While hydrogen tunneling at elevated temperatures has, for instance, often been postulated in biochemical processes, spectroscopic proof is thus far limited to cryogenic conditions, under which thermal reactivity is negligible. We report spectroscopic evidence for H-tunneling in the gas phase at temperatures around 320-350 K observed in the isomerization reaction of a hydroxycarbene into an aldehyde. The charge-tagged carbene was generated in situ in a tandem mass spectrometer by decarboxylation of oxo[4-(trimethylammonio)phenyl]acetic acid upon collision induced dissociation. All ion structures involved are characterized by infrared ion spectroscopy and quantum chemical calculations. The charge-tagged phenylhydroxycarbene undergoes a 1,2-H-shift to the corresponding aldehyde with an half-life of about 10 s, evidenced by isomer-selective two-color (IR-IR) spectroscopy. In contrast, the deuterated (OD) carbene analogue showed much reduced 1,2-D-shift reactivity with an estimated half-life of at least 200 s under the experimental conditions, and provides clear evidence for hydrogen atom tunneling in the H-isotopologue. This is the first spectroscopic confirmation of hydrogen atom tunneling governing 1,2-H-shift reactions at noncryogenic temperatures, which is of broad significance for a range of (bio)chemical processes, including enzymatic transformations and organocatalysis.

We report the first generation and characterization of elusive Breslow intermediates derived from aromatic N-heterocyclic carbenes (NHCs), namely benzimidazolin-2-ylidenes (NMR, X-ray analysis) and thiazolin-2-ylidenes (NMR). In the former case, the diamino enols were generated by reaction of the free N,N-bis(2,6-diisopropylphenyl)- and N,N-bis(mesityl)-substituted benzimidazolin-2-ylidenes with aldehydes while the dimer of 3,4,5-trimethylthiazolin-2-ylidene served as the starting material in the latter case. The unambiguous NMR identification of the first thiazolin-2-ylidene-based Breslow intermediate rests on double C labeling of both the NHC and the aldehyde component. The acyl anion reactivity was confirmed by benzoin formation with excess aldehyde.

Breslow intermediates, first postulated in 1958, are pivotal intermediates in carbene-catalyzed umpolung. Attempts to isolate and characterize these fleeting amino enol species first met with success in 2012 when we found that saturated bis-Dipp/Mes imidazolidinylidenes readily form isolable, though reactive diamino enols with aldehydes and enals. In contrast, triazolylidenes, upon stoichiometric reaction with aldehydes, gave exclusively the keto tautomer, and no isolable enol. Herein, we present the synthesis of the "missing" keto tautomers of imidazolidinylidene-derived diamino enols, and computational thermodynamic data for 15 enol-ketone pairs derived from various carbenes/aldehydes. Electron-withdrawing substituents on the aldehyde favor enol formation, the same holds for N,N'-Dipp [2,6-di(2-propyl)phenyl] and N,N'-Mes [2,4,6-trimethylphenyl] substitution on the carbene component. The latter effect rests on stabilization of the diamino enol tautomer by Dipp substitution, and could be attributed to dispersive interaction of the 2-propyl groups with the enol moiety. For three enol-ketone pairs, equilibration of the thermodynamically disfavored tautomer was attempted with acids and bases but could not be effected, indicating kinetic inhibition of proton transfer.

Untargeted liquid chromatography−mass spectrometry (LC−MS)-based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing "grand challenge" in the utilization of this approach is metabolite identificationconfidently determining the chemical structures of m/z-detected unknowns.Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC−MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for massisolated targets. A significant advantage of this approach is that in silico-predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of α-aminoadipic semialdehyde (α-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While α-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics−IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids and that are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly provided novel insights into the pathophysiology of PDE-ALDH7A1.

The first generation and X‐ray diffraction (XRD) analysis of a crystalline Breslow intermediate (BI) derived from a thiazolin‐2‐ylidene, that is, the aromatic heterocycle present in vitamin B1, is reported. Key to success was the combined use of pentafluorobenzaldehyde and a thiazolin‐2‐ylidene carrying an enol‐stabilizing dispersion energy donor as N‐substituent. A so‐called primary intermediate (PI) could be isolated in protonated form (pPI) as well and analyzed by XRD. Furthermore, the first stable BI derived from an aromatic thiazolin‐2‐ylidene and an aliphatic aldehyde (trifluoroacetaldehyde) was prepared and characterized by NMR spectroscopy in solution. When switching to a saturated thiazolidin‐2‐ylidene, reaction with pentafluorobenzaldehyde afforded a new BI in solution (NMR spectroscopy). Attempts to crystallize the latter BI resulted in the isolation of a novel thiazolidin‐2‐ylidene dimer that had undergone rearrangement to a hexahydro[1,4]‐thiazino[3,2‐b]‐1,4‐thiazine.