Fodder additives represented by dipeptides

FIELD: food industry.

SUBSTANCE: invention relates to fodder additives containing dipeptides or their salts; one amino acid residue of dipeptide is represented by DL-methionine residue; the other amino acid residue of dipeptide is represented by amino acid in L-configuration chosen from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

EFFECT: described are fodder mixtures containing such additives and the said dipeptides production method.

31 cl, 17 dwg, 10 tbl, 25 ex

 

The present invention relates to a new associated with methionine artificial and natural dipeptides essential limiting amino acids such as lysine, threonine and tryptophan, serosoderjaschei amino acids, cysteine and cystine and their synthesis and use as feed additives for feeding production animals, such as chickens, pigs and ruminants, but primarily for feeding of fish and crustaceans, farmed in aquaculture.

The level of technology

Essential amino acids (abbreviated as "EAA"). "essential ammo acids"), which include methionine, lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine and arginine, as well as the two sulfur-containing amino acids cysteine and cystine are the important components of the feed, and play an important role in the industrial cultivation of production animals, such as chickens, pigs and ruminants. At this crucial first of all optimal distribution of essential amino acids and sufficient supply of their animals. Because in the diet based on natural protein sources such as soy, corn and wheat, certain essential amino acids often contain insufficient purposeful addition of this feed synthetic essential amino acids, so the mi, for example, as DL-methionine, L-lysine, L-threonine or L-tryptophan, allows, firstly, to accelerate the growth of animals, respectively, to increase lactation in high producing dairy cows, and secondly, to increase the efficiency of food digestion. This is a significant economic advantage. The market of feed additives are of great industrial and economic importance. In addition, the demand for feed additives is constantly increasing and emerging markets, not least due to the increasing role in the world in such countries as, for example, China and India.

L-Methionine ((S)-2-amino-4-meticiously acid) is for many species the first limiting amino acid among all of the essential amino acids and therefore plays a major role in the diet of animals and as a feed additive (Rosenberg and others, J. Agr. Food Chem., 5, 1957, SS.694-700). However, the classical chemical synthesis of methionine is formed in the form of a racemate, which is a mixture of D - and L-methioninol in the ratio of 50:50. This racemic DL-methionine, however, can be used directly as a feed additive, as in animals of some species in vivo, there is a mechanism for the transformation of unnatural D-enantiomer of methionine in its natural L-enantiomer. When this first happens deamination of D-mate the Nina under the action of nonspecific D-oxidase in α-betamethason, then under the action of L-transaminases turns into L-methionine (Baker D. H., "Amino acids in farm animal nutrition", edited by D Mello J. P. F., Wallingford (UK), CAB International, 1994, cc.37-61). In this way in the body increases the available quantity of L-methionine, which can then be used in the body of the animal for growth. The enzymatic conversion of D-methionine to L-methionine was detected in chickens, pigs and cows, but first and foremost in fish, marine shrimp and freshwater prawns. For example, according to research Sveier and others (Aquacult. Nutr., 7(3), 2001, cc.169-181) and Kim and others (Aquaculture, 101(1-2), 1992, SS.95-103), it was found that the conversion of D-methionine to L-methionine may be carnivorous Atlantic salmon and rainbow trout. The same was found by Robinson and others (J. Nutr., 108(12), 1978, SS.1932-1936) and Schwarz and others (Aquaculture, 161, 1998, SS.121-129) omnivorous fish species, such as Samoobrona and carp. In addition, Forster and Dominy (J. World Aquacult. Soc., 37(4), 2006, SS.474-480) in experiments with feeding omnivorous marine shrimp species Litopenaeus vannamei found that DL-methionine has the same efficiency, and L-methionine. In 2007, around the world produced over 700,000 tons of crystalline DL-methionine, respectively racemic liquid hydroxyanisole methionine (GAM, rat-2-hydroxy-4-(methylthio)butyric acid (GMM) and solid DIN in the form of its calcium salt and successfully used directly edstone as a feed additive in animal breeding mono-gastric, such as agricultural poultry and pigs.

In contrast, methionine, lysine, threonine and tryptophan can be used as feed additives only in the form of L-enantiomers of each of them as D-enantiomers of each of these three essential and limiting amino acids can't turn the body under physiological conditions to the corresponding L-enantiomers. Thus, in particular, the global sales of one only L-lysine, the first limiting amino acid in swine, amounted in 2007 more than one million tonnes. The world sales of the other two limiting essential amino acids - L-threonine and L-tryptophan was in 2007, just over 100,000 tons, respectively, slightly less than 3000 tons

DL-Methionine, DIN, as well as L-lysine, L-threonine and L-tryptophan is usually directly used as additives for animal feed mono-gastric, such as agricultural poultry and pigs. In contrast, the addition of essential amino acids like methionine, lysine, threonine, or GAM, to feed for ruminants to be ineffective, as the basic amount is broken down in the rumen of ruminants under the action of microbes. As a result of such decomposition of the essential amino acids only a small part of their total added to the stern of the number of hits in the subtle symptoms such the IR animal, where usually their absorption into the blood. Among the essential amino acids in the first methionine consumed by ruminants, plays a crucial role, because only the optimal supply of the body of this amino acid provides a high lactation. For efficient support body ruminant methionine it is required to apply in form, is protected from degradation in the rumen. In this regard, there are several opportunities to give D,L-methionine, respectively rat-DIN similar properties. One of these opportunities to give methionine high resistance to degradation in the rumen is applied to methionine corresponding protective layer, respectively in its distribution suitable for use in this purpose, the protective matrix. Because of this methionine may practically without losses to pass through the rumen. In a further protective layer is removed, for example, in the abomasum acid hydrolysis, and release during this methionine can then be absorbed in the small intestine some. For use in such purposes suitable commercially available products, such as product Mepron® company Evonik Degussa and product Smartamine™ company Adisseo. Getting methionine, respectively, causing him coverage in most cases is technically complex and time consuming process is therefore associated with high costs. Additionally deposited on the surface of the finished pellets coating can be easily damaged from mechanical stress and abrasion during processing of feed that may ultimately reduce the effectiveness of protection or even to its complete loss. Therefore, protected methionine granules could not be processed in larger animal feed pellets, as when such processing also would have been the destruction of the protective layer under the action of mechanical loads. This factor severely limits the use of such products. Another possibility to improve the stability of methionine to degradation in the rumen is the chemical formation of derivatives of methionine, respectively GAM. When the functional group of the molecule protects acceptable protective groups. For example, the carboxyl functional group may for these purposes be atrificial alcohols. In this way it is possible to reduce the degradation in the rumen microorganisms. As an example, a commercially available product with chemical protection can be called the product Metasmart™, which represents a racemic isopropyl ether hydroxyanisole methionine (HMBi). According to WO 00/28835 bioavailability HMBi for ruminants is at least 50%. The lack of chemical education derivatives of methionine, respectively, G The M is often their worst bioavailability and relatively low content of the actual active substance.

Along with the problem of decomposition is added to the stern of essential amino acids such as methionine, lysine or threonine, in the rumen of ruminants with various problems you might encounter when adding essential amino acids to feed for fish and crustaceans. As a result of rapid economic development of pisciculture and cultivation of crustaceans in the highly industrialized aquaculture in recent years, an ever increasing role in this area plays a best, cheap and effective Supplement animal diets essential and limiting amino acids (Food and Agriculture Organization of the United Nation (FAO) Fisheries Department, "State of World Aquaculture 2006", 2006, Rome; International Food Policy Research Institute (IFPRI), "Fish to 2020: Supply and Demand in Changing Markets", 2003, Washington, D.C.). However, unlike poultry and pigs in the application of crystalline essential amino acids as additives to feed for certain species of fish and shellfish, you may encounter various problems. Thus, in particular, in the publication Rumsey and Ketola (J. Fish. Res. Bd. Can., 32, 1975, SS.422-426) States that the use of soy flour in combination with individually added crystalline amino acids did not lead to stronger growth of rainbow trout. According to research conducted by Murai and others (Bull. Japan. Soc. Sci. Fsh., 50(11), 1984, S. 1957), was able to establish that the daily feeding of carp feeds with high added thereto crystalline amino acids resulted in the removal of more than 40% of free amino acids from the body through the gills and kidneys. Due to the rapid absorption of added amino acids soon after eating there is a very rapid increase in the concentration of amino acids in blood plasma of fish ("quick response"). However, other amino acids from natural sources of protein, such as soy flour, at this point are not yet in the blood plasma, which can lead to asynchrony in the simultaneous absorption of all the essential amino acids. As a consequence, the part of highly concentrated amino acids rapidly excreted from the body, respectively rapidly metabolized in it and is used, for example, as the only source of energy. The result is that the carp when using crystalline amino acids as feed additives is only a slight rise, respectively, there was no enhancement of growth (AoE etc., Bull. Jap. Soc. Sci. Fish., 36, 1970, SS.407-413). In the case of crustaceans Supplement their feed crystalline essential amino acids can lead to another and other problems. Due to the slow eating poop certain crustaceans, and, for example, as shrimp species Litopenaeus Vannamei, because of the long stay of the feed water added to it water-soluble amino acids are deleted as a result of their dissolution (leaching), which leads to eutrophication of the water body, and not to increase animal growth (Alam and others, Aquaculture, 248, 2005, SS.13-16). Thus the essential amino acids for efficient supply to the body of the fish and shellfish farmed in aquaculture farms require for certain species and certain conditions of their release in a special form, for example in appropriately chemically or physically protected form. Its aim is, firstly, that the product has remained fairly stable in the aquatic environment in the process of feeding the animals and was not removed from the feed as a result of dissolution. Secondly, ultimately it should be possible optimal and highly efficient absorption of amino acid product, consumed by the animal, his body.

Previously there have been numerous efforts to develop acceptable additives to feed for fish and crustaceans, especially feed additives based on the essential amino acids methionine and lysine. For example, in WO 89/06497 describes the use of di - and tripeptides as an additive to feed for fish and crustaceans. Such korovyakovsky should promote growth of animals. In this case, however, used mainly di - and tripeptides of interchangeable and limitiruesh amino acids such as glycine, alanine and series contained in many plant protein sources in more than sufficient quantities.

As methioninamide of dipeptides in the specified publication describes only the DL-alanyl-DL-methionine and DL-methionyl-DL-glycine. However, for this reason, an effective content of active substance in the dipeptide is only 50% (mol/mol), which from an economic point of view should be classified as a serious drawback. In WO 02/088667 described enantioselective synthesis and application of oligomers from the DIN and amino acids such as methionine, as additives to animal feed, including, in particular, and additives to feed for fish and crustaceans. The use of such feed additives should provide the opportunity to accelerate the growth of animals. Described in the publication oligomers formed by flowing enzymatic catalysis reaction obtained as a result of which the individual oligomers have an extremely wide molecular weight distribution (polydispersity). For these reasons, this method leisuretime, roads and difficult to implement and is associated with costly and time-consuming purification of the resulting products. In other Dabrowski in the US 2003/0099689 described the use of synthetic peptides as feed additives to promote growth of aquatic animals. At a mass fraction of peptides can be from 6 to 50% of the total weight of the feed mixture. Such synthetic peptides are mostly composed of essential amino acids. However, enantioselective synthesis of such synthetic oligo - and polypeptides extremely complicated and costly, and the possibility of its implementation on an industrial scale is associated with considerable difficulties. In addition, the efficiency of the polypeptides of individual amino acids is controversial because they are often only very slowly converted or not converted under physiological conditions to the free amino acids. So, for example, Baker and others (J. Nutr., 112, 1982, SS.1130-1132) says that poly-L-methionine, by reason of its complete insolubility in water has no biological value in respect of the chickens, because its absorption in their body impossible.

Along with the use of new chemical derivatives of amino acids, such as methioninamide peptides and oligomers, was also studied various possibilities for the physical protection of essential amino acids, such as, for example, as a coating on the essential amino acid, respectively, its inclusion in a protective matrix.

T is to, for example, Alam and others (Aquacult. Nutr., 10, 2004, SS.309-316, and Aquacultwe, 248, 2005, SS.13-19) found that provided coverage methionine and lysine in contrast to those without coverage have a positive influence on the growth of young kuruma shrimp (Marsupenaeus japonicus). Although the application of special coatings and prevented the leaching of methionine and lysine from feed pellets, however, this approach faces some serious flaws. Obtaining amino acids, respectively, the application of the coating in most cases, is technically difficult and time consuming and therefore costly to implement. In addition, the coating on finally provided them an amino acid, can be easily damaged from mechanical stress and abrasion during processing of feed, which can degrade the effectiveness of the physical protection of amino acids or even a complete loss of such physical protection. To this should be added that the coating or application of the matrix reduces the content of amino acids and therefore is often uneconomical.

The objective of the invention

The present invention was based on the task in the first place to offer feed means, respectively, feed additive for use in animal diets based on the new methioninamide substitute, where m is tionin covalently linked to an essential and limiting amino acid, such as L-lysine, L-threonine and L-tryptophan, and which could be used as a feed additive for feeding production animals, such as chickens, pigs and ruminants, but primarily for feeding of fish and crustaceans, farmed in aquaculture.

Taking into account the above described disadvantages inherent in the prior art, the invention was primarily in the development of chemically-protected product on the basis of a combination of covalently linked DL-methionine and essential amino acids such as L-lysine, L-threonine or L-tryptophan, for use in the diet of different production animals, such as chickens, pigs and ruminants, as well as in the diet of many species are omnivorous, herbivorous and carnivorous fish and crustaceans that live in marine or fresh water. This product, along with the source function of methionine should also function as the source of all other essential amino acids. Such a product must first be slow release mechanism, i.e., the slow and continuous release of free methionine and essential amino acids in physiological conditions. In addition, such a product in a chemically protected form, consisting of methionine and essential amino acids, must be resistant to RA is the situation in the rumen and be thus suitable for use in the diet of all ruminants. For use as an additive to feed for fish and crustaceans such a product, represented in the above form must have a low solubility (vymyvaemosti) in water to prevent removal of the entire feed pellets, respectively, just aft of the extrudate.

Another object of the invention was to provide a substitute crystalline essential amino acids as feed means, respectively, feed additives, which in addition to an exceptionally high biological value and bioavailability would be easy to handle with him and would have high persistence and durability under normal conditions of production of animal feed, especially when pelleting and extrusion.

In this way, for example, chickens, pigs, ruminants, fish and shellfish, along with crystalline essential amino acids should ensure more efficient sources of essential amino acids, which sources would completely absent the disadvantages of the known products or such shortcomings were present only in smaller amounts.

In addition, the invention consisted in the development of various new, flexible ways of synthesis of dipeptides containing only one meinenemy the rest, especially the synthesis of L-EAA-DL-methionine (I) the DL-methionyl-L-EAA (II). One such method of synthesis should allow to use as source material typical of the initial, intermediate and by-products of the industrial process to obtain DL-methionine.

Description of the invention

This problem is solved by using feed additives containing dipeptides or their salts, with one amino acid residue of the dipeptide is a DL-nationally the residue, and the other amino acid residue of the dipeptide is an amino acid in L-configuration selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

In a preferred embodiment, the feed additive comprises a dipeptide of the General formula DL-methionyl-L-EAA (corresponds to a mixture of D-methionyl-L-EAA, L-methionyl-L-EAA) and/or L-EAA-DL-methionine (corresponds to a mixture of L-EAA-D-methionine and L-EAA-L-methionine), where L-EAA represents the amino acid in L-configuration selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

The object of the invention is further feed mixture containing the above-mentioned feed additive.

Feed Supplement containing L-EAA-DL-methionine and/or DL-methionyl-L-EAA and their salts suitable for use as a feed additive in feed is x mixtures for feeding poultry, pigs and ruminants, but primarily for feeding of fish and crustaceans, farmed in aquaculture.

In a preferred embodiment, the feed mixture contains L-EAA-DL-methionine and DL-methionyl-L-EAA in an amount of from 0.01 to 5 wt.%, preferably from 0.05 to 0.5 wt.%.

The use of L-EAA-DL-methionine and DL-methionyl-L-EAA has established itself as the most preferred, because these dipeptides due to its low solubility have an extremely high resistance to leaching.

In addition, this compound exhibits good resistance to granulation and extrusion for the production of feed. The dipeptides L-EAA-DL-methionine and DL-methionyl-L-EAA stable in mixtures with conventional components and feed means, such as, for example, cereals (in particular maize, wheat, triticale, barley, millet, etc.), protein food of vegetable or animal origin (in particular, soybeans and canola, as well as the products of their further processing, legumes (e.g. peas, beans, lupins, and so on), fish meal and other), as well as in combination with added essential amino acids, proteins, peptides, carbohydrates, vitamins, minerals, fats and oils.

Another advantage is that due to the high relative potency - L-EAA-DL-methionine and DL-mation the l-L-EAA - per kg of the substance compared with DL-methionine and L-EAA in mol L-EAA-DL-methionine, respectively, DL-methionyl-L-EAA saved mol of water.

In one of the preferred options proposed in the invention is the use of the feed mixture contains proteins and carbohydrates, preferably based on fish, soy or corn flour, and can be supplemented with essential amino acids, proteins, peptides, vitamins, minerals, carbohydrates, fats, and oils.

