Alkane Activation Initiated by Hydride Transfer: Co‐conversion of Propane and Methanol over H‐ZSM‐5 Zeolite

Co‐conversion of alkane with another reactant over zeolite catalysts has emerged as a new approach to the long‐standing challenge of alkane transformation. With the aid of solid‐state NMR spectroscopy and GC‐MS analysis, it was found that the co‐conversion of propane and methanol can be readily initiated by hydride transfer at temperatures of ≥449 K over the acidic zeolite H‐ZSM‐5. The formation of ¹³C‐labeled methane and singly ¹³C‐labeled n‐butanes in selective labeling experiments provided the first evidence for the initial hydride transfer from propane to surface methoxy intermediates. The results not only provide new insight into carbocation chemistry of solid acids, but also shed light on the low‐temperature transformation of alkanes for industrial applications.     

13C‐Isotope Labeled Surrogate for Estimating Organophosphorus Pesticides in Agricultural Products by Gas Chromatography‐Mass Spectrometry

¹³C‐isotope labeled paraoxon‐ethyl (¹³C₂‐EP) and deuterium‐labeled paraoxon‐methyl (D₆‐MP) were synthesized and employed as the surrogate (SS) and the internal standard (IS) in organophosphorus pesticides (OPs) spiking agricultural QC samples. The residual amounts of OPs were determined with gas chromatography‐mass spectrometry (GC‐MS) method. The isotope‐labeled compounds used in this study could assist the analysts to estimate the appropriateness and the uncertainties produced by pre‐treatment process. It was found that these isotope labeled compounds could improve the accuracy (10% to 40% of quantitative analysis), and provide efficacious calibration for the spiking recoveries of OPs in some agricultural samples.     

Synthesis of Selectively 15N‐Labeled 2′‐O‐{[(Triisopropylsilyl)oxy]methyl}(=tom)‐Protected Ribonucleoside Phosphoramidites and Their Incorporation into a Bistable 32Mer RNA Sequence

We present optimized reaction conditions for the conversion of 2′‐O‐{[(triisopropylsilyl)oxy]methyl}(=tom) protected uridine and adenosine nucleosides into the corresponding protected (3‐¹⁵N)‐labeled uridine and cytidine and (1‐¹⁵N)‐labeled adenosine and guanosine nucleosides 4, 6, 12 , and 18 , respectively (Schemes 1–4). On a DNA synthesizer, the resulting ¹⁵N‐labeled 2′‐O‐tom‐protected phosphoramidite building blocks 19 – 22 were efficiently incorporated into five selected positions of a bistable 32mer RNA sequence 23 (known to adopt two different structures) (Fig. 1). By 2D‐HSQC and HNN‐COSY experiments in H₂O/D₂O 9 : 1, the ¹⁵N‐signals of all base‐paired ¹⁵N‐labeled nucleotides could be identified and attributed to one of the two coexisting structures of 23 .     

A Sequential Homologation of Alkynes and Aldehydes for Chain Elongation with Optional 13C‐Labeling

Terminal alkynes (RCCH) are homologated by a sequence of ruthenium‐catalyzed anti‐Markovnikov hydration of alkyne to aldehyde (RCH₂CHO), followed by Bestmann–Ohira alkynylation of aldehyde to chain‐elongated alkyne (RCH₂CCH). Inverting the sequence by starting from aldehyde brings about the reciprocal homologation of aldehydes instead. The use of ¹³C‐labeled Bestmann–Ohira reagent (dimethyl ((1‐¹³C)‐1‐diazo‐2‐oxopropyl)phosphonate) for alkynylation provides straightforward access to singly or, through additional homologation, multiply ¹³C‐labeled alkynes. The labeled alkynes serve as synthetic platform for accessing a multitude of specifically ¹³C‐labeled products. Terminal alkynes with one or two ¹³C‐labels in the alkyne unit have been submitted to alkyne–azide click reactions; the copper‐catalyzed version (CuAAC) was found to display a regioselectivity of >50 000:1 for the 1,4‐ over the 1,5‐triazine isomer, as shown analytically by ¹³C NMR spectroscopy.     

