• Domestic science and medicine in the 19th - early 20th centuries Chemistry of the 19th century in medicine
• What are peptides? Types and types of peptides. All about peptides and anti-aging cosmetics with peptides Additional peptides are not formed
• Toxic effect on humans of hazardous chemicals
• Radioautography Method of autoradiography in cytology
• Identification of bacteria by antigenic structure
• Organic matter in wastewater What are organic compounds in water
• Hydrogen index (pH factor)
• What is the pH of water and why is it important to know it?
• Redox potential Redox systems
• Reversible redox system Reversible redox systems

Every field of science has its own "blue bird"; cyberneticians dream of "thinking" machines, physicists - of controlled thermonuclear reactions, chemists - of the synthesis of "living matter" - protein. Protein synthesis has long been the subject of science fiction novels, a symbol of the coming power of chemistry. This is explained by the huge role that protein plays in the living world, and by the difficulties that inevitably confronted every daredevil who dared to “fold” an intricate protein mosaic from individual amino acids. And not even the protein itself, but only.

The difference between proteins and peptides is not only terminological, although the molecular chains of both are composed of amino acid residues. At some stage, quantity turns into quality: the peptide chain - the primary structure - acquires the ability to coil into spirals and balls, forming secondary and tertiary structures, already characteristic of living matter. And then the peptide becomes a protein. There is no clear boundary here - a demarcation mark cannot be put on the polymer chain: hitherto - peptide, from here - protein. But it is known, for example, that adranocorticotropic hormone, consisting of 39 amino acid residues, is a polypeptide, and the hormone insulin, consisting of 51 residues in the form of two chains, is already a protein. The simplest, but still protein.

The method of combining amino acids into peptides was discovered at the beginning of the last century by the German chemist Emil Fischer. But for a long time after that, chemists could not seriously think not only about the synthesis of proteins or 39-membered peptides, but even much shorter chains.

Process of protein synthesis

In order to connect two amino acids together, many difficulties must be overcome. Each amino acid, like the two-faced Janus, has two chemical faces: a carboxylic acid group at one end and an amine basic group at the other. If the OH group is taken away from the carboxyl of one amino acid, and an atom is taken away from the amine group of the other, then the two amino acid residues formed in this case can be connected to each other by a peptide bond, and as a result, the simplest of peptides, the dipeptide, will arise. And a water molecule will split off. By repeating this operation, one can increase the length of the peptide.

However, this seemingly simple operation is practically difficult to implement: amino acids are very reluctant to combine with each other. We have to activate them, chemically, and “heat up” one of the ends of the chain (most often carboxylic), and carry out the reaction, strictly observing the necessary conditions. But that's not all: the second difficulty is that not only residues of different amino acids, but also two molecules of the same acid can combine with each other. In this case, the structure of the synthesized peptide will already differ from the desired one. Moreover, each amino acid can have not two, but several "Achilles' heels" - side chemically active groups capable of attaching amino acid residues.

In order to prevent the reaction from deviating from the given path, it is necessary to camouflage these false targets - to “seal” all the reactive groups of the amino acid, except for one, for the duration of the reaction, by attaching the so-called protective groups to them. If this is not done, then the target will grow not only from both ends, but also sideways, and the amino acids will no longer be able to be connected in a given sequence. But this is precisely the meaning of any directed synthesis.

But, getting rid of one trouble in this way, chemists are faced with another: after the end of the synthesis, the protective groups must be removed. In Fischer's time, groups that were split off by hydrolysis were used as "protection". However, the hydrolysis reaction usually turned out to be too strong a “shock” for the resulting peptide: its difficult-to-build “construction” fell apart as soon as the “scaffolding” - protective groups - was removed from it. Only in 1932, Fischer's student M. Bergmann found a way out of this situation: he proposed protecting the amino group of an amino acid with a carbobenzoxy group, which could be removed without damaging the peptide chain.

Protein synthesis from amino acids

Over the years, a number of so-called soft methods have been proposed for "crosslinking" amino acids to each other. However, all of them were in fact only variations on the theme of Fisher's method. Variations in which sometimes it was even difficult to catch the original melody. But the principle itself remained the same. Yet the difficulties associated with protecting vulnerable groups remained the same. Overcoming these difficulties had to be paid for by increasing the number of reaction stages: one elementary act - the combination of two amino acids - was divided into four stages. And each extra stage is an inevitable loss.

Even if we assume that each stage comes with a useful yield of 80% (and this is a good yield), then after four stages these 80% "melt" to 40%. And this is with the synthesis of only a dipeptide! What if there are 8 amino acids? And if 51, as in insulin? Add to this the difficulties associated with the existence of two optical "mirror" forms of amino acid molecules, of which only one is needed in the reaction, add the problems of separating the resulting peptides from by-products, especially in cases where they are equally soluble. What happens in total: Road to nowhere?

And yet these difficulties did not stop chemists. The pursuit of the "blue bird" continued. In 1954, the first biologically active polypeptide hormones, vasopressin and oxytocin, were synthesized. They had eight amino acids. In 1963, a 39-mer ACTH polypeptide, adrenocorticotropic hormone, was synthesized. Finally, chemists in the United States, Germany and China synthesized the first protein - the hormone insulin.

How is it, the reader will say, that the difficult road, it turns out, did not lead to anywhere or anywhere, but to the realization of the dream of many generations of chemists! This is a milestone event! Indeed, this is a landmark event. But let's evaluate it soberly, renouncing sensationalism, exclamation marks and excessive emotions.

Nobody argues: the synthesis of insulin is a huge victory for chemists. This is a colossal, titanic work, worthy of all admiration. But at the same time, the ego is, in essence, the ceiling of the old polypeptide chemistry. This is a victory on the verge of defeat.

Protein synthesis and insulin

There are 51 amino acids in insulin. To connect them in the right sequence, chemists needed to carry out 223 reactions. When, three years after the beginning of the first of them, the last was completed, the yield of the product was less than one hundredth of a percent. Three years, 223 stages, a hundredth of a percent - you must admit that the victory is purely symbolic. It is very difficult to talk about the practical application of this method: the costs associated with its implementation are too high. But in the final analysis, we are not talking about the synthesis of precious relics of the glory of organic chemistry, but about the release of a vital drug that is needed by thousands of people around the world. So the classical method of polypeptide synthesis has exhausted itself on the very first, simplest protein. So, the "blue bird" again slipped out of the hands of chemists?

A new method for protein synthesis

Approximately a year and a half before the world learned about the synthesis of insulin, another message flashed in the press, which at first did not attract much attention: the American scientist R. Maryfield proposed a new method for the synthesis of peptides. Since the author himself at first did not give the method a proper assessment, and there were many flaws in it, it looked in the first approximation even worse than the existing ones. However, already at the beginning of 1964, when Maryfield succeeded in using his method to complete the synthesis of a 9-membered hormone with a useful yield of 70%, scientists were amazed: 70% after all stages is 9% useful yield at each stage of synthesis.

The main idea of ​​the new method is that the growing chains of peptides, which were previously left to the mercy of chaotic movement in the solution, were now tied at one end to a solid carrier - they were, as it were, forced to anchor in the solution. Maryfield took a solid resin and “attached” the first amino acid assembled into a peptide to its active groups by the carbonyl end. The reactions took place inside individual resin particles. In the "labyrinths" of its molecules, the first short shoots of the future peptide first appeared. Then the second amino acid was introduced into the vessel, its carbonyl ends were linked with the free amino ends of the “attached” amino acid, and another “floor” of the future “building” of the peptide grew in the particles. So, stage by stage, the entire peptide polymer was gradually built up.

The new method had undeniable advantages: first of all, it solved the problem of separating unnecessary products after the addition of each amino acid - these products were easily washed off, and the peptide remained attached to the resin granules. At the same time, the problem of solubility of growing peptides, one of the main scourges of the old method, was excluded; earlier, they often precipitated, practically ceasing to participate in the growth process. The peptides “removed” after the end of the synthesis from the solid support were obtained almost all of the same size and structure, in any case, the spread in the structure was less than with the classical method. And accordingly more useful output. Thanks to this method, peptide synthesis - a painstaking, time-consuming synthesis - is easily automated.

