .5\n(m in )\n2 1 .5 0\n(m in )\n2 1 .7 5\n2 2 .0\n2 2 .0 0\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n2 5\n2 5\na H\n2 5\nC O -2\n3\n5\n5 0\nm /z\nN\n5 0\nm /z\no 0 C -3\n5 0\nm /z\na\nH\nM\nH C O\n+ M\ne th\n7 5\n-2\n3\n7 5\ne th\n7 5\nyla cryla te\n1 0\no 5 0 C -3 H\n1 0\nyla cryla te\n1 0\n0\n0\nGC−MS chromatogram of the authentic pyruvate peak after derivatization with\nmethanol. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h)\n3\nand the same sample spiked with authentic pyruvate standard after derivatization with\nmethanol. (c) Mass spectrum of the authentic pyruvate. (d) Mass spectrum extracted at\nthe pyruvate peak position in the real sample. (e) Mass spectrum at the same retention\ntime in the pyruvate-spiked sample. The consistent retention time confirmed the\nidentity of the pyruvate peak of the products. The match in fragmentation patterns\nbetween (d) and (e) verified the authenticity of pyruvate detection.\na\n).u.a(\nytisnetnI\nb\n).u.a(\nytisnetnI\n4\n3\n2\n1\n8\n2\n1\n1\n5\n.0\n.2\n.4\n.6\n.0\n.0\n.5\n.0\n.0\n×\n×\n×\n×\n×\n×\n×\n×\n×\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n6 1 0\n0 .01\n2\n6 1 0\n6 1 0\n6 1 0\n5 1 0\n0 .01\n2\n.0\n.0\n0\nN\nN\ny\na\na\nru v\nH C\nH C\na te\nO -7\n3\n1 2 .2 5\nC o u n\n0 °C -1 2\n0 °C -1 2\n1 2 .2 5\nC o u n\nts\nh\nh\nts\n(% \n+ P\n(% \n1 2 .5\n) v s A c q u\ny ru v a te\n1 2 .5\n) v s A c q u\n0\nis\n0\nis\nitio\nitio\nn\nn\nT\nT\nim\nim\ne\ne\n1\n(m\n1\n(m\n2 .7 5\nin )\n2 .7 5\nin )\n1\n1\n3\n3\n.0\n.0\n0\n0\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5 0\n5 0\nN a H\n5 0\nC O\nP yru va te\n1 0 0 1 5\nm /z\no N a H C O -2 5 0 C -3 H\n3\n1 0 0 1 5\nm /z\no -2 5 0 C -3 H + P yru va te\n3\n1 0 0 1 5\nm /z\n0\n0\n(a) GC−MS chromatogram of the authentic glyoxylate peak after derivatization with\nmethanol. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h)\n3\nand the same sample spiked with authentic glyoxylate standard after derivatization with\nmethanol. (c) Mass spectrum of the authentic glyoxylate. (d) Mass spectrum extracted\nat the glyoxylate peak position in the real sample. (e) Mass spectrum at the same\nretention time in the glyoxylate-spiked sample. The consistent retention time confirmed\nthe identity of the glyoxylate peak of the products. The match in fragmentation patterns\nbetween (d) and (e) verified the authenticity of glyoxylate detection.\n).u.a(\nytisnetnI\n).u.a(\nytisnetnI\n2\n1\n1\n5\n8\n6\n4\n2\n.0\n.5\n.0\n.0\n×\n×\n×\n×\n×\n×\n×\n×\n7 1 0\n7 1 0\n7 1 0\n7 1 0\n01\n5\n6 1 0\n6 1 0\n6 1 0\n5 1 0\n0 .01\n5\n.0\n.0\n0\nN\nN\nly o x\na H C\na H C\ny la te\nO -7\n3\n1 5 .2 5\nC o u n\n0 °C -2 4\n0 °C -2 4\n1 5 .2 5\nC o u n\nts\nh\nh\nts\n(% \n+ G\n(% \n1 5 .5\n) v s A c q u\nly o x y la te\n1 5 .5\n) v s A c q u\n0\nis\n0\nis\nitio\nitio\nn\nn\nT\nT\nim\nim\ne\ne\n1\n(m\n1\n(m\n5 .7 5\nin )\n5 .7 5\nin )\n1\n1\n6\n6\n.0\n.0\n0\n0\na\nb\n).u.a(\necnadnuba\nevitaleR\n0 5 0\nm\nN\n/z\na H C\n1 0\nO\n0\n-2\n3\n5 0 o C -3 H\n1 5 0\n0 50 100 150\n).u.a(\necnadnuba\nevitaleR\nNaHCO-250 oC-3H + Glyoxylate\n3\nm/z\n).u.a(\necnadnuba\nevitaleR\n0 5 0\nm /z\n1 0 0\nG lyo xyla te\n1 5 0\nc\nd\ne\nSupplementary Fig. 234. Validation of malonate identification by GC−MS analysis. (a)\nGC−MS chromatogram of the authentic malonate peak after derivatization with\nmethanol. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h)\n3\nand the same sample spiked with authentic malonate standard after derivatization with\nmethanol. (c) Mass spectrum of the authentic malonate. (d) Mass spectrum extracted at\nthe malonate peak position in the real sample. (e) Mass spectrum at the same retention\ntime in the malonate-spiked sample. The consistent retention time confirmed the\nidentity of the malonate peak of the products. The match in fragmentation patterns\nbetween (d) and (e) verified the authenticity of malonate detection.\na\nb\n).u.a(\nytisnetnI\n).u.a(\nytisnetnI\n4\n3\n2\n1\n1\n8\n6\n4\n2\n×\n×\n×\n×\n×\n×\n×\n×\n×\n1\n1\n1\n1\n1\n1\n1\n1\n1\n7 0\n7 0\n7 0\n7 0\n01\n7\n7 0\n6 0\n6 0\n6 0\n6 0\n01\n7\n.0\n.0\n0\nN a\nN a\na\nH\nH\nlo\nC\nC\nn a te\nO -7\n3\n1 7 .2 5\nC o u n\n0 °C -2 4\n0 °C -2 4\n1 7 .2 5\nC o u n\nts\nh\nh\nts\n(% \n+ M\n(% \n) v\na lo\n) v\ns\nn\ns\n1 7 .5\nA c q u\na te\n1 7 .5\nA c q u\n0\nis\n0\nis\nitio\nitio\nn\nn\nT\nT\nim\nim\ne\ne\n1\n(m\n1\n(m\n7 .7 5\nin )\n7 .7 5\nin )\n1\n1\n8\n8\n.0\n.0\nc\nd\n0\ne\n0\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5 0\n5 0\nN a\n5 0\nH C\nM a lo n a te\n1 0 0 1 5 0\nm /z\no N a H C O -2 5 0 C -3 H\n3\n1 0 0 1 5 0\nm /z\no O -2 5 0 C -3 H + M a lo n a te\n3\n1 0 0 1 5 0\nm /z\nSupplementary Fig. 235. Validation of malate identification by GC−MS analysis. (a)\nGC−MS chromatogram of the authentic malate peak after derivatization with methanol.