In a particularly preferred variant of DL-methionyl-L-EAA, L-EAA-DL-methionine present in the feed mixture individually in the form of D-methionyl-L-EAA, L-methionyl-L-EAA, L-EAA-D-methionine or L-EAA-L-methionine, in the form of a mixture between a, respectively, also in the form of a mixture with D-methionyl-D-EAA, L-methionyl-D-EAA, D-EAA-D-methionine or D-EAA-L-methionine, preferably in each case optionally in a mixture with DL-methionine, which in the preferred embodiment, there are from 0.01 to 90 wt.%, more preferably from 0.1 to 50 wt.%, particularly preferably from 1 to 30 wt.%, and preferably in each case optionally in a mixture with L-EAA, such as L-lysine, the share of which in the preferred embodiment, there are from 0.01 to 90 wt.%, more preferably from 0.1 to 50 wt.%, particularly preferably from 1 to 30 wt.%.

In yet another preferred embodiment of the proposed invention p is imeneniya animals bred and grown in aquaculture farms are freshwater and marine fish and shellfish are selected from the group including carp, trout, salmon, catfish, perch, flatfish, sturgeon, tuna, acne, bream, cod, sea shrimp, krill and freshwater shrimp, particularly preferably from the group comprising white silver carp (Hypophthalmichthys molitrix), carp (Ctenopharyngodon idelld), carp (Cyprinus carpio), bighead carp (Aristichthys nobilis), Golden carp (Carassius carassius), Cuttle (Catia Catid), labeo Roch (Labeo rohita), Pacific and Atlantic salmon (Salmon salar and Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss), channel catfish (Ictalurus punctatus), African catfish (Glorias gariepinus), pangasius (Pangasius bocourti and Pangasius hypothalamus), Nile tilapia (Oreochromis niloticus), milkfish (Chanos chanos), cobey (Rachycentron canadum), Pacific white shrimp (Litopenaeus vannarnei), black tiger shrimp (Penaeus monodon) and giant freshwater prawn (Macrobrachium rosenbergii).

According to the invention L-EAA-DL-methionine (L-EAA-DL-Met) (I) and DL-methionyl-L-EAA (DL-Met-L-EAA) (II) or their salts with alkali and alkaline earth metals, such for example, as a sparingly soluble calcium or zinc salt, is used in the form of D-methionyl-L-EAA, L-methionyl-L-EAA, L-EAA-D-methionine or L-EAA-L-methionine or in the form of mixtures of diastereomers individually or in a mixture with DL-methionine, individually and in a mixture with L-EAA as an additive in feed mixes, preferably as an additive to animal feed for poultry, pigs and ruminants, particularly preferably for fish and crustaceans:

L-EAA-DL-methionine (I) exists in the form of both diastereomers of L-EAA-D-Met (LD-I) and L-EAA-L-Met (LL-I). Likewise dipeptide DL-methionyl-L-EAA (II) exists as two different stereoisomers D-Met-L-EAA (DL-II) and L-Met-L-EAA (LL-II). However, only the two diastereoisomer L-EAA-L-Met (LL-I) and L-Met-L-EAA (LL-II) are natural, whereas both of the other diastereoisomer of L-EAA-D-Met (LD-I) and D-Met-L-EAA (DL-II) are unnatural (see scheme 1).

Scheme 1

The remainder R in the EAA has the following values:

in the formula Ia, ACC. IIa R denotes 1-methylethyl- (valine)

in formula Ib, ACC. IIb R denotes a 2-methylpropyl- (leucine)

in the formula Ic, ACC. IIc R denotes the (1S)-1-methylpropyl- (isoleucine)

in the formula Id, ACC. IId R denotes the (1R)-1-hydroxyethyl (threonine)

in the formula Ie, ACC. IIe R denotes 4-aminobutyl- (lysine)

in the formula If, ACC. IIf R denotes 3-[(aminoiminomethyl)- (arginine)

amino]propyl-

in the formula Ig, ACC. IIg R denotes benzyl (phenylalanine)

in the formula Ih, II. IIh R denotes (1H-imidazol-4-yl)methyl- (histidine)

in the formula Ij, ACC. IIj R denotes (1H-indol-3-yl)methyl- (tryptophan)

This stereoisomers of L-EAA-D-methionine (LD-I), L-EAA-L-m is thionin (LL-I), D-methionyl-L-EAA (DL-II) and L-methionyl-L-EAA (LL-II) can be used individually or as mixtures between them as a feed additive, preferably as an additive to animal feed for poultry, pigs, ruminants, fish, crustaceans, and Pets are welcome.

Along with the development of a new method for the synthesis of L-EAA-DL-methionine (I) and DL-methionyl-L-EAA (II) one of the main objects of the present invention is also the use of compounds of formulas I and II as a mixture of diastereoisomers, which is a mixture of D-methionyl-L-EAA (DL-II) and L-methionyl-L-EAA (LL-II), respectively, a mixture of L-EAA-D-methionine (LD-I) and L-EAA-L-methionine (LL-I), or in the form of an individual diastereoisomer, representing D-methionyl-L-EAA (DL-II), L-methionyl-L-EAA (LL-II), L-EAA-D-methionine (LD-I), respectively, L-EAA-L-methionine (LL-I), as a growth-promoting feed for poultry, pigs and ruminants, and omnivorous, carnivorous and herbivorous fishes and crustaceans in aquaculture farms. In addition, the use of L-EAA-DL-methionine (I), respectively, DL-methionyl-L-EAA (II) as a feed additive can improve lactation in high producing dairy cows.

Thus, in particular, according to the invention it was found that L-EAA-DL-methionine (I), respectively, DL-methionyl-L-EAA (II) as a mixture of diastereoisomers, not only is the fact that a mixture of L-EAA-D-methionine (LD-I) and L-EAA-L-methionine (LL-I) in the ratio 50:50, accordingly, the mixture of D-methionyl-L-EAA (DL-II) and L-methionyl-L-EAA (LL-II) in the ratio 50:50, or in the form of the corresponding individual diastereoisomer may, in physiological conditions, the enzyme to break down in the body of chickens, pigs, cows, fish, such as carp and trout, as well as crustaceans such as Litopenaeus Vannamei (Pacific white shrimp) and Macrobrachium Rosenbergii (giant freshwater prawn), free D-, accordingly, L-methionine and L-EAA (see diagram 2).

For example, chickens are omnivores Karpov, carnivorous trout and omnivorous Pacific white shrimp (Litopenaeus Vannamei) was allocated the appropriate digestive enzymes, which are optimized in experiments in vitro were subjected to conditions comparable to physiological interaction with DL-methionyl-L-EAA (II) as a mixture of diastereoisomers, which is a mixture of D-methionyl-L-EAA (DL-II) and L-methionyl-L-EAA (LL-II) in the ratio 50:50, respectively with L-EAA-DL-methionine (I) representing a mixture of L-EAA-D-methionine (LD-I) and L-EAA-L-methionine (LL-I) in the ratio 50:50, or with each of the respective individual diastereomers, representing D-methionyl-L-EAA (DL-II), L-methionyl-L-EAA (LL-II), L-EAA-D-methionine (LD-I) or L-EAA-L-methionine (LL-I). According to the invention the characteristic cleavage of L-EAA-DL-methionine (I), respectively, DL-methionyl-L-EAA (II) is that, along with two natural diastereomers - L-EAA-L-methionine (LL-I) and L-methionyl-L-EAA (LL-II) under physiological conditions can also split two non-natural diastereoisomer - L-EAA-D-methionine (LD-I) and D-methionyl-L-EAA (DL-II) (see Fig.1-17). This refers to the application of a mixture of D-methionyl-L-EAA (DL-II) and L-methionyl-L-EAA (LL-II), a mixture of D-methionyl-L-EAA (DL-II) and L-EAA-D-methionine (LD-I) (see Fig.12), respectively, a mixture of L-methionyl-L-EAA (LL-II) and L-EAA-D-methionine (LD-I) (see Fig.12), and to the use of the total mixture of all diastereomers, and each of the individual diastereomers (see Fig.1-11 and 13-17).

Scheme 2

Natural dipeptides L-EAA-L-Met (LL-I) and L-Met-L-EAA (LL-II) were digested by the action of digestive enzymes isolated from carnivorous rainbow trout, mirror carp are omnivorous omnivorous Pacific white shrimp and chicken (see table 1).

Table 1


Dipeptide
Trout (carnivorous)Mirror carp (omnivorous)White Pacific shrimp (omnivorous)Chicken (omnivorous)
L-Met-L-Val (LL-IIa)xxx
L-Met-L-Leu (LL-IIb)xxx
L-Met-L-Ile (LL-IIc)xxx
L-Met-L-Thr (LL-IId)xxx
L-Met-L-Lys (LL-IIe)xxx
L-Met-L-Arg (LL-IIf)xxx
L-Met-L-Phe (LL-IIg)xxx
L-Met-L-His (LL-IIh)xxx
L-Met-L-Trp (LL-IIj)xxx
L-Val-L-Met (LL-Ia)xx x
L-Leu-L-Met (LL-Ib)xxx
L-Ile-L-Met (LL-Ic)xxx
L-Thr-L-Met (LL-Id)xxxx
L-Lys-L-Met (LL-Ie)xxxx
L-Arg-L-Met (LL-If)xxx
L-Phe-L-Met (LL-Ig)xxx
L-His-L-Met (LL-Ih)xxx
L-Trp-L-Met (LL-Ij)xxx

To do this, the digestive tract is s fish and marine shrimp were isolated enzymes. Then the solutions of enzymes splits dipeptides L-EAA-L-Met (LL-I) and L-Met-L-EAA (LL-II). For better comparability of digestibility of dipeptides with enzymes isolated from different species of animals, used the same conditions as in the experiments on digestion in vitro (37°C, pH 9).

All natural dipeptides were split under the action of digestive enzymes isolated from carnivorous rainbow trout (see Fig.3 and 4), omnivorous mirror carp (see Fig.1 and 2), omnivorous Pacific white shrimp (see Fig.5 and 6) and chicken (see Fig.16). The process of splitting L-Met-L-EAA (LL-II) usually are slightly faster than the processes of splitting similar dipeptides L-EAA-L-Met (LL-I).

In order to demonstrate the enzymatic cleavage of unnatural dipeptides L - EAA-D-Met (LD-I) and D-Met-L-EAA (DL-II) under the action of digestive enzymes isolated from different fish species, studies were performed in accordance with the following experimental matrix (see table 2).

Table 2
Dipeptide








D-Met-L-Trp (DL-IIj)D-Met-L-Thr (DL-IId)D-Met-L-Lys (DL-IIe)D-Met-L-Leu (DL-IIb)D-MetL-Ile (DL-IIc) D-Met-L-Phe (DL-IIg)L-Trp-D-Met (DL Ij)L-Thr-D-Met (DL-Id)L-Lys-D-Met (DL-Ie)L-Leu-D-Met (DL-Ib)L-Ile-D-Met (DL-Ic)L-Phe-D-Met (DL-Ig)
Trout (carnivorous)xxxxxxxx
Mirror carp (omnivorous)xxxxxxxx
White Amur (herbivorous)xxxxx xxx
White Pacific shrimp (omnivorous)xxxxxxxx
Tilapia (omnivorous)xxxx
Chicken (omnivorous)xxxxxx

For this is from the digestive tracts of fish and marine shrimp were isolated enzymes. Then the solutions of the enzymes were digested chemically synthesized dipeptides L-EAA-D-Met (LD-I) and D-Met-L-EAA (DL-II). For better comparability of digestibility of dipeptides with enzymes isolated from different species of animals, used the same conditions as in the experiments on digestion in vitro (37°C, pH 9).

All unnatural dipeptides L-EAA-D-Met (LD-I) and D-Met-L-EAA (DL-II) are oxidized under the action of digestive enzymes isolated from omnivorous mirror carp (see Fig.7), herbivorous carp (see Fig.8), the carnivorous rainbow trout (see Fig.11), omnivorous Pacific white shrimp (see Fig.10) and chicken (see Fig.17). The process of splitting of D-Met-L-EAA (DL-II) proceed a little slower than the processes of splitting similar dipeptides L-EAA-D-Met (LD-I). Under the same action of digestive enzymes isolated from tilapia (see Fig.9), the splitting of D-Met-L-EAA (DL-II) was faster than the cleavage of the dipeptides L-EAA-D-Met (LD-I). The most quickly digested the dipeptide D-Met-L-Lys (DL-IIe) and L-Lys-D-Met (LD-Ie). The prevailing part of leinstermen of dipeptides under the reaction conditions in vitro was split under the action of all used digestive enzymes already after 5 PM

The results obtained indicate that each used unnatural dipeptide (see Fig.7-11 and 17) can be split under the action of digestive enzymes is Yb different types, marine shrimp and chicken. The use of enzymes isolated from carnivorous rainbow trout, omnivorous mirror carps, tilapias, Pacific white shrimp, herbivorous white cupids, and chickens allowed to confirm experimentally that unnatural dipeptides L-EAA-D-Met (LD-I) and D-Met-L-EAA (DL-II) can be converted in vitro animals of all kinds despite the obvious differences in their digestive systems. The addition of dipeptides L-EAA-D-Met (LD-I) and/or D-Met-L-EAA (DL-II) to the stern allows, therefore, purposefully dose present in it in the lack of essential amino acids (DL-Met and L-EAA).

For example, dipeptides based on the amino acids L-tryptophan and DL-methionine - investigated the decomposition of mixtures of natural and non-natural dipeptides. Mixture of diastereomers, consisting of both non-natural dipeptides L-Trp-D-Met (LD-Ij) and D-Met-L-Trp (DL-IIj), was cut completely, as well as the mixture of natural dipeptide L-Met-L-Trp (LL-IIj) and non-natural dipeptide L-Trp-D-Met (LD-Ij). The effect of "slow release" expressed in a mixture of LD-Ij/DL-IIj much more clearly than the mixture LD-Ij'/LL-IIj, i.e. amino acids tryptophan and methionine released during enzymatic digestion of dipeptides slower one towards the other and over a longer period of time.

Underlying the invention the task is solved with the help of the dipeptide or the th salt of General formula DL-methionyl-DL-EAA or DL-EAA-DL-methionine, where EAA denotes an amino acid, preferably L-configuration selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine. While equally preferred nationally residue in D-, respectively L-configuration. Such dipeptides are Met-Lys, Met-Thr, Met-Trp, Met-His, Met, Val, Met-Leu, Met-LEU, Met-Phe, Met-Arg, Met-Cys and Met-cystine, in each case in configurations DD, LD, DL, and LL, as well as Lys - Met, Thr-Met, Trp-Met, His-Met, Val-Met, Leu-Met, He-Met, Phe-Met, Arg-Met, Cys-Met and cystine-Met, in each case in configurations DD, LD, DL, and LL.

Underlying the invention the problem is solved then use the method of producing the dipeptide containing only one nationally residue of formula DD/LL/DL/LD-I or DD/LL/DL/LD-II:

by the interaction of amino acids with urea derivative of one of General formulas III-V

where R has the following meanings:

in formulas Ia-Va R denotes 1-methylethyl- (valine)

in formulas Ib-Vb R denotes a 2-methylpropyl- (leucine)

in the formulae Ic-Vc R denotes the (1S)-1-methylpropyl- (isoleucine)

in formula Id-Vd R denotes the (1R)-1-hydroxyethyl (threonine)

in formulas Ie-Ve R denotes 4-aminobutyl- (lysine)

in formulas If-Vf R denotes 3-[(aminoiminomethyl)- (arginine)

amino]propyl-

in the formula Ig-Vg R Ref is no benzyl (phenylalanine)

in the formula Ih-Vh R denotes (1H-imidazol-4-yl)methyl- (histidine)

in formulas Ij-Vj R denotes (1H-indol-3-yl)methyl- (tryptophan)

in formulas Ik-Vk R denotes-CH2-SH (cysteine)

in the formula Im-Vm R denotes-CH2-S-S-CH2-CNH2-COOH (cystine)

in the formula IIIn-Vn R denotes-CH2-CH2-3-CH3(methionine)

the residues R and R of urea derivatives of the formula III, IV, and V have the following meanings:

in formulas IIIa-IIIn R1denotes COOH, and R2means NHCONH2,

in formulas IVa-IVn R1represents CONH2and R2means NHCONH2,

in formulas Va-Vn R1-R2mean-CONHCONH-,

when either R is nationally the rest, and added amino acid selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine, or added amino acid is a methionine, and R represents an amino acid residue selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

In one of the preferred options as the original product use or as an intermediate product to form methioninamide or as amino acids selected from the group including lysine, threonine tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

In one of the options proposed in the invention method, it is preferable to subject the solution containing methioninamide (Vn) and water interaction with amino acid in the basic environment, or be subjected to a solution containing as amino acids selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine, and water, interaction with methionine in basic terms.

In yet another embodiment proposed in the invention method thus preferably be used as the source of the product methioninamide (Vn) or intermediate to form as an intermediate product. Below in figure 3 is illustrated the preferred option of receiving DL-methionyl-L-EAA (II) directly from methanimidamide (Vn) and N-carbamoylation (IIIn), respectively amide N-carbamoylation (IVn) in accordance with the method of A.

Scheme 3

Preferably next to set the pH of a solution containing a derivative of urea, 7-14, more preferably 8 to 13, particularly preferably 9-12.

In a preferred embodiment, the reaction is carried out at a temperature of from 30 to 200°C., more prepact the tion from 80 to 170°C., particularly preferably from 120 to 160°C.

In addition, the reaction is preferably carried out under pressure, preferably under a pressure in the range of 2 to 100 bar, more preferably from 4 to 60 bar, particularly preferably from 8 to 40 bar.

