Academic literature on the topic '4-hydroxyisoleucine'

(2S,3R,4S)-4-Hydroxyisoleucine (4-HIL) is a promising drug for treating type II diabetes mellitus. Currently, there are three main methods for obtaining 4-HIL: chemical synthesis, raw material extraction, and biosynthesis. The conventional chemical synthesis, separation, and extraction methods have several disadvantages, including cumbersome steps, high costs, high content of environmental pollution by-products, and poor sustainability. In contrast, the biosynthesis method is characterised by low cost, high sustainability, mild reaction conditions, and environmental friendliness. Therefore, the catalytic synthesis of 4-HIL using the biological enzyme method has become a current research hotspot.

Herbal products are emerging as promising anti-diabetic drugs due to lower cost, lesser side effects and renewable natural sources. 4-hydroxyisoleucine is reported as the bioactive compound, which is responsible for anti-diabetic activity of fenugreek. This review illustrates the overall properties, synthesis, mode of action and pharmacological profile of 4-hydroxyisoleucine. From the seeds of Trigonella foenum graecum an unusual amino acid, 4-hydroxyisoleucine has been isolated which significantly improved lipid profile and it increased glucose-induced insulin release in human and rat pancreatic islet cells. No adverse effect or toxic effect of 4-hydroxyisoleucine has been found. Marketed preparations are also available.

Trigonella foenum- graecum (fenugreek) is a leguminous plant with poor nitrogen fixing ability. The current study deals with the effects of T.viride on growth, yield and 4-hydroxyisoleucine content in fenugreek. 4-hydroxyisoleucine is an antidiabetic compound present only in plants particularly in fenugreek. Treatment of fenugreek seeds with T.viride enhanced the growth, yield and 4-hydroxyisoleucine content significantly compared to control. Seeds were treated with T.viride suspension (107spores/seed) and untreated seeds (1 ml distilled water/ seed) served as control sown in pots filled with potting soil. Several growth parameters like root length and lateral root expansion, shoot length, yield, chlorophyll and nitrogen were estimated. 4-hydroxyisoleucine content in fenugreek seeds was estimated by ninhydrin method. A significant increase of 25 % in root length and 128 % in lateral root expansion and 50 % in shoot length was observed in treated plants over control. The total yield increased by 95 % in treated one. The total chlorophyll content exceeded by 21.86 % and total plant nitrogen by 121.73 %. An increase of 27 % of 4-hydroxyisoleucine content is observed in treated fenugreek plants over control.

Fenugreek is a legume but regarded as a poor nitrogen fixer. The present study deals with the marked effect of Trichoderma viride and rhizobium on quantification of 4-hydroxyisoleucine content and free amino acid composition of fenugreek seeds. 4-hydroxyisoleucine is regarded as an antidiabetic compound present specifically in fenugreek plants. Fenugreek seeds treated with both T.viride and rhizobium boosted free amino acid concentration and 4-hydroxyisoleucine content compared to control and positive control (NPK treated). Seeds were treated with T.viride suspension (107 spores/seed), rhizobium suspension (108 cells/seed) , combination of T.viride and rhizobium suspension in equal amounts (0.5ml of T.viride  0.5ml of rhizobium suspension /seed), NPK suspension ( 1ml NPK suspension / seed) and untreated seeds (1 ml distilled water/ seed) serving as control sown in pots filled with potting soil and also in a randomized plot ( 55 feet) in the field. In free amino acid composition, isoleucine is the precursor for 4-hydroxyisoleucine compound and its concentration was enhanced by 47.79 % (potted plants) and 26.46 % (field plants) over control and by 27.02 % (potted plants) and 13.03 % (field plants) over positive control. 4-hydroxyisoleucine content was quantified using LCMS method in both potted and field plants. An increase of 37.02 % (potted plants) and 23.61 % (field plants) in 4-hydroxyisoleucine concentration in dual treated plants over control and positive control was observed.

