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.
Dissociations of DNA trinucleotide codons as gas-phase singly and doubly protonated ions were studied by tandem mass spectrometry using 15N-labeling to resolve identity in the nucleobase loss and ...backbone cleavages. The monocations showed different distributions of nucleobase loss from the 5′-, middle, and 3′-positions depending on the nucleobase, favoring cytosine over guanine, adenine, and thymine in an ensemble-averaged 62:27:11:<1 ratio. The distribution for the loss of the 5′-, middle, and 3′-nucleobase was 49:18:33, favoring the 5′-nucleobase, but also depending on its nature. The formation of sequence w 2 + ions was unambiguously established for all codon mono- and dications. Structures of low-Gibbs-energy protomers and conformers of dAAA+, dGGG+, dCCC+, dTTT+, dACA+, and dATC+ were established by Born–Oppenheimer molecular dynamics and density functional theory calculations. Monocations containing guanine favored classical structures protonated at guanine N7. Structures containing adenine and cytosine produced classical nucleobase-protonated isomers as well as zwitterions in which two protonated bases were combined with a phosphate anion. Protonation at thymine was disfavored. Low threshold energies for nucleobase loss allowed extensive proton migration to occur prior to dissociation. Loss of the nucleobase from monocations was assisted by neighboring group participation in nucleophilic addition or proton abstraction, as well as allosteric proton migrations remote from the reaction center. The optimized structures of diprotonated isomers for dAAA2+ and dACA2+ revealed combinations of classical and zwitterionic structures. The threshold and transition-state energies for nucleobase-ion loss from dications were low, resulting in facile dissociations involving cytosine, guanine, and adenine.
The insulin receptor (IR, with its isoforms IR-A and IR-B) and the insulin-like growth factor 1 receptor (IGF-1R) are related tyrosine kinase receptors. Recently, the portfolio of solved ...hormone–receptor structures has grown extensively thanks to advancements in cryo-electron microscopy. However, the dynamics of how these receptors transition between their inactive and active state are yet to be fully understood. The C-terminal part of the alpha subunit (
α
CT) of the receptors is indispensable for the formation of the hormone-binding site. We mutated the
α
CT residues Arg717 and His710 of IR-A and Arg704 and His697 of IGF-1R. We then measured the saturation binding curves of ligands on the mutated receptors and their ability to become activated. Mutations of Arg704 and His697 to Ala in IGF-1R decreased the binding of IGF-1. Moreover, the number of binding sites for IGF-1 on the His697 IGF-1R mutant was reduced to one-half, demonstrating the presence of two binding sites. Both mutations of Arg717 and His710 to Ala in IR-A inactivated the receptor. We have proved that Arg717 is important for the binding of insulin to its receptor, which suggests that Arg717 is a key residue for the transition to the active conformation.
Deuterium‐ and tritium‐labeled compounds play a principal role in tracing of biologically active molecules in complicated biochemical systems. The state‐of‐the‐art techniques using noble metal ...catalysts or strong reducing agents often suffers from low functional group tolerances, poor selectivity, tricky or multistep synthesis of reagents, and low specific activity of the labeled product. Herein, we demonstrate a mild and nonmetallic technique of deuteration and tritiation of polarized double bonds, such as carbonyl compounds, yielding labeled alcohols of high specific activities. This one‐pot synthesis uses carrier‐free hydrogen gas in situ activated by a freshly prepared frustrated Lewis pair, generating reducing reagents. This labeling strategy shows better selectivity and functional group tolerances compared with current reductive methods. Reported is an example of the selective reduction of the aldehyde moiety of 3‐acetylbenzaldehyde. What makes this technology groundbreaking is its mildness, selectivity, and generation of limited amount of radioactive waste as almost no byproducts were generated after use of (B(C6F5)33H)(3HTMP) reducing reagent. Radiochemical purity of desired 3H‐labeled product in a crude reaction mixture was determined of over 94%. This work provides, to the community of radiochemists, a practical protocol for frustrated Lewis pairs (FLP)‐assisted deuterium/tritium labeling technology.
