An Efficient Method for the Synthesis of [4–15N]Cytidine, 2′-Deoxy[4–15N]Cytidine, [6–15N]adenosine,and 2′-Deoxy [6–15N]adenosine Derivatives

Abstract

Nucleophilic substitution reactions of 4-azolyl-1 β-P-D-ribofuranosylpyrimidin-2(1H)-one and 6-azolyl-9-β-D-ribofuranosyl-9H-purine derivatives, which were converted from uridine and inosine, with [¹⁵N]phthalimide in the presence of triethylamine or DBU gave N ⁴-phthaloyl[4-¹⁵N]cytidine and N ⁶-phthaloyl[6-¹⁵N]- adenosine derivatives, respectively, in high yields. Similar reactions of those azolyl derivatives with succinimide afforded N ⁴-succinylcytidine and N ⁶-succinyladenosine derivatives in high yields. The corresponding 2′-deoxyribonucleosides were also synthesized efficiently through the same procedure.

     

Depleted 15N in hydrolysable-N of arctic soils and its implication for mycorrhizal fungi–plant interaction

Uptake of nitrogen (N) via root-mycorrhizal associations accounts for a significant portion of total N supply to many vascular plants. Using stable isotope ratios (δ¹⁵N) and the mass balance among N pools of plants, fungal tissues, and soils, a number of efforts have been made in recent years to quantify the flux of N from mycorrhizal fungi to host plants. Current estimates of this flux for arctic tundra ecosystems rely on the untested assumption that the δ¹⁵N of labile organic N taken up by the fungi is approximately the same as the δ¹⁵N of bulk soil. We report here hydrolysable amino acids are more depleted in ¹⁵N relative to hydrolysable ammonium and amino sugars in arctic tundra soils near Toolik Lake, Alaska, USA. We demonstrate, using a case study, that recognizing the depletion in ¹⁵N for hydrolysable amino acids (δ¹⁵N = ?5.6‰ on average) would alter recent estimates of N flux between mycorrhizal fungi and host plants in an arctic tundra ecosystem.     

[15N,1H]/[13C,1H]-TROSY for simultaneous detection of backbone 15N–1H, aromatic 13C–1H and side-chain 15N–1H2 correlations in large proteins

This paper describes a [¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment for the simultaneous acquisition of the heteronuclear chemical shift correlations of backbone amide ¹⁵N–¹H groups, side chain ¹⁵N–¹H₂ groups and aromatic ¹³C–¹H groups in otherwise highly deuterated proteins. The ¹⁵N–¹H and ¹³C–¹H correlations are extracted from two subspectra of the same data set, thus preventing possible spectral overlap of aromatic and amide protons in the ¹H dimension. The side-chain ¹⁵N–¹H₂ groups, which are suppressed in conventional [¹⁵N,¹H)-TROSY, are observed with high sensitivity in the ¹⁵N–¹H subspectrum. [¹⁵N,¹H]/[¹³C,¹H]-TROSY was used as the heteronuclear correlation block in a 3D [¹H,¹H]-NOESY-[¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment with the membrane protein OmpA reconstituted in detergent micelles of molecular weight 80000 Da, which enabled the detection of numerous NOEs between backbone amide protons and both aromatic protons and side chain ¹⁵N–¹H₂ groups.     

Determination of all NOes in 1H–13C–Me-ILV-U−2H–15N Proteins with Two Time-Shared Experiments

We present two time-shared experiments that enable the characterization of all nOes in ¹H–¹³C-ILV methyl-labelled proteins that are otherwise uniformly deuterated and ¹⁵N enriched and possibly selectively protonated for distinct residue types. A 3D experiment simultaneously provides the spectra of a 3D NOESY-HN-TROSY and of a 3D NOESY-HC-PEP-HSQC. Thus, nOes from any protons to methyl or amide protons are dispersed with respect to ¹⁵N and ¹³C chemical shifts, respectively. The single 4D experiment presented here yields simultaneously the four 4D experiments HC-HSQC-NOESY-HC-PEP-HSQC, HC-HSQC-NOESY-HN-TROSY, HN-HSQC-NOESY-HN-TROSY and HN-HSQC-NOESY-HC-PEP-HSQC. This allows for the unambiguous determination of all nOes involving amide and methyl protons. The method was applied to a (¹H,¹³C)-ILV−(¹H)-FY-(U−²H,¹⁵N) sample of a 37 kDa di-domain of the E. coli enterobactin synthetase module EntF.     