In another preferred embodiment, the solution containing methioninamide and water, or a solution containing as amino acids selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine, and water, pre-formed from one or more compounds IIIa-IIIn, IVa-IVn and Va-Vn. In another embodiment, can also be used as precursors as to use the appropriate aminonitriles, cyanhydrin or a mixture of the corresponding aldehyde, hydrogen cyanide and ammonia, respectively, the mixture of the corresponding aldehyde and ammonium and cyanide salts.

In yet another preferred embodiment of the proposed invention the method includes the following steps:

a) interactions derived urea of the formula IIIa-IIIn, IVa-IVn or Va-Vn with amino acid with getting diketopiperazine formula VIa-VIm

where R has the above values

b) interaction of diketopiperazine formula VI with a mixture of dipeptides of formula DD/LLDL/LD-I and DD/LL/DL/LD-II

where R has the above values.

The reaction for converting a derivative of urea of the formula IIIn, IVn and Vn in diketopiperazine formula VIa VIm and subsequent reaction of turning this diketopiperazine with a mixture of diastereomers containing the preferred dipeptides L-EAA-DL-methionine (I) and DL-methionyl-L-EAA (II), illustrated in figure 4.

Scheme 4

The diagram above presents the reaction of turning diketopiperazine VIa VIm in the mixture of the preferred dipeptides L - EAA-DL-methionine (I) and DL-methionyl-L-EAA (II). This option is proposed in the invention method covers presented in figure 4 methods B, C, and G. In these cases diketopiperazine VIa VIm in each case formed as an intermediate product.

Interaction derivative of urea with amino acid with getting diketopiperazine preferably carried out at a temperature in the range from 20 to 200°C., more preferably from 40 to 180°C., particularly preferably from 100 to 170°C.

In one of the preferred embodiments of the above-described way interaction derivative of urea with amino acid with getting diketopiperazine carried out under pressure, preferably under a pressure in the range of 2 to 90 bar, bol is e preferably from 4 to 70 bar, particularly preferably from 5 to 50 bar.

Interaction derivative of urea with amino acid with getting diketopiperazine preferably further performed in the presence of a base. This base preferably be selected from the group comprising nitrogen-containing base, NH4HCO3, (NH4)2CO3Knso3, K2CO3the mixture of NH4OH/CO2, urethane salt, and the Foundation of the alkali and alkaline earth metals.

In yet another preferred embodiment proposed in the invention method, the reaction for obtaining diketopiperazine conduct, or subjecting the derivative of urea of the formula

where R denotes nationally balance, interaction with amino acid selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine, or subjecting the derivative of urea of the formula

where R represents an amino acid residue selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine, interaction with amino acid - methionine.

In one of the preferred options proposed in the invention method, to include truaudio the transformation of the derivative of urea in diketopiperazine interaction with methionine, a derivative of urea and methionine are particularly preferably used in a ratio of between 1:100 to 1:0.5 in.

In yet another preferred embodiment proposed in the invention method diketopiperazine turn in a mixture of dipeptides of formula I and II by acid hydrolysis. By acid hydrolysis, preferably at the same time to turn diketopiperazine in a mixture of L-EAA-DL-methionine (I) and DL-methionyl-L-EAA (II).

Acid hydrolysis is conducted in the presence of acid, preferably selected from the group comprising mineral acids, HCl, H2CO3, CO2/N2O, H2SO4, phosphoric acid, carboxylic acid and hydroxycarbonate acid.

In another preferred embodiment proposed in the invention method diketopiperazine turn in a mixture of dipeptides (I) and (II) by basic hydrolysis. By basic hydrolysis preferably at the same time to turn diketopiperazine in a mixture of L-EAA-DL-methionine (I) and DL-methionyl-L-EAA (II).

Basic hydrolysis preferably at the same time at pH values ranging from 7 to 14, more preferably from 8 to 13, particularly preferably from 9 to 12. This can cause complete racemization. The basic conditions can be created by use of the substance, preferably selected of isgroup, including a nitrogen-containing base, NH4HCO3, (NH4)2CO3the mixture of NH4OH/CO2, urethane salt, knso3, K2CO3, carbonates, and the Foundation of the alkali and alkaline earth metals.

Acid, respectively, the basic hydrolysis is preferably carried out at a temperature in the range from 50 to 200°C., more preferably from 80 to 180°C., particularly preferably from 90 to 160°C.

In one of the preferred variants of the amino acid residue in a derived urea of the formula III-V are presented in D - or L-configuration or a mixture of D - and L-configuration, preferably in a mixture of D - and L-configurations, when a derivative of urea is formed from methionine.

In another preferred embodiment, the amino acid residue in a derived urea of the formula III-V are presented in D - or L-configuration or a mixture of D - and L-configuration, preferably in the L-configuration, when a derivative of urea is formed from the amino acids selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

In the following a preferred embodiment of the proposed invention in the way they get dipeptides, which are presented in the form of a mixture of LL, DL, LD and DD, preferably in the form of a mixture of LL, LD, DL.

In one prefer is lnyh options proposed in the invention method diketopiperazine emit before hydrolysis. Diketopiperazine it is preferable to select from the reaction solution by crystallization, preferably at a temperature in the range from -30 to 120°C, particularly preferably from 10 to 70°C.

For separation of a mixture of diastereoisomers in the form of dipeptides of formula DD/LL/DL/LD-I) and DD/LL/DL/LD-(II), preferably a mixture of diastereoisomers in the form of a mixture of L-EAA-DL-methionine (I) and DL-methionyl-L-EAA (II), the main reaction is acidified solutions of their and get the product by crystallization, respectively deposition. It is preferable to set the pH to a value ranging from 2 to 10, more preferably from 3 to 9, particularly preferably corresponding to the isoelectric point of the particular dipeptide of formula I and II. For acidification and you can use acid, preferably selected from the group comprising mineral acids, HCl, H2CO3, CO2/N2O, H2SO4, phosphoric acid, carboxylic acid and hydroxycarbonate acid.

For separation of a mixture of diastereoisomers in the form of dipeptides of formula DD/LL/DL/LD-I) and DD/LL/DL/LD-(II), preferably a mixture of diastereoisomers in the form of a mixture of L-EAA-DL-methionine (I) and DL-methionyl-L-EAA (II) from acidic reaction solutions of their neutralized by adding bases and get the product by crystallization, respectively deposition. Preferably the mouth of the ways to develop the pH to a value ranging from 2 to 10, more preferably from 3 to 9, particularly preferably corresponding to the isoelectric point of the particular dipeptide of formula I and II. To neutralize this use of the base, preferably selected from the group comprising NH4HCO3, (NH4)2CO3, nitrogen-containing base, NH4OH, urethane salt, knso3, K2CO3, carbonates, and the Foundation of the alkali and alkaline earth metals.

Another alternative proposed in the invention method provides for the synthesis of unnatural dipeptides L - EAA-D-methionine Ia-Ij, respectively D-methionyl-L-EAA IIa-IIj - using the techniques of protective groups. Thus, in particular, for the synthesis of dipeptides L-EAA-D-methionine (LD-I) first, the free amino group of L-EAA defended protective BOC-(tert-butoxycarbonyl-). Alternatively, you can also successfully use the Z protective group (menthoxycarbonyl-). D-Methionine was atrificial methanol, protecting in this way the acid functional group. Then the protected BOC-, respectively, Z is a group of the amino acid L-EAA were subjected to reaction in combination with a methyl ester, D-methionine using dicyclohexylcarbodiimide (DCC) (see scheme 5).

Scheme 5

After cleaning BOC-L-EAA-D-methionine-OMe, respectively Z-L-EAA-D-methionine-About the e first in mild basic conditions were digested methyl ether. Then in the acidic conditions of interaction with HBr in glacial acetic acid was tsalala protective TREATMENT, respectively Z-group and free dipeptide - L-EAA-D-methionine (LD-I) was purified by resultant deposition rates and recrystallization (see diagram 6).

Scheme 6

Alternatively, you can also expose the protected BOC-group of the methyl ester of the dipeptide - BOC-L-EAA-D-methionine-OMe - interaction with HBr in glacial acetic acid to remove the most protective of the BOC-group. After evaporation you can then cleave the methyl ether by the addition of dilute hydrochloric acid. Then the free dipeptide - L-EAA-D-methionine (LD-I) can in this case be cleaned by resultant deposition rates and recrystallization (see diagram 6).

All the above process can also be used in relation to the dipeptides in the form of L-EAA-D-methionine formulae Ia-Ij. Used methyl esters of L-EAA and the protected BOC-, respectively Z-group D-methionine.

All of the above options proposed in the invention method is preferably carried out in aqueous medium.

Besides the above options proposed in the invention method can be carried out in well-known specialists in batch or continuous mode.

Graphics

In Fig.1 graphically presents the network cleavage of the dipeptides L-EAA-L-Met (LL-I) under the action of enzymes, selected from mirror carp.

In Fig.2 graphically presents the cleavage of the dipeptides L-Met-L-EAA (LL-II) under the action of enzymes isolated from mirror carp.

In Fig.3 graphically presents the cleavage of the dipeptides L-EAA-L-Met (LL-I) under the action of enzymes isolated from rainbow trout.

In Fig.4 graphically presents the cleavage of the dipeptides L-Met-L-EAA (LL-II) under the action of enzymes isolated from rainbow trout.

In Fig.5 graphically presents the cleavage of the dipeptides L-EAA-L-Met (LL-I) under the action of enzymes isolated from Pacific white shrimp.

In Fig.6 graphically presents the cleavage of the dipeptides L-Met-L-EAA (LL-II) under the action of enzymes isolated from Pacific white shrimp.

In Fig.7 graphically presents the cleavage of the dipeptides L-EAA-D-Met (LD-I) and D-Met-L-EAA (DL-II) under the action of enzymes isolated from mirror carp.

In Fig.8 graphically presents the cleavage of the dipeptides L-EAA-D-Met (LD-I) and D-Met-L-EAA (DL-II) under the action of enzymes isolated from carp.

In Fig.9 graphically presents the cleavage of the dipeptides L-EAA-D-Met (LD-I) and D-Met-L-EAA (DL-II) under the action of enzymes isolated from tilapia.

In Fig.10 graphically presents the cleavage of the dipeptides L-EAA-D-Met (LD-I) and D-Met-L-EAA (DL-II) under the action of the enzymes isolated from the be the th Pacific shrimp.

In Fig.11 graphically presents the cleavage of the dipeptides L-EAA-D-Met (LD-I) and D-Met-L-EAA (DL-II) under the action of enzymes isolated from rainbow trout.

In Fig.12 graphically presents the decomposition of mixtures of L-Trp-D-Met/D-Met-L-Trp (LD-Ij/DL-IIj) and L-Trp-D-Met/L-Met-L-Trp (LD-Ij/LL-IIj) under the action of enzymes isolated from mirror carp.

In Fig.13 graphically presents the results of experiments in vitro by cleavage of natural dipeptides L-Ile-L-Met (LL-Ic), respectively, L-Met-L-Ile (LL-IIc) under the action of a 1% solution of enzymes isolated from mirror carp, and non-natural dipeptides L-Ile-D-Met (LD-Ic), respectively, D-Met-L-Ile (DL-IIc) under the action of a 10% aqueous solution of enzymes isolated from mirror carp.

In Fig.14 graphically presents the results of experiments in vitro by cleavage of natural dipeptides L-Thr-L-Met (LL-Id), respectively, L-Met-L-Thr (LL-IId) under the action of a 1% solution of enzymes isolated from mirror carp, and non-natural dipeptides L-Thr-D-Met (LD-Id), respectively, D-Met-L-Thr (DL-IId) under the action of a 10% aqueous solution of enzymes isolated from mirror carp.

In Fig.15 graphically presents the results of experiments in vitro by cleavage of natural dipeptides L-Lys-L-Met (LL-Ie), respectively, L-Met-L-Lys (LL-IIe) under the action of a 1% solution of enzymes isolated from mirror carp, and non-natural dipeptides L-Lys-D-Met (LD-Ie), respectively D-Met-L-Ls (DL-IIe) under the action of a 10% aqueous solution of enzymes, selected from mirror carp.

In Fig.16 graphically presents the cleavage of the dipeptides L-Met-L-EAA (LL-II) under the action of enzymes isolated from chicken.

In Fig.17 graphically presents the cleavage of the dipeptides L-EAA-D-Met (LD-I) and D-Met-L-EAA (DL-II) under the action of enzymes isolated from chicken.

Examples

Example 1: General methods of synthesis of unnatural dipeptides L - EAA-D-methionine Ia-Ii, respectively D-methionyl-L-EAA IIa-IIj - using equipment protective groups

For the synthesis of dipeptides L-EAA-D-methionine (LD-I) first, the free amino group of L-EAA defended protective BOC-(tert-butoxycarbonyl-). Alternatively, you can also successfully use the Z protective group (menthoxycarbonyl-). D-Methionine was atrificial methanol, protecting in this way the acid functional group. Then the protected BOC-, respectively, Z is a group of the amino acid L-EAA were subjected to reaction in combination with a methyl ester, D-methionine using dicyclohexylcarbodiimide (DCC) (see scheme 5).

Scheme 5

After cleaning BOC-L-EAA-D-methionine-OMe, respectively Z-L-EAA-D-methionine-OMe, first in mild basic conditions were digested methyl ether. Then in the acidic conditions of interaction with HBr in glacial acetic acid was tsalala protective TREATMENT, respectively Z-group and free dipeptide - L-EAA-D-IU is Jonas (LD-I) - was purified by resultant deposition rates and recrystallization (see diagram 6).

Scheme 6

Alternatively, you can also expose the protected BOC-group of the methyl ester of the dipeptide - BOC-L-EAA-D-methionine-OMe - interaction with HBr in glacial acetic acid to remove the most protective of the BOC-group. After evaporation you can then cleave the methyl ether by the addition of dilute hydrochloric acid. Then the free dipeptide - L-EAA-D-methionine (LD-I) can in this case be cleaned by resultant deposition rates and recrystallization (see diagram 6).

All the above process can also be used in relation to the dipeptides in the form of L-EAA-D-methionine formulae Ia-Ij. Used methyl esters of L-EAA and the protected BOC-, respectively Z-group D-methionine.

Example 2

a) the Technique of synthesis of Z-D-Met

30.0 g (0,201 mole) of D-methionine and 42.4 g (0.4 mol) of Na2CO3was added 200 ml of water and cooled in an ice bath to 0°C. then slowly added of 51.2 g (0,3 mol) of carboxymethyloxime (Cbz-Cl) and the reaction mixture for 3 h and stirred at room temperature. Then was acidified with diluted hydrochloric acid and the reaction solution was extracted three times tert-butyl ether (MTBE) in portions of 50 ml the combined organic phases were dried over MgSO4and concentrated on the ro shall include evaporator. The obtained residue was recrystallized from a mixture of diethyl ether with ethyl acetate and dried in vacuum at 30°C. in This way has allocated of 36.4 g (64%) carboxymethoxy-D-methionine (Z-D-Met) in the form of a white crystalline solid.

b) General methods of synthesis of Z-L-EAA

50 mmol L-EAA and 10.6 g (100 mmol) of Na2CO3added 50 ml of water and cooled in an ice bath to 0°C. then slowly added 12.8 g (75 mmol) of carboxymethyloxime (Cbz-Cl) and the reaction mixture for 3 h and stirred at room temperature. Then was acidified with diluted hydrochloric acid and the reaction solution was extracted three times MTBE portions 25 ml the combined organic phases were dried over MgSO4and concentrated on a rotary evaporator. The obtained residue was recrystallized and dried in vacuum at 30°C.

Example 3: a methodology for the synthesis of D-Met-OMe·HCl

50.0 g (0,335 mole) of D-methionine suspended in 500 ml of methanol and the suspension was barbotirovany gaseous HCl at a moderate speed until saturation. When methionine was dissolved, and the solution was heated to 55°C. then the reaction mixture was left to mix overnight at room temperature. The next morning the mixture was concentrated to dryness on a rotary evaporator at 40°C and the resulting residue is twice recrystallized from diethyl ether. In this way the separation is whether to 47.1 g (86%) of the hydrochloride of the methyl ester of D-methionine in the form of a white crystalline solid.

Example 4: General methods of synthesis of L-EAA-OMe·HCl

of 0.3 mol L-EAA suspended in 500 ml of methanol and the suspension was barbotirovany gaseous HCl at a moderate speed until saturation. When this amino acid was dissolved, and the solution was heated to 50-60°C. After the reaction mixture was left to mix overnight at room temperature. The next morning the mixture was concentrated to dryness on a rotary evaporator at 40°C and the resulting residue is twice recrystallized from diethyl ether or a mixture of diethyl ether with methanol.

Example 5: General methods of synthesis of compounds of group PG-D-Met-L-EAA-OMe (PG-DL-II-OMe) (reaction mix)

20.0 mmol hydrochloride of L-EAA-OMe suspended in a mixture of 30 ml of chloroform and 5 ml of methanol, mixed with 4.15 g (30 mmol) of K2CO3and was stirred for 1 h at room temperature. After that salt was filtered and washed with a small amount of chloroform. After concentration of the filtrate obtained residue was dissolved in 50 ml of tetrahydrofuran, mixed with 4,37 g (21,0 mmol, of 1.05 equiv.) DCC and to 5.66 g (20.0 mmol) of Z-D-methionine and was stirred for 16 h at room temperature. After the reaction mixture was mixed with 3 ml of glacial acetic acid was stirred for 30 min and precipitated precipitated white solid (N,N'-dicyclohexylphosphino) was filtered. The filtrate concentrate who has demonstrated on a rotary evaporator and possibly precipitated precipitated N,N'-dicyclohexylmethane was filtered. The oily residue is then recrystallized twice from a mixture of chloroform with n-hexane and dried in a vacuum generated by an oil pump.