A Non‐Heme Iron Photocatalyst for Light‐Driven Aerobic Oxidation of Methanol

Non‐heme (L)Fe^III and (L)Fe^III‐O‐Fe^III(L) complexes (L=1,1‐di(pyridin‐2‐yl)‐N,N‐bis(pyridin‐2‐ylmethyl)ethan‐1‐amine) underwent reduction under irradiation to the Fe^II state with concomitant oxidation of methanol to methanal, without the need for a secondary photosensitizer. Spectroscopic and DFT studies support a mechanism in which irradiation results in charge‐transfer excitation of a Fe^III?μ‐O?Fe^III complex to generate [(L)Fe^IV=O]²⁺ (observed transiently during irradiation in acetonitrile), and an equivalent of (L)Fe^II. Under aerobic conditions, irradiation accelerates reoxidation from the Fe^II to the Fe^III state with O₂, thus closing the cycle of methanol oxidation to methanal.     

18F‐Labeling of Aryl‐SCF3, ‐OCF3 and ‐OCHF2 with [18F]Fluoride

We report that halogenophilic silver(I) triflate permits halogen exchange (halex) nucleophilic ¹⁸F‐fluorination of aryl‐OCHFCl, ‐OCF₂Br and ‐SCF₂Br precursors under mild conditions. This Ag^I‐mediated process allows for the first time access to a range of ¹⁸F‐labeled aryl‐OCHF₂, ‐OCF₃ and ‐SCF₃ derivatives, inclusive of [¹⁸F]riluzole. The ¹⁸F‐labeling of these medicinally important motifs expands the radiochemical space available for PET applications.     

On the Solution Structure of PHB: Preparation and NMR Analysis of Isotopically Labeled Oligo[(R)‐3‐hydroxybutanoic Acids] (OHBs)

While the chain conformation of poly‐ and oligo[(R)‐3‐hydroxybutanoate] (PHB, OHB) is known to be 2₁‐ and 3₁‐helical in stretched fibers and in the crystalline state, respectively (Fig. 2), the structure in solution is unknown. To be able to determine the NMR‐solution structure, specifically labeled linear oligomers have been prepared: a 16‐mer consisting of alternating pairs of fully ¹³C‐labeled and non‐labeled residues ( 1 ) and a 20‐mer containing an O‐¹³CH(¹³CH₂D)‐¹³CHD^Si‐¹³CO residue in position 9 (from the O‐terminus) and a fully ¹³C‐labeled residue in position 12 ( 2 ), both with (t‐Bu)Ph₂Si protection at the O‐ and Bn protection at the C‐terminus. The labeled (R)‐3‐hydroxybutanoic acid building blocks were prepared by Noyori hydrogenation of the ethyl ester of fully ¹³C‐labeled acetoacetic acid, and the D‐atoms were incorporated by D₂/Pd‐C reduction of a previously reported dibromo‐1,3‐dioxinone 8 (Scheme 1). The oligomers were obtained by a series of fragment couplings (Schemes 2 and 3). 600‐MHz NMR COSY, HSQC, ROESY, and cross‐correlated relaxation measurements (Figs. 4 – 6, 9, and 12, and Tables 1 – 3) at different temperatures and interpretations thereof led to assignments of all resonances, including those from the diastereotopic C(2)H₂ protons, and to determination of the conformationally averaged dihedral angles ϕ₂ and ϕ₃ (Figs. 2, 7, and 8) in the chain of the oligoester. The conclusions are: all but five or six terminal residues adopt the same conformation; the 2₁ helix is not the predominant secondary structure; the structure of the HB chain is averaged, even at –30°. Our investigation confirms the high flexibility of the polyester chain, a property that has been deduced previously from biological studies of PHB in membranes, in ion channels, and as appendage of proteins.     

Designing Point Mutants to Detect Structural Coupling in a Heterotrimeric G Protein α‐subunit by NMR Spectroscopy†

