Azulene-based Protecting Groups

<p>Protecting groups form an indispensable part of modern organic synthetic chemistry. Besides the benefits of selectively passivating certain reactive functionalities, they often provide handling benefits – such as a decrease in the polarity of the compound that facilitates purification, an increase in the structural order of a compound that allows for easier crystallisation, and chromophores that enable easy visualisation on fluorescent TLC plates under UV light. Coloured protecting groups offer additional advantages in synthetic chemistry. They expedite purification by allowing the material to be tracked visually. Phase separation and column chromatography are easier to perform, and reduce the need for the collection of large numbers of fractions, while small-scale loss of material (left behind on taps or in flasks during routine handling) and spillages are much more readily apparent. Despite these advantages, only a few coloured protecting groups have been reported in the literature. The azulenes are a class of compounds with several attractive qualities that can be exploited for use as protecting groups. They are coloured, but not overwhelmingly so. The colour is tunable through placement of electron-donating or electron-withdrawing groups at positions on the ring system, which further allows for protection/deprotection reactions to be designed that incorporate a colour change. Azulene itself is both non-polar and structurally compact, unlike many other organic chromophores such as triarylmethane dyes and carotenoids. Furthermore, azulene’s ability to stabilise both positive and negative charges through resonance with tropylium and cyclopentadienide motifs allows for unusual chemistry, and therefore potentially orthogonal modes of deprotection. Four protecting group candidates incorporating azulene were devised. The 1-azulenylmethylene amine 79 and the 1-azulenesulfonamide 82 protecting group candidates for amines had fatal flaws that were discovered early, such as a tendency to rapidly degrade in open air. The 1-azulenecarboxylate protecting group candidate 74 for alcohols showed some promise, with a high-yielding protection reaction, but none of the deprotection conditions that were developed were sufficiently mild to be usable in a late-stage deprotection strategy on a complex target molecule. The final protecting group candidate, 6-(2-[oxycarbonyl]ethyl)azulene 89, can be used for the protection of carboxylic acids, amines and alcohols as esters, carbamates and carbonates, respectively. The substitution at the 6-position of azulene allows for deprotection through an E1cB mechanism with mild base, involving a cyclopentadienide-stabilised carbanion intermediate, in a similar fashion to the FMOC protecting group. Mild conditions for the protection of all three were found: for carboxylic acids Steglich esterification is employed, and for alcohols and amines coupling with CDI is used. A selection of mild protocols for deprotection were developed, using bases such as DBU or TBAF, or involving two-step activation-deprotection procedures. Finally, the compatibility of the protecting group 89 (dubbed Azul) with common and representative procedures in synthetic chemistry was investigated, such as with bases and with reaction conditions such as oxidations, reductions, cross-couplings, etc. Orthogonality with other common protecting groups (such as TBS, MOM, FMOC) was also explored. Some incompatibilities were found with strongly acidic conditions, high-temperature Suzuki cross-coupling reactions and Swern oxidations, but otherwise the Azul protecting group shows promise as a protecting group that expedites total synthesis through its colourful properties.</p>