Maryfield built a simple machine that itself, according to a given program, did all the necessary operations - supplying reagents, mixing, draining, washing, measuring a dose, adding a new portion, and so on. If according to the old method, it took 2-3 days to add one amino acid, then Maryfield connected 5 amino acids in a day on his machine. The difference is 15 times.

What are the difficulties in protein synthesis

Maryfield's method, called solid-phase, or heterogeneous, was immediately adopted by chemists around the world. However, after a short time it became clear that the new method, along with major advantages, also has a number of serious drawbacks.

As the peptide chains grow, it may happen that in some of them, say, the third “floor” is missing - the third amino acid in a row: its molecule will not reach the junction, getting stuck somewhere along the road in the structural “wilds” solid polymer. And then, even if all the other amino acids, starting with the fourth, line up in the proper order, this will no longer save the situation. The resulting polypeptide in its composition and, consequently, in its properties will have nothing to do with the substance obtained. The same thing happens as when dialing a phone number; it is worth skipping one digit - and the fact that we have typed all the rest correctly will no longer help us. It is practically impossible to separate such false chains from the “real” ones, and the drug turns out to be clogged with impurities. In addition, it turns out that the synthesis cannot be carried out on any resin - it must be carefully selected, since the properties of the growing peptide depend to some extent on the properties of the resin. Therefore, all stages of protein synthesis must be approached as carefully as possible.

DNA protein synthesis, video

And in the end, we bring to your attention an educational video on how protein synthesis occurs in DNA molecules.

First synthesis
peptide hormone oxytocin

In 1953, the American scientist Vincent Du Vigno, together with his colleagues, found out the structure of oxytocin, a cyclic polypeptide. Among the known natural compounds, such cyclic structures have not been encountered before. The following year, the scientist for the first time carried out the synthesis of this substance. This was the first time that a polypeptide hormone was synthesized under in vitro conditions.

Du Vignot is known in the scientific world for his research at the intersection of chemistry and medicine. In the mid 1920s. the subject of his scientific interest was the study of the function of sulfur in insulin - hormone 1 of the pancreas, which regulates the process of carbohydrate metabolism and maintains a normal level of sugar (glucose) in the blood. The young man's interest in the chemistry of insulin arose, according to his recollections, after one of the lectures given by Professor William C. Rose immediately after the discovery of this substance by Frederick G. Banting 2 and John J. R. Macleod. So when, after graduating from university, John R. Murlin of the University of Rochester invited him to study the chemical nature of insulin, the young scientist considered it a destined proposal. “The chance to work on the chemistry of insulin crossed out all my other scientific expectations,” Du Vignot later noted, “so I immediately accepted Professor Murlin’s offer.”

The article was published with the support of the company "vivozmysora.ru". The company offers garbage disposal services in Moscow and the Moscow region, ordering a container. Affordable prices, arrival of the car at the specified time, transportation of waste in containers of 8-27 cubic meters, export is carried out to specialized landfills. Professional drivers with extensive experience, quality service. Detailed information can be found on the company's website page.

During his work at the University of Rochester, Du Vignot managed to make the first assumptions about the chemical composition of insulin, which were largely reflected in his dissertation "Sulfur of Insulin", defended in 1927. According to Du Vignot's views, insulin was one of the derivatives of the amino acid cystine. He identified insulin as a sulfur-containing compound in which the sulfur fragments are disulfide bridges. He also expressed considerations about the peptide 3 nature of insulin.
It should be noted that Du Vignot's data that insulin is a sulfur-containing compound were in good agreement with the main conclusions of the work carried out at that time in this direction by Professor John Jacob Abel and colleagues at Johns Hopkins University. Therefore, the scholarship of the National Research Council, which the young scientist received immediately after defending his dissertation, turned out to be very useful. Thanks to her, Du Vigno worked for some time under the guidance of Professor Abel at the Johns Hopkins University School of Medicine.
Professor Abel, a recognized authority on the study of hormone chemistry, held the view at that time that insulin was a protein compound. Such views ran counter to the ideas that dominated those years. As Du Vignot himself recalled, "it was a time when both chemists and biologists could not accept the fact that an enzyme could be a protein compound." Shortly before this, Professor Abel was able to isolate insulin in crystalline form for the first time (1926). Du Vigno's plans, when he got an internship with Abel, included the following: to isolate the amino acid cystine from insulin crystals and try to study its structure. He accomplished this very quickly. As a result of research, together with the professor's staff and with his direct assistance, the young scientist clearly demonstrated the formation of a number of amino acids during the breakdown of the insulin molecule. One of them was just the sulfur-containing amino acid cystine. At the same time, experiments have shown that the sulfur content in insulin is directly correlated with the sulfur content in cystine. But the results achieved required the study of other sulfur-containing amino acids.
The continuation of financial support from the National Research Council for another year allowed Du Vignot to visit the famous scientific biochemical schools of Western Europe (Dresden, Edinburgh, London), where he was able to gain additional experience in the study of peptides and amino acids.
Upon returning to the United States, the scientist first worked at the University of Illinois, and three years later moved to the medical school of the George Washington University. Here he continued his research on insulin. Particularly interesting were his studies on the effect of disulfide bonds in cystine on the hypoglycemic effect of insulin (lowering blood sugar). Work in the field of insulin also stimulated a new line of research - the study of pituitary hormones 4 .
An important direction of his work at the George Washington University was the study of the mechanism of conversion of methionine to cystine in living organisms. In subsequent years, it was these studies that led him to the problem of studying biological transmethylation (the transfer of methyl groups from one molecule to another).
In 1938, the scientist was invited to the Medical College of Cornell University. Here he continued to study insulin and launched research on the hormones of the posterior pituitary gland.
During the Second World War, these studies had to be interrupted for a while. The scientist and his collaborators worked on the synthesis of penicillin. At the end of the war, Du Vignot was able to return to his previous studies. He was particularly intensive in his work on the isolation of a number of hormones from commercially available extracts of the pituitary gland and tissues of the pituitary gland of a bull and a pig.
The posterior lobe of the pituitary gland produces a number of hormones, two of which had by then been isolated in pure form. One of them is oxytocin, which stimulates the smooth muscles of the uterus, the other is vasopressin, a hormone that contracts peripheral arterioles and capillaries, thereby causing an increase in blood pressure. These hormones have proven to be very difficult to distinguish because they have similar physical properties. Because of this, until the mid-1920s. physicians and biochemists considered them to be one substance with a wide spectrum of biological activity. Thanks to the improvement of methods of chemical analysis, in
in particular fractional precipitation, chromatography and electrophoresis, by the 1940s. these hormones were partially separated.
In 1949, Du Vignot, using the "countercurrent distribution" method for a commercial extract with an oxytocin activity of 20 U/mg, obtained a drug with an activity of 850 U/mg. This prompted the scientist to attempt to study the structure of matter. To this end, he carried out the fragmentation of the polypeptide chain. As a result of the complete hydrolysis of the oxytocin preparation and the analysis of its amino acid composition by Du Vignot, the presence of eight different amino acids in an equimolecular ratio was established. The amount of released ammonia corresponded to three amide groups of the type
–CONH 2 , molecular weight – to monomeric octapeptide. One of the eight amino acid residues has been identified as cystine. Experiments on the oxidation of cystine in oxytocin showed that the disulfide bridge in cystine, previously discovered by Du Vignot, is part of the oxytocin ring system.
The sequence of eight amino acids in oxytocin was finally established by Du Vigneau and his coworkers only in 1953. It should be noted that in parallel with Du Vigneau's group, Professor Hans Tuppi (University of Vienna) worked on the same problems in Vienna, who also in 1953 independently of Du Vigneau established the sequence of amino acids in oxytocin using the Sanger method 5 in his work.
Du Vigno went a slightly different way. He and his collaborators relied not primarily on the analysis of terminal amino acids, but on the identification of the components of a large number of lower peptides. They also studied the reaction of oxidized oxytocin with bromine water, which resulted in the formation of a heptapeptide and a brominated peptide. The study of the structure of the latter showed that the sequence of amino acids in the corresponding dipeptide: cystine - tyrazine (see the table for designations).
Further, by the dinitrophenyl method, it was found that the N-terminal amino acid in the heptapeptide is isoleucine. Du Vignot concludes that the N-terminal sequence in oxidized oxytocin is:

HO 3 S - cis - tyr - izl.

