Control of stereogenic oxygen in a helically chiral oxonium ion.

The control of tetrahedral carbon stereocentres remains a focus of modern synthetic chemistry and is enabled by their configurational stability. By contrast, trisubstituted nitrogen1, phosphorus2 and sulfur compounds3 undergo pyramidal inversion, a fundamental and well-recognized stereochemical phenomenon that is widely exploited4. However, the stereochemistry of oxonium ions-compounds bearing three substituents on a positively charged oxygen atom-is poorly developed and there are few applications of oxonium ions in synthesis beyond their existence as reactive intermediates5,6. There are no examples of configurationally stable oxonium ions in which the oxygen atom is the sole stereogenic centre, probably owing to the low barrier to oxygen pyramidal inversion7 and the perception that all oxonium ions are highly reactive. Here we describe the design, synthesis and characterization of a helically chiral triaryloxonium ion in which inversion of the oxygen lone pair is prevented through geometric restriction to enable it to function as a determinant of configuration. A combined synthesis and quantum calculation approach delineates design principles that enable configurationally stable and room-temperature isolable salts to be generated. We show that the barrier to inversion is greater than 110 kJ mol-1 and outline processes for resolution. This constitutes, to our knowledge, the only example of a chiral non-racemic and configurationally stable molecule in which the oxygen atom is the sole stereogenic centre.

Enantioselective Synthesis of Sealutomicin C.

The sealutomicins are a family of anthraquinone antibiotics featuring an enediyne (sealutomicin A) or Bergman-cyclized aromatic ring (sealutomicins B-D). Herein we report the development of an enantioselective organocatalytic method for the synthesis of dihydroquinolines and the use of the developed method in the total synthesis of sealutomicin C which features a transannular cyclization of an aryllithium onto a γ-lactone as a second key step.

Glutathione Transferase Photoaffinity Labeling Displays GST Induction by Safeners and Pathogen Infection.

Glutathione transferases (GSTs) represent a large and diverse enzyme family involved in the detoxification of small molecules by glutathione conjugation in crops, weeds and model plants. In this study, we introduce an easy and quick assay for photoaffinity labeling of GSTs to study GSTs globally in various plant species. The small-molecule probe contains glutathione, a photoreactive group and a minitag for coupling to reporter tags via click chemistry. Under UV irradiation, this probe quickly and robustly labels GSTs in crude protein extracts of different plant species. Purification and mass spectrometry (MS) analysis of labeled proteins from Arabidopsis identified 10 enriched GSTs from the Phi(F) and Tau(U) classes. Photoaffinity labeling of GSTs demonstrated GST induction in wheat seedlings upon treatment with safeners and in Arabidopsis leaves upon infection with avirulent bacteria. Treatment of Arabidopsis with salicylic acid (SA) analog benzothiadiazole (BTH) induces GST labeling independent of NPR1, the master regulator of SA. Six Phi- and Tau-class GSTs that are induced upon BTH treatment were identified, and their labeling was confirmed upon transient overexpression. These data demonstrate that GST photoaffinity labeling is a useful approach to studying GST induction in crude extracts of different plant species upon different types of stress.

Does the feedback of blood results in observational studies influence response and consent? A randomised study of the Understanding Society Innovation Panel.

BACKGROUND: While medical studies generally provide health feedback to participants, in observational studies this is not always the case due to logistical and financial difficulties, or concerns about changing observed behaviours. However, evidence suggests that lack of feedback may deter participants from providing biological samples. This paper investigates the effect of offering feedback of blood results on participation in biomeasure sample collection. METHODS: Participants aged 16 and over from a longitudinal study - the Understanding Society Innovation Panel-were randomised to three arms - nurse interviewer, interviewer, web survey - and invited to participate in biomeasures data collection. Within each arm they were randomised to receive feedback of their blood results or not. For those interviewed by a nurse both venous and dried blood samples (DBS) were taken in the interview. For the other two arms, they were asked if they would be willing to take a sample, and if they agreed a DBS kit was left or sent to them so the participant could take their own sample and return it. Blood samples were analysed and, if in the feedback arms, participants were sent their total cholesterol and HbA1c results. Response rates for feedback and non-feedback groups were compared: overall; in each arm of the study; by socio-demographic and health characteristics; and by previous study participation. Logistic regression models of providing a blood sample by feedback group and data collection approach controlling for confounders were calculated. RESULTS: Overall 2162 (80.3% of individuals in responding households) took part in the survey; of those 1053 (48.7%) consented to provide a blood sample. Being offered feedback had little effect on overall participation but did increase consent to provide a blood sample (unadjusted OR 1.38; CI: 1.16-1.64). Controlling for participant characteristics, the effect of feedback was highest among web participants (1.55; 1.11-2.17), followed by interview participants (1.35; 0.99 -1.84) and then nurse interview participants (1.30; 0.89-1.92). CONCLUSIONS: Offering feedback of blood results increased willingness to give samples, especially for those taking part in a web survey.