Azulene-based Protecting Groups

<p>Protecting groups form an indispensable part of modern organic synthetic chemistry. Besides the benefits of selectively passivating certain reactive functionalities, they often provide handling benefits – such as a decrease in the polarity of the compound that facilitates purification, an increase in the structural order of a compound that allows for easier crystallisation, and chromophores that enable easy visualisation on fluorescent TLC plates under UV light. Coloured protecting groups offer additional advantages in synthetic chemistry. They expedite purification by allowing the material to be tracked visually. Phase separation and column chromatography are easier to perform, and reduce the need for the collection of large numbers of fractions, while small-scale loss of material (left behind on taps or in flasks during routine handling) and spillages are much more readily apparent. Despite these advantages, only a few coloured protecting groups have been reported in the literature. The azulenes are a class of compounds with several attractive qualities that can be exploited for use as protecting groups. They are coloured, but not overwhelmingly so. The colour is tunable through placement of electron-donating or electron-withdrawing groups at positions on the ring system, which further allows for protection/deprotection reactions to be designed that incorporate a colour change. Azulene itself is both non-polar and structurally compact, unlike many other organic chromophores such as triarylmethane dyes and carotenoids. Furthermore, azulene’s ability to stabilise both positive and negative charges through resonance with tropylium and cyclopentadienide motifs allows for unusual chemistry, and therefore potentially orthogonal modes of deprotection. Four protecting group candidates incorporating azulene were devised. The 1-azulenylmethylene amine 79 and the 1-azulenesulfonamide 82 protecting group candidates for amines had fatal flaws that were discovered early, such as a tendency to rapidly degrade in open air. The 1-azulenecarboxylate protecting group candidate 74 for alcohols showed some promise, with a high-yielding protection reaction, but none of the deprotection conditions that were developed were sufficiently mild to be usable in a late-stage deprotection strategy on a complex target molecule. The final protecting group candidate, 6-(2-[oxycarbonyl]ethyl)azulene 89, can be used for the protection of carboxylic acids, amines and alcohols as esters, carbamates and carbonates, respectively. The substitution at the 6-position of azulene allows for deprotection through an E1cB mechanism with mild base, involving a cyclopentadienide-stabilised carbanion intermediate, in a similar fashion to the FMOC protecting group. Mild conditions for the protection of all three were found: for carboxylic acids Steglich esterification is employed, and for alcohols and amines coupling with CDI is used. A selection of mild protocols for deprotection were developed, using bases such as DBU or TBAF, or involving two-step activation-deprotection procedures. Finally, the compatibility of the protecting group 89 (dubbed Azul) with common and representative procedures in synthetic chemistry was investigated, such as with bases and with reaction conditions such as oxidations, reductions, cross-couplings, etc. Orthogonality with other common protecting groups (such as TBS, MOM, FMOC) was also explored. Some incompatibilities were found with strongly acidic conditions, high-temperature Suzuki cross-coupling reactions and Swern oxidations, but otherwise the Azul protecting group shows promise as a protecting group that expedites total synthesis through its colourful properties.</p>

Automated Home-Cage Monitoring During Acute Experimental Colitis in Mice

For ethical and legal reasons it is necessary to assess the severity of procedures in animal experimentation. To estimate the degree of pain, suffering, distress or lasting harm, objective methods that provide gradebale parameters need to be tested and validated for various models. In this context, automated home-cage monitoring becomes more important as a contactless, objective, continuous and non-invasive method. The aim of this study was to examine a recently developed large scale automated home-cage monitoring system (Digital Ventilated Cage, DVC®) with regard to the applicability and added value for severity assessment in a frequently used acute colitis mouse model. Acute colitis was induced in female C57BL/6J mice by varying doses of DSS (1.5 and 2.5%), matched controls received water only (0%). Besides DVC® activity monitoring and nest scoring, model specific parameters like body weight, clinical colitis score, and intestinal histo-pathology were used. In a second approach, we questioned whether DVC® can be used to detect an influence of different handling methods on the behavior of mice. Therefore, we compared activity patterns of mice that underwent tunnel vs. tail handling for routine animal care procedures. In DSS treated mice, disease specific parameters confirmed induction of a graded colitis. In line with this, DVC® revealed reduced activity in these animals. Furthermore, the system displayed stress-related activity changes due to the restraining procedures necessary in DSS-treatment groups. However, no significant differences between tunnel vs. tail handling procedures were detected. For further analysis of the data, a binary classifier was applied to categorize two severity levels (burdened vs. not burdened) based on activity and body weight. In all DSS-treatment groups data points were allocated to the burdened level, in contrast to a handling group. The fraction of “burdened” animals reflected well the course of colitis development. In conclusion, automated home-cage monitoring by DVC® enabled severity assessment in a DSS-induced colitis model equally well as gold standard clinical parameters. In addition, it revealed changes in activity patterns due to routine handling procedures applied in experimental model work. This indicates that large scale home-cage monitoring can be integrated into routine severity assessment in biomedical research.