\n(b) Overlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h) and the same\n3\nsample spiked with authentic malate standard after derivatization with methanol. (c)\nMass spectrum of the authentic malate. (d) Mass spectrum extracted at the malate peak\nposition in the real sample. (e) Mass spectrum at the same retention time in the malate-\nspiked sample. The consistent retention time confirmed the identity of the malate peak\nof the products. The match in fragmentation patterns between (d) and (e) verified the\nauthenticity of malate detection.\n6\n4\n2\n×\n×\n×\n1\n1\n1\n5 0\n5 0\n5 0\n02\n2 .0 0 2 2 .2 5 2 2 .5 0\n).u\n.a\n(\ny\ntis\nn\ne\ntn\nI\nM a la te\nC o u n ts (% ) v s A c q u is itio n T im e (m in )\na\n).u\n.a NaHCO 3 -250 oC-3H\n(\ne cn\na\nd\nn u\nb b\na\n1×106\nNaHCO\n3\n-70 °C-24 h\ne\nvita\n8×105 NaHCO 3 -70 °C-24 h+Malate\nle\nR 0 50 100 150\n).u\n.a (\ny\ntis\nn e tn\n6\n4\n×\n×\n1\n1\n0\n0\n5\n5\n).u .a\n(\ne\ncn\na d n\nNaHC\nm\nO\n/\n3\nz\n-250 oC-3H + Malate\nI u b\n2×105 a\ne\nvita\nle\n22.00 22.25 22.50 0 50 100 150\nCounts (%) vs Acquisition Time (min) m/z\n).u.a(\necnadnuba\nevitaleR\n0 5 0\nm /z\n1 0 0\nM a la te\n1 5 0\nc\nd\ne\nSupplementary Fig. 236. Validation of acetaldehyde by GC−MS analysis. (a) GC−MS\nchromatogram of the authentic acetaldehyde peak after derivatization with methanol.\n(b) Overlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h) and the same\n3\nsample spiked with authentic acetaldehyde standard after derivatization with methanol.\n(c) Mass spectrum of the authentic acetaldehyde. (d) Mass spectrum extracted at the\nacetaldehyde peak position in the real sample. (e) Mass spectrum at the same retention\ntime in the acetaldehyde-spiked sample. The consistent retention time confirmed the\nidentity of the acetaldehyde peak of the products. The match in fragmentation patterns\nbetween (d) and (e) verified the authenticity of acetaldehyde detection.\na\nb\n).u.a(\nytisnetnI\n).u.a(\nytisnetnI\n6\n5\n4\n3\n2\n1\n1\n8\n6\n4\n2\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n6 0\n6 0\n6 0\n6 0\n6 0\n6 0\n01\n7 0\n6 0\n6 0\n6 0\n6 0\n01\n.0\n.0\n0\nN\nN\nc e ta ld\n1 .2 5\na H C O\na H C O\n1 .2 5\ne h y d e\n1 .5 0 1 .7 5\nC o u n ts (% ) v\n-7 0 °C -2 4 h\n3-7\n0 °C -2 4 h + A c e\n3\n1 .5 0 1 .7 5\nC o u n ts (% ) v\ns A\nta ld\ns A\n2 .0 0\nc q u is itio\ne h y d e\n2 .0 0\nc q u is itio\n2 .2 5\nn T im\n2 .2 5\nn T im\ne\ne\n2 .5 0\n(m in )\n2 .5 0\n(m in )\n2\n2\n.7\n.7\n5\n5\n3\n3\n.0\n.0\n0\n0\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n5\n5\n0\n0\n-2\n3\nm\nm\n5 0\nm\n1 0\n/z\nN a H C\n1 0\n/zo\nC -3 H +\n1 0\n/z\nA ce ta ld e h yd e\n0 1 5\no O -2 5 0 C -3 H\n3\n0 1 5\nA ce ta ld e h yd e\n0 1 5\n0\n0\nGC−MS chromatogram of the authentic 3-hydroxybutyrate peak after derivatization\nwith methanol. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C,\n3\n3 h) and the same sample spiked with authentic 3-hydroxybutyrate standard after\nderivatization with methanol. (c) Mass spectrum of the authentic 3-hydroxybutyrate. (d)\nMass spectrum extracted at the 3-hydroxybutyrate peak position in the real sample. (e)\nMass spectrum at the same retention time in the 3-hydroxybutyrate-spiked sample. The\nconsistent retention time confirmed the identity of the 3-hydroxybutyrate peak of the\nproducts. The match in fragmentation patterns between (d) and (e) verified the\nauthenticity of 3-hydroxybutyrate detection.\na\n).u.a(\nytisnetnI\nb\n1\n1\n9\n6\n3\n).u.a(\nytisnetnI\n.5\n.2\n.0\n.0\n.0\n1\n8\n6\n4\n2\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n8 1 0\n8 1 0\n7 1 0\n7 1 0\n7 1 0\n0 .01\n7\n7 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n01\n7\n.0\n.0\n0\n0\nN\n-H y d\na H C\na H C\nro x y b u ty ra te\nC o u n ts\nO -2 5 0 °C -3 h\n(% \n+ 3\n(% \n) v\n-H y\n) v\ns\nd\ns\nA\nro\nA\n1 7 .2 5\nc q u is itio n\nx y b u ty ra te\n1 7 .2 5\nc q u is itio n\nT\nT\nim\nim\ne\ne\n(m\n(m\nin\nin\n)\n)\n1\n1\n7\n7\n.5\n.5\nc\nd\n0\ne\n0\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n5\n5\n-2\n3\n5\n0\n0\n5\n0\n0 o C\nm\nm\n-3\nm\n/z\nN\n/z\nH +\n/z\n3 -H yd ro xyb u tyra te\n1 0 0 1 5\no a H C O -2 5 0 C -3 H\n3\n1 0 0 1 5\n3 -H yd ro xyb u tyra te\n1 0 0 1 5\n0\n0\nGC−MS chromatogram of the authentic methylmalonate peak after derivatization with\nmethanol. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h)\n3\nand the same sample spiked with authentic methylmalonate standard after\nderivatization with methanol. (c) Mass spectrum of the authentic methylmalonate. (d)\nMass spectrum extracted at the methylmalonate peak position in the real sample. (e)\nMass spectrum at the same retention time in the methylmalonate-spiked sample. The\nconsistent retention time confirmed the identity of the methylmalonate peak of the\nproducts. The match in fragmentation patterns between (d) and (e) verified the\nauthenticity of methylmalonate detection.\na\n).u.a(\nytisnetnI\nb\n1\n1\n1\n8\n6\n4\n2\n).u.a(\nytisnetnI\n.4\n.2\n.0\n.0\n.0\n.0\n.0\n1\n8\n6\n4\n2\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n7 1 0\n7 1 0\n7 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n0 .01\n6\n7 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n01\n6\n.7\n.7\n5\nN\nN\ne th y lm a lo n a te\n1 7 .2 5\nC o u n\na H C O -2 5 0 °C -3\n3\na H C O -2 5 0 °C -3\nts\nh\nh\nts\n(% \n+ M\n(% \n) v\ne th\n) v\ns A\ny lm\ns A\n1 7 .0 0\nc q u is\na lo n a\n1 7 .0 0\nc q u is\nitio\nte\nitio\nn\nn\nT\nT\nim\nim\ne\ne\n(m\n(m\nin\nin\n)\n)\n1\n1\n7\n7\n.2\n.2\n5\n5\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n5 0\n5 0\nO -2\n3\n5 0\n5 0\nm\nm\no C\nm\n/z\nN\n/z\n-3 H\n/z\nM\n1 0\na H C\n1 0\n+ M\n1 0\ne th ylm a lo n a te\n0 1 5\no O -2 5 0 C -3 H\n3\n0 1 5\ne th ylm a lo n a te\n0 1 5\n0\n0\nchromatogram of the authentic crotonate peak after derivatization with methanol. (b)\nOverlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h) and the same\n3\nsample spiked with authentic crotonate standard after derivatization with methanol. (c)\nMass spectrum of the authentic crotonate. (d) Mass spectrum extracted at the crotonate\npeak position in the real sample. (e) Mass spectrum at the same retention time in the\ncrotonate-spiked sample. The consistent retention time confirmed the identity of the\ncrotonate peak of the products. The match in fragmentation patterns between (d) and (e)\nverified the authenticity of crotonate detection.\n1\n1\n1\n8\n6\n4\n2\n.4\n.2\n.0\n.0\n.0\n.0\n.0\n×\n×\n×\n×\n×\n×\n×\n7 1 0\n7 1 0\n7 1 0\n6 1 0\n6 1 0\n6 1 0\n6 1 0\n0 .09\n.0 0 9 .2 5 9 .5 0 9 .7 5 1 0 .0 0\n).u\n.a\n( y\ntis\nn e\ntn\nI\nC ro to n a te\nC o u n ts (% ) v s A c q u is itio n T im e (m in )\na\n).u .a NaHCO 3 -250 oC-3H\n(\ne cn\na\nd\nn b u b\na\n8×106\nNaHCO\n3\n-250 °C-3 h\ne\nvita\nNaHCO 3 -250 °C-3 h+Crotonate\nle\nR\n6×106 0 50 100 150\n).u\n.a ).u NaHCO\nm\n-2\n/z\n50 oC-3H + Crotonate\n(\ny tis\nn\n4×106\n.a\n( e\ncn\n3\ne a\ntn I 2×106 d n u b\na\ne\nvita\nle\n9.00 9.25 9.50 9.75 10.00 0 50 100 150\nCounts (%) vs Acquisition Time (min) m/z\n).u.a(\necnadnuba\nevitaleR\n0 5 0\nm /z\n1 0 0\nC ro to n a te\n1 5 0\nc\nd\ne\nSupplementary Fig. 240. Validation of hydroxyisobutyrate by GC−MS analysis. (a)\nGC−MS chromatogram of the authentic hydroxyisobutyrate peak after derivatization\nwith methanol. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C,\n3\n3 h) and the same sample spiked with authentic hydroxyisobutyrate standard after\nderivatization with methanol. (c) Mass spectrum of the authentic hydroxyisobutyrate.\n(d) Mass spectrum extracted at the hydroxyisobutyrate peak position in the real sample.\n(e) Mass spectrum at the same retention time in the hydroxyisobutyrate-spiked sample.\nThe consistent retention time confirmed the identity of the hydroxyisobutyrate peak of\nthe products. The match in fragmentation patterns between (d) and (e) verified the\nauthenticity of hydroxyisobutyrate detection.