Department of Chemistry, B. N. Bandodkar College of Science, Thane-400 601, Maharashtra, India <em>E-mail </em>: atulbedekar@ gmail.com <em>Manuscript received 20 December 2005, revised 7 August 2006, accepted 23 October 2008</em> 4-Hydroxylsoleucine is reported as bioactive compound, which is responsible for antidiabetic activity of Fenugreek. Our present work includes standardization and preparation of different extracts, which are enriched with 4- hydroxyisoleucine. The paper Includes some unexpected observation in extraction pattern of 4-hydroxyisoleucine with different solvents. The quantitative estimation of 4-hydroxyisoleucine is done by spectrophotometry on the basis of its reaction with ninhydrin.

Fenugreek (Trigonella foenum-graecum) is a plant extensively cultivated in Middle Eastern and South Asian countries where its seeds have been traditionally used as flavoring agents in food and in folk medicine for a variety of ailments, including diabetes. Its active ingredients include proteins, saponins, polyphenols, alkaloids, and flavonoids. Extracts were found rich in different phytoconstituents, such as trigonelline, diosgenin, 4-hydroxyisoleucine, 3-hydroxyisoleucine, fenugreekine, saponins, and alkaloids, of which, 4-hydroxyisoleucine and saponins were focused as bioactive molecules. Fenugreek extracts and ingredients exhibit antioxidant, antidiabetic, hepatoprotective, antihyperlipidemic, antimicrobial, and anti-inflammatory activities. Even though fenugreek is known for its thrombolytic action, antidiabetic and hypocholesterolemic effects are often cited as its most important therapeutic effects. Furthermore, some of the active principles and hypoglycemic activity of fenugreek have been characterized and reported. The present article aims to provide an overview of the active ingredients and antidiabetic activity of fenugreek.

In the present study the combined therapeutic approach of using an isolated compound from a herb and an oral hypoglycemic drug in alloxan-induced diabetic mice is investigated. Herbal drugs can have beneficial effects in terms of reducing the dosage, side effects and duration of action of synthetic drugs. The results show that combined therapy with 4- hydroxyisoleucine and pioglitazone is more beneficial than pioglitazone alone and also more beneficial than the combination of 4 hydroxyisoleucine and glyburide in the treatment of diabetes. (Int J Diabetes Metab 14: 104-105, 2006)

The objective of the present investigation is to extract, quantify and characterize the active constituent, 4- hydroxyisoleucine in Trigonella foenum graecum seeds using HPLC and HPTLC methods. Aqueous extract of crushed seeds of Trigonella foenum graecum was prepared and subjected to HPLC and HPTLC analysis. HPLC analysis was performed using C18 reverse phase column and Fluorescence detector with an elution gradient composed of 65 mmol Sodium acetate, l.5% tetrahydrofurane (pH 5.7) and methanol. HPTLC analysis was performed using semiautomatic TLC sampler Linomat V (CAMAG) controlled by WinCATS software. A constant application rate of 80 nLs was employed with a band width of 3.0mm and distance between two bands was 6.2 mm. The plates were developed in 20 × 10 cm twin trough glass chamber containing 25mL of mobile phase as mixture of butanol: acetic acid: water (4: 1: 1, v/v/v) and detected at λ 254nm and 570 nm. HPLC analysis of Standard concentrations of 4-hydroxyisoleucine exhibited a linear plot with average retention time of 8.13 min and sample analysis revealed a retention time of 8.19 min. HPTLC analysis revealed Rf value of 0.45 for both standard and the seed extract at λ 254nm and 570 nm. Keywords: Trigonella, 4-hydroxyisoleucine, HPLC, HPTLC, Fluorescence detector, Retention time, Rf value.

ABSTRACTWe determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependentl-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine inBacillus thuringiensis2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphaticl-amino acids, as well asl-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product ofl-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found inB. thuringiensis2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containingl-amino acids and generatedl-methionine sulfoxide andl-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst.

