Enrichment of hepatic glycogen and plasma glucose from H218 O informs gluconeogenic and indirect pathway fluxes in naturally feeding mice.

Deuterated water (2 H2 O) is a widely used tracer of carbohydrate biosynthesis in both preclinical and clinical settings, but the significant kinetic isotope effects (KIE) of 2 H can distort metabolic information and mediate toxicity. 18 O-water (H2 18 O) has no significant KIE and is incorporated into specific carbohydrate oxygens via well-defined mechanisms, but to date it has not been evaluated in any animal model. Mice were given H2 18 O during overnight feeding and 18 O-enrichments of liver glycogen, triglyceride glycerol (TG), and blood glucose were quantified by 13 C NMR and mass spectrometry (MS). Enrichment of oxygens 5 and 6 relative to body water informed indirect pathway contributions from the Krebs cycle and triose phosphate sources. Compared with mice fed normal chow (NC), mice whose NC was supplemented with a fructose/glucose mix (i.e., a high sugar [HS] diet) had significantly higher indirect pathway contributions from triose phosphate sources, consistent with fructose glycogenesis. Blood glucose and liver TG 18 O-enrichments were quantified by MS. Blood glucose 18 O-enrichment was significantly higher for HS versus NC mice and was consistent with gluconeogenic fructose metabolism. TG 18 O-enrichment was extensive for both NC and HS mice, indicating a high turnover of liver triglyceride, independent of diet. Thus H2 18 O informs hepatic carbohydrate biosynthesis in similar detail to 2 H2 O but without KIE-associated risks.

Metabolic incorporation of H218 O into specific glucose-6-phosphate oxygens by red-blood-cell lysates as observed by 13 C isotope-shifted NMR signals.

Water enriched with oxygen-18 (H218 O) is a potential tracer for evaluating the sources of glucose and glycogen synthesis since it is incorporated into specific sites of glucose-6-phosphate via specific enzyme-mediated exchange/addition mechanisms. Unlike 2 H, 18 O does not experience significant isotope effects for any of these processes. Therefore, H218 O might provide more precise estimates of endogenous carbohydrate synthesis compared with deuterated water provided that positional 18 O enrichments of glucose can be measured. As a proof of concept, H218 O was incorporated into a well characterized hemolysate model of sugar phosphate metabolism and 13 C NMR was applied to quantify positional 18 O enrichment of glucose-6-phosphate oxygens. Human erythrocyte hemolysate preparations were incubated overnight at 37 °C with a buffer containing sugar phosphate precursors and 20% (n = 5) and 80% (n = 1) H218 O. Enrichment of glucose-6-phosphate was analyzed by 13 C NMR analysis of 18 O-shifted versus unshifted signals following derivatization to monoacetone glucose (MAG). 13 C NMR MAG spectra from hemolysate revealed resolved 18 O-shifted signals in Positions 1-5. Mean 18 O enrichments were 16.4 ± 1.6% (Position 1), 13.3 ± 1.3% (Position 2), 4.1 ± 1.1% (Position 3), 12.6 ± 0.8% (Position 4), 10.7 ± 1.4% (Position 5), and no detectable enrichment of Position 6. No 18 O-shifted glucose-6-phosphate signals were detected in preparations containing sugar phosphate precursors only. H218 O is incorporated into Positions 1-5 of glucose-6-phosphate in accordance with spontaneous aldose hydration and specific enzymatic reaction mechanisms. This provides a basis for its deployment as a tracer for glucose and glycogen biosynthesis.

Isotope-Edited Infrared Spectroscopy for Efficient Discrimination between Pharmaceutical Salts and Cocrystals.

Isotope-edited infrared spectroscopy using carboxylic acids selectively labeled with 13C is proposed herein for the efficient discrimination of pharmaceutical salts and cocrystals, whereby proton-transfer probe vibrations are highlighted by isotope shifts. This new technique can accurately discriminate even a confusing salt from a cocrystal for the traditional method, highlighting the diagnostic peaks. In addition, the established technique also provided the OH in-plane bending vibrations corresponding to intermolecular hydrogen bonding at the carbonyl oxygens of the cocrystals. The technique will accelerate the discrimination, which is a critical process in cocrystal development.

Mass selective laser cooling of 229Th3+ in a multisectional linear Paul trap loaded with a mixture of thorium isotopes.

We consider an experiment on trapping and laser cooling of 229Th3+ ions in a linear Paul trap in the presence of undesirable impurities such as ions of the radioactive isotope 228Th3+. We suggest a method of separating these impurities by means of selective laser cooling utilizing the isotope shift of cooling transitions in 229Th3+ and 228Th3+ ions. According to our estimation, the isotope shift is equal to 3.4 GHZ and makes laser separation of these isotopes possible.

Ultra-high resolution band-selective HSQC for nanomole-scale identification of chlorine-substituted 13 C in natural products drug discovery.