Influence of mycorrhizal associations on foliar δ15N values of legume and non-legume shrubs and trees in the fynbos of South Africa: Implications for estimating N2 fixation using the 15N natural abundance method

In this study, we examined the use of the ¹⁵N natural abundance method to quantify the percentage N derived from fixation of atmospheric N₂ in honeybush (Cyclopia spp.) shrubs and trees in the fynbos, South Africa. Non-fixing shrubs and trees of similar phenology to the Cyclopia species were chosen as reference plants. These reference plants were selected to cover a range of mycorrhizal associations (ericoid mycorrhizal, arbuscular mycorrhizal and non-mycorrhizal). Isotopic analysis revealed a wide range of foliar ¹⁵N values for the reference plants, including many very negative values. The marked differences in ¹⁵N values were defined by the mycorrhizal status of the reference plant species, with the ericoid and arbuscular mycorrhizal plants showing lower foliar ¹⁵N values relative to their non-mycorrhizal counterparts. In contrast, the ¹⁵N values of the N₂-fixing Cyclopia species were uniformly clustered around zero, from –0.11 to –1.43. These findings are consistent with the observation that mycorrhizal fungi discriminate against the heavier ¹⁵N isotope during transfer of N from the fungus to the host plant, leaving the latter depleted in ¹⁵N (i.e. with a more negative ¹⁵N value). However, a major assumption of the ¹⁵N natural abundance method for estimating N₂ fixation is that both legume and reference plant should have the same level of fractionation associated with N uptake. But, because mycorrhizal associations may strongly affect the level of fractionation during N uptake and transfer, the test legume should belong to the same mycorrhizal group as the chosen reference plant species. As shown in this study, if the mycorrhizal status of the legume and the reference plant differs, or cannot be assessed, then the ¹⁵N natural abundance technique cannot be used to quantitatively estimate N₂ fixation.     

NMR assignment of polymerase β labeled with 2H, 13C, and 15N in complex with substrate DNA

DNA Polymerase β is a multifunctional enzyme involved in base excision repair of nuclear DNA in vertebrate cells. We present here the first assignments of the full-length protein (335 residues, 39 kDa) in the presence of a double gap—double hairpin DNA (22 nucleotides, 7 kDa).     

The McClure and Weiss models of Fe–O2 bonding for oxyhemes, and the HbO2 + NO reaction

For the Fe–O₂(S = 0) linkages of oxyhemes, valence bond (VB) structures are re-presented for the McClure [Fe^II(S = 1) + O₂(S = 1)], Pauling–Coryell [Fe^II(S = 0) + O₂*(S = 0)], and Weiss [Fe^III(S = ½) + O₂ ^?(S = ½)] models of bonding. The VB structures for the McClure and Weiss models are of the increased-valence type, with more electrons participating in bonding than occur in their component Lewis structures. The Fe–O bond number and O–O bond order for the McClure structure are correlated with measured Fe–O and O–O bond lengths for oxymyoglobin. Back-bonding from O ₂ ^? to Fe^III of the Weiss structure gives a restricted form of the McClure structure. The McClure and Weiss increased-valence structures are used to provide VB formulations of mechanisms for the oxyhemoglobin + NO reaction. The products of these two formulations are Hb⁺ and NO₃ ^? (where Hb is hemoglobin) and Hb⁺ and OONO^?, respectively. Because Hb⁺ and NO₃ ^? are the observed products, they provide an experimental procedure for distinguishing the McClure and Weiss models. It is also shown that the same type of agreement between McClure-type theory and experiment occurs for oxycoboglobin + NO, cytochrome P450 monooxygenases, and related hydrogen atom transfer reactions. In the appendices, the results of density functional theory and multireference molecular orbital calculations for oxyhemes are related to one formulation of the increased-valence wavefunction for the McClure model, and theory is presented for the calculation of approximate weights for the Lewis structures that are components of the McClure increased-valence structure.     