Amino acids from the hormone oxytocin

Of the thirteen peptides listed below, the first four were obtained by partial hydrolysis of the heptapeptide, the second group, by hydrolysis of oxytocin (in this case, cysteine ​​residues were converted into alanine residues). Then the neutral fraction was separated and treated with bromine water to oxidize the cysteine ​​unit to the cysteic acid unit; the resulting acidic peptide was separated from the neutral peptide on ion exchange resins. The third group of peptides was obtained by hydrolysis of oxytocin desulfurized on Raney nickel. In the formulas below, if the sequence of amino acids in the peptides is known, the amino acid symbols are separated by a dash; if the sequence is unknown, then the characters are separated by a comma.

From heptapeptide:

1. (asp - cis - SO 3 H).
2. (cis - SO 3 H, pro).
3. (cis - SO 3 H, pro, leu).
4. (cis - SO 3 H, pro, leu, gly).

From oxytocin:

5. (lei, gli, pro).
6. (tire, cis - S - S - cis, asp, glu, ley, izl).
7. (tyr, cis - S - S - cis, asp, glu).
8. (cis - S - S - cis, asp, glu).
9. (cis - SO 3 H, asp, glu).

From desulfurized oxytocin:

10. (ala, asp).
11. (ala, asp, glu).
12. (glue, izl).
13. (ala, asp, glu, lei, izl).

Taking into account the structure of the resulting peptides and using the overlay of individual components of the peptides, Du Vignot and co-workers deduced the following amino acid sequence in oxytocin:

cystine - tyrazine - isoleucine - glutamine - NH 2 - asparagine - NH 2 - cystine - proline - leucine - glycine - NH 2.

The structure of oxytocin established by them is shown in fig. one.

It should be noted that, simultaneously with Du Vignot's oxytocin, the structure of another hormone of the posterior pituitary gland, vasopressin, was determined.
The structure of the hormone oxytocin was confirmed by its chemical synthesis in 1954, which was the first complete synthesis of natural peptides. The synthesis included the condensation of N-carbobenoxy-S-benzyl dipeptide (I) with heptapeptide triamide (II) using tetraethylpyrophosphite. After removing the carbobenzoxy and benzyl groups that protected the amino and sulfhydryl groups in both peptides, respectively, the resulting nonapeptide was oxidized with air, resulting in oxytocin (Fig. 2).
Thus, the first structural analysis and the first synthesis of a polypeptide hormone were carried out - an outstanding achievement in biochemistry and medicine. The era of chemical synthesis of biologically active natural peptides began in science with the works of Du Vigneau.

Fig.2. General scheme for the synthesis of oxytocin according to Du Vignot

As is known, in 1955 Du Vigneau was awarded the Nobel Prize in Chemistry "for his work with biologically active compounds, and above all for the first synthesis of a polypeptide hormone."

1 According to the classical point of view, hormones are biologically active substances - regulators of endogenous origin, i.e. synthesized in the body, and not introduced from outside. The chemical nature of hormones is different. These are proteins, peptides, amino acid derivatives, steroids, lipids.
2 In 1922, F. Banting and his co-workers isolated pure insulin for the first time.
3 Peptides are organic natural or synthetic substances whose molecules are built from a-amino acid residues interconnected by C (O)–NH peptide bonds. According to the number of these residues, dipeptides, tripeptides, etc. are distinguished. Long chain peptides are called polypeptides.
4 The pituitary gland is the central endocrine gland. Endocrine glands secrete their metabolic products into the blood.
5 In the polypeptide chain of a protein, on the one hand, there is an amino acid residue bearing a free a-amino group (amino or N-terminal residue), and on the other, a residue with a free a-carboxyl group (carboxyl or C-terminal residue). The analysis of terminal residues plays an important role in the process of determining the amino acid sequence of a protein. For example, at the first stage of the study, it makes it possible to estimate the number of polypeptide chains that make up a protein molecule and the degree of homogeneity of the drug being studied. The first method for identifying terminal amino groups in peptides (dinitrofluorobenzyl method) was developed by Frederick Senger in 1945.

LITERATURE

Plane R. Interview with Vincent du Vigneaud. Journal of Chemical Education, 1976, v. 53, no. 1, p. 8–12;
Du Vigneaud V. A Trail of Research in Sulfur Chemistry and Metabolism and Related Fields. Ithaca, New York: Cornell University Press, 1952;
Bing F. Vincent du Vigneaud. Journal of Nutrition, 1982, v. 112, p. 1465–1473;
Du Vigneaud V., Melville D.B., Gyo..rgy P., Rose K.S. Identity of Vitamin H with Biotin. Science, 1940, v. 92, p. 62–63; Nobel Prize Winners. Encyclopedia. Per. from English. T. 2. M.: Progress, 1992.