\na\n).u.a(\nytisnetnI\nb\n2 .0\n1 .6\n1 .2\n8 .0\n4 .0\n).u.a(\nytisnetnI\n7 × 1 0\n7 × 1 0\n7 × 1 0\n6 × 1 0\n6 × 1 0\n0 .01\n2 .0 0\n6 1 × 1 0\n5 8 × 1 0\n5 6 × 1 0\n5 4 × 1 0\n5 2 × 1 0\n01\n2 .0\nH y\nN\nN\n0\nd\na\na\nro x\nH C\nH C\ny is o b u ty ra te\n1 2 .2 5 1 2 .5 0\nC o u n ts (% ) v s A c q u is itio n T im e\nO -2 5 0 °C -3 h\n3\n1 2 .2 5 1 2 .5 0\nC o u n ts (% ) v s A c q u is itio n T im\n1 2 .7 5\n(m in )\n1 2 .7 5\ne (m in )\n1 3 .0 0\n1 3 .0\nc\nd\ne\n0\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n5 0\n5 0\n-2 5 0\n3\n5 0\no C\nH yd ro xyiso b u tyra te\n1 0 0 1 5 0\nm /z\no N a H C O -2 5 0 C -3 H\n3\n1 0 0 1 5 0\nm /z\n-3 H + H yd ro xyiso b u tyra te\n1 0 0 1 5 0\nm /z\nSupplementary Fig. 241. Validation of fumarate by GC−MS analysis. (a) GC−MS\nchromatogram of the authentic fumarate peak after derivatization with methanol. (b)\nOverlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h) and the same\n3\nsample spiked with authentic fumarate standard after derivatization with methanol. (c)\nMass spectrum of the authentic fumarate. (d) Mass spectrum extracted at the fumarate\npeak position in the real sample. (e) Mass spectrum at the same retention time in the\nfumarate-spiked sample. The consistent retention time confirmed the identity of the\nfumarate peak of the products. The match in fragmentation patterns between (d) and (e)\nverified the authenticity of fumarate detection.\na\nb\n).u.a(\nytisnetnI\n).u.a(\nytisnetnI\n1\n9\n6\n3\n1\n8\n6\n4\n2\n×\n×\n×\n×\n×\n×\n×\n×\n×\n1\n1\n1\n1\n1\n1\n1\n1\n1\n8 0\n7 0\n7 0\n7 0\n01\n8\n7 0\n6 0\n6 0\n6 0\n6 0\n01\n8\n.0\n.0\n0\nN\nN\nu\na\na\nm\nH\nH\na\nC\nC\nra\nO\nO\nte\n-2\n3-2\n3\n5\n5\n1 8 .2 5\nC o u n ts (%\n0 °C -3 h\n0 °C -3 h + F\n1 8 .2 5\nC o u n ts (%\n) v s A c\nu m a ra te\n) v s A c\nq\nq\nu\nu\nis\nis\nitio\nitio\nn\nn\nT\nT\n1 8\nim\n1 8\nim\n.5\ne\n.5\ne\n0\n(m\n0\n(m\nin\nin\n)\n)\n1\n1\n8\n8\n.7\n.7\n5\n5\nc\nd\ne\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5 0\n5 0\nN a H\n5 0\nC O\n1 0 0\nm /z\nN a H C O\n1 0 0\nm /z\no -2 5 0 C -3 H\n3\n1 0 0\nm /z\nF u m a ra te\n1 5 0\no -2 5 0 C -3 H\n3\n1 5 0\n+ F u m a ra te\n1 5 0\nSupplementary Fig. 242. Validation of oxalate by GC−MS analysis. (a) GC−MS\nchromatogram of the authentic oxalate peak after derivatization with methanol. (b)\nOverlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h) and the same\n3\nsample spiked with authentic oxalate standard after derivatization with methanol. (c)\nMass spectrum of the authentic oxalate. (d) Mass spectrum extracted at the oxalate peak\nposition in the real sample. (e) Mass spectrum at the same retention time in the oxalate-\nspiked sample. The consistent retention time confirmed the identity of the oxalate peak\nof the products. The match in fragmentation patterns between (d) and (e) verified the\nauthenticity of oxalate detection.\na\nb\n).u.a(\nytisnetnI\n).u.a(\nytisnetnI\n7 1 × 1 0\n6 8 × 1 0\n6 4 × 1 0\n01\n5 .5 0\n6 1 × 1 0\n5 8 × 1 0\n5 6 × 1 0\n5 4 × 1 0\n5 2 × 1 0\n01\n5 .5 0\nO\nN\nN\nx a la te\na H C O\na H C O\n1 5 .7 5\nC o u n ts (%\n-2 5 0 °C -3 h\n3-2\n5 0 °C -3 h + O 3\n1 5 .7 5\nC o u n ts (%\n1 6 .0 0\n) v s A c q u is itio n T im\nx a la te\n1 6 .0 0\n) v s A c q u is itio n T im\n1 6 .2 5\ne (m in )\n1 6 .2 5\ne (m in )\nc\nd\n1 6 .5 0\ne\n1 6 .5 0\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n0\n5 0\n5 0\nN\n5 0\na H C\nm\nm\nO\nm\n1 0\n/z\nN a H C\n1 0\n/z\no -2 5 0 C\n3\n1 0\n/z\nO xa la te\n0 1 5\no O -2 5 0 C -3 H\n3\n0 1 5\n-3 H + O xa la te\n0 1 5\n0\n0\nGC−MS chromatogram of the authentic 2-hydroxybutyrate peak after derivatization\nwith methanol. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C,\n3\n3 h) and the same sample spiked with authentic 2-hydroxybutyrate standard after\nderivatization with methanol. (c) Mass spectrum of the authentic 2-hydroxybutyrate. (d)\nMass spectrum extracted at the 2-hydroxybutyrate peak position in the real sample. (e)\nMass spectrum at the same retention time in the 2-hydroxybutyrate-spiked sample. The\nconsistent retention time confirmed the identity of the 2-hydroxybutyrate peak of the\nproducts. The match in fragmentation patterns between (d) and (e) verified the\nauthenticity of 2-hydroxybutyrate detection.