Ultra-high resolution band-selective HSQC (bsHSQC) has been employed for detection of 35 Cl-37 Cl isotope shifted 13 C NMR signals for assignment of regioisomerism in bromo-chloro natural products. Optimum pulse sequence and instrumental parameters for maximization of detection of the isotope shifts were explored. The chlorine isotope shifts (Δδ) were detected within crosspeaks and were shown to vary with hybridization of 13 C, substitution of 13 C, presence of ß-chloro substituents, and their relative configuration. Deconvolution of Cl-substituted CH bsHSQC crosspeaks may provide other useful information, including a potentially MS-independent method for quantitating 37 Cl/35 C isotopic fractionation during the biosynthesis of halogenated natural products. Copyright © 2016 John Wiley & Sons, Ltd.

The pKa values of the catalytic residues in the retaining glycoside hydrolase T26H mutant of T4 lysozyme.

T4 phage lysozyme (T4L) is an enzyme that cleaves bacterial cell wall peptidoglycan. Remarkably, the single substitution of the active site Thr26 to a His (T26H) converts T4L from an inverting to a retaining glycoside hydrolase with transglycosylase activity. It has been proposed that T26H-T4L follows a double displacement mechanism with His26 serving as a nucleophile to form a covalent glycosyl-enzyme intermediate (Kuroki et al., PNAS 1999; 96:8949-8954). To gain further insights into this or alternative mechanisms, we used NMR spectroscopy to measure the acid dissociation constants (pKa values) and/or define the ionization states of the Asp, Glu, His, and Arg residues in the T4L mutant. Most notably, the pKa value of the putative nucleophile His26 is 6.8 ± 0.1, whereas that of the general acid Glu11 is 4.7 ± 0.1. If the proposed mechanism holds true, then T26H-T4L follows a reverse protonation pathway in which only a minor population of the free enzyme is in its catalytically competent ionization state with His26 deprotonated and Glu11 protonated. Our studies also confirm that all arginines in T26H-T4L, including the active site Arg145, are positively charged under neutral pH conditions. BRIEF STATEMENT: The replacement of a single amino acid changes T4 lysozyme from an inverting to a retaining glycoside hydrolase. Using NMR spectroscopy, we measured the pKa values of the ionizable residues in the active site of this mutant enzyme. Along with previously reported data, these results provide important constraints for understanding the catalytic mechanisms by which the wild-type and mutant form of T4 lysozyme cleave bacterial peptidoglycan.

High-Accuracy Mass Spectrometry Based Screening Method for the Discovery of Cysteine Containing Peptides in Animal Venoms and Toxins.

Venom and toxin samples derived from animal origins are a rich source of bioactive peptides. A high proportion of bioactive peptides that have been identified in venom contain one or more disulfide bridges, which are thought to stabilize tertiary structure, and therefore influence the peptides' specificity and activity. In this chapter, we describe a label-free mass spectrometry-based screening workflow specifically to detect peptides that contain inter- and intramolecular disulfide bonds, followed by elucidation of their primary structure. This method is based on the determination of the normalized isotope shift (NIS) and the normalized mass defect (NMD) of peptides, two parameters which are heavily influenced by the presence of sulfur in a peptide, where cysteines are the main contributing residues. Using ant defensive secretions as an example, we describe the initial fractionation of the venom on strong cation exchange followed by nanoflow HPLC and mass spectrometry. High resolution zoom scan spectra of high-abundance peptides are acquired, allowing an accurate determination of both monoisotopic and average mass, which are essential for calculation of NMD and NIS. Candidate peptides exhibiting relative low NMD and high NIS values are selected for targeted de novo sequencing. By fine-tuning the collision energy for optimal fragmentation of each selected precursor ions, the full sequence of several novel inter- and intramolecular disulfide bond containing ant defensive peptides can be established.

1,3,5-Tri(iodoethynyl)-2,4,6-trifluorobenzene: halogen-bonded frameworks and NMR spectroscopic analysis.

Halogen bonding is the non-covalent interaction between the region of positive electrostatic potential associated with a covalently bonded halogen atom, named the σ-hole, and a Lewis base. Single-crystal X-ray diffraction structures are reported for a series of seven halogen-bonded cocrystals featuring 1,3,5-tris(iodoethynyl)-2,4,6-trifluorobenzene (1) as the halogen-bond donor, and bromide ions (as ammonium or phosphonium salts) as the halogen-bond acceptors: (1)·MePh3PBr, (1)·EtPh3PBr, (1)·acetonyl-Ph3PBr, (1)·Ph4PBr, (1)·[bis(4-fluorophenyl)methyl]triphenylphosphonium bromide, and two new polymorphs of (1)·Et3BuNBr. The cocrystals all feature moderately strong iodine-bromide halogen bonds. The crystal structure of pure [bis(4-fluorophenyl)methyl]triphenylphosphonium bromide is also reported. The results of a crystal engineering strategy of varying the size of the counter-cation are explored, and the features of the resulting framework materials are discussed. Given the potential utility of (1) in future crystal engineering applications, detailed NMR analyses (in solution and in the solid state) of this halogen-bond donor are also presented. In solution, complex 13C and 19F multiplets are explained by considering the delicate interplay between various J couplings and subtle isotope shifts. In the solid state, the formation of (1)·Et3BuNBr is shown through significant 13C chemical shift changes relative to pure solid 1,3,5-tris(iodoethynyl)-2,4,6-trifluorobenzene.