The abbreviation "PG" denotes a protective group (protective Z or BOC-group).

5A) Z-D-Met-L-Val-OMe (Z-DL-IIa-OMe)

Total formula: C19H28N2O5S (396,50 g/mol), yield: 4,60 g (58%), purity: 97%, white solid.

1H-NMR spectrum of the compound Z-D-Met-L-Val-OMe (Z-DL-IIa-OMe) (500 MHz, CDCl3):δ=0,88(d3J=6.8 Hz, 3H, CH3); 0,93 (d3J=6.8 Hz, 3H, CH3); 1,90-of 2.20 (m, 3H, SCH2CH2CH(CH3)2); 2,10 (s, 3H, SCH3); 2,50-of 2.64 (m, 2H, SCH2); to 3.73 (s, 3H, och3); to 4.38-of 4.44 (m, 1H, CH); 4,48-of 4.54 (m, 1H, CH); 5,08-by 5.18 (m, 2H, OCH2); 5,49 (Shir. s, 1H, NH); 6,58 (Shir. s, 1H, NH); 7.24 to 7,38 (m, 5H, Ph)

13C-NMR spectrum of the compound Z-D-Met-L-Val-OMe (Z-DL-IIa-OMe) (125 MHz, CDCl3):δ=15,26; 17,74; 19,01; 30,13; 31,16; 31,67; 52,21; 57,24; 67,22; 128,16; 128,27; 128,58; 136,16; 156,13; 171,01; 171,95

5B) Z-D-Met-L-Leu-OMe (Z-DL-IIb-OMe)

Total formula: C20H30N2O5S (410,53 g/mol), yield: of 5.40 g (66%), purity: 97%, white solid.

1H-NMR spectrum of the compound Z-D-Met-L-Leu-OMe (Z-DL-IIb-OMe) (500 MHz, D6-DMSO):δ=0,90-0,95 (m, 6H, CH(CH3)2); 1,50-1,72 (m, 3H, CH2CH(CH3)2);

1,90-of 2.15 (m, 2H, SCH2CH2); is 2.09 (s, 3H, SCH3); 2,48-of 2.64 (m, 2H, SCH2); 3,71 (s, 3H, och3); 4,36-of 4.44 (m, 1H, CH); 4,56-to 4.62 (m, 1H, C); 5,12 (s, 2H, och2); 5,56 (d3J=7,6 Hz, 1H, OC(=O)NH); 6,59 (Shir. s, 1H, NH); 7,26 and 7.36 (m, 5H, Ph)

13C-NMR spectrum of the compound Z-D-Met-L-Leu-OMe (Z-DL-IIb-OMe) (125 MHz, D6-DMSO):δ=15,27; 21,86; 22,78; 24,95; 30,11; 31,62; 33,96; 41,35; 50,86; 52,33; 67,20; 128,09; 128,25; 128,57; 156,97; 170,95; 173,01

5B) Z-D-Met-L-Ile-OMe (Z-DL-IIc-OMe)

Total formula: C20H30N2O5S (410,53 g/mol), yield: 5,09 g (62%), purity: 97%, white solid.

1H-NMR spectrum of the compound Z-D-Met-L-Ile-OMe (Z-DL-IIc-OMe) (500 MHz, CDCl3):δ=0,86-of 0.94 (m, 6H, CH(CH3)CH2CH3); 1,10-of 1.45 (m, 2H, CH2CH3); 1,84-of 1.94 (m, 1H, CH(CH3); 1,94-of 2.16 (m, 2H, SCH2CH2); 2,10 (s, 3H, SCH3); 2,49-of 2.64 (m, 2H, SCH2); and 3.72 (s, 3H, och3); 4,36-of 4.44 (m, 1H, CH); to 4.52-4,58 (m, 1H, CH); 5,08-by 5.18 (m, 2H, OCH2); 5,46 (Shir. s, 1H, NH); 6,58 (Shir. s, 1H, NH); 7,28-7,38 (m, 5H, Ph)

13C-NMR spectrum of the compound Z-D-Met-L-Ile-OMe (Z-DL-IIc-OMe) (125 MHz, CDCl3):δ=11,55; 15,26; 15,54; 25,19; 30,12; 31,70; 33,96; 37,79; 52,15; 45,07; 56,55; 67,18; 128,12; 128,24; 128,56; 156,13; 170,92; 171,96

5g) Z-D-Met-L-Thr-OMe (Z-DL-IId-OMe)

Total formula: C18H26N2O6S (398,47 g/mol), yield: 2.14 g (36%), purity: 95%, pale yellow solid.

1H-NMR spectrum of the compound Z-D-Met-L-Thr-OMe (Z-DL-IId-OMe) (500 MHz, CDCl3):δ=1,10-1,25 (m, 3H, SNSN3); 1,95-of 2.20 (m, 2H, SCH2CH2); is 2.09 (s, 3H, SCH3); 2,49 (Shir. s, 1H, HE); 2,52-2,62 (m, 2H, SCH2); 3,74 (s, 3H, och3); 4,30-4,56 (m, 3H, 3×CH); 5,12 (s, 2H, och2; 5,70-5,78 (m, 1H, NH); 7,03 (d3J=8,9 Hz, 1H, NH); 7,28-7,38 (m, 5H, Ph)

13C-NMR spectrum of the compound Z-D-Met-L-Thr-OMe (Z-DL-IId-OMe) (125 MHz, CDCl3):δ=15,15; 20,05; 30,10; 31,91; 52,66; 54,37; 57,44; 67,23; 67,82; 128,17; 128,26; 128,57; 136,16; 156,18; 171,25; 171,87

5D) Z-D-Met-L-Lys(BOC)-OMe (Z-DL-IIe(BOC)-OMe)

Total formula: C25H39N3O7S (525,66 g/mol), yield: 10,86 g (33%), purity: 95%, pale yellow solid.

1H-NMR spectrum of the compound Z-D-Met-L-Lys(BOC)-OMe (Z-DL-IIe(BOC)-OMe) (500 MHz, CDCl3):δ=1.25 and 1,90 (m, 6H, 3×CH2(Lys)); USD 1.43 (s, 9H, C(CH3)3); 1,92-of 2.16 (m, 2H, SCH2CH2); is 2.09 (s, 3H, SCH3); 2,48-2,62 (m, 2H, SCH2); 3,02 - of 3.12 (m, 2H, NCH2); and 3.72 (s, 3H, och3); 4,35 with 4.65 (m, 3H, 2×CH, NH); to 5.13 (s, 2H, och2); 5,58 (d3J=7.5 Hz, 1H, NH); 6.75 in (lat. s, 1H, NH); 7,28 and 7.36 (m, 5H, Ph)

13C-NMR spectrum of the compound Z-D-Met-L-Lys(BOC)-OMe (Z-DL-IIe(BOC)-OMe) (125 MHz, CDCl3):δ=15,31; 22,44; 28,45; 29,47; 30,12; 31,82; 52,08; 52,45; 67,20; 79,15; 128,08; 128,25; 128,34; 128,57; 156,07; 170,97; 172,38

5e) Z-D-Met-L-Phe-OMe (Z-DL-IIg-OMe)

Total formula: C23H28N2O5S (444,54 g/mol), yield: of 3.73 g (42%), purity: 95% (liquid chromatography high resolution (IHVR)), white solid.

1H-NMR spectrum of the compound Z-D-Met-L-Phe-OMe (Z-DL-IIg-OMe) (500 MHz, D6-DMSO/CDCl3):δ=1,72-of 1.94 (m, 2H, SCH2CH2); a 2.01 (s, 3H, SCH3); 2,30-of 2.38 (m, 2H, SCH2); 2,94-3,14 (m, 2H, CH2Ph); 3,70 (s, 3H, och3); 4,25-4,32 (m, 1H, CHCH2CH 2S); 4,70-4,78 (m, 1H, CHCH2Ph); 5,00-5,10 (Shir. s, 2H, OCH2Ph); 6,60-6,70 (m, 1H, NH); 7,10-to 7.35 (m, 10H, 2×Ph); 7,75-7,80 (Shir. s, 1H, NH)

Z) Z-D-Met-L-His-OMe (Z-DL-IIh-OMe)

Total formula: C20H26N4O5S (434,51 g/mol), yield: 2.35 g (27%), purity: 95% (IHVR), pale yellow solid.

1H-NMR spectrum of the compound Z-D-Met-L-His-OMe (Z-DL-IIh-OMe) (500 MHz, CDCl3):δ=1,88 with 2.14 (m, 2H, SCH2CH2); is 2.05 (s, 3H, SCH3); 2,44-of 2.56 (m, 2H, SCH2); 3,06-3,14 (m, 2H, CH2-imidazolyl); 3,68 (s, 3H, och3); 4,20-and 4.40 (m, 2H, NH, CH); 4,70 was 4.76 (m, 1H, CH); 5,11 (s, 2H, OCH2); 5,91 (d3J=7,6 Hz, 1H, NH); 6,76 (Shir. s, 1H, CH(imidazolyl); 7,26 was 7.45 (m, 5H, Ph); 7,73 (Shir. s, 1H, CH(imidazolyl)); of 9.30 (Shir. s, 1H, NH)

13C-NMR spectrum of the compound Z-D-Met-L-His-OMe (Z-DL-IIh-OMe) (125 MHz, CDCl3):δ=15,27; 29,94; 31,81; 33,92; 52,46; 67,14; 116,88; 128,02; 128,12; 128,23; 128,49; 128,58; 133,23; 135,20; 136,21; 156,97; 171,17; 171,57

Z) Z-D-Met-L-Trp-OMe (Z-DL-IIj-OMe)

Total formula: C25H29N3O5S (483,58 g/mol), yield: 5,71 g (59%), purity: 98% (IHVR), pale yellow solid.

1H-NMR spectrum of the compound Z-D-Met-L-Trp-OMe (Z-DL-IIj-OMe) (500 MHz, D6-DMSO):δ=1,60-1,80 (m, 2H, SCH2CH2); 1,95 (s, 1H, SCH3); of 2.25 to 2.35 (m, 2H, SCH2); 3,02-3,20 (m, 2H, CH2-indolyl); of 3.60 (s, 3H, och3); 4,10-4,16 (m, 1H, CH); 4,50-4,60 (m, 1H, CH); 4,98-5,08 (m, 2H, OCH2); 6,94 is 7.50 (m, 12H, indolyl, Ph, OC(=O)NH); of 8.25 (d,3J=8.6 Hz, 1H, CONH-Trp)

13C-NMR spectrum of the compound Z--Met-L-Trp-OMe (Z-DL-IIj-OMe) (125 MHz, D6-DMSO):δ=14,42; 27,01; 29,40; 31,59; 51,75; 52,78; 53,60; 65,36; 109,16; 111,31; 117,84; 118,31; 120,86; 123,60; 126,90; 127,59; 127,68; 128,21; 136,02; 136,89; 155,81; 171,32; 172,06

Example 6: General methods of synthesis of compounds of group PG-L-EAA-D-Met-OMe (PG-LD-1-OMe) (reaction mix)

3,99 g (20.0 mmol) of methyl ester hydrochloride D-methionine suspended in a mixture of 30 ml of chloroform and 5 ml of methanol, mixed with 4.15 g (30 mmol) of K2CO3and was stirred for 1 h at room temperature. After that salt was filtered and washed with a small amount of chloroform. After concentration of the filtrate obtained residue was dissolved in 50 ml of tetrahydrofuran, mixed with 4,37 g (21,0 mmol, of 1.05 equiv.) DCC and 20.0-mmol of the corresponding PG-L-EAA (PG-L-amino acids) and was stirred for 16 h at room temperature. After the reaction mixture was mixed with 3 ml of glacial acetic acid was stirred for 30 min and precipitated precipitated white solid (N,N'-dicyclohexylphosphino) was filtered. The filtrate was concentrated on a rotary evaporator and possibly precipitated precipitated N,N'-dicyclohexylmethane was filtered. The oily residue is then recrystallized twice from a mixture of chloroform with n-hexane and dried in a vacuum generated by an oil pump.

The abbreviation "PG" denotes a protective group (protective Z or BOC-group).

6A) Z-L-Val-D-Met-OMe (Z-LD-Ia-OMe)

Total formula: C19H28N2O5S (396,50 g/mol), yield: a 3.01 g (38%), purity: 95% (IHVR), white solid.

1H-NMR spectrum of the compound Z-L-Val-D-Met-OMe (Z-LD-Ia-OMe) (500 MHz, CDCl3):δ=0,92 (d3J=6.9 Hz, 3H, CH3); 0,99 (d3J=6.9 Hz, 3H, CH3); 1,90 was 2.25 (m, 3H, SCH2CH2CH(CH3)2); 2,07 (s, 3H, SCH3); 2,44-of 2.54 (m, 2H, SCH2); 3,74 (s, 3H, och3); 4,04-4,10 (m, 1H, CH); 4,67-4,74 (m, 1H, CH); 5,12 (s, 2H, och2); 5,28 (Shir. s, 1H, NH); of 6.65 (d,3J=7.5 Hz, 1H, NH); 7,28-7,38 (m, 5H, Ph)

13C-NMR spectrum of the compound Z-L-Val-D-Met-OMe (Z-LD-Ia-OMe) (125 MHz, CDCl3):δ=15,45; 17,46; 19,30; 29,96; 30,87; 31,40; 51,57; 52,55; 60,37; 67,18; 128,08; 128,24; 128,57; 136,19; 156,38; 171,04; 172,04

6b) Z-L-Leu-D-Met-OMe (Z-LD-Ib-OMe)

Total formula: C20H30N2O5S (410,53 g/mol), yield: 4,48 g (55%), purity: 96% (IHVR), white solid.

1H-NMR spectrum of the compound Z-L-Leu-D-Met-OMe (Z-LD-Ib-OMe) (500 MHz, CDCl3):δ=0,94 (d3J=6.3 Hz, 6N, CH(CH3)2); 1,48-1,72 (m, 3H, CH2CH(CH3)2); 1,90-of 2.20 (m, 2H, SCH2CH2); 2,07 (s, 3h, SCH3); 2,42-2,52 (m, 2H, SCH2); to 3.73 (s, 3H, och3); 4,20-4,30 (m, 1H, CH); with 4.64-4.72 in (m, 1H, CH); 5,12 (s, 2H, OCH2); 5,23 (d3J=7.9 Hz, 1H, NH); 6,84 (d3J=7.2 Hz, 1H, NH); 7,28-7,38 (m, 5H, Ph)

13C-NMR spectrum of the compound Z-L-Leu-D-Met-OMe (Z-LD-Ib-OMe) (125 MHz, CDCl3):δ=15,47; 22,97; 24,81; 29,97; 31,46; 51,58; 52,55; 67,23; 128,09; 128,26; 128,58; 136,16; 156,23; 172,02; 172,09

6b) Z-L-Ile-D-Met-OMe (Z-LD-Ic-OMe)

img src="https://img.russianpatents.com/1189/11890422-s.jpg" height="41" width="68" />

Total formula: C20H30N2O5S (410,53 g/mol), yield: 3,89 g (47%), purity: 97% (IHVR), white solid.

1H-NMR spectrum of the compound Z-L-Ile-D-Met-OMe (Z-LD-Ic-OMe) (500 MHz, CDCl3):δ=0,91 (t3J=7,1 Hz, 3H, CH2CH3); 0,96 (d3J=7,1 Hz; 3H, CH(CH3); 1,08 is 1.16 (m, 1H, SN H"CH3); 1,46-and 1.54 (m, 1H, SN H"CH3); 1,88-of 2.20 (m, 3H, CH(CH3), SCH2CH2); 2,07 (s, 3H, SCH3); 2,44-2,52 (m, 2H, SCH2); to 3.73 (s, 3H, och3); 4,08-4,16 (m, 1H, CH); 4,66-4,74 (m, 1H, CH); 5,11 (s, 2H, OCH2); 5,34 (d3J=7,6 Hz, 1H; NH); 6,74 (d3J=8.0 Hz, 1H, NH); 7,28-7,38 (m, 5H, Ph)

13C-NMR spectrum of the compound Z-L-Ile-D-Met-OMe (Z-LD-Ic-OMe) (125 MHz, CDCl3):δ=11,54; 15,46; 15,68; 24,66; 29,96; 31,42; 37,36; 51,59; 52,57; 59,83; 67,19; 128,10; 128,25; 128,58; 136,20; 156,34; 170,99; 172,03

6g) Z-L-Thr-D-Met-OMe (Z-LD-Id-OMe)

Total formula: C18H26N2O6S (398,47 g/mol), yield: 2,47 g (31%), purity: 99% (IHVR), pale yellow solid.

1H-NMR spectrum of the compound Z-L-Thr-D-Met-OMe (Z-LD-Id-OMe) (500 MHz, CDCl3):δ=1,19 (d3J=6.4 Hz, 3H, CH3); 1,94-of 2.20 (m, 2H, SCH2CH2); to 2.06 (s, 3H, SCH3); 2,45 is 2.55 (m, 2H, SCH2); to 3.73 (s, 3H, och3); 4,18 (Shir. s, 1H, CH); 4,39 (Shir. s, 1H; CH); 4,66-4,74 (m, 1H, CH); 5,10-by 5.18 (m, 2H, OCH2); 5,85 (Shir. s, 1H, OC(=O)NH); 7,21 (Shir. s, 1H, NH); 7,28-7,38 (m, 5H, Ph)

13C-NMR spectrum of the compound Z-L-Thr-D-Met-OMe (Z-LD-Id-OMe) (125 MHz, CDCl3):δ=15,43; 18,48; 30,10; 30,91; 51,80; 52,66; 59,16; 66,99; 67,36; 128,04; 128,29; 128,59; 136,08; 156,94; 171,27; 172,25

6D) of BOC-L-Lys(BOC)-D-Met-OMe (BOC-LD-Ie(BOC)-OMe)

Total formula: C22H41N3O7S (491,64 g/mol), yield: 5,22 g (53,1%), purity: 97% (IHVR), white amorphous solid.