L’étude des stéréoisomères de la 4-hydroxyisoleucine a montré que l’isomère (2S,3R,4S) stimule efficacement la sécrétion d’insuline, expliquant les propriétés hypoglycémiantes de cette plante. La synthèse de la 4-hydroxyisoleucine et de ses analogues est un domaine d’étude pour lutter contre le diabète. Cette thèse concerne les synthèses de la 4-hydroxyisoleucine et ses analogues selon trois voies différentes. La chiralité du D-glucose a été exploitée par des réactions contrôlables, douces et stéréosélectives permettant la synthèse de 4 acides aminés et 3 hydroxy-acides. Une deuxième voie considérée concerne la cycloaddition 1,3-dipolaire de nitrones dérivées de (–)- et (+)-menthone sur des alcènes mono- et disubstitués pour créer simultanément 3 centres asymétriques. Cette voie a conduit à la (2S,3R,4R)-4-hydroxyisoleucine et à 12 autres aminoacides. Une troisième voie vise l’attaque nucléophile de cétones par un carbanion fonctionnalisé issu de l’isocyanoacétate d’éthyle pour donner des précurseurs insaturés permettant l’accès aux aminoacides. Cette méthodologie tout juste abordée a mené à la synthèse racémique d’un acide aminé dihydroxylé. Des tests préliminaires suggèrent que deux de ces produits pourraient favoriser la sécrétion d’insuline<br>The study of the stereoisomers of 4-hydroxyisoleucine has shown that the isomer (2S,3R,4S) stimulates the insulin secretion effectively, explaining its the hypoglycaemic properties of the plant. The synthesis of 4-hydroxyisoleucine and its analogues opens a new therapeutic approach of type 2 diabetes. This thesis focuses on the syntheses of 4-hydroxyisoleucine and its analogues according to three different ways. The chirality of D-glucose was exploited by mild and stereoselective reactions allowing the synthesis of 4 amino-acids and 3 hydroxy-acids. The second way rests on the 1,3-dipolar cycloaddition of nitrones derived from (–)- and (+)- menthone on mono- and di-substituted olefin hydrocarbons, to create simultaneously 3 asymmetric centers. This way led to (2S,3R,4R)-4-hydroxyisoleucine and 12 other amino-acids. The third route is based on the nucleophilic attack of the carbanion derived from ethyl isocyanoacetate on a ketone, to provide unsaturated precursors of amino-acids. This methodology led to the racemic synthesis of a dihydroxy-amino-acid. Preliminary biological evaluations suggest that two of these products could enhance the insulin secretion

Nous avons envisagé la synthèse de la 4-hydroxyisoleucine, antidiabétique naturel non insulino-dépendant. A partir des -alcoxyaldéhydes, nous avons pu isoler les -aminotriles et -amino--lactones par réaction de Strecker et les -amino--hydroxyacides par réaction de Jocic. Cependant le contrôle de la stéréochimie par le substrat est faible. La possibilité d'accéder à des -alcoxyaldéhydes de structures variées et stéréochimie contrôlée permet d'envisager une étude structure-activité.

Le diabète de type II concerne des millions de personnes dans le monde. Les médicaments existants montrent des limites dans leur capacité de contrôler efficacement la maladie et leurs effets secondaires sont une des préoccupations importantes. La synthèse de la (2S,3R,4S)-4-OH-Ile dont l’activité antidiabétique a été prouvée, et de ses analogues, ont déjà fait l’objet d’études dont les procédés ne sont pas adaptables à l’échelle industrielle. 3 approches synthétiques ont été développées. La première repose sur une synthèse en version racémique basée sur un intermédiaire clef de type isoxazole. La seconde stratégie, adaptée à une application industrielle est basée sur la réaction énantiosélective de Barbas III entre un α-imino-ester et la butanone catalysée par la L-proline. Cette voie conduit en 6 étapes à la (2S,3R,4S)-4-OH-Ile avec un rendement global de 22%. La troisième approche, toujours en cours d’étude, repose sur la réaction d’hydrogénation de Noyori sur des composés de type (trichlorométhyl)carbinol. Différents types d’analogues ont été synthétisés. Un composé racémique fonctionnalisé en position 4 par un groupement aldéhydique. Deux analogues gem diméthylé en position 3 et 4, obtenus via la réaction de Barbier et une réaction de iodolactonisation diastéréosélective, ont été obtenus. La synthèse de bioisostères particuliers a été développée: un composé monofluoré en position 4, obtenu via la réaction de Barbas III ; un composé gem difluoré en position 3 obtenu via la réaction de Réformatsky et enfin des isostères de la fonction acide (phosphonate et tétrazole). Les tests biologiques de ces analogues sont en cours et deux brevets sont actuellement déposés<br>Diabetes II relate too million people in the world. The existing drugs show limits in their capacity to effectively control the disease and their side effects are one of the important concerns. Synthesis of (2S,3R,4S)-4-OH-Ile, whose antidiabetic activity was proven, and of its analogues is already developped by process which can’t be set up on an industrial scale. Three original synthetic approaches were developped. In a first part, we developped a racemic synthesis based on isoxazole, an intermediate key. The second strategy, adapted to an industrial application is based on the enantioselective reaction of Barbas III between an α--imino-ester and methyl ethyl ketone catalysed by L-proline. This way leads in 6 steps to the (2S,3R,4S)-4-OH-ILe with 22% over yield. The third approach rests on the reaction of hydrogenation of Noyori on (trichloromethyl)carbinol compounds. This pathway is still studied at the laboratory. Second, we investigated the synthesis of analogs of (2S,3R,4S)-4-OH-Ile. A racemic compound functionalized in 4 position by an aldehydic group is synthetised according to the Barbas III reaction. Then, we synthesized gem dimethyl analogs in 3 and 4 positions, whose synthesis is based on Barbier reaction and a diastereoselective iodolactonisation reaction. We synthesized bioisosteres compounds, particularly, synthesis of a mono fluorinated compound in 4 position, according to the reaction of Barbas III, then a gem dlifluorinated compound in 3 position based on the Reformatsky reaction and phosphonate and tetrazole, isosteric group of the acid function. The biological tests of these analogs are in hand and two patents are currently in the course of deposit