To better understand the mechanism by which the activating signal is transmitted from the receptor‐interacting regions on the G protein α‐subunit (G_α) to the guanine nucleotide‐binding pocket, we generated and characterized mutant forms of G_α with alterations in switch II (Trp‐207→Phe) and the carboxyl‐terminus (Phe‐350→Ala). Previously reported bacterial expression methods for the high‐level production of a uniformly isotope‐labeled G_tα/G_i1α chimera, ChiT, were successfully used to isolate milligram quantities of ¹⁵N‐labeled mutant protein. NMR analysis showed that while the GDP/Mg²⁺‐bound state of both mutants shared an overall conformation similar to that of the GDP/Mg²⁺‐bound state of ChiT, formation of the “transition/activated” state in the presence of aluminum fluoride (AlF₄^?) revealed distinct differences between the wild‐type and mutant G_α subunits, particularly in the response of the ¹HN, ¹⁵N cross‐peak for the Trp‐254 indole in the Trp‐207→Phe mutant and the ¹HN, ¹⁵N cross‐peak for Ala‐350 in the Phe‐350→Ala mutant. Consistent with the NMR data, the F350→Ala mutant showed an increase in intrinsic fluorescence that was similar to G_tα and ChiT upon formation of the “transition/activated” state in the presence of AlF₄^?, whereas the intrinsic fluorescence of the Trp‐207→Phe mutant decreased. These results show that the substitution of key amino acid positions in G_α can effect structural changes that may compromise receptor interactions and GDP/GTP exchange.     

HPLC‐DAD and HPLC‐ESI‐MS separation,determination and identification of the spin‐labeled diastereoisomers of podophyllotoxin

Spin‐labeled nitroxide derivatives of podophyllotoxin had better antitumor activity and less toxicity than that of the parent compounds. However, the 2‐H configurations of these spin‐labeled derivatives cannot be determined by nuclear magnetic resonance (NMR) methods. In the present paper, a high‐performance liquid chromatography‐diode array detection (HPLC‐DAD) and a high‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI/MS/MS) method were developed and validated for the separation, identification of four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 position. In the HPLC‐ESI/MS spectra, each pair of diastereoisomers of the spin‐labeled derivatives in the mixture was directly confirmed and identified by [M+H]⁺ ions and ion ratios of relative abundance of [M‐ROH+H]⁺ (ion 397) to [M+H]⁺. When the [M‐ROH+H]⁺ ions (at m/z 397) were selected as the precursor ions to perform the MS/MS product ion scan. The product ions at m/z 313, 282, and 229 were the common diagnostic ions. The ion ratios of relative abundance of the [M‐ROH+H]⁺ (ion 397) to [M+H]⁺, [A+H]⁺ (ion 313) to [M‐ROH+H]⁺, [A+H‐OCH₃]⁺ (ion 282) to [M‐ROH+H]⁺ and [M‐ROH‐ArH+H]⁺ (ion 229) to [M‐ROH+H]⁺ of each pair of diastereoisomers of the derivatives specifically exhibited a stereochemical effect. Thus, by using identical chromatographic conditions, the combination of DAD and MS/MS data permitted the separation and identification of the four pairs of diastereoisomers of spin‐labeled derivatives of podophyllotoxin at C‐2 in the mixture.     

NMR‐Structural Investigations of a β3‐Dodecapeptide with Proteinogenic Side Chains in Methanol and in Aqueous Solutions

The structural properties of an all‐β³‐dodecapeptide with the sequence H‐β‐HLys(N^ε‐CO(CH₂)₃‐S Acm)‐β‐HPhe‐β‐HTyr‐β‐HLeu‐β‐HLys‐β‐HSer‐β‐HLys‐β‐HPhe‐β‐HSer‐β‐HVal‐β‐HLys‐β‐HAla‐OH ( 1 ) have been studied by two‐dimensional homonuclear ¹H‐NMR and by CD spectroscopy. In MeOH solution, high‐resolution NMR spectroscopy showed that the β‐dodecapeptide forms an (M)‐3₁₄‐helix, and the CD spectrum corresponds to the pattern expected for an (M)‐3₁₄‐helical secondary structure. In aqueous solution, however, the peptide adopts a predominantly extended conformation without regular secondary‐structure elements, which is in agreement with the absence of the characteristic trough near 215 nm in the CD spectrum. The NMR and CD measurements with solutions of 1 in MeOH containing 3M urea further indicated that the peptide retains the regular secondary structural elements under these conditions, whereas, after addition of 40% (v/v) H₂O to the MeOH solution, the large ¹H‐chemical‐shift dispersion indicative of a defined spatial peptide fold was lost. The β³‐dodecapeptide is – so far – the longest β‐peptide shown to adopt a regular (M)‐3₁₄‐helix conformation in an organic solvent. The observation that the structure of this long β³‐peptide is not maintained in aqueous solution indicates that the (M)‐3₁₄‐fold is primarily stabilized by short‐range interactions.     