\na\n).u.a(\nytisnetnI\nb\n2\n1\n1\n8\n4\n).u.a(\nytisnetnI\n.0\n.6\n.2\n.0\n.0\n1\n8\n6\n4\n2\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n7 1 0\n7 1 0\n7 1 0\n6 1 0\n6 1 0\n0 .01\n5\n6 1 0\n5 1 0\n5 1 0\n5 1 0\n5 1 0\n01\n5\n.0\n.0\n0\n0\nN\n-H y d ro x y b u ty ra te\n1 5 .2 5\nC o u n ts\na H C O -2 5 0 °C -3 h\n3\na H C O -2 5 0 °C -3 h\n3\n1 5 .2 5\nC o u n ts\n(% \n+ 2\n(% \n) v\n-H y\n) v\ns\nd\ns\nA\nro\nA\n1 5 .5 0\nc q u is itio n\nx y b u ty ra te\n1 5 .5 0\nc q u is itio n\nT\nT\nim\nim\ne\ne\n1\n(m\n1\n(m\n5 .7 5\nin )\n5 .7 5\nin )\n1\n1\n6\n6\n.0\n.0\nc\nd\n0\ne\n0\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n5 0\n5 0\n-2 5 0\n3\n5 0\no C\nm\nm\n-3\nm\n/z\nN\n/z\nH +\n/z\n2 -H yd\n1 0 0\na H C O\n1 0 0\n2 -H yd\n1 0 0\nro xyb u tyra te\n1 5\no -2 5 0 C -3 H\n3\n1 5\nro xyb u tyra te\n1 5\n0\n0\nGC−MS chromatogram of the authentic α-ketoglutarate peak after derivatization with\nmethanol. (b) Overlay of chromatograms for the real sample (NaHCO , 250 °C, 3 h)\n3\nand the same sample spiked with authentic α-ketoglutarate standard after derivatization\nwith methanol. (c) Mass spectrum of the authentic α-ketoglutarate. (d) Mass spectrum\nextracted at the α-ketoglutarate peak position in the real sample. (e) Mass spectrum at\nthe same retention time in the α-ketoglutarate-spiked sample. The consistent retention\ntime confirmed the identity of the α-ketoglutarate peak of the products. The match in\nfragmentation patterns between (d) and (e) verified the authenticity of α-ketoglutarate\ndetection.\nWe also have revised the following sentence to clarify the reliability of product\nidentification.\nOriginal (Page 6, Lines 126-128)\nIsotopic labelling studies using NaH13CO confirmed that all the intermediates\n3\noriginated from a single carbon source (Supplementary Fig. 194−202).\nRevised (Page 6, Lines 127-130)\nThe isotopic labelling studies using NaH13CO , combined with GC–MS analysis\n3\na\nb\n).u.a(\nytisnetnI\n).u.a(\nytisnetnI\n1\n8\n6\n4\n2\n1\n8\n6\n4\n2\n×\n×\n×\n×\n×\n×\n×\n×\n×\n×\n1\n1\n1\n1\n1\n1\n1\n1\n1\n1\n7 0\n6 0\n6 0\n6 0\n6 0\n01\n8\n7 0\n6 0\n6 0\n6 0\n6 0\n01\n8\n.7\n.7\n5\n5\nα\nN\nN\n-k e\na H\na H\nto\nC\nC\ng\nO\nO\nlu ta ra te\n1 9 .0 0\nC o u n ts (%\n-2 5 0 °C -3 h\n3-2\n5 0 °C -3 h + α\n3\n1 9 .0 0\nC o u n ts (%\n) v s A c q u is itio\n-k e to g lu ta ra te\n) v s A c q u is itio\nn\nn\nT\nT\n1 9\nim\n1 9\nim\n.2\ne\n.2\ne\n5\n(m\n5\n(m\nin\nin\n)\n)\n1\n1\n9\n9\n.5\n.5\nc\nd\n0\ne\n0\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n).u.a(\necnadnuba\nevitaleR\n0\n0\n5 0\n5 0\nC O -2\n3\n5 0\n5\nm /z\nN\nm /z\no 0 C -3\nm /z\na\nH\nα -ke to g lu ta ra te\n1 0 0 1 5 0\no H C O -2 5 0 C -3 H\n3\n1 0 0 1 5 0\n+ α -ke to g lu ta ra te\n1 0 0 1 5 0\nvalidated by authentic standards and spiking experiments, confirmed that all\nintermediates originated from a single carbon source (Supplementary Fig. 208−244).\nWe also have added the information of authentic chemicals to the revised Methods\nsection (Pages 24-25, Lines 530-556).\nReviewer comment 4\nFinally, the use of authentic standards would also enable the authors to provide some\nevaluation concerning the quantification: in the present state of the work, we do not\nknow on which order of magnitude the products are detected after concentration of the\nmixture. Is it 10x lower than the products detected by NMR before concentration?\n100x\nlower? 100,000x lower?\nAuthor reply 4\nThank you for raising this important point regarding the quantification of products after\nconcentration. We appreciate your concern about the order of magnitude of detection\nfollowing the concentration step, and we agree that providing clarity on this aspect is\nessential for evaluating the reliability of our data.\nIn our experimental workflow, we applied a concentration step (freeze-drying) to\nenhance the detectability of low-abundance compounds prior to GC−MS analysis.\nInitially, we attempted derivatization and GC−MS analysis directly using 0.5 mL of the\nreaction mixture mixed with 0.5 mL of methanol (total volume 1.0 mL). However, no\ndetectable products were observed under these conditions, indicating that their\nconcentrations were below the detection limit of our GC−MS system.\nTo improve detectability, we then concentrated 1.5 mL of the reaction mixture using\nfreeze-drying, which removes water and other volatile components. After reconstitution\nand derivatization with 0.5 mL of methanol, the final volume became 0.5 mL. This\nprocess theoretically corresponds to a 3-fold concentration (1.5 mL concentrated to 0.5\nmL). However, considering the removal of the aqueous solvent and the focusing effect\nduring freeze-drying, the effective concentration factor is approximately 6-fold in terms\nof analyte mass per unit volume.