The Mitsunobu reaction basically consists in the conversion of an alcohol into an ester under inversion of configuration, employing a carboxylic acid and a pair of two auxiliary reagents, mostly triphenylphosphine and a dialkyl azodicarboxylate. This reaction has been frequently used in carbohydrate chemistry for the modification of sugar hydroxy groups. Modification at the anomeric position, leading mainly to anomeric esters or glycosides, is of particular importance in the glycosciences. Therefore, this review focuses on the use of the Mitsunobu reaction for modifications of sugar hemiacetals. Strikingly, unprotected sugars can often be converted regioselectively at the anomeric center, whereas in other cases, the other hydroxy groups in reducing sugars have to be protected to achieve good results in the Mitsunobu procedure. We have reviewed on the one hand the literature on anomeric esterification, including glycosyl phosphates, and on the other hand glycoside synthesis, including S- and N-glycosides. The mechanistic details of the Mitsunobu reaction are discussed as well as this is important to explain and predict the stereoselectivity of anomeric modifications under Mitsunobu conditions. Though the Mitsunobu reaction is often not the first choice for the anomeric modification of carbohydrates, this review shows the high value of the reaction in many different circumstances.

Both metabolism and transport are key elements defining the bioavailability and biological activity of molecules, i.e. their adverse and therapeutic effects. Structured and high quality experimental data stored in a suitable container, such as a relational database, facilitates easy computational processing and thus allows for high quality information/knowledge to be efficiently inferred by computational analyses. Our aim was to create a freely accessible database that would provide easy access to data describing interactions between proteins involved in transport and xenobiotic metabolism and their small molecule substrates and modulators. We present Metrabase, an integrated cheminformatics and bioinformatics resource containing curated data related to human transport and metabolism of chemical compounds. Its primary content includes over 11,500 interaction records involving nearly 3,500 small molecule substrates and modulators of transport proteins and, currently to a much smaller extent, cytochrome P450 enzymes. Data was manually extracted from the published literature and supplemented with data integrated from other available resources. Metrabase version 1.0 is freely available under a CC BY-SA 4.0 license at http://www-metrabase.ch.cam.ac.uk.

Understanding which physicochemical properties, or property distributions, are favorable for successful design and development of drugs, nutritional supplements, cosmetics, and agrochemicals is of great importance. In this study we have analyzed molecules from three distinct chemical spaces (i) approved drugs, (ii) human metabolites, and (iii) traditional Chinese medicine (TCM) to investigate four aspects determining the disposition of small organic molecules. First, we examined the physicochemical properties of these three classes of molecules and identified characteristic features resulting from their distinctive biological functions. For example, human metabolites and TCM molecules can be larger and more hydrophobic than drugs, which makes them less likely to cross membranes. We then quantified the shifts in physicochemical property space induced by metabolism from a holistic perspective by analyzing a data set of several thousand experimentally observed metabolic trees. Results show how the metabolic system aims to retain nutrients/micronutrients while facilitating a rapid elimination of xenobiotics. In the third part we compared these global shifts with the contributions made by individual metabolic reactions. For better resolution, all reactions were classified into phase I and phase II biotransformations. Interestingly, not all metabolic reactions lead to more hydrophilic molecules. We were able to identify biotransformations leading to an increase of logP by more than one log unit, which could be used for the design of drugs with enhanced efficacy. The study closes with the analysis of the physicochemical properties of metabolites found in the bile, faeces, and urine. Metabolites in the bile can be large and are often negatively charged. Molecules with molecular weight >500 Da are rarely found in the urine, and most of these large molecules are charged phase II conjugates.

