18O Labeled Nucleosides. 2. A General Method for the Synthesis of Specifically Labeled Pyrimidine Ribonucleosides1

Abstract

A facile method for the synthesis of highly enriched ¹⁸O labeled pyrimidine ribonucleosides is described using uridine as a model compound. The isotopic label may be selectively incorporated into the base moiety at O² or into the ribose portion of the molecule at the 5′ position. In addition, both positions may be labeled and this is the first report of a method for labeling of both the base and sugar moieties of pyrimidine ribonucleosides. The site and level of isotope incorporation may be determined mass spectrometrically.     

15N-Labeled Oligodeoxynucleotides -Useful Probes for 1H-NMR Investigations

Abstract

The amino protons of ¹⁵N-labeled deoxyoligonucleotides were studied as possible structural probes for NMR investigations of the interaction between DNA and regulatory proteins. To apply this strategy, 6-¹⁵NH₂-2′ -deoxyadenoslne, 4-¹⁵NH -2′-deoxycytidine and 2-¹⁵NH -2′-deoxyguanosine were chemically synthesized. The labeled nucleosides were introduced into distinct positions of oligodeoxy-nucleotides by large-scale DNA synthesis. The behaviour of the ¹⁵N-coupled cytidine amino protons in a 18 base pair (bp) lac operator sequence were investigated using H-¹⁵N INDOR spectroscopy .     

Synthesis of Stable Isotope Labeled Analogs of the Anti-Hepatitis C Virus Nucleotide Prodrugs PSI-7977 and PSI-352938

In order to support bioanalytical LC/MS method development and plasma sample analysis in preclinical and clinical studies of the anti-hepatitis C-virus nucleotides, PSI-7977 and PSI-352938, the corresponding stable isotope labeled forms were prepared. These labeled compounds were prepared by addition reaction of the freshly prepared Grignard reagent ¹³CD₃MgI to the corresponding 2 ′-ketone nucleosides followed by fluorination of the resulting carbinol with DAST. As expected, these 2 ′-C-(trideuterated-¹³C-methyl) nucleotide prodrugs showed similar anti-HCV activity to that of the corresponding unlabeled ones.     

Incorporation of 13C,15N-labeled nucleosides and measurement of RNA synthesis and turnover in sea urchin embryos

The uptake of nucleosides into sea urchin embryos and their subsequent incorporation into RNA increases with increasing external nucleoside concentration. When embryos are incubated with high concentrations of ¹³C,¹⁵N-labeled nucleosides, newly synthesized RNA becomes sufficiently labeled with heavy isotope to be separated from unlabeled RNA on cesium formate equilibrium gradients. High concentrations of nucleosides do not affect development of embryos or rates of RNA synthesis. The extent of density-labeling of precursor pools increases with incubation time, and only levels off after many hours. During incubations with high concentrations of nucleosides, ATP pools expand up to twofold. Using density-labeling to circumvent precursor pool measurements, a method is presented to study the synthesis and decay of pulse-labeled RNA. The instantaneous rate of synthesis of total RNA at the blastula stage is 9.3 × 10^?15 mol of total nucleotide/embryo per minute and the average half-life of total RNA is 23 minutes.     

NMR Resonance Assignment Methodology: Characterizing Large Sparsely Labeled Glycoproteins

Characterization of proteins using NMR methods begins with assignment of resonances to specific residues. This is usually accomplished using sequential connectivities between nuclear pairs in proteins uniformly labeled with NMR active isotopes. This becomes impractical for larger proteins, and especially for proteins that are best expressed in mammalian cells, including glycoproteins. Here an alternate protocol for the assignment of NMR resonances of sparsely labeled proteins, namely, the ones labeled with a single amino acid type, or a limited subset of types, isotopically enriched with ¹⁵N or ¹³C, is described. The protocol is based on comparison of data collected using extensions of simple two-dimensional NMR experiments (correlated chemical shifts, nuclear Overhauser effects, residual dipolar couplings) to predictions from molecular dynamics trajectories that begin with known protein structures. Optimal pairing of predicted and experimental values is facilitated by a software package that employs a genetic algorithm, ASSIGN_SLP_MD. The approach is applied to the 36-kDa luminal domain of the sialyltransferase, rST6Gal1, in which all phenylalanines are labeled with ¹⁵N, and the results are validated by elimination of resonances via single-point mutations of selected phenylalanines to tyrosines. Assignment allows the use of previously published paramagnetic relaxation enhancements to evaluate placement of a substrate analog in the active site of this protein. The protocol will open the way to structural characterization of the many glycosylated and other proteins that are best expressed in mammalian cells.     