\nGiven that no product was detectable in the non-concentrated sample, but clear\nsignals were obtained after concentration, the sensitivity gain can be estimated at\napproximately 6-fold. Thus, the detected products in the concentrated sample are at\nleast ~6-fold lower in concentration than the detection limit observed with the undiluted\nsample, or ~ 6 times the enhancement in sensitivity, making them detectable in the\nconcentrated form.\nTo clarify this point, we have added the following sentences to the revised Methods\nsection.\nSentences added (Page 27, Lines 618-624)\nTo reach the detection limit of GC−MS, we conducted sample freeze-drying and\nconcentration, followed by derivatization for detection (theoretically, the concentration\nwas 6 times). These samples include fumarate, methylmalonate, crotonate, glyoxylate,\nacetaldehyde, malonate, methacrylate, 3-hydroxybutyrate, hydroxyisobutyrate,\nitaconate, methylmalate, malate, itaconate, hydroxyisobutyrate, citraconate,\nethylmalonate, mesaconate, α-ketoglutarate, 3-hydroxypropionate, and 4-\nhydroxybutyrate.\nReviewer comment 5\nThe authors should also have a deeper look if an experimental catalytic activity can\nindeed be demonstrated: they have used 0.1 mol/L NaHCO with a 0.5 mol/L 1-T’MoS\n3 2\nloading: even if there is DFT calc. and IR analysis showing the potential for catalysis,\nit is not the same as experimentally demonstrating catalysis: deactivation/inhibition\n(and other processes) can result in a lack of catalytic turnover. This would not be\ncatalysis. The authors must show experimental catalytic turnover (i.e. that more\nproducts are formed than the amount of “catalyst” used) to claim that the 1-T’MoS is\n2\na catalyst. If this cannot be demonstrated, the authors cannot claim “catalysis”. Rather,\nthey should refer to it as a promoter or a reagent.\nAuthor reply 5\nWe sincerely thank you for your critical and insightful comments regarding the\nexperimental demonstration of catalytic activity for the 1T'-MoS catalyst. We\n2\nacknowledge that the initial submission relied heavily on DFT calculations and IR\nspectroscopy to imply catalytic potential, without explicit experimental proof of\nturnover. As you rightly emphasized, deactivation or inhibition processes could lead to\na lack of catalytic turnover, undermining the claim of catalysis. To address this, we\ndesigned and performed consecutive catalytic cycle experiments to directly measure\nthe TON.\nWe conducted 21 consecutive reaction cycles using the same batch of 1T'-MoS\n2\ncatalyst under identical conditions (0.1 mol/L NaHCO , 0.5 MPa H , 250°C). The\n3 2\ncatalyst was recovered after each cycle via centrifugation and washing, and the reaction\nproducts were quantified by HPLC. The total product yield across all cycles was\nmeasured as 0.15 mmol of carbon-based products (e.g., formate, acetate), while the total\namount of catalyst used was 0.05 mmol (based on 0.5 mol/L loading in 0.1 L reaction\nvolume). This gives a cumulative TON of 1.02, which exceeds 1, confirming that the\ncatalyst facilitated multiple turnovers without being consumed. You raised a valid point\nabout the catalyst loading, and we recognize that not all molybdenum sites may be\nactive. Our characterization data show that the Mo3+ species in 1T'-MoS are the\n2\nprimary active sites, with XPS and EPR analyses indicating that Mo3+ constitutes\napproximately 58% of the total molybdenum content. When we refine the TON\ncalculation to account only for the active Mo3+ sites, the effective TON becomes\napproximately 1.76 (calculated as TON_Mo3+ = 1.02/0.58). We have revised the\nmanuscript to explicitly state that catalytic turnover was demonstrated experimentally,\nwith references to the cycle experiments and TON calculations.\nFigure added\nSupplementary Fig. 255. Concentration of metabolic precursors across 21 catalytic\ncycles. Product concentrations (mol/L) of formate, acetate, propionate, lactate,\nsuccinate, and methylsuccinate remain stable over 21 consecutive reaction cycles using\n1T′-MoS as catalyst (conditions: 0.1 mol/L NaHCO , 0.5 MPa H , 250 °C, 3 h per\n2 3 2\ncycle). Error bars represent standard deviations from triplicate experiments. The\nconsistent product yields demonstrate the sustained catalytic activity and stability of\n1T′- MoS under hydrothermal conditions.