Backbone 1H, 13C and 15N resonance assignments of dengue virus NS2B–NS3p in complex with aprotinin

Dengue virus, belongs to Flaviviridae, is an arthropod transmitted virus that threatens millions of people’s lives. As with other flaviviruses, a positive single-stranded 11-kilobases RNA in the dengue virus genome encodes three structural proteins (capsid protein C, membrane protein M, and envelope protein E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The two component protease NS2B–NS3p is essential for viral replication and is believed to be a potential antiviral drug target. Aprotinin, a native inhibitor, is proved to retard the activity of NS2B–NS3p. The backbone assignments of NS2B–NS3p will be essential for determining the high resolution solution structure of NS2B–NS3p and screening new antiviral drugs. Herein, we report the backbone ¹H, ¹⁵N, ¹³C resonance assignments of the N terminal fragment of NS2B (4.8 kDa) and NS3p (18.5 kDa) in complex with aprotinin (6.5 kDa) by high resolution NMR.     

An aqueous–organic two-phase bioprocess for efficient production of the natural aroma chemicals 2-phenylethanol and 2-phenylethylacetate with yeast

The natural aroma chemicals 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEAc) are of high industrial relevance and can be produced from l-phenylalanine in a yeast-based process with growth-associated product formation. Due to product inhibition, in situ product removal is mandatory to obtain economically interesting concentrations. A fed-batch approach using polypropylene glycol 1200 as in situ extractant and the precursor in a saturated concentration led to the highest 2-PE productivity reported for a bioprocess so far. With Kluyveromyces marxianus CBS 600, 26.5 g/l 2-PE and 6.1 g/l 2-PEAc in the organic phase were obtained, corresponding to space–time yields of 0.33 and 0.08 g/l h, respectively.     

Bilateral C1–C2 Transarticular Screw and C1 Laminar Hook Fixation and Bone Graft Fusion for Reducible Atlantoaxial Dislocation: A Seven-Year Analysis of Outcome

Background

Bilateral C1-2 transarticular screw and C1 laminar hook fixation was developed on the basis of transarticular screws fixation. The modified technique has showed a better biomechanical stability than established techniques in previous study. However, long-term (minimum follow-up 7 years) outcomes of patients with reducible atlantoaxial dislocation who underwent this modified fixation technique have not still been reported.

Methods

A retrospective study was conducted to evaluate the outcome of 36 patients who underwent this modified technique. Myelopathy was assessed using the Ranawat myelopathy score and Myelopathy Disability Index. Pain scores were assessed using Visual Analogue Scale. Radiological imaging was assessed and the following data were extracted: the atlantodental intervals, the space available for cord, presence of spinal cord signal change on T2 weighted image, C1–C2 angle, C2–C7 angle and fusion rates.

Findings

All patients achieved a minimum seven-year follow up. 95% patients with neck and suboccipital pain improved after surgery; in their Visual Analogue pain scores, there was a greater than 50% improvement in their VAS scores with a drop of 5 points on the VAS (P<0.05). 92% of patients improved in the Ranawat myelopathy grade; the Myelopathy Disability Index assessment showed a preoperative mean score of 35.62 with postoperative mean 12.75(P<0.05). There was not any significant atlantoaxial instability at each follow-up time. The space available for cord increased in all patients. Postoperative sagittal kyphosis of the subaxial spine was not observed. After six months after surgery, bone grafts of all patients were fused. No complications related to surgery were found in the period of follow-up.

Conclusions

The long-term outcomes of this case series demonstrate that under the condition of thorough preoperative preparations, bilateral C1–C2 transarticular screw and C1 laminar hook fixation and bone graft fusion is a reliable posterior atlantoaxial fusion technique for reducible atlantoaxial dislocation.     