1H-NMR spectrum of the compound BOC-L-Lys(BOC)-D-Met-OMe (BOC-LD-Ie(BOC)-OMe) (500 MHz, CDCl3):δ=1.32 to to 1.42 (m, 2H, CH2(Lys)); the 1.44 (s, 9H, C(CH3)3); 1,45 (s, 9H, C(CH3)3); 1,46-of 1.56 (m, 2H, CH2(Lys)); 1,60-1,72 (m, 1H, CHCH'H(Lys)); 1,82-of 1.92 (m, 1H, CHCH'CH(Lys); 1,92-2,03 (m, 1H, SCH2CHH'H"); is 2.09 (s, 3H, SCH3); 2,12-2,22 (m, 1H, SCH2CH H"); of 2.51 (t,3J=7,4 Hz, 2H, SCH2); is 3.08-and 3.16 (m, 2H, NCH2); of 3.75 (s, 3H, och3); as 4.02-4,12 (m, 1H, CH); 4,54-to 4.62 (m, 1H, NH); 4,66-4,74 (m, 1H, CH): 5,06-to 4.14 (m, 1H, NH); for 6.81 (d,3J=7,4 Hz, 1H,NH)

6e) Z-L-Phe-D-Met-OMe (Z-LD-Ig-OMe)

Total formula: C23H28N2O5S (444,54 g/mol), yield: 3.51 g (40%), purity: 99% (IHVR), white solid.

1H-NMR spectrum of the compound Z-L-Phe-D-Met-OMe (Z-LD-Ig-OMe) (500 MHz, CDCl3):δ=1,78-2,04 (m, 2H, SCH2CH2); 2,02 (s, 3H, SCH3); 2,20-of 2.30 (m, 2H, SCH2); 3,02-3,14 (m, 2H, CH2Ph); 3,71 (s, 3H, och3); 4,40-4,50 (m, 1H, CH); 4,60-of 4.66 (m, 1H, CH); 5,09 (s, 2H, och2); 5,31 (Shir. s, 1H, OC(=O)NH); 6.42 per (d3J=7,6 Hz, 1H, NH); 7,16 and 7.36 (m, 10H, 2×Ph)

13C-NMR spectrum of the compound Z-L-Phe-D-Met-OMe (Z-LD-Ig-OMe) (125 MHz, CDCl3):δ=15,37; 29,67; 31,35; 38,63; 51,52; 52,53; 56,36; 67,15; 127,18; 128,06; 128,24; 128,57; 128,83; 129,26; 136,13; 136,30; 155,90; 170,63; 171,88

G) BOC-L-Phe-D-Met-OMe (BOC-LD-Ig-OMe)

Total formula: C20H30N2O5S (410,53 g/mol), yield: a 4.03 g (49%), purity: 98% (IHVR), white solid.

1H-NMR spectrum of the compound BOC-L-Phe-D-Met-OMe (BOC-LD-Ig-OMe) (500 MHz, CDCl3):δ=of 1.42 (s, 9H, C(CH3)3); 1,80-of 2.08 (m, 2H, SCH2CH2); 2,04 (s, 3H, SCH3); 2,24-of 2.34 (m, 2H, SCH2); 3,07 (d3J=7.2 Hz, 2H, CH2Ph); to 3.73 (s, 3H, och3); 4,30 was 4.42 (m, 1H, CH); 4,60-and 4.68 (m, 1H, CH); 4,90-5,02 (Shir. s, 1H, NH); 6,44 (d3J=7.9 Hz, 1H, NH); 7,18-7,34 (m, 5H, Ph)

13C-NMR spectrum of the compound BOC-L-Phe-D-Met-OMe (BOC-LD-Ig-OMe) (125 MHz, CDCl3):δ=15,39; 28,29; 29,67; 31,51; 38,42; 51,47; 52,50; 56,00; 80,38; 127,07; 128,79; 129,27; 136,60; 156,42; 171,00; 171,94

Z) Z-L-His-D-Met-OMe (Z-LD-Ih-OMe)

Total formula: C20H26N4O5S (434,51 g/mol), yield: 1.65 g (19%), purity: 95% (IHVR), pale yellow solid.

1H-NMR spectrum of the compound Z-L-His-D-Met-OMe (Z-LD-Ih-OMe) (500 MHz, D6-DMSO/CDCl3):δ=1,82-to 1.98 (m, 2H, SCH2CH2); a 2.01 (s, 3H, SCH3); 2,30 is 2.44 (m, 2H, SCH2); was 2.76 vs. 2.94 (m, 2H, CH2-imidazolyl); 3,63 (s, 3H, och3); 4,28 was 4.42 (m, 2H, 2×CH); free 5.01 (s, 2H, OCH2); 6,78 (Shir. s, 1H, CH(imidazolyl)); 7,25-7,37 (m, 6H, Ph, NH); 7,50 (Shir. s, 1H, CH(imidazolyl)); 8,27 (Shir. s, 1H, NH); 11,76 (Shir. s, 1H, NH(imidazolyl))

13C-NMR spectrum of the compound Z-L-His-D-Met-OMe (Z-LD-Ih-OMe) (125 MHz, D6-DMSO/CDCl3):δ=14,54; 29,40; 30,52; 50,78; 51,79; 54,61; 65,35; 127,47; 127,61; 128,20; 134,53; 136,92; 155,57; 171,39; 171,94

6I) Z-L-Trp-D-Met-OMe (Z-LD-Ii-OMe)

Total formula: C25H29N3O5S (483,58 g/mol), yield: 5.50 g (57%), purity: 99% (IHVR), pale yellow solid.

1H-NMR spectrum of the compound Z-L-Trp-D-Met-OMe (Z-LD-Ij-OMe) (500 MHz, CDCl3):δ=1,68-of 1.92 (m, 2H, SCH2CH2); of 1.97 (s, 3H, SCH3); 2,08 with 2.14 (m, 2H, SCH2); 3,14-to 3.34 (m, 2H, CH2-indolyl); to 3.64 (s, 3H, och3); 4,50-to 4.62 (m, 2H, 2×CH); 5,10 (s, 2H, och2); 5,44 (Shir. s, 1H, NH); 6,32 (Shir. s, 1H, NH); 7,00-7,38, 10H; aromatic.); 8,17 (Shir. s, 1H, NH)

13C-NMR spectrum of the compound Z-L-Trp-D-Met-OMe (Z-LD-Ij-OMe) (125 MHz, CDCl3):δ=15,31; 29,48; 31,26; 33,97; 51,48; 52,45; 55,65; 67,10; 1101,37; 111,34; 118,77; 119,94; 122,44; 123,14; 127,32; 128,09; 128,22; 128,56; 136,20; 136,28; 155,99; 171,15; 171,80

6K) BOC-L-Trp-D-Met-OMe (BOC-LD-Ii-OMe)

Total formula: C22H31N3O5S (449,56 g/mol), yield: 5,91 g (66%), purity: 99% (IHVR), white solid.

1H-NMR spectrum of the compound BOC-L-Trp-D-Met-OMe (BOC-LD-Ij-OMe) (500 MHz, CDCl3):δ=1,42 (s, 8H, C(CH3)3); 1.70 to to 1.98 (m, 2H, SCH2CH2); to 1.99 (s, 3H, SCH3); 2,10-of 2.20 (m, 2H, SCH2); 3,14-to 3.34 (m, 2H, CH2-indolyl); 3,66 (s, 3H, och3); of 4.44-to 4.52 (m, 1H, CH); 4,56-to 4.62 (m, 1H, CH); 5,12 (Shir. s, 1H, NH); 6,39 (d3J=8.0 Hz, 1H, NH);? 7.04 baby mortality-7,38 (m, 5H, indolyl-CH); 8,17 (d3J=7.9 Hz, 1H, NH)

13C-NMR spectrum of the compound BOC-L-Trp-D-Met-OMe (BOC-LD-Ij-OMe) (125 MHz, CDCl3):δ=15,28; 28,27; 29,43; 31,36; 33,93; 52,38; 55,25; 80,19; 110,54; 111,25; 118,78; 119,80; 122,31; 123,06; 127,40; 136,25; 155,40; 171,53; 171,85

Example 7: General procedure for the synthesis of compounds of gr is PPI PG-L-EAA-D-Met (PG-LD-I) and PG-D-Met-L-EAA (PG-DL-II) (the splitting of the methyl ester)

10.0 mmol PG-L-EAA-D-Met-OMe (PG-LD-1-OMe) or PG-D-Met-L-EAA-OMe (PG-DL-11-OMe) suspended in 15 ml of water and 200 ml of methanol and mixed with 1.2 EQ. (12.0 mmol) of NaOH (12.0 ml 1N. NaOH). After two hours of stirring the homogeneous reaction solution was acidified with diluted hydrochloric acid and methanol drove on a rotary evaporator. Vegascasinoonline this white solid was filtered off, washed with 20 ml of water and recrystallized.

The abbreviation "PG" denotes a protective group (protective Z or BOC-group).

Example 8: General methods of synthesis of compounds of the group of L-EAA-D-Met (LD-I) and D-Met-L-EAA (PL-11) (removal of N-terminal Z protective group)

5.0 mmol of Z-L-EAA-D-Met (Z-LD-I) or Z-D-Met-L-EAA (Z-LD-II) was dissolved in 50 ml of glacial acetic acid and mixed with 18.5 ml (15.6 g, 250 mmol, 50 EQ.) dimethyl sulfide and 5.0 g (3.6 ml) and 33% HBr in acetic acid (1.65 g, 4.0 EQ.). Upon completion of the reaction, the reaction solution was concentrated on a rotary evaporator. The residue was dissolved in about 50 ml of methanol and mixed with 3.5 g (50 mmol, 10 EQ.) methanolate sodium. After 20 minutes stirring at room temperature the solution was neutralized with concentrated hydrochloric acid and then concentrated on a rotary evaporator. The residue was dissolved in 40 ml of water and was extracted three times with diethyl ether in portions of 40 ml of the Aqueous phase was concentrated on a rotary evaporate the e, that was accompanied by the precipitation of a white friable solid. The resulting dipeptide was separated by vacuum filtration, washed with a small amount of water and dried in vacuum.

Example 9: General methods of synthesis of compounds of the group of L-EAA-D-Met (LD-I) and D-Met-L-EAA (DL-II) (removal of N-terminal protective BOC-group)

5.0 mmol of BOC-L-EAA-D-Met (BOC-LD-I) or BOC-D-Met-L-EAA (BOC-DL-II) was dissolved in 50 ml of glacial acetic acid and mixed with 5.0 g (3.6 ml) and 33% HBr in acetic acid (1.65 g (4.0 EQ.)). Upon completion of the reaction, the reaction solution was concentrated on a rotary evaporator. The residue was dissolved in 40 ml of water and was extracted three times with diethyl ether in portions of 40 ml of the Aqueous phase with constant cooling in an ice bath was slowly neutralized 20% NaOH solution. Then, the solution washed three times with diethyl ether in 40 ml and the aqueous phase was concentrated on a rotary evaporator, followed by precipitation of a white friable solid. The resulting dipeptide was separated by vacuum filtration, washed with a small amount of water and dried in vacuum.

9a) D-Met-L-Leu (DL-IIb)

Output: 860 mg (66%), purity: 98% (IHVR), white friable solid.

1H-NMR spectrum of the compound H-D-Met-L-Leu (DL-IIb) (500 MHz, D6-DMSO+HCl):δ=0,85 (d3J=6.3 Hz, 3H, CH3); 0,90 (d3J=6.3 Hz, 3H, CH3); 1,50-1,70 (m, 3H,SCH 2CH2CH(CH3)2); 2,00-2,10 (m, 5H, SCH3CH2CH); 2,45 is 2.55 (m, 2H, SCH2); 3,88-of 3.94 (m, 1H, CH); 4,22-4,30 (m, 1H, CH); 8,40 at 8.60 (m, 3H),NH3+); 8,95 (d3J=8,3 Hz, 1H, NH)

13C-NMR spectrum of the compound D-Met-L-Leu (DL-IIb) (500 MHz, D6-DMSO+HCl):δ=14,56; 21,16; 22,95; 24,50; 28,21; 31,22; 50,66; 51,77; 168,16; 173,50

Mass spectrometry high resolution (msvr) (pESI) for C11H23N2O3S (MN+):

calculated: 263,14294

found: 263,14224

9b) D-Met-L-Ile (DL-IIc)

Yield: 900 mg (69%), purity: 99% (IHVR), white friable solid.

1H-NMR spectrum of the compound D-Met-L-Ile (DL-IIc) (500 MHz, D6-DMSO+HCl):δ=0,82-of 0.90 (m, 6H, 2×CH3); 1,16-of 1.44 (m, 2H, SCH2CH3); 1,80-1,90 (m, 1H, CH); 2,00 is 2.10 (m, 2H, CH2); is 2.05 (s, 3H, SCH3); 2,46-of 2.54 (m, 2H, SCH2); 3,96-was 4.02 (m, 1H, CH); 4,24-4,30 (m, 1H, CH); 8.36-8,44 (m, 3H,NH3+); 8,79 (d3J=8.5 Hz, 1H, NH)

13C-NMR spectrum of the compound D-Met-L-Ile (DL-IIc) (500 MHz, D6-DMSO+HCl):δ=11,44; 14,86; 15,96; 24,95; 28,58; 31,71; 36,75; 52,00; 56,82; 168,64; 172,74

Msvr (pESI) for C11H23H2O3S (MN+):

calculated: 263,14294

found: 263,14215

9b) D-Met-L-Thr (DL-IId)

Output: 640 mg (51%), purity: 98% (IHVR), white R is Chloe solid.

1H-NMR spectrum of the compound D-Met-L-Thr (DL-IId) (500 MHz, D6-DMSO+HCl):δ=1,10 (d3J=6.2 Hz, 3H, SNSN3); to 2.06 (s, 3H, SCH3); 2.06 to and 2.14 (m, 2H, SCH2CH2); 2,48-2,60 (m, 2H, SCH2); 4,00-to 4.28 (m, 4H, 2×CH, CHOH); 8,40-8,46 (m, 3H,NH3+); 8,77 (d3J=8.6 Hz, 1H, NH)

13C-NMR spectrum of the compound D-Met-L-Thr (DL-IId) (500 MHz, D6-DMSO+HCl):δ=15,14; 20,94; 28,74; 31,94; 52,44; 58,81; 66,97; 169,22; 172,20

Msvr (pESI) for C9H19N2O4S (MH+):

calculated: 251,10655

found: 251,10583

9D) D-Met-L-Lys·2HCl (DL-IIe-2HCl)

Output: 613 mg (49%), purity: 97% (IHVR), yellowish solid.

1H-NMR spectrum of the compound D-Met-L-Lys·2HCl (DL-IIe-2HCl) (500 MHz, DMSO):δ=1.32 to to 1.42 (m, 2H, CH2(Lys); 1,52-of 1.62 (m, 2H, CH2(Lys); 1,64 and 1.80 (m, 2H, CH2(Lys); 2,00-2,10 (m, 5H, SCH2CH2, SCH3); 2,46-of 2.56 (m, 2H, SCH2); 2,70-2,82 (m, 2H, NCH2); to 3.92-4.00 points (m, 1H, CH); 4,16-4,24 (m, 1H, CH); 7,9 (Shir. s, 3H,NH3+); 8.3 (the Shire. s, 3H,NH3+); of 8.92 (d,3J=7.7 Hz, 1H, NH)

MCBP (pESI) for C11H24O3S (MN+):

calculated: 278,15384

found: 278,15288

9D) D-Met-L-Phe (DL-IIg)

p> Output: 930 mg (63%), purity: 98% (IHVR), white friable solid.

1H-NMR spectrum of the compound D-Met-L-Phe (DL-IIg) (500 MHz, D6-DMSO+HCl):δ=1,64-to 1.82 (m, 2H, SCH2CH2); 1,95 (s, 3H, SCH3); 2,10-of 2.26 (m, 2H, SCH2); 2,80-3,20 (m, 2H, CH2Ph); 3,70 (t3J=6,1 Hz, 1H, CHCH2Ph); 4,42-4,50 (m, 1H, CHCH2CH2S); 7,16-7,28 (m, 5H, Ph); 8,50 at 8.60 (Shir. s, 1H, NH)

13C-NMR spectrum of the compound D-Met-L-Phe (DL-IIg) (500 MHz, D6-DMSO+HCl):δ=14,28; 28,08; 31,63; 37,03; 51,84; 53,78; 126,28; 127,97; 129,08; 137,69; 168,90; 172,65

MCBP (pESI) for C14H21N2O3S (MH+):

calculated: 297,12729

found: 297,12643

9F) D-Met-L-Trp (DL-IIj)

Output: 1,38 g (82%), purity: 98% (IHVR), white friable solid.