Judgement Bias in Miniature Donkeys: Conditioning Factors and Personality Links

Expectation-related bias may configure individuals’ perception of their surrounding environment and of the elements present in it. This study aimed to determine the repercussions of environmental (weather elements) or subject-inherent factors (sex, age, or personality features) on judgment bias. A cognitive bias test was performed in eight Miniature jennies and four jacks. Test comprised habituation, training and testing phases during which subjects were trained on how to complete the test and scored based on their latency to approach an ambiguous stimulus. A questionnaire evaluating eleven personality features was parallelly completed by three caretakers, five operators and two care assistants to determine the links between personality features and judgment bias. Adjusted latencies did not significantly differ between sexes (Mann–Whitney test, p > 0.05). Although Miniature donkeys can discriminate positive/negative stimuli, inter-individual variability evidences were found. Such discrimination is evidenced by significant latency differences to approach positive/negative stimuli (33.7 ± 43.1 vs. 145.5 ± 53.1 s) (Mann–Whitney test, p < 0.05). Latencies significantly increased with patience, indicative of an expression of pessimism. Better understanding judgement bias mechanisms and implications may help optimize routine handling practices in the framework of animal welfare.

The utility of voluntary weighing in captive group-living rhesus macaques (Macaca mulatta)

Bodyweight is an important health and welfare indicator for captive non-human primates (NHPs). Bodyweight can be measured during routine handling procedures, which cause stress. Alternatively, animals can be trained to step onto a scale, but training success varies greatly between individuals. Being able to weigh animals regularly without having to handle or train them is thus desirable for monitoring animal health and welfare. This study investigates the utility, ie the participation, reliability and time investment, of voluntary weighing in captive NHPs living in large social groups. Subjects of the study were 92 rhesus macaques (Macaca mulatta) housed in four social groups at the Biomedical Primate Research Centre in Rijswijk, The Netherlands. A scale was placed in their home enclosure during several sessions. Individuals were unwilling to step onto an unbaited scale. When likeable food items were used to attract individuals to the scale, 68% of them stepped onto the scale. Age and dominance rank did not affect stepping onto the scale, whereas exploratory tendency and social group did. The level of agreement between bodyweight by voluntary weighing and bodyweight measured during sedation was very high. These results show that the majority of rhesus macaques in social groups can be weighed voluntarily and that voluntary weighing is reliable. When optimising and further developing the method, voluntary weighing can form a valuable tool in the captive management of NHPs.

Effects of farm location on Vibrio parahaemolyticus and V. vulnificus levels in oysters after desiccation and resubmersion in the northern Gulf of Mexico

Desiccation is a routine farming practice utilized in off-bottom oyster aquaculture to reduce biofouling organisms and improve shell quality. This practice can increase Vibrio parahaemolyticus and V. vulnificus levels, leading to increased risk of illness for raw oyster consumers. Previous resubmersion studies were performed in geographic proximity to one another, so to better understand the broader applicability of resubmersion, the next step was to perform concurrent studies in multiple geographic locations within a region. This study evaluated the effect of variations in geographic location on the recovery time needed for elevated vibrio levels to return to ambient levels in desiccated oysters after resubmersion at Gulf Coast farms. Two trials were performed between May-August 2019 at sites spanning ~100 km: three in Alabama and one in Florida. Oysters were deployed in OysterGro cages at each location, two weeks prior to each trial, then either desiccated for 24 h or remained submersed as controls. Triplicate samples were taken prior to and immediately following the desiccation period, as well as 7 and 14 d post-resubmersion. Total and pathogenic ( tdh +/ trh +) V. parahaemolyticus , and V. vulnificus levels were determined using most probable number (MPN) real-time PCR. Vibrio levels increased by 0.23-3.50 log MPN/g after desiccation. Recovery times varied among geographic locations by trial and Vibrio spp., with all vibrio counts recovering to levels not significantly higher than those in control oysters within 7-14 days of resubmersion (p≥0.06). These results suggest a 14-day resubmersion period of cultured oysters allowed vibrio levels, elevated due to routine handling, to return to ambient levels at all farm sites studied.