The impact of simulated chronic nitrogen deposition on the biomass and N2-fixation activity of two boreal feather moss–cyanobacteria associations

Bryophytes achieve substantial biomass and play several key functional roles in boreal forests that can influence how carbon (C) and nitrogen (N) cycling respond to atmospheric deposition of reactive nitrogen (N_r). They associate with cyanobacteria that fix atmospheric N₂, and downregulation of this process may offset anthropogenic N_r inputs to boreal systems. Bryophytes also promote soil C accumulation by thermally insulating soils, and changes in their biomass influence soil C dynamics. Using a unique large-scale (0.1 ha forested plots), long-term experiment (16 years) in northern Sweden where we simulated anthropogenic N_r deposition, we measured the biomass and N₂-fixation response of two bryophyte species, the feather mosses Hylocomium splendens and Pleurozium schreberi. Our data show that the biomass declined for both species; however, N₂-fixation rates per unit mass and per unit area declined only for H. splendens. The low and high treatments resulted in a 29% and 54% reduction in total feather moss biomass, and a 58% and 97% reduction in total N₂-fixation rate per unit area, respectively. These results help to quantify the sensitivity of feather moss biomass and N₂ fixation to chronic N_r deposition, which is relevant for modelling ecosystem C and N balances in boreal ecosystems.     

The effect of ethanol on the formation of N 2-ethylidene-dG adducts in mice: implications for alcohol-related carcinogenicity of the oral cavity and esophagus

The present study aimed to experimentally confirm that long-term alcohol drinking causes a high risk of oral and esophageal cancer in aldehyde dehydrogenase 2 (ALDH2)-deficient individuals. Aldh2 knockout mice, an animal model of ALDH2-deficiency, were treated with 8% ethanol for 14 months. Levels of acetaldehyde-derived DNA adducts were increased in esophagus, tongue and submandibular gland. Our finding that a lack of Aldh2 leads to more DNA damage after chronic ethanol treatment in mice supports epidemiological findings on the carcinogenicity of alcohol in ALDH2-deficient individuals who drink chronically.     

Synthesis and biological activity of new series of N-modified analogues of the N/OFQ(1–13)NH2 with aminophosphonate moiety

New series of N-modified analogues of the N/OFQ(1-13)NH(2) with aminophosphonate moiety have been synthesized and investigated for biological activity. These peptides were prepared by solid-phase peptide synthesis-Fmoc-strategy. The N/OFQ(1-13)NH(2) analogues were tested for agonistic activity in vitro on electrically stimulated rat vas deferens smooth-muscle preparations isolated from Wistar albino rats. Our study has shown that the selectivity of the peptides containing 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids to the N-side of Phe is not changed-they remain selective agonists of NOP receptors. The derivative with the largest ring (NOC-6) demonstrated efficacy similar to that of N/OFQ(1-13)NH(2), but in a 10-fold higher concentration. The agonistic activity of newly synthesized N-modified analogues of N/OFQ(1-13)NH(2) with aminophosphonate moiety was investigated for the first time.     

The Correlation of RNase A Enzymatic Activity with the Changes in the Distance between Nε2-His12 and Nδ1-His119 Upon Addition of Stabilizing and Destabilizing Salts

The effect of stabilizing and destabilizing salts on the catalytic behavior of ribonuclease A (RNase A) was investigated at pH 7.5 and 25°C, using spectrophotometric, viscometric and molecular dynamic methods. The changes in the distance between N_ε2 of His₁₂ and N_δ1 of His₁₁₉ at the catalytic center of RNase A upon the addition of sodium sulfate, sodium hydrogen sulfate and sodium thiocyanate were evaluated by molecular dynamic methods. The compactness and expansion in terms of Stokes radius of RNase A upon the addition of sulfate ions as kosmotropic salts, and thiocyanate ion as a chaotropic salt, were estimated by viscometric measurements. Enzyme activity was measured using cytidine 2′, 3′-cyclic monophosphate as a substrate. The results from the measurements of distances between N_ε2 of His₁₂ and N_δ1 of His₁₁₉ and Stokes radius suggest (i) that the presence of sulfate ions decreases the distance between the catalytic His residues and increases the globular compactness, and (ii) that there is an expansion of the enzyme surface as well as elongation of the catalytic center in the presence of thiocyanate ion. These findings are in agreement with activity measurements.     

Comparison of procainamide and 2-aminobenzamide labeling for profiling and identification of glycans by liquid chromatography with fluorescence detection coupled to electrospray ionization–mass spectrometry

One of the most widely used methods for glycan analysis is fluorescent labeling of released glycans followed by hydrophilic interaction chromatography–(ultra-)high-performance liquid chromatography [HILIC–(U)HPLC]. Here, we compare the data obtained by (U)HPLC–fluorescence (FLR) coupled to electrospray ionization–mass spectrometry (ESI–MS) for procainamide and 2-aminobenzamide (2-AB)-labeled N-glycans released from human immunoglobulin G (IgG). Fluorescence profiles from procainamide show comparable chromatographic separation to those obtained for 2-AB but gave higher fluorescence intensity as well as significantly improved ESI efficiency (up to 30 times that of 2-AB). Thus, labeling with procainamide increases the ability to identify minor glycan species that may have significant biological activity.     