1H-NMR spectrum of the compound D-Met-L-Trp (DL-IIj) (500 MHz, D6-DMSO+HCl):δ=1,50-1,80 (m, 2H, SCH2CH2); of 1.93 (s, 3H, SCH3); 2,30-to 2.40 (m, 2H, SCH2); 3,02-up 3.22 (m, 2H, CH2); 3,34 is 3.40 (m, 1H, SCH2CH2CH); to 4.38-and 4.40 (m, 1H, CH); 6,90-of 7.60 (m, 5H, indolyl); 8,05-8,15 (Shir. s, 1H, CONH); 10,80 (Shir. s, 1H, NH)

13C-NMR spectrum of the compound D-Met-L-Trp (DL-IIj) (500 MHz, D6-DMSO+HCl):δ=14,37; 27,38; 29,12; 33,28; 53,00; 53,49; 110,26; 111,17; 118,07; 118,26; 120,64; 123,36; 127,52; 135,98; 171,87; 173,53

MCBP (pESI) for C16H22H3O3S (MN+):

calculated: 336,13819

found: 336,13718

G) L-Leu-D-Met (LD-Ib)

Output: 710 mg (54%), purity: 99% (IHVR), white friable solid.

1H-NMR-spectrum soy is inane H-L-Leu-D-Met (LD-Ib) (500 MHz, D6-DMSO+HCl):δ=0,91 (t3J=5.4 Hz, 6N, 2×CH3); 1,62 (t3J=6,8 Hz, 2H, CH2CH(CH3)2); 1,60-1,75 m, 1H, CH(CH3)2); 1,88-2,04 (m, 2H, SCH2CH2); 2,04 (s, 3H, SCH3); 2,40-of 2.54 (m, 2H, SCH2); 3,78-3,86 (m, 1H, CH); 4,32-and 4.40 (m, 1H, CH); at 8.36 (d,3J=4.0 Hz, 3H,NH3+); 9,03 (d3J=7.8 Hz, 1H, NH)

13C-NMR spectrum of the compound H-L-Leu-D-Met (LD-Ib) (500 MHz, D6-DMSO+HCl):δ=14,56; 22,78; 23,33; 23,93; 29,89; 30,58; 41,03; 51,40; 51,56; 169,41; 173,03

MCBP (pESI) for C11H23N2O3S (MH+):

calculated: 263,14294

found: 263,14218

S) L-Ile-D-Met (LD-Ic)

Output: 790 mg (59%), purity: 97% (IHVR), white friable solid.

1H-NMR spectrum of the compound L-Ile-D-Met (LD-Ic) (500 MHz, D6-DMSO):δ=0,82 (t3J=7,4 Hz, 3H, CH3CH2); 0,86 (2,3J=6.6 Hz, 3H, CH3CH); 1,02 by 1.12 (m, 1H, CH3SN N"); 1,36 of 1.46 (m, 1H, CH3SN N"); 1,64-1,72 (m, 1H, CH3CH); 1,80-to 1.98 (m, 2H, SCH2CH2); 2,00 (s, 3H, SCH3); 2,36 is 2.44 (m, 2H, SCH2); 3,27 (d3J=5,1 Hz, 1H, CH); 3,99 (t3J=5.3 Hz; 1H, CH); 7,92 (Shir. s, 1H, NH)

13C-NMR spectrum of the compound L-Ile-D-Met (LD-Ic) (500 MHz, D6-DMSO):δ=11,57; 14,54; 15,60; 23,58; 29,69; 32,42; 37,90; 53,06; 58,79; 172,09; 173,37

MCBP (pESI) for C11H23N2O3S (MH+):

calculated: 263,14294

found: 263,14224

9) L-Thr-D-Met (LD-Id)

Yield: 690 mg (55%), purity: 99% (IHVR), white friable solid.

1H-NMR spectrum of the compound L-Thr-D-Met (LD-Id) (500 MHz, D6-DMSO+CDCl3):δ=1,08 (d3J=6.6 Hz, 3H, CH3); 1,82-of 2.08 (m, 2H, SCH2CH2), 2,02 (s, 3H, SCH3); 2,38-of 2.50 (m, 2H, SCH2); 3,06 (d3J=4,2 Hz, 1H, CH); 3,88-of 3.94 (m, 1H, CH); 3,98-Android 4.04 (m, 1H, CH); to $ 7.91 (d,3J=7,3 Hz, 1H, NH)

13C-NMR spectrum of the compound L-Thr-D-Met (LD-Id) (500 MHz, D6-DMSO+CDCl3):δ=14,75; 19,70; 30,07; 32,45; 53,71; 60,22; 67,45; 172,58; 174,24

Msvr (pESI) for C9H19N2O4S (MH+):

calculated: 251,10655

found: 251,10586

9K) L-Lys-D-Met·2HCl (LD-Ie-2HCl)

Output: 676 mg (54%), purity: 96% (IHVR), colorless crystals.

1H-NMR spectrum of the compound L-Lys-D-Met·2HCl (LD-Ie-2HCl) (500 MHz, D6-DMSO):δ=1.30 and the 1.44 (m, 2H, CH2(Lys)); 1,54-of 1.64 (m, 2H, CH2(Lys)); 1,72-of 1.84 (m, 1H, CH2(Lys)); 1,90-2,04 (m, 2H, SCH2CH2); is 2.05 (s, 3H, SCH3); 2,44-of 2.58 (m, 2H, SCH2); 2,70 is 2.80 (m, 2H, NCH2); 3,82-3,90 (m, 1H, CH); 4,34 was 4.42 (m, 1H, CH); 7,9 (Shir. s, 3H),NH3+); 8.3 (the Shire. s, 3H),NH3+); 8,91 (d3J=7.9 Hz, 1H, NH)

Msvr (pESI) for C11H24O3S (MN+):

calculated: 278,15384

found: 278,15290

9L) L-Phe-D-Met (LD-Ig)

Output: 880 mg (59%), purity: 98% (IHVR), white friable solid.

1H-NMR spectrum of the compound L-Phe-D-Met (LD-Ig) (500 MHz, D6-DMSO+D2O):δ=1.60-to 2,02 (m, 4H, SCH2CH2); is 2.05 (s, 3H, SCH3); is 3.08-of 3.32 (m, 2H, PhCH2); 4,12-4,16 (m, 1H, CH); 4,20-4.26 deaths (m, 1H, CH); 7,30-to 7.50 (m, 5H, Ph)

13C-NMR spectrum of the compound L-Phe-D-Met (LD-Ig) (500 MHz, D6-DMSO+D2O):δ=15,37; 30,72; 32,10; 38,09; 55,40; 55,96; 129,24; 130,50; 130,71; 136,55; 169,47; 178,42

Msvr (pESI) for C14H21N2O3S (MH+):

calculated: 297,12729

found: 297,12646

9) L-Trp-D-Met (LD-Ij)

Yield: 1.40 g (83%), purity: 98% (IHVR), white friable solid.

1H-NMR spectrum of the compound L-Trp-D-Met (LD-Ij) (500 MHz, D6-MCO):δ=1,68-of 1.88 (m, 2H, SCH2CH2); of 1.94 (s, 3H, SCH3); 2,24 (d3J=7.9 Hz, 2H, SCH2); 2,80-is 2.88 (m, 1H, CH); 3,10-and 3.16 (m, 1H, CH); 3,70 is 3.76 (m, 1H, CH); 4,00-4,06 (m, 1H, CH); 6,90-of 7.60 (m, 5H, indolyl); 8,10 (Shir. s, 1H, NH); 10,90 (Shir. s, 1H, NH)

13C-NMR spectrum of the compound L-Trp-D-Met (LD-Ij) (500 MHz, D6-DMSO):δ=14,51; 29,56; 29,90; 32,09; 52,78; 54,59; 109,82; 111,26; 118,15; 118,30; 120,80; 123,82; 127,20; 136,16; 172,03; 173,02

Msvr (pESI) for C16H22N3O3S (MH+):

calculated: 336,13819

found: 336,13724

Example 10: the Chemical synthesis of a mixture of diastereoisomers Met-Ile (IIc) of 5-[2-(methylthio)ethyl]-2,4-imidazolidinedione (methanimidamide) (Vn) and L-isoleucine using KON

of 11.8 g (of 0.09 mole) of L-isoleucine, and 17.2 g (of 0.09 mol, purity: 91) 5-[2-(methylthio)ethyl]-2,4-imidazolidinedione (Vn) and 11.9 g (to 0.8 mole) of 85% KOH was dissolved in 150 ml of water and during 5 h and stirred at 150°C in 200-ml steel autoclave firm Roth, equipped with magnetic stirrer, during which the pressure was increased to 8 bar. Upon completion of the reaction, the autoclave was cooled, precipitated precipitated solid was filtered and washed it with a small amount of water. The filtrate was barbotirovany CO2served with moderate consumption. Fallen thus precipitated solid was again separated by vacuum filtration, washed with a little cold water and within a few hours was dried at 30°C in a vacuum generated by an oil pump. Output: 7,3 g (31% of theory) of a white solid.1H-NMR-spectrum coincided with superimposed1H-NMR spectra of compounds L-Met-L-Ile (LL-IIc) and D-Met-L-Ile (DL-IIc) (see example 9b).

Example 11: the Chemical synthesis of a mixture of diastereoisomers Met-Ile (IIc) of N-carbamoylation (IIIn) and L-isoleucine using KON

of 11.8 g (of 0.09 mole) of L-isoleucine, 17,5 g (of 0.09 mol, purity: 99%) of N-carbamoylation (IIIn) and 11.9 g (0,18 mol) of 85% KOH was dissolved in 150 ml of water and during 5 h and stirred at 150°C in 200-ml steel autoclave firm Roth, equipped with a magnetic stirrer, during which the pressure was increased to 7 bar. Upon completion of the reaction, the autoclave was cooled, precipitated precipitated solid was filtered and washed it with a small amount of water. The filtrate was neutralized with 10% sulfuric acid, precipitated in the sediment solid vases is separated in a vacuum-filtering, washed it with a small amount of cold water and within a few hours was dried at 30°C in a vacuum generated by an oil pump. Yield: 6.4 g (27% of theory) of a white solid.1H-NMR coincided with superimposed1H-NMR spectra of compounds L-Met-L-Ile (LL-IIc) and D-Met-L-Ile (DL-IIc) (see example 9b).

Example 12: the Chemical synthesis of a mixture of diastereoisomers Met-Ile (IIc) of amide 2-[(aminocarbonyl)amino]-4-(methylthio)butyric acid (amide N-carbamoylation) (IVn) and L-isoleucine using KON

of 11.8 g (of 0.09 mole) of L-isoleucine, 17,4 g (90 mmol, purity: 98.5 per cent) amide 2-[(aminocarbonyl)amino]-4-(methylthio)butyric acid (IVn) and 11.9 g (to 0.8 mole) of 85% K IT was dissolved in 150 ml of water and during 5 h and stirred at 150°C in 200-ml steel autoclave firm Roth, equipped with a magnetic stirrer, during which the pressure was increased to 7 bar. Upon completion of the reaction, the autoclave was cooled, precipitated precipitated solid was filtered and washed it with a small amount of water. The filtrate was neutralized policecontributing hydrochloric acid, precipitated thus precipitated solid was separated by vacuum filtration, washed with a little cold water and within a few hours was dried at 30°C in a vacuum generated by an oil pump. Yield: 8.0 g (34% of theory) of a white solid.1H-NMR coincided with superimposed 1H-NMR spectra of compounds L-Met-L-Ile (LL-IIc) and D-Met-L-Ile (DL-IIc) (see example 9b).

Example 13: the Chemical synthesis of 3-[2-(methylthio)ethyl]-6-(1-(methyl)propyl)-2,5-piperazinediones (VIc) of 5-[2-(methylthio)ethyl]-2,4-imidazolidinedione (methanimidamide) (Vn) and L-isoleucine

of 11.8 g (of 0.09 mole) of L-isoleucine, and 17.2 g (of 0.09 mol, purity: 91%) 5-[2-(methylthio)ethyl]-2,4-imidazolidinedione (Vn) and 7.1 g (0,9 mol) (NH4)HCO3was dissolved in 150 ml of water and during 5 h and stirred at 150°C in 200-ml steel autoclave firm Roth, equipped with a magnetic stirrer, which was accompanied by increased pressure. By periodic release of gas pressure is maintained constant at the level of 8 bar. Upon completion of the reaction, the autoclave was cooled in an ice bath. The resulting suspension is then filtered, the filtered solid was repeatedly washed with water and within a few hours was dried him at 30°C in a vacuum generated by an oil pump. Output: 9,9 g (45% of theory) of the compound VIc in the form of a white solid.

1H-NMR spectrum of 3-[2-(methylthio)ethyl]-6-(1-(methyl)propyl)-2,5-piperazinediones (VIc) (500 MHz, D6-DMSO):δ=0,85 (t3J=7,4 Hz, 3H, CH2CH3); 0,90 (d3J=7,4 Hz, 3H, SNSN3); 1,10-1,50 (m, 2H, SCH2CH2); 1,80-1,90 (m, 1H, CH); 1,90-2,00 (m, 2H, CH2); 2,04 (s, 3H, SCH3); 2,42-of 2.58 (m, 2H, SCH2); 3,64-3,68 (m, 1H, CH); 3,94-3,98 (m, 1H, CH); 8.08-8,16 (, 2H, 2×NH)

13C-NMR spectrum of 3-[2-(methylthio)ethyl]-6-(1-(methyl)propyl)-2,5-piperazinediones (VIc) (500 MHz, D6-DMSO+HCl):δ=12,02; 14,85; 15,27; 24,61; 28,74; 32,15; 39,90; 52,92; 59,34; 167,90; 168,10

Example 14: the Chemical synthesis of 3-[2-(methylthio)ethyl]-6-(1-methyl)propyl)-2,5-piperazinediones (VIc) of N-carbamoylation (IIIn) and L-isoleucine

of 11.8 g (of 0.09 mole) of L-isoleucine, 17,5 g (of 0.09 mol, purity: 99%) of N-carbamoylation (IIIn) and 7.1 g (0,9 mol) (NH4)HCO3was dissolved in 150 ml of water and during 5 h and stirred at 150°C in 200-ml steel autoclave firm Roth, equipped with a magnetic stirrer, which was accompanied by increased pressure. By periodic release of gas pressure is maintained constant at the level of 8 bar. Upon completion of the reaction, the autoclave was cooled in an ice bath. The resulting suspension is then filtered, the filtered solid was repeatedly washed with water and within a few hours was dried at 30°C in a vacuum generated by an oil pump. Yield: 9.1 g (41,3% of theory) of the compound VIc in the form of a white solid. The NMR spectrum was identical with the NMR spectrum of example 13.

Example 15: the Chemical synthesis of 3-[2-(methylthio)ethyl]-6-(1-methyl)propyl)-2,5-piperazinediones (VIc) of amide 2-[(aminocarbonyl)amino]-4-(methylthio)butyric acid (amide N-carbamoylation) (IVn) and L-isoleucine

of 11.8 g (of 0.09 mole) of L-isoleucine, 17,4 g (90 mmol, purity: 98.5 per cent) amide 2-[(aminocarbonyl the l)amino]-4-(methylthio)butyric acid (IVn) and 7.1 g (0,9 mol) (NH 4)HCO3was dissolved in 150 ml of water and during 5 h and stirred at 150°C in 200-ml steel autoclave firm Roth, equipped with a magnetic stirrer, which was accompanied by increased pressure. By periodic release of gas pressure is maintained constant at the level of 8 bar. Upon completion of the reaction, the autoclave was cooled in an ice bath. The resulting suspension is then filtered, the filtered solid was repeatedly washed with water and within a few hours was dried at 30°C in a vacuum generated by an oil pump. Yield: 10.3 g (47% of theory) of a white solid substance IVc. The NMR spectrum was identical with the NMR spectrum of example 13.

Example 16: Synthesis of a mixture of diastereoisomers Ile-Met (Ic) and Met-Ile (IIc) of 3-[2-(methylthio)ethyl]-6-(1-methyl)propyl)-2,5-piperazinediones (VIc) using concentrated hydrochloric acid

24.4 g (100 mmol) 3-[2-(methylthio)ethyl]-6-(1-methyl)propyl)-2,5-piperazinediones (VIc) suspended in 66 g of water. Next, while stirring slowly dropwise added 11 g of concentrated hydrochloric acid and then gently with very vigorous stirring, they were heated to a temperature of distillation. After the reaction mixture for 8 h was heated under reflux, during which all the solid is passed into the solution. During subsequent cooling to precipitate fell out a small amount neprology avego of diketopiperazine, which was filtered. Then in the beaker from the ice bath, the pH of the filtrate was adjusted at 5-6 by addition of 32% ammonia water. In the sediment fell mixture of DL-Met-DL-Ile (a mixture of diastereomers of compound IIc) and DL-Ile-DL-Met (a mixture of diastereomers of compounds Ic) in the form of a white friable solid. This solid was dried in a drying Cabinet at 40°C in vacuum, created a water vacuum pump. Output: 21,5 g (82,0%).

Example 17: Synthesis of a mixture of diastereoisomers Ile-Met (Ic) and Met-Ile (IIc) of 3-[2-(methylthio)ethyl]-6-(1-methyl)propyl)-2,5-piperazinediones (VIc) under alkaline conditions using ammonia

19.6 g (0,8 mol) of 3-[2-(methylthio)ethyl]-6-(1-methyl)propyl)-2,5-piperazinediones (VIc), of 22.4 ml of 25% ammonia solution and 160 ml of water was heated in an autoclave to 150°C with a dwell time at this temperature for 2 hours, After cooling, unreacted diketopiperazine was separated by vacuum filtration. It can be reused in the next reaction mixture. The filtrate was concentrated on a rotary evaporator at a water temperature of 80°C until precipitation of the first crystals. After cooling, the mixture was left to stand overnight, after which by filtration and drying was found to be a mixture of DL-Met-DL-Ile (a mixture of diastereomers of compound IIc) and DL-Ile-DL-Met (a mixture of diastereomers of compounds Ic) in the form of a white friable solid. Yield: 12.2 g (58%).