DU VIGNO Vincent(18.V.1901 - 11.XII.1978) was born in Chicago (Illinois). His father, Alfred J. Du Vigno, was an inventor, design engineer. The boy showed interest in the natural sciences quite early. Already in his school years, he set up experiments in chemistry and physics in the home laboratory of one of his comrades.
In 1918, with the financial support of his sister Beatrice, Vincent began his studies at the University of Illinois with a degree in engineering chemistry. But soon organic chemistry became his subject of interest, and then biochemistry (under the influence of H. B. Lewis). In 1923, the young man received a bachelor's degree (supervisor - Professor K.S. Marvel), and the following year - a master's degree in chemistry, having completed work on the synthesis of one of the medicinal compounds that has a local anesthetic and vasopressor (causing an increase in blood pressure ) action.
It should be noted that the years of study at the university for Vincent were not easy financially. In parallel with his studies, he had to work hard: first as a waiter, then as an instructor for lieutenants in the US military cavalry reserve. While teaching lieutenants, he met an English major, a young girl named Zella Zon Ford, who, upon graduation from the university, became the wife of Du Vigno. Under the influence of her future spouse, Zella attended courses in mathematics and chemistry. Therefore, in the first years of her marriage, she worked as a teacher of natural sciences. Subsequently, the couple had a daughter, Marilyn, and a son, Vincent, who became a doctor.
Immediately after graduation, Du Vignot made several attempts to get a job in some pharmaceutical company, because his scientific interest for life became, as he later called, "the study of the relationship between the chemical structure of organic compounds and their biological activity." But in the beginning, nothing came of it, and the young scientist worked for half a year in the analytical laboratory of the Du Pont company. Then, with the support of his former supervisor, Dr. Marvel, he managed to get a job at a Philadelphia military hospital. At the hospital, Du Vignot was finally able to conduct scientific research in the field of clinical chemistry and at the same time begin teaching at the medical school at the University of Pennsylvania. At the same time, there was the possibility of entering the graduate school of this university. But in the spring of 1925, the young scientist unexpectedly received a tempting offer from Professor J.R. Murlin - to study the chemistry of insulin at the newly opened medical school at the University of Rochester. An important role in this was played by the recommendations of his former university mentors Professors Lewis and Marvel.
In 1927, the scientist received a doctorate in chemistry from the University of Rochester.
In 1928, he went to Germany, to Dresden, to the laboratory of Professor Max Bergmann (a student of Emil Fischer), who at that time was already a recognized authority in the field of amino acid and peptide chemistry. With him, Du Vigno trained in the field of peptide synthesis. M. Bergman liked the results of Du Vigno's research, and he invited the young trainee to become his assistant. But Du Vigno, having rejected the tempting offer, went on an internship to Scotland, to the University of Edinburgh, to professor of medical chemistry George Barger, and then to the clinic of the University of London to professor C. R. Harrington.
After some time, I had to think about returning to my homeland and taking a permanent job at a university. After sending out letters offering his candidacy to the staff of a number of universities, Du Vigno soon received several offers at once. He recalled this turning point in his life this way: “I got one offer
a) from Professor Murlin of Rochester, b) from Professor Abel of the School of Pharmacy at Johns Hopkins University,
c) a place at the University of Pennsylvania and finally d) a place in New York in clinical chemistry. In addition to this, there was also an offer from Illinois from Professor Rose and Roger Adams, who offered a place in the Department of Physiological Chemistry. At this time, I already knew for sure that I wanted to be a biochemist, while I want to combine research work with teaching in the field of biochemistry. Therefore, I accepted the offer from Illinois, although in terms of money it did not meet my needs.
In Illinois, the scientist worked for three years, and very successfully. But then came an offer from the George Washington University School of Medicine (Washington State), where Du Vignot immediately received a professorship and headed the biochemistry department. Many of the researchers from his working group also followed him to the new university. Here the scientist continued his studies of insulin and partially cystine. An important direction of his activity at the George Washington University was also his research in the field of biotin chemistry.
In the 1920s - early 1930s. many researchers noted that rats fed only egg white and did not receive other proteins had some neurological problems, in addition, their skin condition worsened significantly. A balanced diet solved these problems. The vitamin that the rats lacked so much in the first diet was called vitamin H. The well-known biochemist Paul Gyo..rgy turned to Du Vigno with a request to identify this substance. In 1936, a similar substance was unexpectedly isolated by other researchers and identified as a derivative of biotin (a sulfur-containing substance necessary for yeast cell division). Du Vigno's successive experiments in this direction showed that biotin secreted from liver and milk tissue is a coenzyme. It takes part in cellular respiration, and is identical in structure and properties to the substance known as vitamin H. Biotin was immediately added to the list of vital B vitamins. As it turned out, there is a protein in eggs, avidin, which binds tightly to biotin and thus prevents its absorption by living organisms.
At George Washington University, Du Vignot's work was also focused on creating a new biochemistry curriculum for medical students.
Since 1938, the scientific activity of the scientist moved to the walls of Cornell University in New York, where he was invited to the post of professor of biochemistry and dean of the Faculty of Biochemistry of the Medical College. This medical center became a real scientific home for him for the remainder of his academic career. Here he took with him five employees from the George Washington University to continue his research. In his memoirs, the scientist noted that each time he moved from one university to another, he took with him employees from the old place of work, in his figurative expression, "it's like transplanting a tree - it must be with a piece of land from the old place."
It was at Cornell University that the scientist carried out his most recognized work by the scientific community on the determination of the structure and synthesis of oxytocin. The hormone synthesized by him was successfully tested in clinical conditions on women to stimulate labor. He carried out further research in the field of biologically active hormones to establish the possibility of substituting one amino acid for another in a number of the structures he studied. In parallel, he continued to study biotin, amino acid metabolism, etc.
The work of a scientist at Cornell University was marked by the highest awards: the Nichols Medal of the American Chemical Society (1945), the Borden Prize in Medical Sciences, the Osborne and Mendel Prizes of the American Institute of Nutrition (1953), the Charles Frederick Chandler Medal of Columbia University (1956), the Willard Gibbs Medal (1956) and the Nobel Prize.
From 1967 to 1975, the scientist was a professor of chemistry at Cornell University in Ithaca. Du Vigno has also served on the boards of the Rockefeller Institute for Medical Research, the National Institute of Arthritis and Metabolic Diseases, and the New York Health Research Institute, President of the Harvey Society, the American Society for Biological Chemistry, and Chairman of the Board of the Federation of American Societies for Experimental Biology.

Polypeptide chains are known to be the basis of proteins. The polypeptide chain can be represented by a generalized structure (83):

The end link with the NH 2 group is called the N-terminus, the other end link with the COOH group is called the C-terminus. Polypeptides - a special case polyamides, the CO-NH bonds connecting the elementary units of the polypeptide chain are called peptide connections.

Monomers for the synthesis of polypeptide chains - α-amino acids; all of them, except one, can be represented by formulas (84)-(84'); one - proline - by formulas (85) - (85 '):

In environments close to neutral, amino acids exist almost entirely in the form of bipolar ions (84') and (85'). Radicals R I can be aliphatic, aromatic, heterocyclic, many of them contain a variety of functional groups: OH, NH 2 , COOH, SH, etc. To designate α-amino acids in the literature, three letters (Latin) names are used (most often the first three, but not always), for example gly (glycine), Val (valine), trp(tryptophan).

Non-template syntheses of polypeptide chains from α-amino acids are based on several targeted modifications of functional groups; these modifications ensure the flow at each stage the only reactions - interactions of the carboxyl function of the previous link with the amino group of the next one (if you count from the N-terminus). The need for such a modification can be illustrated by the simplest example of the synthesis of a dimer, a dipeptide, for which formal synthesis from monomers:

For preparative synthesis of dipeptide (88) it is necessary to: A. Protect the NH 2 group of amino acid (86) to avoid interaction variants (86)-(86) and (87)-(86); B. Activate the carboxyl function of the amino acid (86), since the carboxyl group itself is inactive in reactions with nucleophiles; B. Protect the COOH group of the amino acid (87); it is necessary for this amino acid was not in the form of a bipolar ion type (84'); in this form, the amino group is not nucleophilic and therefore inactive.

Polycondensation leading to the synthesis of a peptide chain with a given primary structure can be represented by the following scheme:

where Z is a protective group for the amino group; X is the activating group for the first carboxyl function; Y is the protecting group for the second carboxyl function.

After the formation of the dipeptide protected at both ends (89), the protective group is removed either from its N-terminus ( 1 ), or from its C-terminus ( 2 ) (combining deprotection with activation). Further, the liberated NH 2 group in the dipeptide (90) or the activated carboxyl function in the dipeptide (91) is used to carry out the next stage - the reaction with the next modified monomer to form the tripeptide; this pattern is repeated. In the variant ( 1 ) the peptide chain is extended from the C-terminus, in the variant ( 2 ) - from the N-terminus. Not necessarily modified monomers can be introduced into the reaction, but peptides can also be "cross-linked" with each other.

The scheme shown here is simplified - in fact, it is also necessary to protect some functional groups located in the side groups R i , for example, the NH 2 group in the side radical of lysine.

A. Protecting groups. Basic requirements for protecting groups: a. They must completely prevent participation of the protected group in the ongoing reactions (block the protected group); b. After the reaction, they fairly easy to remove with the regeneration of the protected group and without affecting other fragments of the reaction product (in particular, in the synthesis of peptides - without breaking the peptide bonds).

1. NH 2 -Protective groups(Z groups). Now a large number of options for effective protection of the NH 2 group are known; several types of protecting groups are used. Here we restrict ourselves to the most widely used type - urethane protecting groups. To set them up, a compound containing an NH 2 group is reacted with a carbonic acid monoester derivative, for example, acid chloride (chlorocarbonate ester, chlorocarbonate):

In addition to acid chlorides, azides or anhydrides can be used. The grouping RO-CO-NH- is called urethane, hence the name of the defense. Installation of urethane protection - analogue acylation amino groups; conventional acylation with carboxylic acid derivatives is not applicable, because acyl protecting groups are poorly removed; on the contrary, the urethane protection is easily removed under mild conditions, and under different conditions, depending on the nature of the R radical. Here are three examples:

a. R=C 6 H 5 CH 2 ; the protecting group is called benzyloxycarbonyl(carbobenzyloxy-protection, Z-protection); this is historically the first example of an NH 2 group urethane protection (M. Bergman, L. Zervas, 1932). After the necessary reaction, the benzyloxycarbonyl protection is easily removed by mild catalytic hydrogenation (more precisely, by hydrogenolysis):

The products of hydrogenolysis of the protective group—toluene and CO2—are easily removed from the reaction medium.

b. R = (CH 3 ) 3 C; protective group - tert- butyloxycarbonyl, Boc-protection ( B utyl- o xy c arbonyl); this protection is easily removed by mild acid treatment, for example, by the action of trifluoroacetic acid:

Here, both deprotection products are gaseous, making their removal even easier.

B. R \u003d CH 3 SO 2 CH 2 CH 2 - methylsulfonylethyloxycarbonyl protection (Msc-protection); this protection is removed by NaOH under mild conditions (pH 10-12.0°C).