Synthesis and Bioimaging of Positron‐Emitting 15O‐Labeled 2‐Deoxy‐D‐glucose of Two‐Minute Half‐Life

In positron emission tomography (PET), which exploits the affinity of a radiopharmaceutical for the target organ, a systematic repertoire of oxygen‐15‐labeled PET tracers is expected to be useful for bioimaging owing to the ubiquity of oxygen atoms in organic compounds. However, because of the 2‐min half‐life of ¹⁵O, the synthesis of complex biologically active ¹⁵O‐labeled organic molecules has not yet been achieved. A state‐of‐the‐art synthesis now makes available an ¹⁵O‐labeled complex organic molecule, 6‐[¹⁵O]‐2‐deoxy‐D ‐glucose. Ultrarapid radical hydroxylation of 2,6‐dideoxy‐6‐iodo‐D ‐glucose with molecular oxygen labeled with ¹⁵O of two‐minute half‐life provided the target ¹⁵O‐labeled molecule. The labeling reaction with ¹⁵O was complete in 1.3 min, and the entire operation time starting from the generation of ¹⁵O‐containing dioxygen by a cyclotron to the purification of the labeled sugar was 7 min. The labeled sugar accumulated in the metabolically active organs as well as in the bladder of mice and rats. ¹⁵O‐labeling offers the possibility of repetitive scanning and the use of multiple PET tracers in the same body within a short time, and hence should significantly expand the scope of PET studies of small animals.     

Simultaneous detection of stable isotope‐labeled and unlabeled l‐tryptophan and of its main metabolites,l‐kynurenine,serotonin and quinolinic acid,by gas chromatography/negative ion chemical ionization mass spectrometry

A method for the detection of unlabeled and ¹⁵N₂‐labeled l ‐tryptophan (l ‐Trp), l ‐kynurenine (l ‐Kyn), serotonin (5‐HT) and quinolinic acid (QA) in human and rat plasma by GC/MS is described. Labeled and unlabeled versions of these four products were analyzed as their acyl substitution derivatives using pentafluoropropionic anhydride and 2,2,3,3,3‐pentafluoro‐1‐propanol. Products were then separated by GC and analyzed by selected ion monitoring using negative ion chemical ionization mass spectrometry. l ‐[¹³C₁₁, ¹⁵N₂]‐Trp, methyl‐serotonin and 3,5‐pyridinedicarboxylic acid were used as internal standards for this method. The coefficients of variation for inter‐assay repeatability were found to be approximately 5.2% for l ‐Trp and ¹⁵N₂‐Trp, 17.1% for l ‐Kyn, 16.9% for 5‐HT and 5.8% for QA (n = 2). We used this method to determine isotope enrichments in plasma l ‐Trp over the course of a continuous, intravenous infusion of l ‐[¹⁵N₂]Trp in pregnant rat in the fasting state. Plasma ¹⁵N₂‐Trp enrichment reached a plateau at 120 min. The free Trp appearance rate (Ra) into plasma was 49.5 ± 3.35 µmol/kg/h. The GC/MS method was applied to determine the enrichment of ¹⁵N‐labeled l ‐Trp, l ‐Kyn, 5‐HT and QA concurrently with the concentration of non‐labeled l ‐Trp, l ‐Kyn, 5‐HT and QA in plasma. This method may help improve our understanding on l ‐Trp metabolism in vivo in animals and humans and potentially reveal the relative contribution of the four pathways of l ‐Trp metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

NMR “Crystallography” for Uniformly (13C, 15N)‐Labeled Oriented Membrane Proteins

In oriented‐sample (OS) solid‐state NMR of membrane proteins, the angular‐dependent dipolar couplings and chemical shifts provide a direct input for structure calculations. However, so far only ¹H–¹⁵N dipolar couplings and ¹⁵N chemical shifts have been routinely assessed in oriented ¹⁵N‐labeled samples. The main obstacle for extending this technique to membrane proteins of arbitrary topology has remained in the lack of additional experimental restraints. We have developed a new experimental triple‐resonance NMR technique, which was applied to uniformly doubly (¹⁵N, ¹³C)‐labeled Pf1 coat protein in magnetically aligned DMPC/DHPC bicelles. The previously inaccessible ¹H_α–¹³C_α dipolar couplings have been measured, which make it possible to determine the torsion angles between the peptide planes without assuming α‐helical structure a priori. The fitting of three angular restraints per peptide plane and filtering by Rosetta scoring functions has yielded a consensus α‐helical transmembrane structure for Pf1 protein.     