Example 18: the United ity in vitro with the digestion of L-EAA-L-Met (LL-I), accordingly, L-Met-L-EAA (LL-II) under the action of digestive enzymes isolated from omnivorous cyprinid

a) the Secretion of digestive enzymes from the mirror carp (Cyprinus carpio morpha noblis)

Digestive enzymes were allocated in accordance with the method described in EID and MATTY (Aquaculture, 79, 1989, SS.111-119). To do this, five annual mirror carp (Cyprinus carpio morpha noblis) extracted the intestines, washed with water, cut along with bowel scraped mucosa. Together with crushed ice crushed in a blender. The resulting suspension was treated with an ultrasonic rod to destroy even whole cells. For separating cellular components and grease the suspension for 30 min, centrifuged at 4°C, the homogenate was separated by decanting and sterilized by his stripes thimerosal. 5 mirror carp got 296,3 ml solution of enzymes of the intestinal mucosa and the solution was stored in the dark at 4°C.

b) Conducting experiments in vitro with digestion

L-Met-L-EAA (LL-II), respectively, L-EAA-L-Met (LL-I) was dissolved in a buffer solution of Tris/HCl and mixed with a solution of enzymes. For comparison and to assess the rate exclusively chemical cleavage in each case carried out on a "dummy" experience without mortar enzymes (see table 3). Periodically, a sample was taken and quantitatively evaluated its composition by her anal is calibrated for GHUR-chromatograph. The degree of conversion was determined by dividing the methionine content in the content of L-Met-L-EAA (LL-II), respectively, L-EAA-L-Met (LL-I) (see Fig.1 and 2).

Table 3
SampleThe value in the "idle" experience
Raw materialsThe substrate (LL-I, respectively LL-II)0.15 mmole0.15 mmole
Buffer solution Tris/HCl, pH 9,57.5 ml8,1 ml
The start of the reactionThe solution of enzyme (corresponds to 1.5% of the resultant solution of enzymes isolated from Karpov)589 μl---
Reaction37°C37°C
Termination reactions0.2 ml of the reaction solution was dissolved in 9.8 ml of 10% aqueous solution of N3RHO4

Example 19: in vitro Experiments with the digestion of L-EAA-D-Met (LD-I). accordingly, D-Met-L-EAA (DL-II) by the action of digestive enzymes, selected from omnivorous cyprinid

a) the Secretion of digestive enzymes from the mirror carp (Cyprinus carpio morpha noblis)

Digestive enzymes were allocated in accordance with the method described in EID and MATTY (Aquaculture, 79, 1989, SS.111-119). To do this, five annual mirror carp (Cyprinus carpio morpha noblis) removed the intestines and processed it described in example 18 method.

b) Conducting experiments in vitro with digestion

D-Met-L-EAA (DL-II), respectively, L-EAA-D-Met (LD-I) was dissolved in a buffer solution of Tris/HCl and mixed with a solution of enzymes. For comparison and to assess the rate exclusively chemical cleavage in each case carried out on a "dummy" experience without mortar enzymes (see table 4). Periodically, a sample was taken and quantitatively evaluated its members through its analysis on the calibrated GHUR-chromatograph. The degree of conversion was determined by dividing the area under the curve obtained for methionine, the area under the curve obtained for D-Met-L-EAA (DL-II), respectively, L-EAA-D-Met (LD-I) (see Fig.7).

Table 4
SampleThe value in the "idle" experience
Raw materials The substrate (LD-I, respectively, DL-II)0.15 mmole0.15 mmole
Buffer solution Tris/HCl, pH 9,57.5 ml13.4 ml
The start of the reactionThe solution of enzyme (corresponding to 15% of the resultant solution of enzymes isolated from Karpov)of 5.89 ml---
Reaction37°C37°C
Termination reactions0.2 ml of the reaction solution was dissolved in 9.8 ml of 10% aqueous solution of N3RHO4

Example 20: in vitro Experiments with the digestion of L-EAA-L-Met (LL-I). accordingly, L-Met-L-EAA (LL-II) under the action of digestive enzymes isolated from carnivorous trout

a) the Secretion of digestive enzymes of rainbow trout (Oncorhynchus mykiss)

Digestive enzymes were allocated in accordance with the method described in EID and MATTY (Aquaculture, 79, 1989, SS.111-119). To do this, six annual rainbow trout (Oncorhynchus mykiss) were extracted intestine and processed it described in example 18 method.

b) Conducting experiments in vitro with digestion

In vitro experiments with digesting prospect who drove analogously to example 18 (see table 5, Fig.3 and 4).

Table 5
SampleThe value in the "idle" experience
Raw materialsThe substrate (LL-I, respectively LL-II)0.15 mmole0.15 mmole
Buffer solution Tris/HCl, pH 9,57.5 ml7.9 ml
The start of the reactionThe solution of enzyme (corresponding to 1.0% of the resultant solution of enzymes isolated from trout)424 μl---
Reaction37°C37°C
Termination reactions0.2 ml of the reaction solution was dissolved in 9.8 ml of 10% aqueous solution of N3RHO4

Example 21: in vitro Experiments with the digestion of L-EAA-D-Met (LD-I), respectively, D-Met-L-EAA (DL-II) under the action of digestive enzymes isolated from carnivorous trout

a) the Secretion of digestive enzymes from R. durnyh trout (Oncorhynchus mykiss)

Digestive enzymes were allocated in accordance with the method described in EID and MATTY (Aquaculture, 79, 1989, SS.111-119). To do this, six annual rainbow trout (Oncorhynchus mykiss) were extracted intestine and processed it described in example 18 method.

b) Conducting experiments in vitro with digestion

In vitro experiments with digestion was carried out similarly to example 19 (see table 6, Fig.11).

Table 6
SampleThe value in the "idle" experience
Raw materialsThe substrate (LD-I, respectively, DL-II)0,143 mmole (40,1 mg)0,143 mmole (40,1 mg)
Buffer solution Tris/HCl, pH 9,55,7 mlto 9.9 ml
The start of the reactionThe solution of enzyme (corresponds to 10% of the resultant solution of enzymes isolated from trout)4,2 ml---
Reaction37°C37°C
Termination reactions0.2 ml of the reaction solution was dissolved in 9.8 ml of 10% aqueous solution of N3RHO4

Example 22: in vitro Experiments with the digestion of L-EAA-L-Met (LL-I), respectively, L-Met-L-EAA (LL-II) under the action of digestive enzymes isolated from omnivorous marine shrimp

a) the Secretion of digestive enzymes from Pacific white shrimp (Litopenaeus Vannamei)

Digestive enzymes were allocated in accordance with the method described in Ezquerra and Garcia-Carreno (J. Food Biochem., 23, 1999, cc.59-74). To do this, from five pounds Pacific white shrimp (Litopenaeus Vannamei) were removed liver and together with crushed ice crushed in a blender. Further processing was performed analogously to example 18.

b) Conducting experiments in vitro with digestion

In vitro experiments with digestion was carried out similarly to example 18 (see table 7, Fig.5 and 6).

Table 7
SampleThe value in the "idle" experience
Raw materialsThe substrate (LL-I, respectively LL-II)0.15 mmole0.15 mmole
Buffer solution Tris/HCl, pH 9,57.5 ml7,8 ml
The start of the reactionThe solution of enzyme (corresponding to 2 shrimp)258 ál---
Reaction37°C37°C
Termination reactions0.2 ml of the reaction solution was dissolved in 9.8 ml of 10% aqueous solution of N3RHO4

Example 23: in vitro Experiments with the digestion of L-EAA-D-Met (LD-I), respectively, D-Met-L-EAA (DL-II) under the action of digestive enzymes isolated from omnivorous marine shrimp

a) the Secretion of digestive enzymes from Pacific white shrimp (Litopenaeus Vannamei)

Digestive enzymes were allocated in accordance with the method described in Ezquerra and Garcia-Carreno (J. Food Biochem., 23, 1999, cc.59-74). To do this, from five pounds Pacific white shrimp (Litopenaeus Vannamei) were removed liver and together with crushed ice crushed in a blender. Further processing was performed analogously to example 18.

b) Conducting experiments in vitro with digestion

In vitro experiments with digestion was carried out similarly to example 19 (see table 8, Fig.10).

Table 8
SampleThe value in the "idle" experience
Raw materialsThe substrate (LD-I, respectively, DL-II)0,143 mmole (40,1 mg)0,143 mmole (40,1 mg)
Buffer solution Tris/HCl, pH 9,55,7 ml7.9 ml
The start of the reactionThe solution of enzyme (corresponding to 8 shrimp)2.2 ml---
Reaction37°C37°C
Termination reactions0.2 ml of the reaction solution was dissolved in 9.8 ml of 10% aqueous solution of N3RHO4

Example 24: in vitro Experiments with the digestion of L-EAA-L-Met (LL-I). accordingly, L-Met-L-EAA (LL-II) under the action of digestive enzymes isolated from chickens

a) the Secretion of digestive enzymes from chickens

Digestive enzymes were allocated in accordance with the method described in EID and MATTY (Aquaculture, 79, 1989, cc.111-119. To do this, one chicken extracted the intestines, washed with water, cut along with bowel scraped mucosa. Together with crushed ice crushed in a blender. The resulting suspension was treated with an ultrasonic rod to destroy even whole cells. For separating cellular components and grease the suspension for 30 min, centrifuged at 4°C, the homogenate was separated by decanting and sterilized by his stripes thimerosal. From one chicken got 118,9 ml solution of enzymes of the intestinal mucosa and the solution was stored in the dark at 4°C.

b) Conducting experiments in vitro with digestion

L-Met-L-EAA (LL-II), respectively, L-EAA-L-Met (LL-I), was dissolved in a buffer solution of Tris/HCl and mixed with a solution of enzymes. For comparison and to assess the rate exclusively chemical cleavage in each case carried out on a "dummy" experience without mortar enzymes. Periodically, a sample was taken and quantitatively evaluated its members through its analysis on the calibrated GHUR-chromatograph. The degree of conversion was determined by dividing the methionine content in the content of L-Met-L-EAA (LL-II), respectively, L-EAA-L-Met (LL-I) (see table 9, Fig.16).

Sample
Table 9
The value in the "idle" experience
Raw materialsThe substrate (LL-I, respectively LL-II)0.15 mmole0.15 mmole
Buffer solution Tris/HCl, pH 9,511.3 ml12.5 ml
The start of the reactionThe solution of enzyme (corresponding to 1.0% of the resultant solution of enzymes isolated from chicken)1,19 ml---
Reaction37°C37°C
Termination reactions0.2 ml of the reaction solution was dissolved in 9.8 ml of 10% aqueous solution of N3RHO4

Example 25: in vitro Experiments with the digestion of L-EAA-D-Met (LD-I), respectively, D-Met-L-EAA (DL-II), under the action of digestive enzymes isolated from chickens

a) the Secretion of digestive enzymes from chickens

Digestive enzymes were allocated in accordance with the method described in EID and MATTY (Aquaculture, 79, 1989, cc.111-119). To do this, one chicken extracted the intestines and processed it described in example 24 method.

b) the Conduct is of the in vitro experiments with digestion

D-Met-L-EAA (DL-II), respectively, L-EAA-D-Met (LD-I), was dissolved in a buffer solution of Tris/HCl and mixed with a solution of enzymes. For comparison and to assess the rate exclusively chemical cleavage in each case carried out on a "dummy" experience without mortar enzymes. Periodically, a sample was taken and quantitatively evaluated its members through its analysis on the calibrated GHUR-chromatograph. The degree of conversion was determined by dividing the area under the curve obtained for methionine, the area under the curve obtained for D-Met-L-EAA (DL-II), respectively, L-EAA-D-Met (LD-I), (see table 10, Fig.17).

Table 10
SampleThe value in the "idle" experience
Raw materialsThe substrate (LD-I, respectively, DL-II)0.15 mmole0.15 mmole
Buffer solution Tris/HCl, pH 9,511.3 ml12.5 ml
The start of the reactionThe solution of enzyme (corresponds to 1% of the resultant solution of enzymes isolated from chicken) 1,19 ml---
Reaction37°C37°C
Termination reactions0.2 ml of the reaction solution was dissolved in 9.8 ml of 10% aqueous solution of N3RHO4.

1. Feed additive containing dipeptides or their salts, with one amino acid residue of the dipeptide is a DL-nationally the residue, and the other amino acid residue of the dipeptide is an amino acid in L-configuration selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

2. Feed additive under item 1, containing dipeptides of General formula DL-methionyl-L-EAA and/or L-EAA-DL-methionine, where L-EAA represents the amino acid in L-configuration selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

3. The feed mixture containing feed additive under item 1 or 2.

4. The feed mixture under item 3, containing DL-methionyl-L-EAA and/or L-EAA-DL-methionine individually in the form of D-methionyl-L-EAA, L-methionyl-L-EAA, L-EAA-D-methionine or L-EAA-L-methionine, in the form of a mixture between a, respectively, also in the form of a mixture with D-methionyl-D-EA is, L-methionyl-D-EAA, D-EAA-D-methionine or D-EAA-L-methionine.

5. The feed mixture under item 4, optionally contained in a mixture with methionine.

6. The feed mixture under item 5, contained in a mixture with D,L-methionine with its content in the range from 0.01 to 90 wt.%.

7. The feed mixture under item 6, contained in a mixture with D,L-methionine content of from 0.1 to 50 wt.%.

8. The feed mixture under item 7 contained in a mixture with D,L-methionine content of from 1 to 30 wt.%.

9. The feed mixture under item 4, optionally contained in a mixture with L-EAA.

10. The feed mixture under item 9, where L-EAA is L-lysine.

11. The feed mixture under item 9, where L-EAA is contained in the range from 0.01 to 90 wt.%.

12. The feed mixture on p. 11, L-EAA is contained in the range from 0.1 to 50 wt.%.

13. The feed mixture under item 12, where L-EAA is contained in the range from 1 to 30 wt.%.

14. Dipeptide or its salt of the General formula DL-methionyl-DL-EAA or DL-EAA-DL-methionine, where EAA is an amino acid selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

15. The method of producing the dipeptide containing only one nationally residue of formula DD/LL/DL/LD-I and/or DD/LL/DL/LD-II:

by the interaction of amino acids with urea derivative of one of General formulas III-V
,
where
R the following values:

in formulas Ia-VaR denotes 1-methylethyl-(valine)
in formulas Ib-VbR denotes a 2-methylpropyl-(leucine)
in the formulae Ic-VcR denotes the (1S)-1-methylpropyl-(isoleucine)
in formula Id-VdR denotes the (1R)-1-hydroxyethyl-(threonine)
in formulas Ie-VeR denotes 4-aminobutyl-(lysine)
in formulas If-VfR denotes 3-[(aminoiminomethyl)-(arginine)
amino]propyl-
in the formula Ig-VgR denotes benzyl-(phenylalanine)
in the formula Ih-VhR denotes (1H-imidazol-4-yl)methyl-(histidine)
in formulas Ij-Vj R denotes (1H-indol-3-yl)methyl-(tryptophan)
in formulas Ik-VkR denotes-CH2-SH(cysteine)
in the formula Im-VmR denotes-CH2-S-S-CH2-C(H)NH2-COOH(cystine)
in the formula IIIn-VnR denotes-CH2-CH2-S-CH3(methionine)

the remains of R1and R2in the urea derivatives of the formula III, IV, and V have the following meanings:
in formulas IIIa-IIIn R1denotes COOH, a R2means NHCONH2,
in formulas IVa-IVn R1represents CONH2, a R2means NHCONH2,
in formulas Va-Vn R1-R2mean-CONHCONH-,
when either R is nationally the rest, and added amino acid selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine, or added amino acid is a methionine, a, R represents an amino acid residue selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

16. The way p is p. 15, in the exercise of which as of the original product is used either as an intermediate product to form methioninamide or as amino acids selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

17. The method according to p. 15 or 16, in the exercise of which the solution containing methioninamide and water, is subjected to the interaction with the amino acid in the basic environment or solution containing as amino acids selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine, and water, is subjected to the interaction with methionine in basic terms.

18. The method according to p. 15 or 16, in the exercise of which the pH value of the solution containing a derivative of urea, set at a value ranging from 7 to 14 and/or the reaction is carried out at a temperature in the range from 30 to 200°C and/or the reaction is carried out under pressure in the range of 2 to 100 bar.

19. The method according to any of paragraphs.15-16, at which the solution containing methioninamide and water, or a solution containing as amino acids selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine, and water, after the way the van of one or more compounds III, IV and V.

20. The method according to p. 15, involving the following stages:
a) interactions derived urea of the formula III, IV or V with an amino acid with getting diketopiperazine formula VI
,
where R is specified in the letter of 15 values,
b) interaction of diketopiperazine formula VI with a mixture of dipeptides of formula DD/LL/DL/LD-I and DD/LL/DL/LD-II
,
where R is specified in the letter of 15 values.

21. The method according to p. 20, at which the interaction derivative of urea with amino acid with getting diketopiperazine carried out at a temperature in the range from 20 to 200°C and/or under pressure, preferably under a pressure in the range of 2 to 90 bar.