In this study, two probabilistic machine-learning algorithms were compared for in silico target prediction of bioactive molecules, namely the well-established Laplacian-modified Naïve Bayes classifier (NB) and the more recently introduced (to Cheminformatics) Parzen-Rosenblatt Window. Both classifiers were trained in conjunction with circular fingerprints on a large data set of bioactive compounds extracted from ChEMBL, covering 894 human protein targets with more than 155,000 ligand-protein pairs. This data set is also provided as a benchmark data set for future target prediction methods due to its size as well as the number of bioactivity classes it contains. In addition to evaluating the methods, different performance measures were explored. This is not as straightforward as in binary classification settings, due to the number of classes, the possibility of multiple class memberships, and the need to translate model scores into "yes/no" predictions for assessing model performance. Both algorithms achieved a recall of correct targets that exceeds 80% in the top 1% of predictions. Performance depends significantly on the underlying diversity and size of a given class of bioactive compounds, with small classes and low structural similarity affecting both algorithms to different degrees. When tested on an external test set extracted from WOMBAT covering more than 500 targets by excluding all compounds with Tanimoto similarity above 0.8 to compounds from the ChEMBL data set, the current methodologies achieved a recall of 63.3% and 66.6% among the top 1% for Naïve Bayes and Parzen-Rosenblatt Window, respectively. While those numbers seem to indicate lower performance, they are also more realistic for settings where protein targets need to be established for novel chemical substances.

Understanding the metabolic fate of small organic molecules is of fundamental importance to the successful design and development of drugs, nutritional supplements, cosmetics and agrochemicals [1,2]. In the current study we investigated how the products of metabolism differ from their parent molecules by analysing a large dataset of experimentally determined metabolic transformations (Figure ​(Figure1).1). This dataset was split into three specific chemical domains representing approved drug molecules, human metabolites and molecules from traditional Chinese medicines to allow individual analysis. We also quantified the impact of individual Phase I and Phase II metabolic reactions on calculated chemical descriptors using MetaPrint2D [3] and suggest new approaches to utilise metabolism for the design of drugs and cosmetics. The last section of this study investigates the properties of metabolites found in the bile, faeces and urine and analyses their commonalities and differences. Figure 1 Four important questions pertinent to the design and development of new molecules with favourable ADME properties addressed in this work. d, approved drugs; h, human metabolites; t, molecules from traditional Chinese medicines; MW, molecular weight.

The use of heme analogues from vegetable origin could provide an alternative iron source of potentially high bioavailability. Sodium iron chlorophyllin is a water-soluble semisynthetic chlorophyll derivative where the magnesium in the porphyrin ring has been substituted by iron. We have used an in vitro model that combines gastric and intestinal digestion followed by intestinal iron uptake in Caco-2 cells to determine the bioavailability of iron from sodium iron chlorophyllin. Our results demonstrate that sodium iron chlorophyllin is stable under simulated gastrointestinal conditions and is able to deliver bioavailable iron to Caco-2 cells. Similar to the heme, the bioavailability of iron from sodium iron chlorophyllin is dependent on the food matrix, and it was inhibited by calcium. Potentially, sodium iron chlorophyllin could be used as an iron fortificant from vegetable origin with high bioavailability.

As consumers become aware of the role functional foods can play in their personal health, the differentiating aspects of such functional foods need to be determined. One such aspect is the information provided on the bioavailability of the active ingredients responsible for functionality, which plays a role in marketing and the regulatory process. Regulators are starting to develop guidelines and criteria that take bioavailability into account. Although bioavailability is not a new term for foods, the many parameters which determine bioavailability in a food context are not understood in full. On one hand, bioavailability is controlled by the active ingredient itself, as determined by its physicochemical characteristics and the response of the human physiology upon exposure. Understanding the basic principles and physiology is, however, too simple an approach. On the other hand, for food-specific processing, the food matrix itself and the response of human physiology towards a food product and meal intake also play a role. They determine to a great extent the gastrointestinal milieu from which the active ingredient needs to be absorbed. This interplay of ingredient, matrix, gastrointestinal condition and body response, including post-absorption, has to be taken into account in determining bioavailability of a functional ingredient or in developing new functional food products. Thus the models that predict bioavailability based on physicochemical parameters should be used cautiously as other factors can be dominant. Within foods there is a need to expand these predictive models into the relevant chemical space. To test these predictions or food concepts, in vitro models are suitable as long as they reflect the actual intestinal conditions in the fasted or fed state. In this area considerable progress has been made, resulting in proper biorelevant models. One aspect, which is still little understood, is that of meal composition and meal conditions (e.g. is the active ingredient taken with the meal, or before or after) on bioavailability. No relevant standards have been agreed on experimental conditions, nor are all critical limits known for the physiology triggers and their inter- and intra-individual variations. To guide the functional foods researcher in this complex area, examples are given and a simple logic scheme is provided as a start in developing a bioavailability research strategy.