Asymmetric Synthesis of Cyclopropyl Carbocyclic Nucleosides

Abstract

A number of nucleosides have been synthesized as potential antiviral and antitumor agents.¹ More recently, various dideoxynucleosides have been synthesized and found to be potent anti-HIV agents.² As a part of our drug discovery program for the treatment of HIV and HBV, we have initiated to synthesize cyclopropyl carbocyclic nucleosides as potential antiviral agents. Several papers regarding the synthesis of cyclopropyl carbocyclic nucleosides have appeared in the literature.^3–5 However, they are all reported as racemic mixtures. In this abstract, we wish to report the asymmetric synthesis of cylopropyl carbocyclic nucleosides from optically active common intermediates, 6 and 11.     

7-Carbapurine Ribofuranosides: Synthesis by Solid-Liquid Phase-Transfer Glycosylation and 15N-NMR Spectra

Abstract

The synthesis of pyrrolo [2,3-d]pyrimidine ribofuranosides by solid-liquid phase-transfer glycosylation is described and ¹⁵N NMR spectra of 7-carbapurine nucleosides are reported.     

Lyophilization of TC-99m-Hynic Labeled Peg-Liposomes

Abstract

The development of long circulating liposomes represented a major step forward towards the use of radiolabeled liposomes in nuclear medicine. The long circulation property markedly improves their uptake and consequently visualization of sites of infection and inflammation. Previously, we have developed a rapid and convenient method to label polyethylene glycol (PEG)-lipo-somes with technetium-99m (Tc-99m). PEG-liposomes containing the technetium-chelator hydrazino nicotinamide (HYNIC) could be labeled with Tc-99m with high efficiency. We showed that these Tc-99m-HYNIC labeled PEG-liposomes have excellent in vivo imaging characteristics in several pre-clinical and clinical studies. However, an important limitation associated with the use of HYNIC-PEG-liposome formulation as radiopharmaceutical is that their labeling efficiency decreases markedly within 3 months. In this paper we present a lyophilization method for HYNIC-PEG-liposomes using sucrose as a lyopro-tectant. The long-term stability of these liposomes in terms of the particle size and labeling efficiency upon reconstitution were determined. Additionally, the in vivo behavior of reconstituted radiolabeled liposomes in a rat model of focal infection was studied at two time-points after preparation.

Increasing the duration of the dehydration step significantly reduced the mean particle size upon reconstitution. Increasing the storage temperature from -20^°C to +4^°C also improved the particle size distribution upon reconstitution. The labeling efficiency for both freeze-dried preparations remained high during the 1 year-storage period and was always higher than 86%, but decreased for the control liposomes. Eight months after preparation, these liposomes had a labeling efficiency as low as 6%, whereas both freeze-dried preparations could still be labeled with an efficiency of 90%. The in vivo studies showed that there was no major difference in the biodistribution of the radiolabeled liposomes between 3 and 30 weeks post-preparation in rats with an Staphylococcus aureus abscess, indicating an acceptable long-term shelf-life of both freeze-dried liposome preparations. Abscesses were visualized from 2 hours post injection onwards.

In conclusion, a freeze-drying method which improved the long term shelf-life of HYNIC-PEG-liposomes is presented. The in vivo behavior of Tc-99m-PEG-liposomes, reconstituted 30 weeks after preparation, was similar to the biodistribution obtained with the non-freeze-dried preparation. The splenic uptake of these liposomes was slightly increased.     

Effective Synthesis of Fluorescently Labeled Morpholino Nucleoside Triphosphate Derivatives

Morpholino nucleoside triphosphates (A, U, G, C, T) bearing the active functional amino group tethered to morpholine residue and their fluorescently labeled derivatives were synthesized. All compounds were characterized by ¹H, ¹³C, and ³¹P NMR, and mass spectrometry. A possibility of using fluorescently labeled morpholino nucleoside triphosphates as chain terminators in DNA sequencing is discussed.     

Synthesis and Cellular Uptake of Fluorescently Labeled Oligodeoxynucleotide Prodrugs

Abstract

In this paper we described the synthesis on solid support of 5′-fluorescein labeled dodecathymidylates having interucleoside phosphodiester or phosphorothioate linkages temporary masked with enzymolabile S-pivaloyl-2-thioethyl (tBuSATE) groups.     

Rapid Preparation of Stable Isotope Labeled Peptides that Bind to Target Proteins by a Phage Library System

We have developed a system for directly isolating foreign peptides displayed on the N-terminus of the major coat protein of bacteriophage M13. The phage particle in this system is formed as a mixture of wild type and modified coat proteins. The N-terminal segment of the modified coat protein was mutated for chemical cleavage, in order to obtain the displayed peptide from the major coat protein. Using ¹³C, ¹⁵N- labeled medium, we introduced stable isotopes, ¹³C and/or ¹⁵N, into the coat proteins. The NMR spectra for the cleaved peptides from the phage particles could be recorded within a few days after the selection of the phage clone.     