The difference in the conditions for removing the above protections makes it possible to protect the α-NH 2 group of the amino acid and the NH 2 group in the side radical of lysine in different ways. Then one protection (α-NH 2 groups) can be removed, and the other (“lysine”) can be left (protection of the side groups is usually removed after the completion of the formation of the polypeptide chain).

There are several more options for urethane protection, as well as several other types of protection of the NH 2 group - formyl, phthalyl, trifluoroacetyl; information about these methods can be found in the literature on bioorganic chemistry.

2. COOH - Protective groups. The most commonly used is the formation of benzyl or tert- butyl ethers:

B
enzyl esters are usually obtained by direct esterification, tert- butyl - by adding isobutylene during acid catalysis (esterification tert- butanol is sterically hampered). The protective groups are removed under mild conditions, similar to the conditions for removing the corresponding urethane protective groups.

Sometimes a simple salt formation is used to protect the COOH group:

COOH → -COO‾.

B. Activating groups (X groups). Peptide bond formation reactions are referred to as acylation reactions; the main stage of such reactions is the nucleophilic addition (in this case, the NH 2 group) to the C=O bond of the carboxyl function. As already mentioned, the COOH group is rather inactive in acylation reactions, since the lone pair of electrons of the oxygen atom of the OH group largely compensates for the deficit of electron density on the carbonyl carbon atom:

The activating group (X) must be electron-withdrawing, to make the carbon atom of the carboxyl group more electrophilic and facilitate the attack of the amino group to form a peptide bond.

Quite a lot of derivatives of carboxylic acids containing electron-withdrawing groups are known, but not all of them can be used; for example, the most obvious activating group, C1, is unsuitable (i.e. no acid chlorides are used), because in this case, the configuration of the amino acid is not preserved (racemization occurs). The following are commonly used activation options.

BUT. Formation of activated esters (X = OR) . In this variant, aryl esters of acids are obtained, which contain electron-withdrawing groups in the aromatic radical (for example, pair-nitrophenyl or pentafluorophenyl):

B. Formation of acid azides(X = N 3):

Acid azides are obtained via esters and hydrazides; the azide group has a strong electron-withdrawing effect

AT. Formation of mixed anhydrides. Commonly used mixed ethers α-amino acids and derivatives of carbonic (92) or phosphoric (93) acids:

The preparation of mixed anhydrides with carbonic acid derivatives is convenient in that during the subsequent formation of a peptide bond, the activating group is removed in the form of alcohol and CO 2, which is convenient for preparative purposes:

The formation of mixed anhydrides of α-amino acids with a derivative of phosphoric acid (aminoacyladenylates) is an important reaction that precedes the process of protein biosynthesis - translation.

G. Use of carbodiimides The use of carbodiimides R-N=C=N-R 1 allows the activation of the carboxyl group and the formation of a peptide bond in one stage, without isolating the activated amino acid (or peptide). If, for example, carbodiimide is added to a mixture of an NH 2 -protected first amino acid and a COOH-protected second amino acid, then two successive reactions occur:

First, the carbodiimide reacts with the carboxyl group of the first amino acid to form its activated derivative (94) (resembling a mixed anhydride); then this derivative reacts with the NH 2 group of the second amino acid, and a peptide is formed, and the activating group is removed in the form sym. disubstituted urea.

One of the most widely used reagents of this type is dicyclohexylcarbodiimide(DCC) (R=R 1=cyclohexyl); during peptide synthesis, it forms sym. dicyclohexylurea, insoluble in most organic solvents and easily separated by filtration. Also widely used water soluble carbodiimides [for example, R = Et, R 1 = (CH 2) 3 N(CH 3) 2 ].

Carbodiimides are used not only in peptide synthesis, but also in the synthesis in vitro polynucleotides (see below).

D. UsageN-carboxyanhydrides. This option allows combine protection of the amino group and activation of the carboxyl function. N-Carboxyanhydrides (Leichs anhydrides) are formed by the interaction of α-amino acids with phosgene:

P
and this is combined group protectionNH 2 urethane type and activation of the carboxyl groups according to the type of formation of a mixed anhydride with a carbonic acid derivative. The formation of polypeptides using N-carboxyanhydrides proceeds as follows:

The interaction of N-carboxyanhydride with the salt of the second amino acid at precisely established a pH value of 10.2 leads to the formation of a peptide bond and the production of a salt of a dipeptide derivative (95) containing a carbamic acid salt moiety. With weak acidification (pH 5), the resulting fragment of carbamic acid immediately decarboxylated(carbamic acid derivatives with a free COOH group are very easily decarboxylated), i.e. deprotection occurs at the N-terminus of the dipeptide. Next, the obtained dipeptide (96) is reacted with the next N-carboxyanhydride at pH 10.2, etc.

This variant, in principle, makes it possible to reduce the number of stages of peptide synthesis, but it requires accurate compliance with the conditions, in particular, maintaining an accurate pH value. Under other conditions, in particular, the formation homopolymers homopolypeptides from N-carboxyanhydrides according to the scheme:

Such homopolypeptides can serve as models (albeit rather approximate ones) of natural polypeptides, so their preparation was of practical use.

Peptide synthesis on polymeric carriers. As can be seen from the above, the synthesis of polypeptide chains of any considerable length includes a large number of separately carried out stages (tens or even hundreds). This is a very laborious process; in addition, the highest efficiency of each stage is required, minimizing the loss of the resulting peptides. Efficiency is largely determined by the relative solubility of the peptides and other reaction products to be separated from the peptide: if the solubility is different, separation and purification are simplified.

The technique of peptide synthesis on a polymer carrier greatly simplifies the synthesis procedure and, in particular, radically solves the problem of solubility, which makes it possible to increase the synthesis efficiency. The idea of ​​synthesis is that the formed polypeptide chain from the very beginning of synthesis, it is bound to the macromolecule of the carrier polymer and only at the end of the synthesis is separated from it.

The most common use is insoluble carrier polymer ( solid phase peptide synthesis); this technique was first proposed by R. Merrifield in 1963. A partially chloromethylated styrene copolymer with a small amount of 1,4-divinylbenzene is usually used as a carrier polymer; it is a spatial polymer with rare cross-links between the chains and a certain number of CH 2 C1 groups:

P
eptide synthesis on a carrier proceeds according to the scheme:

First, the first amino acid (NH 2 -protected, most often with Boc protection) is “attached” to the carrier polymer due to the interaction of the chloromethyl group with the carboxyl group of the amino acid (more precisely, the carboxylate group, into which it is converted in the presence of triethylamine); the amino acid is attached to the polymer, forming a benzyl ester with it (97). Next, the NH 2 group is deprotected, a second NH 2 -protected amino acid is added (usually in the presence of carbodiimide); an N-protected dipeptide attached to the polymer is formed (98). Then the cycle is repeated: protection Z is removed, a third amino acid is added, etc.; there is an increase in the peptide chain from the C-terminus according to the scheme of linear synthesis.

Growing peptide chain from the very beginning(from the first link) insoluble, because is covalently bound to a three-dimensional polymer, which is by definition insoluble [at the same time, the three-dimensional network rare; so the polymer can to swell in a solvent, and the reagents have free access to the N-terminus of the growing chain]. That's why all by-products(primarily excess reagent) easily removed washing, extraction or filtration of the polymer [reagents at each stage take in great abundance to ensure the completeness of each reaction]. This significantly increases the efficiency of the synthesis.

Upon completion of the formation of the desired peptide chain, it is detached from the carrier polymer (for example, by the action of a mixture of HBr-CF 3 COOH under mild conditions); at the same time, protection is removed from the N-terminus (if it is Vos-protection):

Solid-phase synthesis of peptides is automated and carried out on special devices - synthesizers. The greatest success has been achieved in the synthesis oligopeptides(about 8-15 links); however, high molecular weight polypeptides can also be obtained by this method; in particular, one of the first significant advances in solid phase synthesis was the synthesis of the enzyme ribonuclease containing 124 units.