22. The method according to p. 20 or 21, at which the interaction derivative of urea with amino acid with getting diketopiperazine carried out in the presence of a base, preferably in the presence of a base selected from the group comprising nitrogen-containing base, NH4HCO3, (NH4)2CO3Knso3, K2CO3the mixture of NH4OH/CO2, urethane salt, and the Foundation of the alkali and alkaline earth metals.

23. The method according to p. 20 or 21, at which the reaction for obtaining diketopiperazine conduct, or exposing derived mo is eveny formula
,
where R denotes nationally balance, interaction with amino acid selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine, or subjecting the derivative of urea of the formula
,
where R represents an amino acid residue selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine, interaction with amino acid - methionine.

24. The method according to p. 20 or 21, at which diketopiperazine turn in a mixture of dipeptides of formula I and II by acid hydrolysis, preferably in the presence of acid selected from the group comprising mineral acids, HCl, H2CO3, CO2/N2O, H2SO4, phosphoric acid, carboxylic acid and hydroxycarbonate acid.

25. The method according to p. 20 or 21, at which diketopiperazine turn in a mixture of dipeptides of formula I and II by basic hydrolysis, preferably at pH values ranging from 7 to 14, preferably using a base selected from the group comprising nitrogen-containing base, NH4HCO3, (NH4)2CO3the mixture of NH4OH/CO2, urethane salt, knso32CO3, carbonates, and the Foundation of the alkali and alkaline earth metals.

26. The method according to p. 15 or 16, in the exercise of which is derived urea III-V are presented in the D-configuration, the L-configuration or a mixture of D - and L-configuration, preferably in a mixture of D - and L-configurations, when a derivative of urea is formed from methionine (IIIn-Vn), or a derivative of urea III-V are presented in the D-configuration, the L-configuration or a mixture of D - and L-configuration, preferably in the L-configuration, when a derivative of urea III-V are formed from amino acids, selected from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

27. A way of separating the mixture of diastereoisomers of the dipeptides of formula I and II by crystallization of the main reaction solutions obtained by carrying out the method according to p. 16, preferably by setting the pH value of the solution to a value ranging from 2 to 10, preferably from 3 to 9, particularly preferably corresponding to the isoelectric point of the particular dipeptide of formula I and II, the addition of acid, preferably selected from the group comprising mineral acids, HCl, H2CO3, CO2/N2O, H2SO4, phosphoric acid, carboxylic acid and hydroxycarbonate acid.

28. Way you the population of a mixture of diastereoisomers of the dipeptides of formula I and II by crystallization of the acid reaction solution, obtained by carrying out the method according to p. 24, by setting the pH value of the solution to a value ranging from 2 to 10, by adding a base selected from the group including NH4HCO3, (NH4)2CO3, nitrogen-containing base, NH4OH, urethane salt, knso3, K2CO3, carbonates, and the Foundation of the alkali and alkaline earth metals.

29. The method according to p. 28, where the specified pH of the solution has a value of from 3 to 9.

30. The method according to p. 29, where the specified value corresponds to the isoelectric point of the particular dipeptide of formula I, II respectively.

31. The use of compounds of formula I and/or II under item 15 as a feed additive for feeding mainly poultry, pigs, ruminants, freshwater or marine fish, crustaceans or Pets.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to compounds, which can be used as inhibitors of protease of hepatitis C virus, pharmaceutical compositions, containing the said compounds, and methods of their application.

EFFECT: obtaining compounds which can be used as inhibitors of protease of hepatitis C virus.

41 cl, 10 dwg, 7 tbl, 26 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to compounds, which can be used as inhibitors of protease of hepatitis C virus, to pharmaceutical compositions, which contain said compounds, and to methods of their application for treatment of diseases mediated by protease of hepatitis C virus.

EFFECT: obtaining compounds, which can be used as inhibitors of protease of hepatitis C virus.

37 cl, 22 dwg, 7 tbl, 34 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention described the macrocyclic compounds of formula wherein the radical values are presented in the patent claim. The above compounds are serine protease inhibitors wherein serine protease is hepatitis C virus (HCV) NS3 protease. This invention also discloses pharmaceutical compositions having antiviral activity against HCV, containing the claimed compounds, and one or more pharmaceutically acceptable carriers, as well as a method for preparing these compositions. The present invention describes a method of inhibiting the hepatitis C virus replication in a host, and a method of inhibiting activity of hepatitis C virus serine protease.

EFFECT: there are also presented a method of treating or preventing HCV infection, and a method of treating, preventing or ameliorating one or more symptoms of a liver disease or disorder associated with HCV infection in an individual.

69 cl, 39 ex, 8 tbl, 4 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of formula (I) a pharmaceutically acceptable salt thereof wherein each dash line (represented as ---) represents a double bond; X represents N or CH; R1a and R1b independently represent hydrogen or C1-6-jalkyl; L represents -O-; R2 represents hydrogen; R3 represents hydrogen or C1-6-alkyl; R4 represents quinolinyl substituted by one, two or three substitutes specified in C1-6-alkyl, C1-6-alkyloxy, thiazolyl or pyrazolyl, wherein said thiazolyl or pyrazolyl are substituted on any carbon atom by C1-6-alkyl; n is equal to 3, 4, 5 or 6; p is equal to 1 or 2. The invention refers to a pharmaceutical composition possessing the properties of KS3/4a-protease HCV inhibitors, containing a carrier, and an virally effective amount of the compound of formula (I) as an active ingredient. The method for preparing the compound of formula (I), wherein the above method involves forming an amide bond of an intermediate product (2a) and sulphonylamide (2b), as presented by the diagram, wherein G represents a group Also, the invention refers to alternative methods for preparing the compound of formula (I).

EFFECT: there are presented macrocyclic compounds possessing inhibitory activity on hepatitis C virus (HCV) replication.

13 cl, 1 tbl, 17 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: application describes prodrugs being 2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxyethoxy)-phenyl]pyridine-3,5-dicarbonitryl derivatives, and a method for preparing them.

EFFECT: invention can find application in treating and/or preventing cardiovascular diseases.

8 cl, 4 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing pure crystalline D-isoglutamyl-D-trytophan which involves a step of removing protection from essentially pure N-tert-butoxycarbonyl-D-isoglutamyl-D-tryptophan or diester thereof to yield essentially pure D-isoglutamyl-D- tryptophan. An amorphous ammonium alt of D-isoglutamyl-D- tryptophan (1:1) is also disclosed. Also disclosed is a method of producing a pure monoammonium salt of D-isoglutamyl-D-tryptophan from essentially pure N-tert-butoxycarbonyl-D- isoglutamyl-D-tryptophan. Disclosed is a compound H-D-Glu-(γ-D-Trp-OR2)-α-OR1 and pharmaceutically acceptable acid addition salts thereof. Disclosed is a solid pharmaceutical composition and use thereof as an immunodepressant or anti-psoriasis agent.

EFFECT: improved method.

51 cl, 14 ex, 8 dwg, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to organic chemistry, namely new 3,8-diaminotetrahydroquinoline derivatives of formula (1a) or to their pharmaceutically acceptable salts wherein X represents CH2, C=O or CH-OR; m is 1 or 2; Ar represents a phenyl group or a 5-merous or 6-merous aromatic heterocyclic group having one element specified in S and N, (wherein the phenyl group may be substituted by 1-2 halogen atoms); each R1 and R2 represents a hydrogen atom; R3 represents a C1-C6 alkyl group or indolyl-C1-4 alkyl group (the indolyl group is optionally substituted by a C1-C6 alkyl group or a halogen atom), n is 0; R4 and R5 which may be identical or different, each represents a hydrogen atom or a C1-C6 linear or branched alkyl group; each R6 and R7 represents a hydrogen atom; and R represents a hydrogen atom. Also, the present invention refers to a drug preparation and a pharmaceutical composition of the basis of the compound of formula (1a), to the compound of formula (F1), to a method for preparing an intermediate compound (e).

EFFECT: there are prepared new 3,8-diaminotetrahydroquinoline derivatives which possess high GHS-R antagonist activity.

10 cl, 1 tbl, 124 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to crystalline modifications: 1 (polymorphous form F), 2 (polymorphous form I) and 3 (polymorphous form X) of monosodium salt of D-isoglytamyl-D-tryptophan (1:1) characterised by powder X-ray pattern peaks presented in the application materials, as well as to pharmaceutical compositions containing them. The invention describes their use for treating various diseases and body conditions of at least one autoimmune diseases specified in a group consisting of psoriasis, atopic dermatitis and rheumatoid arthritis.

EFFECT: present invention describes the methods for producing the declared crystalline modifications of monosodium salt of D-isoglytamyl-D-tryptophan (1:1).

42 cl, 4 ex, 9 dwg

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing lipodipeptides based on L-glutamic acid or L-glutamine and L-ornithine, L-lysine or L-arginine. L-glutamic acid or L-glutamine derivatives esterified with fatty alcohol residues are obtained by fusing an amino acid with a corresponding alcohol in the presence of a strongly acidic ion-exchange resin in H+ form. Amino groups of L-ornithine, L-lysine or L-arginine are protected and then activated with carboxyl groups. Further, a reaction takes place between esterified derivatives of L-glutamic acid or L-glutamine and the protected derivatives of L-ornithine, L-lysine or L-arginine to form lipodipeptides. The protective groups are then removed.

EFFECT: invention simplifies the process at the esterification step, reduces reaction temperature and reaction time to 2 hours.

3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel polyfunctional fullerene C60 amino acid derivatives of formula (1) , wherein R is H, mono- or dinitroxyC1-6alkyl, maleinimide; N-Z denotes a α, β, γ, ω-amino acid fragment of general formula where m=2-5 and M is a nitroxyC1-6alkyl group, a C1-6alkyl group or an alkali metal salt, having biological activity, as well as methods for production thereof and a method for covalent bonding of fullerene derivatives with SH-containing proteins. The invention also relates to the use of nitroxyalkyl-N-(fullerenyl)amino acids as nitrogen monoxide donors and to use of nitroxyalkyl-N-(fullerenyl)amino acids as quick-acting vasodilatators for antihypertensive therapy. The invention also relates to a method of inhibiting a metastasis process and a method of enhancing antileukemic activity of cyclophosphamide. Disclosed nitroxyalkyl-N-fullerenyl amino acid derivatives have an effect on coronary, contractile and pumping ability of the isolated heart of Vistar rats and are quick-acting vasodilatators which reduce arterial pressure and heart rate and cause relaxation of coronary vessel with less depressive effect on myocardial function compared to nitroglycerine.

EFFECT: disclosed compounds considerably intensify antileukemic activity of cyclophosphamide, increase chemosensitising activity when combined with cyclophosphamide.

9 cl, 8 ex, 3 tbl, 3 dwg

FIELD: chemistry.

SUBSTANCE: method includes steps of: (1) feeding a raw compound of formula I into a macroporous adsorption resin; (2) washing the macroporous adsorption resin with water, an organic solvent or a mixed solution of an organic solvent and water as the washing liquid; and (3) elution of the compound of formula I from the macroporous adsorption resin with water, an organic solvent or a mixed solution of an organic solvent and water as an eluent, where at step (1) the solution containing a raw compound of formula I contains ionisable salts; and the macroporous adsorption resin is selected from a nonpolar aromatic adsorption resin polymerised from styrene and divinyl benzene, or a methacrylic adsorption resin of medium polarity with methacrylate residues in the structure.

EFFECT: purification method has the advantages of using a small amount of organic solvents without using silica gel and low environmental impact; the collected compound also has better purity compared to previous methods.

13 cl, 2 tbl, 2 dwg, 8 ex

FIELD: metallurgy.

SUBSTANCE: invention relates to casein succinylate of iron (III) wherein iron content varies from 4.5 wt % to 7 wt %, water solubility exceeds 92% while phosphorus-to-nitrogen ratio exceeds 5 wt %.

EFFECT: additionally, invention relates to production of iron (III) and to pharmaceutical composition containing casein succinylate of iron (III).

17 cl, 4 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: invention represents a method of obtaining collagen from biological material, which includes milling of a raw material, liquid processing of the biological material with obtaining a collagen-containing substance, separated into a sediment and liquid fraction, characterised by the fact that as the biological material applied is a medusa, preferably Rhopilema, preferably its cupola, which is crushed preferably to 1-2 mm, and for obtaining the collagen-containing substance the material prepared in such a way is mixed with drinking water with a ratio by the raw material weight to water as 1:2 and extracted at a temperature preferably of 15-18°C for 6-12 hours with periodical mixing, after that, the obtained extract is separated into the liquid fraction and collagen-containing sediment, which is after that dehydrated to moisture weight in it not more than 10%, after which it is pre-packed and packed.

EFFECT: extension of an arsenal of methods for obtaining neutral collagen.

FIELD: chemistry.

SUBSTANCE: invention relates to a method for chemical conversion of a peptide chain into a peptide thioether. A -C(=X)-R1 group is incorporated into a thiol group of a cysteine residue and the obtained peptide then reacts in an organic solvent with a compound having a substituted group of formula: NH-C(=Y)NHR3, and a -NH-C(=Y)NHR3 group binds in an addition reaction with the carboxyl group of the peptide bond at the N-terminal side of the cysteine residue, through which the peptide bond is broken and the peptide moiety at the C-terminal end is cut off. When the obtained peptide chain, having a -NH-C(=Y)NHR3 group, reacts with thiol in a buffer solution, a thiol exchange reaction occurs, specifically the thiol group of the thiol compound binds with the carbon of the carbonyl to which the -NH-C(=Y)NHR3 was bound, thereby removing the -NH-C(=Y)NHR3 group.

EFFECT: achieving conversion to peptide thioether.

14 cl, 4 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of production of casein calcium chloride of technical casein by precipitation, and can be used in microbiological studies for production of components of storing media of cultures of microorganisms, and also production of calcium co-precipitates for food industry.

EFFECT: improvement of the method.

2 cl, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of purifying a compound of formula 1 which includes the following steps: (1) adding a raw compound 1 to a macroporous adsorption resin, (2) washing the macroporous adsorption resin with an aqueous solution, an organic solvent or a mixed solution of an organic solvent and water, (3) elution using the aqueous solution, organic solvent or mixed solution of an organic solvent and water.

EFFECT: improved method.

9 cl, 7 dwg, 12 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of purifying daptomycin, which includes steps a) loading partially purified daptomycin into an anion-exchange chromatographic column and subsequent purification steps b) and c) in reversed-phase chromatographic columns, where the elution buffer at step a) is a monovalent salt solution and the elution buffer at step b) and c) is an aqueous alcohol.

EFFECT: improved method.

16 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science and concerns a method for producing the purified antigen of Dirofilaria immitis. The presented method involves mechanical homogenisation, centrifugation of a homogenate, collection of a supernatant to be used as an antigen; the homogenisation involves a 2-cm head end of a mature female of Dirofilaria immitis placed in an aqueous solution of saccharose 0.25 M in a ratio of 1:3, frozen at a temperature of -18°C, that is followed by mechanical homogenisation and protein extraction in the aqueous solution of saccharose 0.25 M at 4°C for 12 hours, wherein 3 cycles of five 30-second ultrasonic homogenisations of the supernatant is performed at 70 kHz every 30 seconds at 0°C; the supernatant prepared after ultrasonic homogenisation is dissolved in cooled acetone at a temperature of 0°C in a ratio of 1:20 with exposition for 1 hour at a temperature of 4°C.

EFFECT: presented invention enables producing the high-sensitivity and specificity antigen and can be used in diagnosis of dirofilariasis in humans and animals.

2 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology, more specifically to allergen modifications to reduce an allergenic capacity thereof, and may be used in medicine. A modified allergen is prepared by sequential modification of all primary amino groups or a portion thereof of lysine and arginine residues of an allergen molecule with using potassium cyanate and phenylglyoxal and used as an ingredient of a pharmaceutical composition for treating allergy.

EFFECT: invention provides preparing the modified allergen possessing the reduced allergenic capacity as compared to a respective native allergenic material and allergoids prepared by modification by either cyanate, or phenylglyoxal.

9 cl, 12 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and represents a method of obtaining a preparation of an antibody or its antigen-binding site with reduced content of host cell proteins (HCP) from a sample mixture. The claimed invention can be used to obtain the antibody preparation. The method includes bringing pH to values 3-4 to form an initial separated sample. After that, pH of the initial separated sample is brought to values 6-8. The initial separated sample is brought in contact with resin for protein A-based affinity chromatography. The resin for affinity chromatography is washed out with a buffer solution. Sampling is carried out after affinity chromatography. The sample after affinity chromatography is brought in contact with an ion-exchange resin, after ion-exchange sampling is carried out. After the ion exchange sample is brought in contact with the resin for hydrophobic interaction chromatography (HIC) with sampling after HIC.

EFFECT: invention makes it possible to obtain the preparation of the antibody or its antigen-binding site with reduced HCP content.

25 cl, 12 dwg, 8 tbl, 4 ex

Calf liquid fodder // 2527507

FIELD: food industry.

SUBSTANCE: invention relates to farm industry, in particular, to fodder production. The calf liquid fodder includes ripened raw materials with usage of a kumiss starter consisting of Bulgarian and acidophilous lactic bacilli and yeast. The raw materials are represented by a mixture of milled synanthropic flies larvae and water. The said components are used at the following ratio: commercial kumiss starter - 15.0 - 30.0 wt %, milled synanthropic flies larvae - 7.0 - 8.5 wt %, water - 63.0 - 76.5 wt %.

EFFECT: invention allows to enhance synanthropic flies larvae palatability to calves, exclude preserving agents and increase the fodder biological value.

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