An efficient 3D NMR technique for correlating the proton and15N backbone amide resonances with the α-carbon of the preceding residue in uniformly15N/13C enriched proteins

Summary A 3D NMR technique is described which correlates the amide proton and nitrogen resonances of an amino acid residue with the C chemical shift of its preceding residue. The technique uses a relay mechanism, transferring magnetization from¹⁵N to¹³C via the intervening carbonyl nucleus. This method for obtaining sequential connectivity is less sensitive to large line widths than the alternative HNCA experiment. The technique is demonstrated for the protein calmodulin, complexed with a 26 amino acid fragment of skeletal muscle myosin light chain kinase.Abbreviations CaM Calmodulin - HCACO -proton to -carbon to carbonyl correlation - H(CA)NHN -proton (via -carbon) to nitrogen to amide proton correlation - HMQC heteronuclear multiple quantum correlation - HNCA amide proton to nitrogen to C -carbon correlation - M13 a 26-residue fragment of the CaM-binding domain of skeletal muscle myosin light chain kinase comprising residues 577–602.     

Phase II Study of Pseudomonas aeruginosa-Mannose-Sensitive Hemagglutinin in Combination with Capecitabine for Her-2–Negative Metastatic Breast Cancer Pretreated with Anthracycline and Taxane

Purpose

Metastatic breast cancer (MBC) remains an incurable disease despite major therapeutic advances. Pseudomonas aeruginosa–mannose-sensitive hemagglutinin (PA-MSHA) has been established to have anti-proliferative effects against breast cancer cells in preclinical experiments, and is indicated for treatment of cancer in China. We performed a phase II trial combining PA-MSHA with capecitabine in patients with heavily pretreated MBC.

Methods

Eligibility criteria included human epidermal growth factor receptor 2–negative MBC, prior therapy with anthracyclines and taxanes, at least one prior chemotherapy regimen for metastatic disease or early relapse after a taxane plus anthracycline adjuvant regimen, and adequate organ function and performance status. PA-MSHA 1 mg was administered subcutaneously every other day and capecitabine 1000 mg/m² orally twice a day for 2 weeks on, 1 week off. The primary end point was progression-free survival.

Results

A total of 97 patients were enrolled. Median progression-free survival (PFS) was 4.0 months [95 % confidence interval (CI) 3.0–4.9], which was not significantly different from that in historical controls. However, median PFS was significantly longer (8.2 months; 95 % CI 6.7–9.7) in 24 patients with moderate immune-related adverse events (irAEs) such as fever or skin induration at the injection site than in those with no or mild irAEs (3.1 months, 95 % CI 2.5–3.6; p = 0.003). Overall survival was also improved in these patients (25.4 vs. 16.4 months; p = 0.044). PA-MSHA has a good safety profile, with only 6 patients (6.2 %) discontinuing treatment. PA-MSHA did not increase capecitabine-related toxicities such as hand-foot syndrome, nausea, and vomiting.

Conclusion

Adding PA-MSHA to capecitabine has a good safety profile in patients with heavily pre-treated MBC, although benefit from this regimen might occur only in patients with moderate PA-MSHA–related adverse events.

Trial Registration

ClinicalTrials.gov NCT01380808     

A triple-resonance pulse scheme for selectively correlating amide1HN and15N nuclei with the1Hα proton of the preceding residue

Summary A 3D¹H–¹⁵N–¹³C triple resonance experiment is presented that contains exclusively cross peaks between the¹H^N and¹⁵N nuclei of one residue with the H of the preceding residue. The pulse sequence, designed to minimize the time coherence, is transverse on nuclei with short T₂ values. The experiment consists of coherence transfers via one-bond couplings from the H^N via N, CO, C to the H and back to the H^N for detection; it is called HN(COCA)HA. The experiment was tested on uniformly¹⁵N- and¹³C-enriched T4 lysozyme.