Selected di- and tripeptides exhibit angiotensin-I converting enzyme (ACE) inhibitory activity in vitro. However, the efficacy in vivo is most likely limited for most peptides due to low bioavailability. The purpose of this study was to identify descriptors of intestinal stability, permeability, and ACE inhibitory activity of dipeptides. A total of 228 dipeptides were synthesized; intestinal stability was obtained by in vitro digestion, intestinal permeability using Caco-2 cells and ACE inhibitory activity by an in vitro assay. Databases were constructed to study the relationship between structure and activity, permeability, and stability. Quantitative structure-activity relationship (QSAR) modeling was performed based on computed models using partial least squares regression based on 400 molecular descriptors. QSAR modeling of dipeptide stability revealed high correlation coefficients (R > 0.65) for models based on Z and X scales. However, amino acid (AA) clustering showed the best results in describing stability of dipeptides. The N-terminal AA residues Asp, Gly, and Pro as well as the C-terminal residues Pro, Ser, Thr, and Asp stabilize dipeptides toward luminal enzymatic peptide hydrolysis. QSAR modeling did not reveal significant correlation models for intestinal permeability. 2D-fingerprint models were identified describing ACE inhibitory activity of dipeptides. The intestinal stability of 12 peptides was predicted. Peptides were synthesized and stability was confirmed in simulated digestion experiments. Based on the results, specific dipeptides can be designed to meet both stability and activity criteria. However, postabsorptive ACE inhibitory activities of dipeptides in vivo are most likely limited due to the very low intestinal permeability of dipeptides.

Iron deficiency is the most common nutritional disorder in the world. It is estimated that 2 billion people are anaemic. Approximately 50% of infants, school-age children and women of reproductive age suffer from iron deficiency anaemia in some countries of South Asia, compared to about 25% in Latin America and 10% in the industrialised countries of Europe.Iron deficiency is a result of the imbalance between the amount of iron absorbed and iron excreted. Such an imbalance may be the result of low iron intake due to low iron content of the diet or to low bioavailability of the dietary iron to compensate for the losses. Certain compounds such as ascorbic acid are well known for increasing iron bioavailability. However, not many other compounds have been found with a similar effect. In this review, we evaluate the methods employed in the search for compounds that could enhance iron bioavailability from iron fortified foods. All the methods are designed to screen for natural compounds that can form complexes with iron to guarantee product stability and acceptable taste, but still able to increase the concentration of soluble iron in the proximity of the intestinal brush border membrane, allowing for efficient iron uptake into the intestinal cells.When used as a prelude of human trials, this screening strategy might enable improved design and productivity of the more expensive human experiment. Applications of this model include product development, but also screening of plant cultivars for improved bioavailability, development of improved supplements and studies of the precise factors that promote iron uptake.

