In situ Generated Iridium Nanoparticles as Hydride Donors in Photoredox-Catalyzed Hydrogen Isotope Exchange Reactions with Deuterium and Tritium Gas.

We have studied the photoredox-catalyzed hydrogen isotope exchange (HIE) reaction with deuterium or tritium gas as isotope sources and in situ formed transition metal nanoparticles as hydrogen atom transfer pre-catalysts. By this means we have found synergistic reactivities applying two different HIE mechanisms, namely photoredox-catalyzed and CH-functionalization HIE leading to the synthesis of highly deuterated complex molecules. Finally, we adopted these findings successfully to tritium chemistry.

Small-scale two-dimensional liquid chromatography for a preparative re-purification of a highly labile tritium-labeled compound.

Tritium-labeled compounds are generally less stable than their non-labeled counterparts. This requires storage at low temperatures, a constant workflow of quality checks, and subsequent re-purifications. As the amount of tritium-labeled material is typically purified in the µg range, repeated injections on analytical-scale ultra high-performance liquid chromatography systems can provide high-resolution re-purification results. Yet, degradants can be undesirably included in the compound isolation because the amount of decomposition can vary dramatically depending on the structure. We report a case of a sensitive molecule that could not be isolated in pure form even though the chromatographic separation was successful. In this case, the use of a small-scale two-dimensional preparative liquid chromatography approach with a direct transfer interface to a second (trapping) column resulted in a highly pure compound (>98% radiochemical purity). This approach combines high chromatographic resolution, accurate control over the re-purification process, minimal sample manipulation, and higher overall safety for the handling of radioactive samples.

Synthesis and analysis of isotopically stable labeled nitrosamines as mass spectrometry standards for drug impurity quality control.

We describe a simple and easy pathway to synthesize nitrosamine mass spectrometry standards in good to moderate yields. N-alkylation of Boc-protected primary or secondary amines using stable isotope labeled alkyl halides yielded the key intermediates that were deprotected, and then, the nitrosamine was formed with sodium nitrite and sodium hydrogensulfate. Special attention to safety, disposal of waste, and surface cleaning was carried throughout.

Photoredox-Mediated Hydrogen Isotope Exchange Reactions of Amino-Acids, Peptides, and Peptide-Derived Drugs.

Hydrogen isotopically labelled compounds are essential diagnostic tools in drug research and development, as they provide vital information about the biological metabolism of drug candidates and their metabolites. Herein we report a photoredox-initiated hydrogen atom transfer (HAT) protocol which efficiently and selectively introduces deuterium or tritium at C(sp3 )-H bonds, utilizing heavy water (D2 O or T2 O) as the hydrogen isotope source, and a guanidine base. This protocol has been successfully applied to the incorporation of deuterium in several amino acids (lysine, glycine and proline) and small peptides. Finally, the method has been applied to tritium, because tritium-labelled peptides are essential for application in biological experiments, such as ligand-binding assays, or absorption, distribution, metabolism, and excretion (ADME) studies.

Side chain removal from corticosteroids by unspecific peroxygenase.

Two unspecific peroxygenases (UPO, EC 1.11.2.1) from the basidiomycetous fungi Marasmius rotula and Marasmius wettsteinii oxidized steroids with hydroxyacetyl and hydroxyl functionalities at C17 - such as cortisone, Reichstein's substance S and prednisone - via stepwise oxygenation and final fission of the side chain. The sequential oxidation started with the hydroxylation of the terminal carbon (C21) leading to a stable geminal alcohol (e.g. cortisone 21-gem-diol) and proceeded via a second oxygenation resulting in the corresponding α-ketocarboxylic acid (e.g. cortisone 21-oic acid). The latter decomposed under formation of adrenosterone (4-androstene-3,11,17-trione) as well as formic acid and carbonic acid (that is in equilibrium with carbon dioxide); fission products comprising two carbon atoms such as glycolic acid or glyoxylic acid were not detected. Protein models based on the crystal structure data of MroUPO (Marasmius rotula unspecific peroxygenase) revealed that the bulky cortisone molecule suitably fits into the enzyme's access channel, which enables the heme iron to come in close contact to the carbons (C21, C20) of the steroidal side chain. ICP-MS analysis of purified MroUPO confirmed the presence of magnesium supposedly stabilizing the porphyrin ring system.

Contribution of MATE1 to Renal Secretion of the NMDA Receptor Antagonist Memantine.