Synthesis of Bridged Pyrimidine Nucleosides and Triazo [4, 3-c] Pyrimidine Nucleoside Analogues

Abstract

The synthesis and the spectral characterization of a number of N⁴-N⁴ bridged pyrimidine nucleosides and triazo [4, 3-c] pyrimidine nucleoside analogues are reported.     

A Convenient Synthesis of D-Psicofuranosyl Nucleosides1

Abstract

As part of our studies on the synthesis of conformationally restricted nucleosides of types 1 and 2, where X = CH₂, O or S, we required access to differentially substituted D-psicofuranosyl nucleosides such as 3. As shown in the table, we have developed a convenient approach to such compounds that depends on the direct condensation of the 1,2:3,4-di-O-isopropylidene-β-D-psicofuranose derivative 4 with an appropriate silylated purine or pyrimidine base.² Although the α and β anomers of 3 are formed in a 1:1 ratio, the yields of the β anomers are generally comparable with earlier condensation methods that use psicofuranosyl- halide², 2-benzoates⁴ or 2-nitro derivative⁵. However, the present method has the advantage that the starting sugar 4 is more readily accessible. The precursor 6′-alcohol can be prepared in very large amounts from D-fructose using the method of Prisbe et al.⁴     

Original Articles: Dynamic Redistribution of Isotopically Labeled Cohorts of Nitrogen Inputs in Two Temperate Forests

We compared simulated time series of nitrogen-15 (¹⁵N) redistribution following a large-scale labeling experiment against field recoveries of ¹⁵NH₄ ⁺ and ¹⁵NO₃ ⁻ in vegetation tissues. We sought to gain insight into the altered modes of N cycling under long-term, experimentally elevated N inputs. The study took place in two contrasting forests: a red pine stand and a mixed deciduous stand (predominantly oak) at the Harvard Forest, Massachusetts, USA. We used TRACE, a dynamic simulation model of ecosystem biogeochemistry that includes ¹⁵N/¹⁴N ratios in N pools and fluxes. We simulated input–output and internal fluxes of N, tracing the labeled cohorts of N inputs through ecosystem pools for one decade. TRACE simulated the peaks and timing of ¹⁵N recovery in foliage well, providing a key link between modeling and field studies. Recovery of tracers in fine roots was captured less well. The model was structured to provide rapid, initial sinks for ¹⁵NO₃ ⁻ and ¹⁵NH₄ ⁺ in both forests, as indicated by field data. In simulations, N in litter turned over rapidly, even as humus provided a long-term sink for rapidly cycling N. This sink was greater in the oak forest. Plant uptake fluxes of N in these fertilized plots were on the same order of magnitude as net assimilation fluxes in forest-floor humus. A striking result was the small rate of incorporation of N in humus resulting from the transfer of litter material to humus, compared with large fluxes of N into humus and its associated microorganisms through direct transfers from pools of inorganic N in soils. Received 19 May 1998; accepted 30 September 1998     

Europium Cryptate Labeled Deoxyuridine-Triphosphate Analog: Synthesis and Enzymatic Incorporation

Abstract

The synthesis of an europium tris-bipyridine cryptate labeled 2′-deoxyuridine-5′-triphosphate analog (K-11-dUTP) is described. This labeled triphosphate was incorporated into DNA through enzymatic reactions with terminal transferase and DNA polymerases. The enzymatic reactions were monitored by TRACE (Time Resolved Amplification of Cryptate Emission), a homogeneous method using Fluorescence Resonance Energy Transfer (FRET) from an europium cryptate as donor to a modified allophycocyanine as acceptor.     

The Effect of Universal Fluorinated Nucleobases on the Catalytic Activity of Ribozymes

Abstract

Four fluoro modified universal nucleobases have been synthesized. The universal nucleobases 1 and 2 , containing a 2,4-difluorobenzene as nucleobase and a 4,6-difluorobenzimidazole, respectively, were chemically incorporated into a selected hammerhead ribozyme sequence which has already been retrovirally expressed as an anti-HIV ribozyme to investigate their effect on the catalytic activity of the ribozymes. The substitution of the natural nucleosides with either 1 or 2 results only in a small decrease of the catalytic activity. The K_m value for the monosubstituted ribozyme with a 2,4-difluorobenzene is 309 nM^?1, the corresponding k_cat is 2.91 · 10^?3 min^?1. A disubstituted hammerhead ribozyme carrying one of each modification has also been synthesized. For a further stabilization of the ribozyme/substrate complex 2′-(β-aminoethoxy) modified fluorinated nucleosides 15 and 16 have been developed.     