Triazine Probes Target Ascorbate Peroxidases in Plants.

Though they are rare in nature, anthropogenic 1,3,5-triazines have been used in herbicides as chemically stable scaffolds. Here, we show that small 1,3,5-triazines selectively target ascorbate peroxidases (APXs) in Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), rice (Oryza sativa), maize (Zea mays), liverwort (Marchantia polymorpha), and other plant species. The alkyne-tagged 2-chloro-4-methyl-1,3,5-triazine probe KSC-3 selectively binds APX enzymes, both in crude extracts and in living cells. KSC-3 blocks APX activity, thereby reducing photosynthetic activity under moderate light stress, even in apx1 mutant plants. This suggests that APX enzymes in addition to APX1 protect the photosystem against reactive oxygen species. Profiling APX1 with KCS-3 revealed that the catabolic products of atrazine (a 1,3,5-triazine herbicide), which are common soil pollutants, also target APX1. Thus, KSC-3 is a powerful chemical probe to study APX enzymes in the plant kingdom.

Synthesis, Characterization, and Reactivity of Complex Tricyclic Oxonium Ions, Proposed Intermediates in Natural Product Biosynthesis.

Reactive intermediates frequently play significant roles in the biosynthesis of numerous classes of natural products although the direct observation of these biosynthetically relevant species is rare. We present here direct evidence for the existence of complex, thermally unstable, tricyclic oxonium ions that have been postulated as key reactive intermediates in the biosynthesis of numerous halogenated natural products from Laurencia species. Evidence for their existence comes from full characterization of these oxonium ions by low-temperature NMR spectroscopy supported by density functional theory (DFT) calculations, coupled with the direct generation of 10 natural products on exposure of the oxonium ions to various nucleophiles.

Structure Determination of a Chloroenyne from Laurencia majuscula Using Computational Methods and Total Synthesis.

Despite numerous advances in spectroscopic methods through the latter part of the 20th century, the unequivocal structure determination of natural products can remain challenging, and inevitably, incorrect structures appear in the literature. Computational methods that allow the accurate prediction of NMR chemical shifts have emerged as a powerful addition to the toolbox of methods available for the structure determination of small organic molecules. Herein, we report the structure determination of a small, stereochemically rich natural product from Laurencia majuscula using the powerful combination of computational methods and total synthesis, along with the structure confirmation of notoryne, using the same approach. Additionally, we synthesized three further diastereomers of the L. majuscula enyne and have demonstrated that computations are able to distinguish each of the four synthetic diastereomers from the 32 possible diastereomers of the natural product. Key to the success of this work is to analyze the computational data to provide the greatest distinction between each diastereomer, by identifying chemical shifts that are most sensitive to changes in relative stereochemistry. The success of the computational methods in the structure determination of stereochemically rich, flexible organic molecules will allow all involved in structure determination to use these methods with confidence.

A Total Synthesis of Salinosporamide†A.

Salinosporamide A is a ß-lactone proteasome inhibitor currently in clinical trials for the treatment of multiple-myeloma. Herein we report a short synthesis of this small, highly functionalized, biologically important natural product that uses an oxidative radical cyclization as a key step and allows for the preparation of gram quantities of advanced synthetic intermediates.

Short, Tin-Free Synthesis of All Three Inthomycins.

The inthomycins are a family of structurally and biologically rich natural products isolated from Streptomyces species. Herein the implementation of a modular synthetic route is reported that has enabled the enantioselective synthesis of all three inthomycins. Key steps include Suzuki and Sonogashira cross-couplings and an enantioselective Kiyooka aldol reaction.

A Short Synthesis of Aphanamol I in Both Racemic and Enantiopure Forms.

A short synthesis of the biologically active sesquiterpene natural product (+)-aphanamol I in both racemic and enantiopure forms is reported. Key steps include: a catalytic enantioselective conjugate addition, an oxidative radical cyclization, and a ring-expanding Claisen rearrangement.

Synthesis of bicyclic tetrahydrofurans from linear precursors using manganese(III) acetate.

We have recently developed methodology based on oxidative radical reactions for the synthesis of [3.3.0]-bicyclic lactones containing both cyclopentanes and γ-lactams along with application of this methodology to the synthesis of natural products and complex molecular architectures. Herein we report an extension of this methodology to the synthesis of oxygen heterocycles including bicyclic bis-lactones.

Analysis of four studies in a comparative framework reveals: health linkage consent rates on British cohort studies higher than on UK household panel surveys.