One of the problems faced by solid-phase synthesis is the decrease in the degree of swelling of the polymer as the peptide chain grows; this hinders access to the NH 2 groups of the growing polymer chain. In this case, the reaction of setting up the next link may not be complete, a peptide is partially formed with a “skipped” link, which, as a rule, no longer has the desired biological activity (skipping at least one link in the polypeptide chain changes its spatial organization, and, consequently, biological activity). Therefore, such “false” peptides must be separated from the “correct” ones, which is rather difficult.

The problem is at least partially solved when used as carriers soluble polymers; Linear polymers such as polystyrene, polyethylene glycols, or polyurethanes can be used as such carriers. In this variant, the synthesis is carried out in solution, where the access of reagents to the growing chain is easier compared to solid-phase synthesis. Then the polymer with the growing peptide chain “attached” to it is precipitated with a “bad” solvent, filtered from the rest of the products, dissolved again in a “good” solvent, and the synthesis is continued. This option, proposed by M. M. Shemyakin, is called liquid phase peptide synthesis; it is used for the synthesis of oligopeptides; during the synthesis of high-molecular-weight polypeptides, the solubility of the polymer changes, which creates a number of problems.

Non-matrix laboratory synthesis of peptides (in all variants) is currently used mainly for the synthesis of natural oligopeptides; the synthesis of natural proteins is more efficiently carried out biotechnologically - by embedding genes encoding proteins into recombinant DNA, followed by cloning and expression of these genes.

1. Introduction…………………………………………………………………………3

2. What are peptides? ............................................... ..............................................four

2.1. The structure of peptides………………………………………………………….5

2.2. Synthesis of peptides…………………………………………………………….7

3. Solid phase synthesis of peptides………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………

3.1. Merrinfield method………………………………………………………10

3.2. Solid substrate……………………………………………………….14

3.3. Substrate selection………………………………………………………...14

3.4. Linkers………………………………………………………………….16

4. The first synthesis of a natural hormone - oxytocin……………………….22

5. Synthesis of insulin in the cell……………………………………………………..30

6. Conclusion…………………………………………………………………..34

7. Literature…………………………………………………………………...35

Introduction

In organic chemistry, there is not a single reaction that in practice provides quantitative yields of target products in any case. The only exception seems to be the complete combustion of organic substances in oxygen at a high temperature to CO 2 and H 2 O. Therefore, purification of the target product is a complex and time-consuming task. For example, 100% purification of peptide synthesis products is an intractable problem. Indeed, the first complete synthesis of a peptide, the hormone oxytocin (1953), containing only 8 amino acid residues, was considered an outstanding achievement that brought its author, V. du Vignot, the Nobel Prize in 1955. However, in the next twenty years, the synthesis of polypeptides of this complexity turned into into routine, so that at present the synthesis of polypeptides consisting of 100 or more amino acid residues is no longer considered an insurmountable task.

The purpose of the work: to disassemble and explain: "What caused such dramatic changes in the field of polypeptide synthesis?"

What are peptides?

Peptides are natural or synthetic compounds whose molecules are built from alpha-amino acid residues interconnected by peptide (amide) bonds C (O) NH. They can also contain a non-amino acid component in the molecule (eg, a carbohydrate residue). According to the number of amino acid residues included in the peptide molecules, dipeptides, tripeptides, tetrapeptides, etc. are distinguished. Peptides containing up to 10 amino acid residues are called oligopeptides, those containing more than 10 amino acid residues are called polypeptides. Natural polypeptides with a molecular weight of more than 6 thousand are called proteins.

For the first time, peptides were isolated from enzymatic protein hydrolysates. The term "peptides" was proposed by E. Fisher. The first synthetic peptide was obtained by T. Curtius in 1881. E. Fisher by 1905 developed the first general method for the synthesis of peptides and synthesized a number of oligopeptides of various structures. The current contribution to the development of peptide chemistry was made by E. Fischer's students E. Abdergalden, G. Leike, and M. Bergman. In 1932, Bergman and L. Zervas used the benzyloxycarbonyl group (carbobenoxy group) in the synthesis of peptides to protect the alpha-amino groups of amino acids, which marked a new stage in the development of peptide synthesis. The obtained N-protected amino acids (N-carbobenoxyamino acids) were widely used to obtain various peptides, which were successfully used to study a number of key problems in the chemistry and biochemistry of these substances, for example, to study the substrate specificity of proteolytic enzymes. With the use of N-carbobenoxyamino acids, natural peptides (glutathione, carnosine, etc.) were synthesized for the first time. An important achievement in this area was developed in the early 50s. P. Vaughan et al. Synthesis of peptides by the mixed anhydride method.

In 1953, V. Du Vigno synthesized the first peptide hormone, oxytocin. Based on the concept of solid-phase peptide synthesis developed by P. Merrifield in 1963, automatic peptide synthesizers were created. Methods for controlled enzymatic synthesis of peptides have been intensively developed. The use of new methods made it possible to synthesize the hormone insulin, etc.

Advances in the synthetic chemistry of peptides were prepared by advances in the development of such methods for the separation, purification and analysis of peptides as ion-exchange chromatography, electrophoresis on various media, gel filtration, high performance liquid chromatography (HPLC), immunochemical analysis, etc. methods of end group analysis and methods of stepwise cleavage of peptides. In particular, automatic amino acid analyzers and automatic devices for determining the primary structure of peptides, the so-called sequencers, were created.

Structure of peptides

The peptide bond has the properties of a partially double bond. This is manifested in a decrease in the length of this bond (0.132 nm) compared to the length of a simple C N bond (0.147 nm). The partially double-linked nature of the peptide bond makes it impossible for substituents to rotate freely around it, so the peptide group is planar and usually has a trans configuration (form I). Thus, the backbone of the peptide chain is a series of rigid planes with a movable ("hinged") joint in the place where the asymmetric C atoms are located (indicated by an asterisk in the I section).

Preferential formation of certain conformers is observed in peptide solutions. With chain elongation, ordered elements of the secondary structure acquire more pronounced stability (similar to proteins). The formation of a secondary structure is especially typical for regular peptides, in particular for polyamino acids.

Properties

Oligopeptides are similar in properties to amino acids, polypeptides are similar to proteins. Oligopeptides are, as a rule, crystalline substances that decompose when heated to 200-300 0 C. They are highly soluble in water, dilute acids and alkalis, almost insoluble in organic solvents. Exceptions are oligopeptides built from hydrophobic amino acid residues.

Oligopeptides have amphoteric properties and, depending on the acidity of the medium, can exist in the form of cations, anions, or zwitterions. The main absorption bands in the IR spectrum for the NH group are 3300 and 3080 cm -1 , for the C=O group 1660 cm -1 . In the UV spectrum, the absorption band of the peptide group is in the region of 180-230 nm. The isoelectric point (pI) of peptides varies widely and depends on the composition of amino acid residues in the molecule. The pKa values ​​of the peptides are for a-COOH approx. 3, for -H 2 approx. eight.

The chemical properties of oligopeptides are determined by the functional groups contained in them, as well as by the features of the peptide bond. Their chemical transformations are largely similar to the corresponding reactions of amino acids. They give a positive biuret reaction and a ninhydrin reaction. Dipeptides and their derivatives (especially esters) are easily cyclized, turning into diketopiperazines. Under the action of 5.7 normal hydrochloric acid, the peptides are hydrolyzed to amino acids within 24 hours at 105 0 C.

Synthesis of peptides

In peptide synthesis, reactions known from organic chemistry for the preparation of amides and specially developed methods for the synthesis of peptides are used. For the successful implementation of these syntheses, it is necessary to activate the carboxyl group, i.e. increase the electrophilicity of the carbonyl carbon. This is achieved by chemical modification of the carboxyl group of amino acids. The type of such modification usually determines the name of the method of peptide synthesis.

1. Chloride method.

The method is based on the reaction of obtaining amides by the interaction of acid chlorides with the corresponding amines. It was in this way that the first peptides were obtained. Currently, this method is used extremely rarely, since it is accompanied by the formation of by-products and racemization of peptides.