Small‐Sized Tungsten Nitride Particles Strongly Anchored on Carbon Nanotubes and their Use as Supports for Pt for Methanol Electro‐oxidation

The anchoring of small‐sized WN (tungsten nitride) nanoparticles (NPs) with good dispersion on carbon nanotubes (CNTs) offers an effective means of obtaining promising materials for use in electrocatalysis. Herein, an effective method based on grinding treatment followed by a nitridation process is proposed to realize this goal. In the synthesis, a solution containing H₄[SiO₄(W₃O₉)₄] (SiW₁₂) and CNTs modified with polyethylenimine (PEI‐CNTs) was ground to dryness. Small‐sized WN NPs were anchored onto the CNTs with good dispersion after calcination under NH₃. Under hydrothermal assembly conditions (absence of grinding), WN particles of larger size and with inferior dispersion were obtained, demonstrating the important role of the grinding process. The benefit of the small‐sized WN has been demonstrated by using WN/CNTs as a support for Pt to catalyze the methanol electro‐oxidation reaction. The mass activity of Pt‐WN/CNTs‐G‐70 (where G denotes the grinding treatment, and 70 is the loading amount (%) of WN in the WN/CNTs) was evaluated as about 817 mA mg^?1_Pt, better that those of commercial Pt/C (340 mA mg^?1_Pt) and Pt/CNTs (162 mA mg^?1_Pt). The Pt‐WN/CNTs‐G also displayed good CO tolerance. In contrast, Pt‐WN/CNTs prepared without the grinding process displayed an activity of 344 mA mg^?1_Pt, verifying the key role of grinding treatment in the preparation of WN/CNTs with good co‐catalytic effect.     

A General Copper‐Mediated Nucleophilic 18F Fluorination of Arenes

Molecules labeled with fluorine‐18 are used as radiotracers for positron emission tomography. An important challenge is the labeling of arenes not amenable to aromatic nucleophilic substitution (S_NAr) with [¹⁸F]F^?. In the ideal case, the ¹⁸F fluorination of these substrates would be performed through reaction of [¹⁸F]KF with shelf‐stable readily available precursors using a broadly applicable method suitable for automation. Herein, we describe the realization of these requirements with the production of ¹⁸F arenes from pinacol‐derived aryl boronic esters (arylBPin) upon treatment with [¹⁸F]KF/K₂₂₂ and [Cu(OTf)₂(py)₄] (OTf=trifluoromethanesulfonate, py=pyridine). This method tolerates electron‐poor and electron‐rich arenes and various functional groups, and allows access to 6‐[¹⁸F]fluoro‐L ‐DOPA, 6‐[¹⁸F]fluoro‐m‐tyrosine, and the translocator protein (TSPO) PET ligand [¹⁸F]DAA1106.     

Gold Nanoparticles Doped Three‐Dimensional Sol‐gel Matrix for Amperometric Human Chorionic Gonadotrophin Immunosensor

A novel electrochemical immunosensor for human chorionic gonadotrophin (hCG) was proposed by immobilization of hCG in gold nanoparticles doped three‐dimensional (3D) sol‐gel matrix and an interfacial competitive immunoreaction. The 3D organized composite structure was prepared by assemble of gold nanoparticles into a hydrolyzed (3‐mercaptopropyl)‐trimethoxysilane sol‐gel matrix, which showed good biocompatibility. After the interfacial competitive immunoreaction the formed HRP‐labeled immunoconjugate showed good enzymatic activity for the oxidation of o‐phenylenediamine by H₂O₂. With a competitive format, a method comprising of o‐phenylenediamine‐H₂O₂‐immobilized HRP labeled hCG immunoconjugate system for immunoassay of hCG from 5.0 to 30.0 mIU mL^?1 was developed. The immunosensor showed good precision, high sensitivity, acceptable stability and reproducibility and could be used for detection of hCG in human serum with the consistent results in comparison with those obtained by a commercial analyzer.     

