Palladium-Catalyzed Nondirected Late-Stage C-H Deuteration of Arenes

We describe a palladium catalyzed non-directed late-stage deuteration of arenes. Key aspects include the use of D2O as a convenient and easily available deuterium source and the discovery of highly active N,N-bidentate ligands containing an N-acyl sulfonamide group. The reported protocol enables high degrees of deuterium incorporation via a reversible C-H activation step and features an extraordinary functional group tolerance, allowing for the deuteration of complex substrates. This is exemplified by the late-stage isotopic labelling of various pharmaceutically relevant motifs and related scaffolds. We expect that this method, amongst other applications, will prove useful as a tool in drug development processes and for mechanistic studies.

Light-driven decarboxylative deuteration enabled by a divergently engineered photodecarboxylase

Despite the well-established chemical processes for C-D bond formation, the toolbox of enzymatic methodologies for deuterium incorporation has remained underdeveloped. Here we describe a photodecarboxylase from Chlorella variabilis NC64A (CvFAP)-catalyzed approach for the decarboxylative deuteration of various carboxylic acids by employing D2O as a cheap and readily available deuterium source. Divergent protein engineering of WT-CvFAP is implemented using Focused Rational Iterative Site-specific Mutagenesis (FRISM) as a strategy for expanding the substrate scope. Using specific mutants, several series of substrates including different chain length acids, racemic substrates as well as bulky cyclic acids are successfully converted into the deuterated products (>40 examples). In many cases WT-CvFAP fails completely. This approach also enables the enantiocomplementary kinetic resolution of racemic acids to afford chiral deuterated products, which can hardly be accomplished by existing methods. MD simulations explain the results of improved catalytic activity and stereoselectivity of WT CvFAP and mutants.

Synthesis of α-deuterioalcohols by single-electron umpolung reductive deuteration of carbonyl using D2O as deuterium source

Deuterium incorporation can effectively stabilize the chiral centers of drug and agrochemical candidates that hampered by rapid in vivo racemization. In this work, the synthetically challenging chiral center deuteration of alcohols has been achieved via a single-electron umpolung reductive deuteration protocol using benign D2O as deuterium source and mild SmI2 as electron donor. The broad scope and excellent functional group tolerance of this method has been showcased by the synthesis of 43 respective α-deuterioalcohols in high yields and ≥98% deuterium incorporations. The potential application of this versatile method has been exemplified in the synthesis of 6 deuterated drug derivatives, 1 deuterated human hormone and 3 deuterated natural products. This method using D2O is greener and more efficient compared to traditional pyrophoric metal deuteride mediated reductive deuterations.

Acyl Fluorides as Direct Precursors to Fluoride Ketyl Radicals: Reductive Deuteration using SmI2 and D2O

A highly chemoselective reductive deuteration of acyl fluorides to provide α,α-dideuterio alcohols with exquisite levels of deuterium incorporation was developped using SmI2 and D2O as deuterium source. This method introduces...

Deuterium labelling by electrochemical splitting of heavy water

Deuterium incorporation is crucial in organic synthesis and has wide applications in the pharmaceutical industry. State-of-the-art H/D isotope exchange and chemical defunctionalization for deuterium incorporation suffer from significant drawbacks, including expensive deuterium sources, low deuteration efficiency and poor selectivity. In this perspective, we highlight an alternative pathway for forming C-D bonds by electrocatalytic heavy water splitting (D2O) under mild conditions. In addition, the intrinsic mechanism and examples of the synthesis of deuterated pharmaceuticals are discussed in detail. Finally, we present the challenges facing this field and provide an overall perspective on future research directions.

Hydrogen–Deuterium Exchange Mass Spectrometry Identifies Activated Factor IX-Induced molecular Changes in Activated Factor VIII

Hydrogen–deuterium exchange mass spectrometry (HDX-MS) was employed to gain insight into the changes in factor VIII (FVIII) that occur upon its activation and assembly with activated factor IX (FIXa) on phospholipid membranes. HDX-MS analysis of thrombin-activated FVIII (FVIIIa) revealed a marked increase in deuterium incorporation of amino acid residues along the A1–A2 and A2–A3 interface. Rapid dissociation of the A2 domain from FVIIIa can explain this observation. In the presence of FIXa, enhanced deuterium incorporation at the interface of FVIIIa was similar to that of FVIII. This is compatible with the previous finding that FIXa contributes to A2 domain retention in FVIIIa. A2 domain region Leu631-Tyr637, which is not part of the interface between the A domains, also showed a marked increase in deuterium incorporation in FVIIIa compared with FVIII. Deuterium uptake of this region was decreased in the presence of FIXa beyond that observed in FVIII. This implies that FIXa alters the conformation or directly interacts with this region in FVIIIa. Replacement of Val634 in FVIII by alanine using site-directed mutagenesis almost completely impaired the ability of the activated cofactor to enhance the activity of FIXa. Surface plasmon resonance analysis revealed that the rates of A2 domain dissociation from FVIIIa and FVIIIa-Val634Ala were indistinguishable. HDX-MS analysis showed, however, that FIXa was unable to retain the A2 domain in FVIIIa-Val634Ala. The combined results of this study suggest that the local structure of Leu631-Tyr637 is altered by FIXa and that this region contributes to the cofactor function of FVIII.

Microwave enhanced synthesis of halogenated derivatives of L-tyrosine labeled with deuterium in aromatic ring

Three halogenated derivatives of L-tyrosine, selectively labeled with deuterium in aromatic ring, i.e., 3′-fluoro-[5′-2H]-, 3′-chloro-[5′-2H]-, and 3′-iodo-[2′,5′-2H2]-L-tyrosine, were synthesized using microwave assisted acid-catalyzed isotope exchange between 3′-fluoro-, 3′-chloro- and 3′-iodo-L-tyrosine and heavy water. The degree of deuterium incorporation was confirmed by 1H NMR spectroscopy. The spectroscopic data indicate that isotope exchange depends on the method of heating and the power of microwaves. The deuterium enrichment of 3′-fluoro-[5′-2H]- and 3′-chloro-[5′-2H]-L-tyrosine amounted to 70% and 60%, respectively, while for 3′-iodo-[2′,5′-2H2]-L-tyrosine this value was about 50% and 95% for the 2′- and 5′-position. The isotopomers were obtained in good chemical yields of 50–70%.

Quantification of the necessary labelling input in protein stable isotope probing

Even though protein stable isotope probing is at least 100-times more sensitive than nucleic acid approaches, it is still unknown how much labelling input is necessary to not lose incorporation. Here, I used the average peptide model together with primary incorporation rates from previous experiments to determine the minimally necessary labelling input to detect incorporation of atoms present in proteins. Carbon-13 showed the highest efficiency for incorporation with detection misses only below 20.24 atom% labelling input. Despite the highest abundance in proteins, deuterium incorporation is not lost with at least 32.22 atom% labelling input due to instable acidic protons that undergo HD-exchange. All incorporation of oxygen-18 is detected with at least 28.15 atom% labelling input due to the +2 mass shift between its isotopes making up for its low abundance in proteins. Nitrogen-15 is less abundant in proteins and therefore harbors the most inefficient incorporation, resulting in a minimally necessary labelling input of 73.10 atom% labelling input. Altogether, these results can be used to properly design stable isotope probing experiments with the aim to maximize incorporation that can be extrapolated to other nucleic acid approaches due to similar incorporation routes.