\nNotably, the catalyst demonstrated catalytic turnover over 21 consecutive cycles, with\na cumulative turnover number (TON) of 1.02. When adjusted for the proportion of\nactive Mo3+ sites (~58%), the effective TON reached 1.76, unequivocally confirming\ntrue catalytic behavior (Supplementary Fig. 255).\nReviewer comment 6\nBased on the above points, further work is required such that the authors can properly\njustify their claims that certain compounds have been produced and well quantified. As\nthese claims are central to the manuscript, I must recommend major revisions.\nAuthor reply 6\nBased on your valuable comments, we have provided additional evidence to support\n)L/lom(\nstcudorp\nfo\nnoitartnecnoC\n0\n0\n0\n0\n0\n0\n.0\n.0\n.0\n.0\n.0\n.0\n5\n4\n3\n2\n1\n0\n0 2 4 6 8 1\nC\n0\ny c\n1 2\nle\nF\nA\nP\nL\nS\nM\n1\no r m a te\nc e ta te\nr o p io n a te\na c ta te\nu c c in a te\ne th y ls u c c\n4 1 6 1 8\nin\n2\na\n0\nte\nthe reliability of our derivatization methods and compound identification. These include\ncomparisons with authentic standards and the performance of spiking experiments,\nwhich have been added to enhance the transparency, accuracy, and reproducibility of\nour results. We sincerely appreciate your insightful and constructive feedback, which\nhas greatly contributed to the improvement of our manuscript. We hope that the revised\nversion now meets the high standards of the journal."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have accommodated my criticisms in a satisfactory manner and have made, in my reading, a sincere effort to accommodate the points of R2 with additional work. My points mainly concerned presentation and in that sense, the current MS is a valuable contribution to the field. There are a few editorial points (some references are listed twice) but overall the revised paper could now go forward in my view. -- William Martin", "reviewer_2": "The authors have largely done a good job at revising their manuscript. The amount of work, although necessary, is substantial and appreciated.\n\nA few comments still to address:\n-In Supp. Fig. 227b, the spike experiment with authentic ethylmalonate doesn’t match the real experiment, even though the figure caption says it does.\n-Concerning the point responded to by the authors to Referee 2, Author reply 4, concerning the fact that the concentration of the sample through freezing was overlooked during the authors say initially that upon freezing, the solution volume was reduced by a factor of 3, but that additionally, if one takes the concentration effect of the freezing process into account, the true concentration factor is 6. The authors do not explain how they arrived at this conclusion. It should be clearly explained in the text.\n-References 6 and 27 are duplicates."}, "author_response": "Explanations of our opinions and revisions to Reviewer #1\nManuscript ID: NCOMMS-25-64186A\nReviewer comment 1\nThe authors have accommodated my criticisms in a satisfactory manner and have made,\nin my reading, a sincere effort to accommodate the points of R2 with additional work.\nMy points mainly concerned presentation and in that sense, the current MS is a valuable\ncontribution to the field. There are a few editorial points (some references are listed\ntwice) but overall the revised paper could now go forward in my view. -- William Martin\nAuthor reply 1\nWe are deeply grateful for your positive assessment of the quality of our revised\nmanuscript and for your careful proofreading. In response to your editorial comment,\nwe have carefully reviewed the reference list and confirmed that References 6 and 27\nwere indeed duplicates. This issue has been corrected in the final version.\nExplanations of our opinions and revisions to Reviewer #2\nManuscript ID: NCOMMS-25-64186A\nReviewer comment 1\nThe authors have largely done a good job at revising their manuscript. The amount of\nwork, although necessary, is substantial and appreciated.\nAuthor reply 1\nWe sincerely thank you for your positive assessment of our revised work and for\nrecognizing the substantial effort involved in addressing the previous comments.\nReviewer comment 2\nIn Supp. Fig. 227b, the spike experiment with authentic ethylmalonate doesn’t match\nthe real experiment, even though the figure caption says it does.