Bioavailability is the fraction of an administered dose that reaches the systemic circulation. Health claims for functional foods can only be made if the ingredient reaches the target site to trigger the physiological action, hence substantiation requires good bioavailability. This is also expressed in the European Regulation on Health Claims. Albeit not new at all, a full understanding of the controlling factors for bioavailability in foods is still lacking. Foods are complex systems and can be part of a meal. The impact of product composition and the interplay with human physiology during fed or fasted state has to be understood: a functional ingredient has to be released from the product matrix into a molecularly dispersed state, either in classic solution or in micellar state. Only in the dispersed state can actives cross the gut wall. Release and dissolution are depending on both molecular physicochemical properties of the active and those of the entire product. More complex is the uptake of hydrophobic, poorly water-soluble substrates. As they do not dissolve in the aqueous intestinal environment, presence of fat, release of bile, enzymes, and gut motility to induce lipolysis are required. Surface-active bile salts together with lipolysis products create micelles containing the hydrophobic active. This imposes limitations on the formulation space for hydrophobic compounds. Finally, many ingredients are not fit for straightforward use as they compromise the stability or sensory characteristics of the product. Compartmentalization strategies, like encapsulation, may offer solutions to the problem; it should, however, not be forgotten that encapsulates themselves may effect bioavailability through the changed dynamics of the uptake processes. To explore and build a better understanding of these factors, a range of models are available and used in product formulation and claim substantiation. A structured approach and the selection of proper models will help to improve functional food formulations in the future.

The abundance of different techniques and protocols available reflects the need for reliable in vitro methods to assess intestinal absorption of potentially bioactive compounds. Physicochemical assays try to pinpoint the molecular properties contributing to the absorption process. The end points of biologically based methods, such as cell cultures and excised tissues, account for all processes undergone by a molecule that traverses a ‘living’ biological membrane, a cell or tissue. On top of fundamental physical processes (e.g., solubility, diffusion) such biological methods incorporate physiological responses such as active transport and metabolism. In this review, an account of in vitro methods for the assessment of molecular properties (lipophilicity, solubility, permeability) influencing intestinal absorption is given. Their advantages and limitations and the possibilities offered by this area of research are also evaluated. The combination of results from both classes of assays (physicochemical and biological) and integration with computational models will guide future developments in this field. Finally, possible future developments including stem cell research and multiple-end point assays are discussed.

Cytotoxicity testing allows determining whether a compound or extract contains significant quantities of biologically harmful chemicals. Cytotoxicity test methods are useful for screening because they serve to separate toxic from nontoxic materials, providing predictive evidence of compound safety. However, a wide range of assays measuring different aspects of cell death is available in the market, but it is difficult to determine which one(s) to use when evaluating a selection of compounds. The objective of this study was to compare different commercially available in vitro assays for cytotoxicity in HepG2 cells according to its sensitivity, reproducibility, simplicity, cost, and speed. The assays evaluated included Alamar Blue for the measurement of mitochondrial activity, ATPlite and ViaLight for the determination of cellular adenosine triphosphate (ATP), ToxiLight as an indicator of cellular necrosis, and Caspase-3 Fluorometric Assay, Apo-ONE Caspase-3/7 Homogeneous Assay, and Caspase-Glo for the determination of caspase-3/7 activity. All assays were performed using 4 compounds of previously reported cytotoxic activity: DMSO, butyric acid, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and camptothecine. Overall, it was concluded that the best way to evaluate the potential cytotoxicity of a compound is to employ a battery of assays that focus on different aspects of cell death. In this case, the focus has been on ATP levels, cell necrosis, and capsase-3/7 activation. Many other kits are commercially available in the market for these and other aspects of necrosis and/or apoptosis. However, the use of ViaLight Plus, ToxiLight, and Caspase-3 Fluorometric Assay resulted in the most useful combination when working with HepG2 cells.

Similarity searches using combinations of seven different similarity coefficients and six different representations have been carried out on the Dictionary of Natural Products database. The objective was to discover if any special methods of searching apply to this database, which is very different in nature from the many synthetic databases that have been the subject of previous studies of similarity searching. Search effectiveness was assessed by a recall analysis of the search outputs from sets of pharmacologically active target structures. The different target sets produce exceptional but contradictory results for the Russell-Rao and Forbes coefficients, which have been shown to be due to a dependence on molecular size; these are the coefficients of choice in the case of large and small structures, respectively. Rankings from these results have been combined using a data fusion scheme and some small gains in performance were normally obtained by using substructural fingerprints and molecular holograms in combination with the Squared Euclidean or Tanimoto coefficients.