A High‐Valent Non‐Heme μ‐Oxo Manganese(IV) Dimer Generated from a Thiolate‐Bound Manganese(II) Complex and Dioxygen

This study deals with the unprecedented reactivity of dinuclear non‐heme Mn^II–thiolate complexes with O₂, which dependent on the protonation state of the initial Mn^II dimer selectively generates either a di‐μ‐oxo or μ‐oxo‐μ‐hydroxo Mn^IV complex. Both dimers have been characterized by different techniques including single‐crystal X‐ray diffraction and mass spectrometry. Oxygenation reactions carried out with labeled ¹⁸O₂ unambiguously show that the oxygen atoms present in the Mn^IV dimers originate from O₂. Based on experimental observations and DFT calculations, evidence is provided that these Mn^IV species comproportionate with a Mn^II precursor to yield μ‐oxo and/or μ‐hydroxo Mn^III dimers. Our work highlights the delicate balance of reaction conditions to control the synthesis of non‐heme high‐valent μ‐oxo and μ‐hydroxo Mn species from Mn^II precursors and O₂.     

Ionization‐Induced Solvent Migration in Acetanilide‐Methanol Clusters Inferred from Isomer‐Selective Infrared Spectroscopy

We present the resonance‐enhanced multiphoton ionization, infrared‐ultraviolet hole burning (IR‐UV HB), and IR dip spectra of the trans‐acetanilide–methanol (AA–MeOH) cluster in the S₀, S₁, and cationic ground state (D₀) in a supersonic jet. The IR‐UV HB spectra demonstrate the co‐existence of two isomers in S_0,1, in which MeOH binds either to the NH or the CO site of the peptide linkage in AA, denoted as AA(NH)–MeOH and AA(CO)–MeOH. When AA(CO)–MeOH is selectively ionized, its IR spectrum in D₀ is the same as that measured for AA⁺(NH)–MeOH. Thus, photoionization of AA(CO)–MeOH induces migration of MeOH from the CO to the NH site with 100% yield.     

Supramolecular Complexation in Biological Media: NMR Study on Inclusion of an Anionic Tetraarylporphyrin into a Per‐O‐Methylated β‐Cyclodextrin Cavity in Serum,Blood, and Urine

The supramolecular complexation of 5,10,15,20‐tetrakis(4‐sulfonatophenyl)porphyrin (TPPS) with heptakis(2,3,6‐tri‐O‐methyl)‐β‐cyclodextrin (TMCD) has been known to be highly specific in aqueous media. In this study, we have used NMR spectroscopy to reveal that this supramolecular system also works even in biologically crowded media such as serum, blood, and urine. A ¹³C‐labeled heptakis(2,3,6‐tri‐O‐methyl‐¹³C)‐β‐cyclodextrin (¹³C‐TMCD) was synthesized and studied using one‐dimensional (1D) HMQC spectroscopy in serum and blood. The 1D HMQC spectrum of ¹³C‐TMCD showed clear signals due to the 2‐, 3‐, and 6‐O¹³CH₃ groups, whose chemical shifts changed upon addition of TPPS due to quantitative formation of the ¹³C‐TMCD/TPPS=2/1 inclusion complex in such biological media. The ¹H NMR signals of non‐isotope‐labeled TPPS included by ¹³C‐TMCD were detected using the ¹³C‐filtered ROESY technique. A pharmacokinetic study of ¹³C‐TMCD and its complex with TPPS was carried out in mice using the 1D HMQC method. The results indicated that (1) 1D HMQC is an effective technique for monitoring the inclusion phenomena of ¹³C‐labeled cyclodextrin in biological media and (2) the intermolecular interaction between ¹³C‐TMCD and TPPS is highly selective even in contaminated media like blood, serum, and urine.     

Non‐natural Cofactor and Formate‐Driven Reductive Carboxylation of Pyruvate

A non‐natural cofactor and formate driven system for reductive carboxylation of pyruvate is presented. A formate dehydrogenase (FDH) mutant, FDH*, that favors a non‐natural redox cofactor, nicotinamide cytosine dinucleotide (NCD), for generation of a dedicated reducing equivalent at the expense of formate were acquired. By coupling FDH* and NCD‐dependent malic enzyme (ME*), the successful utilization of formate is demonstrated as both CO₂ source and electron donor for reductive carboxylation of pyruvate with a perfect stoichiometry between formate and malate. When ¹³C‐isotope‐labeled formate was used in in vitro trials, up to 53 % of malate had labeled carbon atom. Upon expression of FDH* and ME* in the model host E. coli, the engineered strain produced more malate in the presence of formate and NCD. This work provides an alternative and atom‐economic strategy for CO₂ fixation where formate is used in lieu of CO₂ and offers dedicated reducing power.