La première partie de ce manuscrit est consacrée à la mise la mise au point d’un procédé d’analyse de bruts réactionnels à haut débit capable d’effectuer très rapidement l’analyse d’un mélange quelle que soit sa composition. La méthode repose sur l’emploi de la spectrométrie de masse comme moyen de détection de substrats fonctionnalisés à l'aide d'un marqueur, chimiquement inerte, possédant des caractéristiques d’ionisation spécifiques. Après avoir défini la structure du marqueur (stabilité chimique, inertie réactionnelle, coefficient d’ionisation. . . ), la méthode a été utilisée pour évaluer l’influence des conditions opératoires sur différentes réactions de couplage, la mise au point d’un couplage décarboxylatif pallado-catalysé, la mise en évidence d’une réaction inattendues et enfin pour suivre l’évolution de produits naturels marqués au sein d’un milieu réactionnel. La seconde partie du manuscrit expose les trois stratégies mise en œuvre pour la production industrielle de la (2S,3R,4S)-4-hydroxy-isoleucine, un produit naturel aux propriétés antidiabétiques. La première voie repose sur la réduction énantiosélective d’un précurseur racémique à l’aide de catalyseurs au ruthénium en présence de ligands chiraux de type 1,2-diamine. La seconde voie repose sur la réduction diastéréosélective d’un intermédiaire achiral de type isoxazole visant l’obtention d’un mélange racémique de 4-hydroxy isoleucine enrichi en diastéréoisomère désiré. La dernière voie repose sur une condensation asymétrique de Mannich catalysée par la proline et conduit sans aucune purification chromatographique à la (2S,3R,4S)-4-hydroxy-isoleucine avec un rendement global de 22%<br>The first part of this paper deals with a high-throughput procedure for crude mixtures capable of rapidly analysing a mixture whatever its composition. This method uses mass spectrometry to detect substrates functionalized with a tag, chemically inert and with specific characteristics. After having defined the tag structure (chemical stability, reaction inertia, ionisation coefficient…), this process was used to evaluate the influence of operating conditions on several coupling reactions, to improve a palladium-catalysed decarboxylative coupling, to highlight an unexpected reaction, and, lastly, to see how tagged natural products evolve in a crude mixture. The second part of this paper sets out the three strategies used in the industrial production of (2S,3R,4S)-4-hydroxyisoleucine, a natural product with anitdiabetic properties. The first pathway is based on an enantioselective reduction of a racemic precursor using ruthenium catalysts in the presence of chiral ligands such as 1,2-diamine. The second pathway is based on the diastereoselective reduction of an achiral isoxazole intermediate to obtain a racemic mixture of a diastereoisomerically enriched 4-hydroxyisoleucine. The last pathway is based on a proline-catalysed asymmetric Mannich condensation which produces (2S,3R,4S)-4-hydroxyisoleucine without any chromatographic purification and with a global yield of 22%

