3HNAAG, N‐acetyl‐l‐aspartyl‐l‐glutamic acid, has been widely used as a substrate in glutamate carboxypeptidase II (GCPII) reactions, either to determine the inhibitory constants at 50% inhibition ...(IC50) of novel compounds or to measure GCPII activities in different tissues harvested from various disease models. The importance of 3HNAAG, combined with its current commercial unavailability, prompted the development of a reliable eight‐step synthetic procedure towards 3H2NAAG starting from commercially available pyroglutamate. Pure 3HNAAG of high molar activity (49.8 Ci/mmol) and desired stereochemistry was isolated in high radiochemical yield (67 mCi) and radiochemical purity (>99%). The identity was confirmed by mass spectrometry and co‐injection with unlabeled reference.
3HNAAG, N‐acetyl‐l‐aspartyl‐l‐glutamic acid, widely used as a substrate in glutamate carboxypeptidase II (GCPII) reactions and no longer commercially available, was prepared by a reliable eight‐step synthetic procedure starting from commercially available pyroglutamate. Pure 3HNAAG of high molar activity (49.8 Ci/mmol) and desired optical configuration was isolated in high radiochemical yield (67 mCi) and radiochemical purity (>99%).
We report a combined experimental and computational study of adenosine cation radicals that were protonated at adenine and furnished with a radical handle in the form of an acetoxyl radical, •CH2COO, ...that was attached to ribose 5′-O. Radicals were generated by collision-induced dissociation (CID) and characterized by tandem mass spectrometry and UV–vis photodissociation action spectroscopy. The acetoxyl radical was used to probe the kinetics of intramolecular hydrogen transfer from the ribose ring positions that were specifically labeled with deuterium at C1′, C2′, C3′, C4′, C5′, and in the exchangeable hydroxyl groups. Hydrogen transfer was found to chiefly involve 3′-H with minor contributions by 5′-H and 2′-H, while 4′-H was nonreactive. The hydrogen transfer rates were affected by deuterium isotope effects. Hydrogen transfer triggered ribose ring cleavage by consecutive dissociations of the C4′–O and C1′–C2′ bonds, resulting in expulsion of a C6H9O4 radical and forming a 9-formyladenine ion. Rice-Ramsperger-Kassel-Marcus (RRKM) and transition-state theory (TST) calculations of unimolecular constants were carried out using the effective CCSD(T)/6-311++G(3d,2p) and M06-2X/aug-cc-pVTZ potential energy surfaces for major isomerizations and dissociations. The kinetic analysis showed that hydrogen transfer to the acetoxyl radical was the rate-determining step, whereas the following ring-opening reactions in ribose radicals were fast. Using DFT-computed energies, a comparison was made between the thermochemistry of radical reactions in adenosine and 2′-deoxyadenosine cation radicals. The 2′-deoxyribose ring showed lower TS energies for both the rate-determining 3′-H transfer and ring cleavage reactions.
The synthesis of tritium-labelled glycine transporter 1 inhibitor Org24598 is reported. Because of the instability of the Org24598 skeleton under hydrogenation conditions, a synthetic approach using ...an in-house prepared tritium-labelled alkylating agent (
HMeI, SA = 26.2 Ci/mmol) was employed. Alternative methods of labelling are discussed.
We adapted a radioligand receptor binding assay for measuring insulin levels in unknown samples. The assay enables rapid and accurate determination of insulin concentrations in experimental samples, ...such as from insulin-secreting cells. The principle of the method is based on the binding competition of insulin in a measured sample with a radiolabeled insulin for insulin receptor (IR) in IM-9 cells. Both key components, radiolabeled insulin and IM-9 cells, are commercially available. The IR binding assay was used to determine unknown amounts of insulin secreted by MIN6 β cell line after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. The experimental data obtained by the IR binding assay were compared to the results determined by RIA kits and both methods showed a very good agreement of results. We observed the stimulation of glucose-induced insulin secretion from MIN6 cells by arginine, weaker stimulation by ornithine, but inhibitory effects of dopamine. Serotonin effects were either stimulatory or inhibitory, depending on the concentration of serotonin used. The results will require further investigation. The study also clearly revealed advantages of the IR binding assay that allows the measuring of a higher throughput of measured samples, with a broader range of concentrations than in the case of RIA kits. The IR binding assay can provide an alternative to standard RIA and ELISA assays for the determination of insulin levels in experimental samples and can be especially useful in scientific laboratories studying insulin production and secretion by β cells and searching for new modulators of insulin secretion.