The weak base memantine is actively secreted into urine, however the underlying mechanisms are insufficiently understood. Potential candidates involved in memantine renal secretion are organic cation transporter 2 (OCT2) and multidrug and toxin extrusion proteins (MATE1, MATE2-K). The aim of this in vitro study was the examination of the interaction of memantine with OCT2 and MATEs. Memantine transporter inhibition and transport were examined in HEK cells expressing human OCT2, MATE1, or MATE2-K. Monolayers of single- (MDCK-OCT2, MDCK-MATE1) and double-transfected MDCK cells (MDCK-OCT2-MATE1) were used for studies on vectorial, basal to apical memantine transport. Memantine inhibited OCT2-, MATE1-, and MATE2-K-mediated metformin transport with IC50 values of 3.2, 40.9, and 315.3 µM, respectively. In HEK cells, no relevant memantine uptake by OCT2, MATE1, or MATE2-K was detected. Vectorial transport experiments, however, indicated a role of MATE1 for memantine export: After memantine administration to the basal side of the monolayers, memantine cellular accumulation was considerably lower (MDCK-MATE1 vs MDCK control cells, P < 0.01) and memantine transcellular, basal to apical transport was higher in MATE1 expressing cells (MDCK-MATE1 vs MDCK control cells, P < 0.001 at 60 and 180 min). Both effects were abolished upon addition of the MATE inhibitor cimetidine. These experiments suggest a relevant role of MATE1 for renal secretion of memantine. In the clinical setting, renal elimination of memantine could be impaired by coadministration of MATE inhibitors.

One-pot synthesis of human metabolites of SAR548304 by fungal peroxygenases.

Unspecific peroxygenases (UPOs, EC 1.11.2.1) have proved to be stable oxygen-transferring biocatalysts for H2O2-dependent transformation of pharmaceuticals. We have applied UPOs in a drug development program and consider the enzymatic approach in parallel to a conventional chemical synthesis of the human metabolites of the bile acid reabsorption inhibitor SAR548304. Chemical preparation of N,N-di-desmethyl metabolite was realized by a seven-step synthesis starting from a late precursor of SAR548304 and included among others palladium catalysis and laborious chromatographic purification with an overall yield of 27%. The enzymatic approach revealed that the UPO of Marasmius rotula is particularly suitable for selective N-dealkylation of the drug and enabled us to prepare both human metabolites via one-pot conversion with an overall yield of 66% N,N-di-desmethyl metabolite and 49% of N-mono-desmethylated compound in two separated kinetic-controlled reactions.

Preparation of labeled human drug metabolites and drug-drug interaction-probes with fungal peroxygenases.

Enzymatic conversion of a drug can be an efficient alternative for the preparation of a complex metabolite compared with a multi-step chemical synthesis approach. Limitations exist for chemical methods for direct oxygen incorporation into organic molecules often suffering from low yields and unspecific oxidation and also for alternative whole-cell biotransformation processes, which require specific fermentation know-how. Stable oxygen-transferring biocatalysts such as unspecific peroxygenases (UPOs) could be an alternative for the synthesis of human drug metabolites and related stable isotope-labeled analogues. This work shows that UPOs can be used in combination with hydrogen/deuterium exchange for an efficient one-step process for the preparation of 4'-OH-diclofenac-d6. The scope of the reaction was investigated by screening of different peroxygenase subtypes for the transformation of selected deuterium-labeled substrates such as phenacetin-d3 or lidocaine-d3. Experiments with diclofenac-d7 revealed that the deuterium-labeling does not affect the kinetic parameters. By using the latter substrate and H2 (18) O2 as cosubstrate, it was possible to prepare a doubly isotope-labeled metabolite (4'-(18) OH-diclofenac-d6). UPOs offer certain practical advantages compared with P450 enzyme systems in terms of stability and ease of handling. Given these advantages, future work will expand the existing 'monooxygenation toolbox' of different fungal peroxygenases that mimic P450 in vitro reactions.

HPLC-SPE-NMR in pharmaceutical development: capabilities and applications.

High-performance liquid chromatography-solid phase extraction-NMR spectroscopy (HPLC-SPE-NMR) has recently become commercially available and has been evaluated with regard to its applicability in a pharmaceutical environment. The addition of an automated SPE unit to an HPLC-NMR system for peak trapping results in an improved NMR signal-to-noise ratio (S/N) and also has other practical advantages. The trapping efficiency is shown to depend on compound polarity and is highest for compounds eluting late on reversed-phase HPLC systems. Multiple peak trapping further increases the S/N, again with the best results for less polar compounds. For polar compounds, multiple peak trapping resulted in no S/N gain as the amount of material retained on the SPE cartridge was equivalent to that from a single injection. When compared with conventional HPLC-NMR, a S/N gain of up to five-fold could be achieved for some compounds in a single trapping step. A major advantage of the technique is the independence of the chromatographic step from the NMR step, resulting in greater versatility than conventional HPLC-NMR in the HPLC solvents and NMR solvents that can be used. Practical applications from both drug metabolite and drug impurity identification are presented.

Direct on-line hyphenation of capillary liquid chromatography to nuclear magnetic resonance spectroscopy: practical aspects and application to drug metabolite identification.

In combining the high peak concentrations of capillary liquid chromatography (CapLC) with the high mass sensitivity of micro scale nuclear magnetic resonance (NMR) the hyphenation of CapLC to micro NMR offers a substantial gain in overall sensitivity. This paper deals with our experiences gained using a commercial CapLC-NMR system which has very recently become available. The limits of detection (SNR > 3) for a test compound of a molecular weight of M 318 were found to be approximately 100 ng (0.35 nmol) within an hour acquisition time and approximately 25 ng over night (85 pmol). Practical aspects such as the feasibility of stopped-flow experiments and sample handling issues are discussed in detail and first possible drug metabolite applications to hepatocyte incubations and direct analysis of plasma samples are presented.