BACKGROUND: A number of cohort studies and longitudinal household panel studies in Great Britain have asked for consent to link survey data to administrative health data. We explore commonalities and differences in the process of collecting consent, achieved consent rates and biases in consent with respect to socio-demographic, socio-economic and health characteristics. We hypothesise that British cohort studies which are rooted within the health sciences achieve higher consent rates than the UK household longitudinal studies which are rooted within the social sciences. By contrast, the lack of a specific health focus in household panel studies means there may be less selectivity in consent, in particular, with respect to health characteristics. METHODS: Survey designs and protocols for collecting informed consent to health record linkage on two British cohort studies and two UK household panel studies are systematically compared. Multivariate statistical analysis is then performed on information from one cohort and two household panel studies that share a great deal of the data linkage protocol but vary according to study branding, survey design and study population. RESULTS: We find that consent is higher in the British cohort studies than in the UK household panel studies, and is higher the more health-focused the study is. There are no systematic patterns of consent bias across the studies and where effects exist within a study or study type they tend to be small. Minority ethnic groups will be underrepresented in record linkage studies on the basis of all three studies. CONCLUSIONS: Systematic analysis of three studies in a comparative framework suggests that the factors associated with consent are idiosyncratic to the study. Analysis of linked health data is needed to establish whether selectivity in consent means the resulting research databases suffer from any biases that ought to be considered.

Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.

Structure reassignment of laurefurenynes A and B by computation and total synthesis.

The originally assigned stereostructures of laurefurenynes A and B have been reassigned on the basis of DFT calculations of NMR chemical shifts, synthesis of model compounds and total synthesis of laurefurenyne B, demonstrating the power of this combined approach for stereostructure elucidation/confirmation.

A randomised study of nurse collected venous blood and self-collected dried blood spots for the assessment of cardiovascular risk factors in the Understanding Society Innovation Panel.

Dried blood spot (DBS) sample collection has been suggested as a less invasive, cheaper and more convenient alternative to venepuncture, which requires trained personnel, making it a potentially viable approach for self-collection of blood on a large scale. We examine whether participants in a longitudinal survey were willing to provide a DBS sample in different interview settings, and how resulting cardiovascular risk biomarkers compared with those from venous blood to calculate clinical risk. Participants of the Understanding Society Innovation Panel, a representative sample of UK households, were randomly assigned to three modes of interview. Most participants (84%) were interviewed in their allocated mode. Participants (n = 2162) were interviewed by a nurse who collected both a blood sample by venepuncture and a DBS card ('nurse collection') or participants were seen by an interviewer or took part in the survey online to self-collect a DBS card ('self-collection'). All DBS cards were returned in the post after the sample had dried. Lipids (total cholesterol, HDL-cholesterol, triglycerides), HbA1c and C-reactive protein were measured in venous and DBS samples and equivalence was calculated. The resultant values were used to confirm equivalent prevalence of risk of cardiovascular disease in each type of blood sample by mode of participation. Of participants interviewed by a nurse 69% consented to venous blood sample and 74% to a DBS sample, while in the self-collection modes, 35% consented to DBS collection. Demographic characteristics of participants in self-collection mode was not different to those in nurse collection mode. The percentage of participants with clinically raised biomarkers did not significantly differ between type of blood collection (for example, 62% had high cholesterol (> 5 mmol/l) measured by venepuncture and 67% had high cholesterol within the self-collected DBS sample (p = 0.13)). While self-collected DBS sampling had a lower response rate to DBS collected by a nurse, participation did not vary by key demographic characteristics. This study demonstrates that DBS collection is a feasible method of sample collection that can provide acceptable measures of clinically relevant biomarkers, enabling the calculation of population levels of cardiovascular disease risk.

Total synthesis and structure confirmation of elatenyne: success of computational methods for NMR prediction with highly flexible diastereomers.

Elatenyne is a small dibrominated natural product first isolated from Laurencia elata. The structure of elatenyne was originally assigned as a pyrano[3,2-b]pyran on the basis of NMR methods. Total synthesis of the originally proposed pyrano[3,2-b]pyran structure of elatenyne led to the gross structure of the natural product being reassigned as a 2,2'-bifuranyl. The full stereostructure of this highly flexible small molecule was subsequently predicted by Boltzmann-weighted DFT calculations of (13)C NMR chemical shifts for all 32 potential diastereomers, with the predicted structure being in accord with the proposed biogenesis outlined below. Herein we report two complementary total syntheses of elatenyne, which confirm the computer-predicted stereostructure. Additionally, the total syntheses of (E)-elatenyne and a related 2,2'-bifuranyl, laurendecumenyne B, are reported. This work has not only allowed the full structure determination of all of these natural products but also provides excellent supporting evidence for their proposed biogenesis. The total synthesis of elatenyne demonstrates that DFT calculations of (13)C NMR chemical shifts coupled with biosynthetic postulates, comprise a very useful method for distinguishing among large numbers of highly flexible, closely related molecules.