2. Azide method

The starting material in this method is most often the ethyl ester of an N-protected amino acid, from which hydrazide is obtained, the latter is converted with sodium nitrite in the presence of hydrochloric acid into acid azide. In the reaction, hydrazine is usually used, in which one of the nitrogens is blocked by a protective group (Z-carbobenoxy or carbotretbutyloxy group), which avoids the formation of side dihydrazides. Azides interact with C-protected amino acids under mild conditions to form peptides.

Racemization in this method is minimized, but side reactions can occur, namely: azides can rearrange to isocyanates, which in turn, when interacting with an alcohol used as a solvent, form urethanes.

3. Mixed anhydrides

Mixed amino acid anhydrides with carbonic acid derivatives, obtained, for example, using isobutyl chlorocarbonate, have found wide application in peptide synthesis:

The reaction in this synthesis is carried out at a low temperature (-10..-20 C), rather quickly, which significantly reduces the possibility of formation of by-products and racemization. The fast stepwise synthesis of peptides using mixed anhydrides is called REMA synthesis. Methods of formation using mixed anhydrides are widely used in solid-phase synthesis of peptides.

Thus, carrying out peptide synthesis requires consideration and strict observance of certain factors. So, in order to reduce the formation of by-products and racemization, the following typical conditions for the reaction of peptide bond formation are recommended:

1) the process must be carried out at low temperatures, the reaction time must be minimal;

2) the reaction mass should have a pH close to neutral;

3) organic bases such as piperidine, morpholine, etc. are used as acid-binding reagents;

4) carrying out the reaction is desirable in anhydrous media.

Solid phase synthesis

Solid-phase synthesis is a methodical approach to the synthesis of oligomers (polymers) using a solid insoluble carrier, which is an organic or inorganic polymer.

In the early 1960s, a new approach was proposed to solve the isolation and purification problems arising in peptide synthesis. Later, the author of the discovery of this approach, R.B. Merrifield, in his Nobel Lecture, described how this happened: “One day I had the idea of ​​how the goal of more efficient peptide synthesis could be achieved. The plan was to assemble the peptide chain in stages, with the chain having one end attached to a solid support during synthesis.” As a result, the isolation and purification of intermediate and target peptide derivatives was reduced to a simple filtration and thorough washing of the solid polymer to remove all excess reagents and by-products remaining in solution. Such a mechanical operation can be performed quantitatively, easily standardized, and can even be automated. Let's consider this procedure in more detail.

Merrifield method

The polymer carrier in the Merrifield method is a granular cross-linked polystyrene containing chloromethyl groups in the benzene rings. These groups transform the polymer into a functional analogue of benzyl chloride and give it the ability to easily form ester bonds upon reaction with carboxylate anions. Condensation of such a resin with N-protected amino acids leads to the formation of the corresponding benzyl esters. Removal of the N-protection from gives a C-protected derivative of the first amino acid covalently linked to the polymer. Aminoacylation of the freed amino group with an N-protected derivative of the second amino acid, followed by removal of the N-protection, results in a similar dipeptide derivative also bound to the polymer:

Such a two-step cycle (deprotection-aminoacylation) can in principle be repeated as many times as required to build up a polypeptide chain of a given length.

The use of a solid carrier alone cannot yet simplify the solution of the problem of separating an n-mer peptide from its (n-1)-membered precursor, since both of them are polymer-bound. However, this approach allows the safe use of large excesses of any reagent necessary to achieve almost 100% conversion of the (n-1)-membered precursor to the n-membered peptide, since the target products bound to the carrier at each stage can be easily and quantitatively released. from excess reagents (which would be very problematic when working in homogeneous systems).

It immediately became clear that the possibility of purifying the product after each reaction by simple filtration and washing, and that all reactions could be carried out in one reaction vessel, were ideal preconditions for mechanization and automation of the process. Indeed, it took only three years to develop an automatic procedure and equipment that would allow the programmed synthesis of polypeptides with a given sequence of amino acid residues. Initially, both the equipment itself (tanks, reaction vessels, hoses) and the control system were very primitive. However, the power and effectiveness of the overall strategy has been convincingly demonstrated by a number of peptide syntheses performed on this equipment. For example, using such a semi-automatic procedure, the synthesis of the natural hormone insulin, built from two polypeptide chains (consisting of 30 and 21 amino acid residues) linked by a disulfide bridge, was successfully performed.

The solid phase technique resulted in significant savings in labor and time required for peptide synthesis. For example, at the cost of considerable effort, Hirschman and 22 collaborators completed an outstanding synthesis of the enzyme ribonuclease (124 amino acid residues) using traditional liquid-phase methods. Almost simultaneously, the same protein was obtained by automated solid phase synthesis. In the second case, the synthesis, which included 369 chemical reactions and 11,931 operations, was performed by two participants (Gatte and Merrifield) in just a few months (on average, up to six amino acid residues per day were added to the growing polypeptide chain). Subsequent improvements made it possible to build a fully automatic synthesizer.

The Merrifield method served as the basis for a new direction in organic synthesis - combinatorial chemistry.

Although sometimes combinatorial experiments are carried out in solutions, they are mainly carried out using solid-phase technology - the reactions proceed using solid substrates in the form of spherical granules of polymer resins. This provides a number of benefits:

1. Various parent compounds can be associated with individual beads. These granules are then mixed and thus all starting compounds can interact with the reagent in one experiment. As a result, reaction products are formed on separate granules. In most cases, the mixing of raw materials in traditional liquid chemistry usually leads to failures - polymerization or gumming of products. Experiments on a solid substrate rule out these effects.

2. Because the raw materials and products are bonded to the solid support, excess reactants and non-support products can be easily washed away from the polymeric solid support.

3. Large excesses of reagents can be used to drive the reaction to completion (greater than 99%) as these excesses are easily separated.

4. By using low batch volumes (less than 0.8 mmol per gram of support), unwanted side reactions can be avoided.

5. The intermediates in the reaction mixture are bound to the granules and do not need to be purified.

6. Individual polymer beads can be separated at the end of the experiment and thus individual products are obtained.

7. The polymer substrate can be regenerated in those cases when the breaking conditions are selected and the appropriate anchor groups - linkers are selected.

8. Automation of solid-phase synthesis is possible.

The necessary conditions for solid-phase synthesis, in addition to the presence of an insoluble polymer substrate that is inert under reaction conditions, are:

1. The presence of an anchor or linker - a chemical function that ensures the connection of the substrate with the applied compound. It is covalently bound to the resin. The anchor must also be a reactive functional group in order for substrates to interact with it.

2. The bond formed between the substrate and the linker must be stable under the reaction conditions.

3. There must be ways to cleave the product or intermediate from the linker.

Let us consider in more detail the individual components of the solid-phase synthesis method: a solid support and a linker.

Solid backing

As stated above, the first types of resins that Merrifield used were polystyrene beads, where the styrene was crosslinked with 1% divinylbenzene. The beads were modified with chloromethyl groups (linker) to which the amino acids could be linked via ester groups. These ester bonds are stable under the reaction conditions used for peptide synthesis.

One disadvantage of polystyrene beads is the fact that they are hydrophobic while the growing peptide chain is hydrophilic. As a result, sometimes the growing peptide chain is not solvated and folds due to the formation of intramolecular hydrogen bonds. This form makes it difficult for new amino acids to reach the end of the growing chain. Therefore, more polar solid substrates such as polyamide resins are often used. Such resins are more suitable for non-peptide combinatorial synthesis.

Choice of solid substrate

Synthetic approaches to library preparation are often determined by the nature of the chosen polymeric support. The granular polymer must meet certain criteria, depending on the synthesis and screening strategies.

For the resulting libraries, the size and uniformity of the granules, as well as the resistance of the resin to the formation of clusters, are important. The ability of a resin to swell in organic and aqueous media is particularly important when mandatory samples are used to screen for structure while still on the bead.