Reliable protocol of the routine use of frustrated Lewis pairs for reducing of polarized double bonds is reported. The described method is selective towards reduction of aldehydes and leaves unaffected nonactivated ketone moieties and other functionalities sensitive to standard hydrogenation methods. Revolutionary is use of ultra‐mild reaction conditions (200 mbar of 3H2), nonmetallic character of very selective reagent providing 94% radiochemical purity of desired 3H‐labeled product in a crude reaction mixture.
Tritiodefluorination of alkyl C–F groups Brož, Břetislav; Marek, Aleš
Journal of labelled compounds & radiopharmaceuticals,
September 2019, 2019-09-00, 20190901, Letnik:
62, Številka:
11
Journal Article
Recenzirano
A straightforward methodology of fluorine substitution by tritium/deuterium is reported. The described method is selective towards the F─C (sp3) group and leaves both the aromatic F─C (sp2) and F2─C ...(sp3) moieties unaffected. Alkylfluorides, readily synthesized from appropriate alcohols by treatment with diethylaminosulfur trifluoride (DAST) reagent in an overall yield up to 76%, undergoes activation with the boron‐based Lewis acid B(C6F5)3, and stoichiometric in situ reduction with a tritide/deuteride reagent—the TMP2(3)H2(3)HB(C6F5)3 system of frustrated Lewis pair. This methodology provides an isolated yield of up to 93% of regio‐specifically labeled small organic compounds with superior 2H‐enrichment of over 95%. The specific activity of prepared 1‐(2‐3H‐ethyl)naphthalene was determined at 29.0 Ci/mmol. The site selectivity of the Lewis acid/ TMP2(3)H2(3)HB(C6F5)3 approach is orthogonal to currently used methods and allows for isotopic labeling of complementary positions in molecules. Reported labeling methodology proceeds well at ultra‐mild reaction conditions (220 mbar of T2), allowing very low consumption of the radioactive source (4.2 Ci/156 GBq), and producing limited amount of radioactive waste.
Reliable protocol for tritiodefluorination of alkylfluorides is reported. The described method is selective towards the F─C (sp3) group and leaves both the aromatic F─C (sp2) and F2─C (sp3) moieties unaffected. The site selectivity of the Lewis acid/ TMP2(3)H2(3)HB(C6F5)3 approach is orthogonal to currently used methods and allows for isotopic labeling of complementary positions in molecules.
The multifunctional radioligand 3HT0901317 (3H1) has been employed as a powerful autoradiographic tool to target several receptors, such as liver X, farnesoid X, and retinoic acid‐related orphan ...receptor alpha and gamma subtypes at nanomolar concentrations. Although 3H1 is commercially available and its synthesis via tritiodebromination has been reported, the market price of this radioligand and the laborious synthesis of corresponding bromo‐intermediate potentially preclude its widespread use in biochemical, pharmacological, and pathological studies in research lab settings. We exploit recent reports on hydrogen‐isotope exchange (HIE) reactions in tertiary benzenesulfonamides where the sulfonamide represents an ortho‐directing group that facilitates CH activation in the presence of homogenous iridium(I) catalysts. Herein, we report a time‐ and cost‐efficient method for the tritium late‐stage labeling of compound 1—a remarkably electron‐poor substrate owing to the tertiary trifluoroethylsulfonamide moiety. Under a straightforward HIE condition using a commercially available Kerr‐type NHC Ir(I) complex, (cod)Ir (NHC)Cl, the reaction with 1 afforded a specific activity of 10.8 Ci/mmol. Additionally, alternative HIE conditions using the heterogeneous catalyst of Ir‐black provided sufficient 0.72 D‐enrichment of 1 but unexpectedly failed while repeating with tritium gas.
Described is a late‐stage tritium labeling of T0901317 using an HIE strategy in the electron‐poor substrate lacking a good ortho‐directing group for C─H activation. Homogeneous catalysis with a Kerr‐type catalyst provided 3HT0901317 with SA of 10.8 Ci/mmol. Unprecedented translation issue of deuterium into tritium experiment during heterogeneous catalysis was observed, whereby efficient deuterium labeling conditions using Ir‐black provided reasonable 0.72 D‐enrichment of T0901317 but turned out to be ineffective when repeated under similar conditions in tritium experiments.