¹9F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR.

Naturally occurring RNA lacks fluorine-19 ((19)F), thus, their specifically fluorinated counterparts are particularly well suited to noninvasively monitoring the dynamic conformational properties and ligand-binding interactions of the RNA. For nuclear magnetic resonance (NMR) spectroscopy, (19)F-NMR of fluorine-substituted RNA provides an attractive, site-specific probe for structure determination in solution. Advantages of (19)F include high NMR sensitivity (83% of (1)H), high natural abundance (100%), and the extreme sensitivity of (19)F to the chemical environment leading to a large range of chemical shifts. The preparation of base-substituted 2-fluoropurine and 5-fluoropyrimidine 5'-triphosphates (2F-ATP/5F-CTP/5F-UTP) can be carried out using efficient enzymatic synthesis methods. Both pyrimidine analogs, 5-fluorouridine and 5-fluorocytidine, as well as, 2-fluoroadenosine are readily incorporated into RNA transcribed in vitro using T7 RNA polymerase.

Identical Hydrogen-Bonding Strength of the Retinal Schiff Base between Primate Green- and Red-Sensitive Pigments: New Insight into Color Tuning Mechanism.

Three aspects are generally considered in the color-tuning mechanism of vision: (I) chromophore distortion, (II) electrostatic interaction between the protonated Schiff base and counterion, and (III) polarity around the ß-ionone ring and polyene chain. Primate green- and red-sensitive proteins are highly homologous but display maximum absorption at 530 and 560 nm, respectively. In the present study, the N-D stretching frequency of monkey green-sensitive protein was identified by using C15-D retinal. The hydrogen-bonding strength between monkey green and red was identical. Together with a previous resonance Raman study, we conclude that the 30 nm difference originates exclusively from the polarity around the ß-ionone ring and polyene chain. Three amino acids (Ala, Phe, and Ala in monkey green and Ser, Tyr, and Thr in monkey red, respectively) may be responsible for color tuning together with protein-bound water molecules around the ß-ionone ring and polyene chain but not at the Schiff base region.

(13)C discrimination between diet, faeces, milk and milk components.

Stable isotope analysis is a fundamental tool in food origin and authenticity testing. Its use in livestock production requires knowledge of isotope discrimination between product and diet. Here, we report (13)C discrimination ((13)Δ) for milk, milk components (fat, casein and lactose) and faeces in eight lactating dairy cows, which grazed pasture or were fed fresh pasture herbage in the stall. Cows were supplemented with grain maize at 1.72 kg d(-1) (dry matter). Feed components were collected daily, and faeces, milk fat, casein, lactose and whole milk 4 times per week during an 8-week-long sampling period. Carbon isotope composition (δ(13)C) of each sample was analysed. δ(13)C was lowest in milk fat (-29.8‰) and highest in casein (-26.4‰). Compared to the diet, whole milk was depleted in (13)C ((13)Δ = 0.4‰) due to a strong (13)C-depletion of fat ((13)Δ = 2.2‰), which was not fully compensated by the (13)C-enrichment of casein ((13)Δ = -1.1‰) and lactose ((13)Δ = -0.7‰). Faeces were also depleted in (13)C ((13)Δ =1.7‰). Influences of feeding environment (stall vs. pasture) and herbage quality were minor (<0.4‰). A review of literature data shows large variation between studies. We consider that the present results are superior, as they are based on a much larger data set regarding the number of cows and milkings (total n = 256) with greater detail in analyses of diet and milk products. Also, the study covered both stall- and pasture-feeding scenarios in realistic settings with long periods of equilibration. This is the first comprehensive analysis of (13)C discrimination between diet and all main milk components (as well as faeces). Thus, the results will improve the use of stable isotope analyses in regard to authenticity testing and proof of origin.

Wavelengths and Energy Levels of Neutral Kr84 and Level Shifts in All Kr Even Isotopes.

Interferometrically-measured wavelengths of 109 lines of neutral Kr84 are compared with those of Kr86. Sixty energy levels of neutral Kr84 derived from those wavelengths and 25 Kr86-Kr84 isotope shifts previously measured are given along with their shifts from the energy levels of Kr86. Twenty levels of each of Kr82, Kr80, and Kr78 are also evaluated using isotope-shift information in the literature. The differences between the experimentally observed shifts and the normal mass shift leave large negative residuals which are accounted for by ionization energy differences and by the specific mass shift. It appears that the volume effect causes only a very small, if any, energy level shift.