Graphical abstract
Synthesis of [13C6]‐ibrutinib Kriegelstein, Michal; Hroch, Miloš; Marek, Aleš
Journal of labelled compounds & radiopharmaceuticals,
November 2021, 2021-11-00, 20211101, Letnik:
64, Številka:
13
Journal Article
Recenzirano
Convenient and straightforward synthesis of ibrutinib labeled by carbon‐13 isotope is reported. Isotopically labeled building block is introduced in the last step of reaction sequence affording ...sufficient isolated yield (7%) of 13C6‐ibrutinib calculated towards starting commercially available 13C6‐bromobenzene.
Convenient and straightforward synthesis of 13C6‐ibrutinib is reported.
Cytidine ribonucleosides were furnished at O5′ with fixed-charge 6-trimethylammoniumhexan-1-aminecarbonyl tags and studied by UV–vis photodissociation action spectroscopy in the gas phase to probe ...isolated nucleobase chromophores in their neutral, protonated, and hydrogen-adduct radical forms. The action spectrum of the doubly charged cytidine conjugate showed bands at 310 and 270 nm that were assigned to the N3- and O2-protonated cytosine tautomers formed by electrospray, respectively. In contrast, cytidine conjugates coordinated to dibenzo-18-crown-6-ether (DBCE) in a noncovalent complex were found to strongly favor protonation at N3, forming a single-ion tautomer. This allowed us to form cytidine N3–H radicals by electron transfer dissociation of the complex and study their action spectra. Cytidine radicals showed only very weak absorption in the visible region of the spectrum for dipole-disallowed transitions to the low (A and B) excited states. The main bands were observed at 360, 300, and 250 nm that were assigned with the help of theoretical vibronic spectra obtained by time-dependent density functional theory calculations of multiple (>300) radical vibrational configurations. Collision-induced dissociations of cytidine radicals proceeded by major cleavage of the N1–C1′ glycosidic bond leading to loss of cytosine and competitive loss of N3-hydrogen atom. These dissociations were characterized by calculations of transition-state structures and energies using combined Born–Oppenheimer molecular dynamics and DFT calculations. Overall, cytidine radicals were found to be kinetically and thermodynamically more stable than previously reported analogous adenosine and guanosine radicals.
Noncanonical nucleobases and nucleosides represent newly discovered species of relevance for DNA ionization. We report a targeted synthesis of gas-phase 9-methylene(1
)adenine cation radical (
) as a ...low-energy isomer of ionized 9-methyladenine. Ion
showed unique collision-induced dissociation and UV-vis photodissociation action spectra that distinguished it from other cation radical isomers. Ab initio energy calculations with coupled cluster theory extrapolated to the complete basis set limit, CCSD(T)/CBS, identified cation radical
as the global energy minimum of the adenine-related C
H
N
isomers. The action spectrum of
was assigned on the basis of vibronic absorption spectra that were calculated with time-dependent density functional theory for multiple vibrational configurations of thermal ions. The major dissociation of
proceeded by hydrogen loss that was elucidated by deuterium labeling at the exchangeable N-1 and NH
positions and C-8 position and by kinetic analysis. The dissociation involved a reversible rearrangement to intermediate dihydropteridine structures, yielding a protonated aminopteridine as the product, which was identified by multistep UV-vis action spectroscopy. We also report a computational study of related noncanonical isomers of 2'-deoxyadenosine cation radical having the radical defect at C-1' that were found to be thermodynamically more stable than the canonical isomer in both the gas phase and aqueous solution. The noncanonical isomers were calculated to have extremely low ion-electron recombination energies of 4.42-5.10 eV that would make them dead-end hole traps if produced by DNA ionization.