有机荧光团及荧光标记细胞的阴极荧光成像研究

目的 阴极荧光（CL）成像是一种以电子束为激发源的高分辨荧光成像技术,但生物材料对电子束的敏感性限制了CL技术在生命科学中的广泛应用。为了研究和发展CL技术在生物样品中的应用,本文旨在通过探究电子辐照引起碳基材料的结构损伤、有机基团的降解及荧光猝灭等问题,深入理解电子源对有机荧光团的激发特性。方法 本研究应用扫描电镜（SEM）和阴极荧光谱仪系统（SEM-CL）,研究电子源对有机荧光团及荧光探针标记细胞的激发特性,观测了有机物的CL信号的发射特性、强度衰减、成像方式及特点。结果 实验结果显示,在低能量（2.5～5 keV）和低束流（～10 pA）电子辐照下,有机荧光微珠发射出较强的荧光,CL像分辨率达到～30 nm。荧光微珠经过12 min辐照,信号强度衰减了25%,CL像仍保持了可接受的发光强度和足够的信噪比。此外,还获得了从细胞表面到内部一定深度内,荧光标记的亚细胞结构信息。结论 在SEM-CL系统中,可以同时获得由电子束激发产生的电子像和CL像,实现阴极荧光与电子显微镜关联（CCLEM）成像。本实验的研究结果为CCLEM技术应用于生物结构研究提供了数据及技术支持。     

PREFERENTIAL LABELING OF RAT LYMPHOCYTES WITH A RAPID RATE OF TURNOVER BY TRITIATED DEOXYCYTIDINE

Lymphocytes in thymic cortex and germinal centers of lymphoid tissues are labeled intensely with generally labeled tritiated deoxycytidine [G-³H]dCyd whereas they are weakly labeled with methyl tritiated deoxythymidine [methyl-³H]dThd of the same specific activity, not only by single injection but also by an intensive injection schedule. [G-³H]dCyd can be used to label short-lived lymphocytes strongly, although not specifically. The distribution patterns of labeled lymphocytes were different depending on the injection schedules of [G-³H]dCyd. [G-³H]dCyd can be used as a precursor molecule for cytosine and also thymine found in DNA. The ratios of radioactive thymine to cytosine measured biochemically on DNA extracted from radioactive lymphocytes labeled by the various schedules indicate strongly that short- and long-lived lymphocyte populations have different abilities to utilize pyrimidine nucleosides for DNA synthesis.     

Production of Specifically Labeled Compounds by Methanobacterium espanolae Grown on H2-CO2 plus [13C]Acetate

Methanobacterium espanolae, an acidiphilic methanogen, required acetate for maximal growth on H₂-CO₂. In the presence of 5 to 15 mM acetate, at a growth pH of 5.5, the μ_max was 0.05 h^-1. M. espanolae consumed 12.3 mM acetate during 96 h of incubation at 35°C with shaking at 100 rpm. At initial acetate levels of 2.5 to 10.0 mM, the amount of biomass produced was dependent on the amount of acetate in the medium. ¹³C nuclear magnetic resonance spectra of protein hydrolysates obtained from cultures grown on [1-¹³C]- or [2-¹³C]acetate indicated that an incomplete tricarboxylic acid pathway, operating in the reductive direction, was functional in this methanogen. The amino acids were labeled with a very high degree of specificity and at greater than 90% enrichment levels. Less than 2% label randomization occurred between positions primarily labeled from either the carboxyl or methyl group of acetate, and very little label was transferred to positions primarily labeled from CO₂. The labeling pattern of carbohydrates was typical for glucogenesis from pyruvate. This methanogen, by virtue of the properties described above and its ability to incorporate all of the available acetate (10 mM or lower) from the growth medium, has advantages over other microorganisms for use in the production of specifically labeled compounds.     

Some Examples of the Use of Trifluoromethane Sulfonic Anhydride in Nucleic Acid Chemistry

Abstract

Trifluoromethane sulfonic anhydride has been used for the inversion of configuration at the 3′-position of 2′-deoxypurine nucleosides, for the modification of the base moiety of purine and pyrimidine nucleosides, for nucleophilic substitution in the sugar moiety, for the synthesis of O²,3′-cyclothymidine and for sugar-base condensation reactions. Reaction can be carried out under very mild conditions. The conditions for these reactions are quite different so that a good selectivity can be obtained when different reactive groups are present.