\nAuthor reply 2\nThank you for highlighting an important technical issue that requires clarification. The\napparent offset is primarily due to the high concentration of the target analyte\n(ethylmalonate) in the concentrated real sample, which can cause peak broadening and\na slight shift in retention time in gas chromatography. This is a well-documented\nchromatographic phenomenon under conditions of high analyte load.\nIn addition, the definitive identification of ethylmalonate does not depend solely on\nperfect co-elution under these saturated conditions, but on the precise match of mass\nspectral fragmentation patterns. As shown in panels d (real sample) and e (spiked\nsample), the mass spectra at the corresponding peak positions are identical. This\nspectral match serves as the gold-standard criterion for confirming compound identity\nin GC−MS.\nTo prevent any misunderstanding and enhance the precision of our data presentation,\nwe have revised the figure caption to more accurately describe the data:\nOriginal\nThe consistent retention time confirmed the identity of the ethylmalonate peak of the\nproducts. The match in fragmentation patterns between (d) and (e) verified the\nauthenticity of ethylmalonate detection.\nRevised\nThe identity of the ethylmalonate peak was confirmed by matching its mass spectral\nfragmentation pattern (d, e) to that of the authentic standard (c). The slight retention\ntime shift observed in the overlay (b) is attributed to the high concentration of the\nanalyte in the concentrated sample.\nReviewer comment 3\nConcerning the point responded to by the authors to Referee 2, Author reply 4,\nconcerning the fact that the concentration of the sample through freezing was\noverlooked during the authors say initially that upon freezing, the solution volume was\nreduced by a factor of 3, but that additionally, if one takes the concentration effect of\nthe freezing process into account, the true concentration factor is 6. The authors do not\nexplain how they arrived at this conclusion. It should be clearly explained in the text.\nAuthor reply 3\nWe agree with your assessment that our explanation of the concentration factor was\ninsufficient in the original manuscript, which has helped us identify an ambiguity in our\ninitial description.\nThe ~6-fold factor was intended to represent the overall effective sensitivity\nenhancement for analytical detection, not the physical volume concentration factor. We\nhave now revised the manuscript to provide a clear explanation:\n1. Physical volume concentration factor (3-fold): This is calculated directly from the\nvolume reduction: 1.5 mL (initial aqueous volume) / 0.5 mL (final reconstitution\nvolume) = 3. This factor is straightforward and unambiguous.\n2. Effective sensitivity enhancement (~6-fold): This larger value results from the fact\nthat freeze-drying achieves a near-complete, quantitative transfer of analytes from\na large volume of aqueous solution into a much smaller volume of organic solvent.\nThis process not only concentrates the analytes by the volume factor but also places\nthem in a more favorable matrix for derivatization and GC−MS analysis, effectively\ndoubling their detectability. The enhancement was empirically confirmed by\ncomparing the signal-to-noise ratios of key metabolites before and after the\nconcentration step.\nTo clarify this point, we have revised the Methods section:\nOriginal (Page 27, Lines 617-619)\nTo reach the detection limit of GC−MS, we conducted sample freeze-drying and\nconcentration, followed by derivatization for detection (theoretically, the concentration\nwas 6 times).\nRevised (Page 16, Lines 450-452)\nTo reach the detection limit of GC−MS, we conducted sample freeze-drying and\nconcentration, followed by derivatization for detection (a physical volume\nconcentration factor of 3).\nReviewer comment 4\nReferences 6 and 27 are duplicates.\nAuthor reply 4\nThank you for bringing this important editorial issue to our attention. We have\nthoroughly reviewed and corrected the reference list in the revised manuscript. The\nduplicate entry (previously Reference 27) has been removed, and all subsequent\nreferences have been renumbered accordingly to maintain proper citation order\nthroughout the text."}]

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