De nos jours, l’un des axes prioritaires de recherche concerne la mise au point de nouvelles approches de synthèse de composés bioactifs présents dans les plantes. Le développement de ces travaux a concouru à l’émergence d’une nouvelle stratégie de synthèse d’un acide aminé naturel extrait d’une Légumineuse d’Asie appelée fenugrec, qui possède des propriétés antidiabétiques : la (2S,3R,4S)-4-hydroxyisoleucine. Plus généralement, les travaux décrits dans ce manuscrit concernent la synthèse totale de la (2S,3R,4S)-4-hydroxyisoleucine et de ses analogues ainsi que la mise au point d’une déprotection de para-méthoxyamines par voie électrochimique. Dans un premier temps, nous avons développé une stratégie de synthèse de l’acide aminé naturel et de ses isomères, basée sur l’étude de la réduction avec différents systèmes catalytiques chiraux et achiraux, d’un intermédiaire -iminoester carbonylé. Une deuxième approche de synthèse en quatre étapes de la (2S,3R,4S)-4-hydroxyisoleucine énantiomériquement pure, a été décrite et s’est avérée être particulièrement bien adaptée pour une utilisation au niveau industriel. Elle repose sur une condensation asymétrique de Mannich catalysée par la proline et une réduction diastéréosélective d’un -oxo--aminoester. La synthèse d’analogues de la (2S,3R,4S)-4-hydroxyisoleucine a été développée dans une troisième partie et a permis de valider la stratégie élaborée précédemment. Les tests biologiques sont actuellement en cours afin d’évaluer leurs propriétés antidiabétiques. Enfin, une déprotection sélective par voie électrochimique de para-méthoxyphénylamines a été mise au point et constitue une alternative à l’utilisation d’oxydants classiques tel que CAN, plus respectueuse de l’environnement<br>Nowadays, the medicinal research takes profit of organic transformations to develop novel synthetic methodologies to afford for example bioactive molecules present in plants. In this manuscript, various facets in organic chemistry have been investigated. The main one was focused on the total synthesis of (2S,3R,4S)-4-Hydroxyisoleucine, a natural product extracted from fenugreek seeds. It is a small amino acid bearing three contiguous stereogenic centers and having some interesting anti-diabetic properties. Two approaches have been designed and successfully validated. The first one is a racemic approach and afforded the (2S,3R,4S)-4-Hydroxyisoleucine and its isomers. It involves a catalytic diastereoselective hydrogenation reaction of an -iminoester precursor. The second strategy aims at developing an industrial scale synthesis of the optically pure natural compound. It is based on the catalytic asymmetric Mannich-Type condensation reaction and the diastereoselective reduction of an N-Protected--keto--aminoester to afford in a four-step synthesis the (2S,3R,4S)-4-Hydroxyisoleucine with a good overall yield and the use of available and cheap starting materials. This synthetic approach also allowed the synthesis of 4-Hydroxyisoleucine’s analogues and their potential anti-diabetic properties are at the moment evaluated in Canada. Besides this project, I studied a regiospecific synthesis of isoxazoles starting from ,-diketonic esters and I examined the use of Rhodium and Ruthenium organometallic complexes in catalytic asymmetric regiospecific hydrogenation of ,-disubstituted-(acetamido)acrylates. I have also worked on the selective deprotection of the para-methoxyphenyl group through anodic oxidation. This electrochemical route can afford primary amine derivatives and constitutes an alternative to the use of classical and environmental unfriendly oxidants such as CAN