Consenting to health record linkage: evidence from a multi-purpose longitudinal survey of a general population.

BACKGROUND: The British Household Panel Survey (BHPS) is the first long-running UK longitudinal survey with a non-medical focus and a sample covering the whole age range to have asked for permission to link to a range of administrative health records. This study determines whether informed consent led to selection bias and reflects on the value of the BHPS linked with health records for epidemiological research. METHODS: Multivariate logistical regression is used, with whether the respondent gave consent to data linkage or not as the dependent variable. Independent variables were entered as four blocks; (i) a set of standard demographics likely to be found in most health registration data, (ii) a broader set of socio-economic characteristics, (iii) a set of indicators of health conditions and (iv) information about the use of health services. RESULTS: Participants aged 16-24, males and those living in England were more likely to consent. Consent is not biased with respect to socio-economic characteristics or health. Recent users of GP services are underrepresented among consenters. CONCLUSIONS: Whilst data could only be linked for a minority of BHPS participants, the BHPS offers a great range of information on people's life histories, their attitudes and behaviours making it an invaluable source for epidemiological research.

Total Synthesis and Structure Confirmation of (E) and (Z)-Ocellenyne.

The (E/Z)-ocellenynes are C15 dibrominated Laurencia natural products whose structures have been subject to several reassignments on the basis of extensive NMR analysis, biosynthetic postulates, and DFT calculations. Herein, we report the synthesis of both (E)- and (Z)-ocellenyne, which, in combination with single crystal X-ray diffraction studies, allows their absolute configuration to be established and defines the configuration of the syn-12,13-dibromide as being (S, S) in keeping with their proposed biogenesis from the (6S, 7S)-laurediols.

Collection of biospecimens from parent-child dyads in a community garden-based nutrition intervention: protocol and feasibility.

BACKGROUND: Non-invasive human biospecimens, including stool, urine, and hair, are important in understanding the relationship between diet and changes in human physiologic processes that affect chronic disease outcomes. However, biospecimen collection can be difficult when collecting samples for research studies that occur away from a centralized location. We describe the protocol and feasibility in collecting stool, urine, and hair biospecimens from parents and their children at a remote location as a part of a summer community garden-based intervention. METHODS: Stool, urine, and hair were collected as a part of the Summer Harvest Adventure (SHA) study, a randomized controlled, community garden-based intervention targeting children (ages 8-11 years) and their parents from low-resource neighborhoods. Biospecimens were collected from willing children and/or their parent/adult caregivers at baseline and post-intervention for evaluation of microbiome, metabolomics, and hair analyses among both intervention and control groups at a location distant from the academic laboratories conducting the analysis. The protocol used to assemble, deliver, collect, and process biospecimens are presented along with the frequencies with which specimens were successfully obtained. RESULTS: One hundred forty six participants (73 parent-child dyads) were part of the larger SHA study and thus eligible to provide a biospecimen. A total of 126 participants, 115 participants, and 127 participants consented to provide their hair, stool and urine samples, respectively. Of the participants that consented to provide a sample, 44 children (69.8%) and 38 parents (60.3%) provided at least one hair sample, 27 children (48.2%) and 37 parents (62.7%) provided at least one stool sample, and 36 children (57.1%) and 42 parents (65.6%) provided at least one urine sample. Sample collection at the offsite location, transport, and handling at the academic center were successful and all biospecimens were deemed adequate for analyses. DNA and metabolomics yield on a subset of stool samples obtained provided excellent results in terms of an abundance of species and metabolities, as would be predicted. Urine and hair analyses are underway. CONCLUSION: Our work is one of the first to describe the feasibility of collecting human biospecimens, specifically stool, urine, and hair, from both parents and their children from low-resourced neighborhoods in a non-traditional garden research setting. Future work will report findings related to mechanisms between diet, microbiome, metabolites, and clinical outcomes.

Forwards and backwards - synthesis of Laurencia natural products using a biomimetic and retrobiomimetic strategy incorporating structural reassignment of laurefurenynes C-F.

Laurefurenynes C-F are four natural products isolated from Laurencia species whose structures were originally determined on the basis of extensive nuclear magnetic resonance experiments. On the basis of a proposed biogenesis, involving a tricyclic oxonium ion as a key intermediate, we have reassigned the structures of these four natural products and synthesized the four reassigned structures using a biomimetic approach demonstrating that they are the actual structures of the natural products. In addition, we have developed a synthesis of the enantiomers of the natural products laurencin and deacetyllaurencin from the enantiomer of (E)-laurefucin using an unusual retrobiomimetic strategy. All of these syntheses have been enabled by the use of tricyclic oxonium ions as pivotal synthetic intermediates.