Sixty DNA trinucleotide cation radicals covering a large part of the genetic code alphabet were generated by electron transfer in the gas phase, and their chemistry was studied by collision-induced ...dissociation tandem mass spectrometry and theoretical calculations. The major dissociations involved loss of nucleobase molecules and radicals, backbone cleavage, and cross-ring fragmentations that depended on the nature and position of the nucleobases. Mass identity in dissociations of symmetrical trinucleotide cation radicals of the (XXX+2H)+• and (XYX+2H)+• type was resolved by specific 15N labeling. The specific features of trinucleotide cation radical dissociations involved the dominant formation of d 2 + ions, hydrogen atom migrations accompanying the formation of ( w 2 +H)+•, ( w 2 +2H)+, and ( d 2 +2H)+ sequence ions, and cross-ring cleavages in the 3′- and 5′-deoxyribose moieties that depended on the nucleobase type and its position in the ion. Born–Oppenheimer molecular dynamics (BOMD) and density functional theory calculations were used to obtain structures and energies of several cation-radical protomers and conformers for (AAA+2H)+•, (CCC+2H)+•, (GGG+2H)+•, (ACA+2H)+•, and (CAA+2H)+• that were representative of the different types of backbone dissociations. The ion electronic structure, protonation and radical sites, and hydrogen bonding were used to propose reaction mechanisms for the dissociations.
Cocaine- and amphetamine-regulated transcript peptide (CARTp) is an anorexigenic neuropeptide whose receptor is undisclosed. Previously, we reported the specific binding of CART(61–102) to ...pheochromocytoma PC12 cells, where CART(61–102) affinity and the number of binding sites per cell corresponded to ligand-receptor binding. Recently, Yosten et al. designated orphan GPR160 as the CARTp receptor, because the GPR160 antibody abolished neuropathic pain and anorexigenic effects induced by CART(55–102) and exogenous CART(55–102) coimmunoprecipitated with GPR160 in KATOIII cells. As no direct evidence that CARTp is a ligand for GPR160 has been described, we decided to verify this hypothesis by testing CARTp affinity to the GPR160 receptor. We investigated the GPR160 expression in PC12 cells since it is cell line known to specifically bind CARTp. Moreover, we examined the specific CARTp binding in THP1 cells, with high endogenous GPR160 expression and GPR160-transfected cell lines U2OS and U-251 MG. In PC12 cells, the GPR160 antibody did not compete for specific binding with 125I-CART(61–102) or with 125I-CART(55–102), and GPR160 mRNA expression and GPR160 immunoreactivity were not detected. Moreover, THP1 cells did not show any 125I-CART(61–102) or 125I-CART(55–102) specific binding despite GPR160 detection by fluorescent immunocytochemistry (ICC). Finally, no 125I-CART(61–102) or 125I-CART(55–102) specific binding in the GPR160-transfected cell lines U2OS and U-251 MG, selected due to their negligible endogenous expression of GPR160, was detected, despite the detection of GPR160 by fluorescent ICC. Our binding studies clearly demonstrated that GPR160 cannot be a receptor for CARTp. Further studies are needed to identify true CARTp receptors.
Fast and reasonable low‐scale (200 nmol) syringe‐made synthesis of 15N‐labeled oligonucleotides representing DNA trinucleotide codons is communicated. All codons were prepared by solid‐phase ...controlled pore glass synthesis column technique via the phosphoramidite method. Twenty‐four labeled oligonucleotides covering the DNA genetic code alphabet were prepared using commercially available reagents and affordable equipment in a reasonably short period of time, with acceptable yields and purity for direct applications in mass spectrometry.
A controlled pore glass (CPG)‐supported oligonucleotide synthesis of DNA trinucleotide codons containing various combinations of 15N‐labeled nucleic bases A, G, C, and T is reported. The cost‐effective protocol uses a simple commercially available equipment and an optimized amount of valuable 15N‐labeled phosphoramidite used for the coupling step.