Diabetes is a progressive multi-factorial metabolic syndrome with serious short and long term complications affecting many of organs with high increasing prevalence in the world. Using herbs and their derivatives for treating diabetes has a long history in many traditional cultures across the world. Molecules and compounds were isolated from herbs are the basis of many therapeutics which we are using in medicine for treating a variety of health conditions. The seeds of fenugreek, Trigonella foenum graecum, commonly used as a spice in Middle Eastern countries and widely used in South Asia and Europe, are known to have anti-diabetic properties. In 1979, Hardman identified an unusual amino acid (2S, 3R, 4S) 4-hydroxyisoleucine (4HO-Ile) in a fenugreek seed extract as an active compound in fenugreek seed. It was so far found only in fenugreek seed, which is responsible for its anti-diabetic properties. Studies on 4-hydroxyisoleucine effects on type 2 diabetes and insulin resistant animal models revealed that it has anti-diabetic properties of enhancing insulin secretion under hyperglycaemic conditions, and increasing insulin sensitivity. Unfortunately, the available published researches for 4-hydroxyisoleucine are limited and its mechanism of actions is not clear. Here we describe for the first time the anti-diabetic activity of 4-hydroxyisoleucine in a model of type 1 diabetes as all the previous works focused on 4-hydroxyisoleucine activity in type 2 diabetes and insulin resistant condition. Treatment of streptozotocin-treated type 1 diabetes rats, where levels of insulin are much reduced, by 65%, compared to normal animals, with daily doses of 4-hydroxyisoleucine at 50 mg/kg/day for four weeks could reduce plasma glucose in the diabetic group. Moreover the high levels of lipids (cholesterol, HDL, III LDL and triglyceride) and uric acid in the diabetic rats, could be restored to levels found in non-diabetic controls by the treatment with 4-hydroxyisoleucine. These results demonstrate that 4-hydroxyisoleucine has significant anti-diabetic activities in type 1 diabetes as well as previously studied type 2 diabetes and insulin resistance model that are independent of insulin. The findings suggest the potential of 4HO-Ile as an adjunct to diabetes treatment and for type 1 as well as type 2 diabetes. To investigate the insulin-independent effects of 4-hydroxyisoleucine further, the cell based experiments were designed to assess the effect of 4-hydroxyisoleucine on cellular glucose uptake and ATP content after one day incubation. Isoleucine was added to the experiment as a positive control because firstly it has some level of anti-diabetic properties according to previously published studies and secondly it has similar molecular backbone as 4-hydroxyisoleucine. BRIN-BD 11, a functional and glucose responsive pancreatic beta cell, was selected as a cell model which is not insulin-responsive and dependent on the insulin signalling pathway for glucose uptake. Use of the model provides the opportunity to study the mechanisms of action of both 4-hydroxyisoleucine and isoleucine independently. We adopted a unique approach using inhibitors to target suggested pathway and molecules within the cell which may be involved in both 4-hydroxyisoleucine and isoleucine mechanism of actions. The results revealed that 4- hydroxyisoleucine and isoleucine could increase glucose uptake in BRIN-BD 11 cells, but as previously suggested, 4-hydroxyisoleucine activity is in direct correlation with glucose concentration. 4-hydroxyisoleucine has higher activity in higher concentrations of glucose. 4-hydroxyisoleucine increased the glucose uptake much greater than isoleucine at 11mM and 22mM concentration of glucose in IV cell culture medium. Endpoint measurements of ATP content of the BRIN-BD11 cells did not show any significant changes between 4-hydroxyisoleucine and isoleucine groups and control as well as an insulin level measurement in culture medium after 24 hours. The results showed that there are substantial differences between isoleucine and 4-hydroxyisoleucine mechanisms of action unlike their similar glucose uptake stimulatory effect which is greater in 4-hydroxyisoleucine. 4-hydroxyisoleucine activity strongly dependent on new protein synthesis and GLUT 1 activity. GLUT 1 is widely available in most of the cells and it controls the basal glucose uptake independent of insulin. The connection between GLUT 1 and 4-hydroxyisoleucine effect in cellular level, supports the idea that 4-hydroxyisoleucine utilises the glucose basal consumption and uptake of cells. Mitochondrial calcium channel signalling inhibition affects 4-hydroxyisoleucine and isoleucine functionality as well as inhibition of mitochondria pyruvate carrier. Real time monitoring of cell metabolism by Seahorse XF-24 autoanalyser after 24 hours incubation with 4-hydroxyisoleucine and isoleucine revealed that both 4-hydroxyisoleucine and isoleucine activities are strongly dependent on mitochondrial respiration but 4-hydroxyisoleucine significantly up-regulates glycolysis which is not affected by isoleucine. The connection between mitochondria calcium signalling and contradictory behaviour of 4-hydroxyisoleucine and isoleucine support the very important role of mitochondria in their mechanisms of action.