Routine handling does not lead to chronic stress in captive green anole (Anolis carolinensis)

Routine handling has been shown to affect stress levels in a variety of animal species. This could result in a general decrease in welfare and may confound the results of scientific experiments or observations on captive study animals. In reptiles, there seems to be variation in the effects of handling on stress levels. Furthermore, most studies on reptiles only look at the effect of handling in the short term. In this study we quantified the physiological and behavioural impact of being held, twice daily, for 1 min at a time over a three-week period on the green anole (Anolis carolinensis). Measurements were collected at the end of the three-week repeated handling period. Our results showed no effect of repeated handling on body mass, tail-base width, heterophil to lymphocyte ratios (H/L ratios), behaviour and faecal corticosterone metabolite (FCM) levels for both males and females in the experimental treatments ('handled', 'unhandled'). Our study animals did score very highly for several stress-indicating variables in the three weeks preceding the experiments — suggesting that they had experienced considerable stress during capture, transport and temporary housing in the pet store.

Laboratory safety: handling B. pseudomallei isolates without a biosafety cabinet

B. pseudomallei is a tier 1 select agent that is associated with laboratory-acquired melioidosis, with international guidelines recommending isolate handling within a class II biosafety cabinet (BSC) in a Biosafety Level (BSL) 3 facility. In low-resource settings this may not be practical, and therefore the authors aimed to assess the risk of laboratory-acquired melioidosis during routine work. Prior exposure to the organism was determined with a questionnaire and concomitant serology. Of 30 laboratory scientists handling B. pseudomallei on 1,267 occasions outside a biosafety cabinet, no infections were documented and all participants remained seronegative. Additionally, we performed controlled environmental air sampling during 78 laboratory handling events including plate opening, oxidase testing, and McFarland suspension creation. None of the experiments demonstrated aerosolization of the organism. This study suggests the risk of laboratory-acquired melioidosis is likely to be low. However, individual laboratories will need to undertake a risk assessment including melioidosis endemicity, availability of resources for containment, the nature of routine handling to be undertaken, and the presence of predisposing risk factors for infection in the staff concerned. Additionally, laboratories should take region-specific guidelines into consideration. Further research is required to better inform on the overall risk of infection in the microbiology laboratory.

The Impact of Long-COVID on Functioning – Results from a Community Survey in Patients After Mild and Moderate SARS-CoV-2-Infection in Germany

BackgroundIn COVID-19 survivors a relatively high number of long-term symptoms have been observed. Besides impact on quality of life, these symptom (now called long-COVID) may have an impact on functioning and my hinder affected people to participate in social life. However, little is known if and to what extent patients with mild and moderate COVID-19 who did not need hospitalization or intensive care develop such a syndrome.MethodsA cross-sectional study in 1027 patients with mild or moderate COVID-19 has been performed in two communities in Bavaria, Germany. The Rehabilitation-Needs-Survey (RehabNeS) that includes Short Form 36 health questionnaire (SF-36) on health-related quality of life, was performed. Descriptive statistics were calculated. Results97.5 % of patients reported one symptom in infection phase such as fatigue, respiratory problems, limitations of the sense of taste and smell, as well as fear and anxiety and other symptoms. In this phase 84.1% of participants experienced activity limitations and participation restrictions such as carrying out daily routine, handling stress, getting household tasks done, care/support for others, and relaxing and leisure.61.9% of participants reported persisting symptoms after more than 3 months after infection. These were among others fatigue, sleep disturbances, respiratory problems pain, fears and anxiety, and restrictions in movement. 49% of the participants reported on activity limitations and participation restrictions. Predominately these were handling stress, carrying out daily routine, looking after one’s health, relaxing and leisure activities as well as doing house work.The impact on quality of life and vocational performance were rather low.ConclusionThe results show that long-term symptoms after mild and moderate COVID-19 are common and lead to limitations of activities and participation. However, it seems that in most cases they are not very severe and do not lead to frequent or severe issues with quality of live or work ability.