Gas-phase dissociations were investigated for several peptide ions containing the Gly-Leu* N-terminal motif where Leu* was a modified norleucine residue containing the photolabile diazirine ring. ...Collisional activation of gas-phase peptide cations resulted in facile N
2
elimination that competed with backbone dissociations. A free lysine ammonium group can act as a Brønsted acid to facilitate N
2
elimination. This dissociation was accompanied by insertion of a lysine proton in the side chain of the photoleucine residue, as established by deuterium labeling and gas-phase sequencing of the products. Electron structure calculations were used to provide structures and energies of reactants, intermediates, and transition states for Gly-Leu*-Gly-Gly-Lys amide ions that were combined with RRKM calculations of unimolecular rate constants. The calculations indicated that Brønsted acid-catalyzed eliminations were kinetically preferred over direct loss of N
2
from the diazirine ring. Mechanisms are proposed to explain the proton-initiated reactions and discuss the reaction products. The non-catalyzed diazirine ring cleavage and N
2
loss is proposed as a thermometer dissociation for peptide ion dissociations.
Fig. a
ᅟ
Experimental data from ion mobility measurements and electron transfer dissociation were combined with extensive computational analysis of ion structures and dissociation energetics for ...Gly-Leu-Gly-Gly-Lys cations and cation radicals. Experimental and computational collision cross sections of (GLGGK + 2H)2+ ions pointed to a dominant folding motif that is represented in all low free-energy structures. The local folding motifs were preserved in several fragment ions produced by electron transfer dissociation. Gradient optimizations of (GLGGK + 2H)+• cation-radicals revealed local energy minima corresponding to distonic zwitterionic structures as well as aminoketyl radicals. Both of these structural types can isomerize to low-energy tautomers that are protonated at the radical-containing amide group forming a new type of intermediates, −C•O–NH2 +– and −C•(OH)NH2 +–, respectively. Extensive mapping with B3LYP, M06-2X, and MP2(frozen core) calculations of the potential energy surface of the ground doublet electronic state of (GLGGK + 2H)+• provided transition-state and dissociation energies for backbone cleavages of the N–Cα and amide C–N bonds leading to ion–molecule complexes. The complexes can undergo facile prototropic migrations that are catalyzed by the Lys ammonium group and isomerize enolimine c -type fragments to the more stable amide tautomers. In contrast, interfragment hydrogen atom migrations in the complexes were found to have relatively high transition energies and did not compete with fragment separation. The extensive analysis of the intermediate and transition-state energies led to the conclusion that the observed dissociations cannot proceed competitively on the same potential energy surface. The reactive intermediates for the dissociations originate from distinct electronic states that are accessed by electron transfer.
Radiolabeled hormones in insulin research, a minireview Jiráček, Jiří; Žáková, Lenka; Marek, Aleš
Journal of labelled compounds & radiopharmaceuticals,
December 2020, 2020-12-00, 20201201, Letnik:
63, Številka:
14
Journal Article
Recenzirano
Preparation of both 125I‐labeled insulin and insulin‐like growth factor 1 (IGF‐1) was critical because it enabled a detailed characterization of binding properties of these important hormones towards ...their cognate transmembrane receptors. Binding modes of hundreds of hormone derivatives were analyzed using competition radioligand binding assays. This effort has resulted in development of six insulin analogs that are today clinically used for the treatment of diabetes. Here, we will briefly summarize a history of insulin research employing iodinated hormones.
This minireview briefly summarizes a history of research employing 125I‐labeled insulin and insulin‐like growth factor 1.
CART (cocaine‐ and amphetamine‐regulated transcript) peptides are involved in food intake regulation, stress, and other physiological functions. Although CART peptides have been known for over 25 ...years, their receptor(s) have not yet been characterized. In this short review, we will summarize our previous studies, where we reported specific binding of 125I‐CART(61‐102) to PC12 rat pheochromocytoma cells. Competitive binding experiments performed with mono‐ and di‐iodinated peptides and their isoforms with oxidized Met67 resulted in nanomolar binding affinity. Moreover, in our previous study, CART(61‐102), as well as di‐iodinated CART(61‐102), have shown a strong anorexigenic effect in fasted lean mice after intracerebroventricular administration. In conclusion, from our previous studies, iodination of CART(61‐102) resulted in mono‐ and di‐iodinated analogs with or without oxidized Met67. All analogs revealed a high affinity to binding sites at PC12 cells and preserved biological activity.