The main types of polymer resins for combinatorial synthesis currently used are:

1. Polystyrene cross-linked with 0.5-2% divinylbenzene (StratoSpheres)

2. Polyethylene glycol grafted onto a crosslinked polystyrene-1% divinylbenzene copolymer (TentaGel, AgroGel, NovaGel)

3. Polyethylene glycol grafted onto 1% cross-linked polystyrene (PEG-PS)

4. Polystyrene macroporous resin with high degree of cross-linking (AgroPore, TentaPore)

5. Bis-2-acrylamide-polyethylene glycol-monoacrylamido-polyethylene glycol (PEGA) copolymer

6. Dimethylacrylamide deposited on a macroporous matrix of diatomaceous earth (Pepsyn K)

7. Dimethylacrylamide deposited on a macroporous matrix - cross-linked 50% polystyrene-divinylbenzene (Polyhipe)

Although classic granular resins are more suitable for the combinatorial synthesis of compound libraries, alternative supports are sometimes used.

For example, cellulose is a good support for multiple “droplet synthesis” of peptides or for the synthesis of libraries on paper. “Drip” syntheses are carried out by dropping solutions of protected amino acids onto modified paper in the presence of an activating reagent. Here, the carrier itself is the reaction vessel and there is no need for manipulations typical of liquid media during synthesis (usually shaking in the case of solid phase synthesis). The reaction proceeds due to the diffusion of the liquid in the carrier. This principle of internal bulk synthesis was tested using polymeric supports on a synthesizer using centrifugation to eliminate liquid. The drip technique has been found to be comparable to the classical solid phase operation in peptide synthesis.

It has also been found that cotton, as the purest form of cellulose, can serve as a convenient solid phase support, especially for multiple synthesis or library generation.

Although pellets are the most common form of solid support, other types (such as needles) can also be used for combinatorial synthesis. The modified glass surface can also be used for oligonucleotide synthesis.

Linkers

A linker is a molecular fragment covalently bonded to a solid support. It contains reactive functional groups with which the first reactant interacts and which, as a result, becomes associated with the resin. The resulting bond should be stable under the reaction conditions, but easily broken at the final stage of the synthesis.

Different linkers are used depending on which functional group is present in the substrate and which functional group is to be formed at the end of the procedure.

In the practice of combinatorial synthesis, the following linkers are most often used:

• Chloromethyl (-CH 2 Cl),
• Hydroxyl (-OH),
• Amine (-NH 2),
• Aldehyde (-CHO),
• Silyl (-OSiR 3).

Linker type	resin type	What attaches	What synthesizes	What makes the break
halomethyl		Carboxylic acids, alcohols, phenols, thiols, amines	Acids, alcohols, esters, thioethers	TFMSA, H 2 /Pd, i-Bu 2 AlH, MeONa, HF
halomethyl		Alkyl and arylamines	Anilides and sulfamides	CF 3 COOH, SOCl 2 /CF 3 COOH
halomethyl		Alcohols, acids, phenols, thiols, amines	Alcohols, acids, thiols, amines, esters	1-5% CF 3 COOH, 30% hexafluoroisopropanol
Hydroxyl		Alcohols, acids	Alcohols, acids, amides	CF 3 COOH, amine/AlCl 3 , i-Bu 2 AlH
Hydroxyl		Alcohols, acids	Alcohols, acids	5% CF3COOH, 10% AcOH
Hydroxyl		acids	acids	Light with a wavelength of 365 nm. Linker is stable to CF 3 COOH and piperidine
Hydroxyl		acids	Acid amides, alcohols, esters, hydrazides	Nucleophiles (NaOH, NH 3 / MeOH, NaBH 4 / EtOH, MeOH / CF 3 COOH, NH 2 NH 2 / DMF
Hydroxyl		Protected peptides, acid-slots	Cyclic peptides, ureas	25% CF 3 COOH, hydrazides
Hydroxyl Linker Rinker		Alcohols, acids, phenols	Alcohols, acids, phenols	1-5% CF3COOH
Amino		acids	Carboxamides	95%CF3COOH
Amino		acids	Protected amides	1% CF3COOH
Amino		acids	Aldehydes and ketones	LiAlH 4 and Grignard reagents
Amino		carboxylic acids	Amides or carboxylic acids	Activation of a sulfonamide with diazomethane or bromoacetonitrile followed by nucleophile attack on an amine or hydroxide
Aldehyde		Primary or secondary alcohols	Alcohols	95% CF 3 COOH/H 2 O or CF 3 COOH/CH 2 Cl 2 /EtOH
Aldehyde		Amines	Carboxamides, sulfonamides	CF3COOH

Wang resins can be used in peptide synthesis via an N-protected amino acid ester-linked to a linker. This ester bond is resistant to the coupling and deprotection step, but can be broken with trifluoroacetic acid to remove the final peptide from the resin bead.

Substrates with a carboxyl group can be linked to the Rink resin via an amide bond. Once the procedure is complete, reaction with trifluoroacetic acid liberates the product with a primary amide group.

Primary and secondary alcohols can be associated with the resin modified with dihydropyran. The binding of the alcohol occurs in the presence of 4-toluenesulfonate in dichloromethane. The product is removed using trifluoroacetic acid.

The first synthesis of the peptide hormone - oxytocin

In 1953, the American scientist Vincent Du Vigno, together with his colleagues, found out the structure of oxytocin, a cyclic polypeptide. Among the known natural compounds, such cyclic structures have not been encountered before. The following year, the scientist for the first time carried out the synthesis of this substance. This was the first time that a polypeptide hormone was synthesized under in vitro conditions.

Du Vignot is known in the scientific world for his research at the intersection of chemistry and medicine. In the mid 1920s. the subject of his scientific interest was the study of the function of sulfur in insulin, a pancreatic hormone that regulates the process of carbohydrate metabolism and maintains a normal level of sugar (glucose) in the blood. The young man's interest in the chemistry of insulin arose, according to his recollections, after one of the lectures given by Professor William C. Rose immediately after the discovery of this substance by Frederick G. Banting and John J.R. MacLeod. So when, after graduating from university, John R. Murlin of the University of Rochester invited him to study the chemical nature of insulin, the young scientist considered it a destined proposal. “The chance to work on the chemistry of insulin crossed out all my other scientific expectations,” Du Vignot later noted, “so I immediately accepted Professor Murlin’s offer.”
During his work at the University of Rochester, Du Vignot managed to make the first assumptions about the chemical composition of insulin, which were largely reflected in his dissertation "Sulfur of Insulin", defended in 1927. According to Du Vignot's views, insulin was one of the derivatives of the amino acid cystine. He identified insulin as a sulfur-containing compound in which the sulfur fragments are disulfide bridges. He also expressed considerations about the peptide nature of insulin.
It should be noted that Du Vignot's data that insulin is a sulfur-containing compound were in good agreement with the main conclusions of the work carried out at that time in this direction by Professor John Jacob Abel and colleagues at Johns Hopkins University. Therefore, the scholarship of the National Research Council, which the young scientist received immediately after defending his dissertation, turned out to be very useful. Thanks to her, Du Vigno worked for some time under the guidance of Professor Abel at the Johns Hopkins University School of Medicine.
Professor Abel, a recognized authority on the study of hormone chemistry, held the view at that time that insulin was a protein compound. Such views ran counter to the ideas that dominated those years. As Du Vignot himself recalled, "it was a time when both chemists and biologists could not accept the fact that an enzyme could be a protein compound." Shortly before this, Professor Abel was able to isolate insulin in crystalline form for the first time (1926). Du Vigno's plans, when he got an internship with Abel, included the following: to isolate the amino acid cystine from insulin crystals and try to study its structure. He accomplished this very quickly. As a result of research, together with the professor's staff and with his direct assistance, the young scientist clearly demonstrated the formation of a number of amino acids during the breakdown of the insulin molecule. One of them was just the sulfur-containing amino acid cystine. At the same time, experiments have shown that the sulfur content in insulin is directly correlated with the sulfur content in cystine. But the results achieved required the study of other sulfur-containing amino acids.
The continuation of financial support from the National Research Council for another year allowed Du Vignot to visit the famous scientific biochemical schools of Western Europe (Dresden, Edinburgh, London), where he was able to gain additional experience in the study of peptides and amino acids.
Upon returning to the United States, the scientist first worked at the University of Illinois, and three years later moved to the medical school of the George Washington University. Here he continued his research on insulin. Particularly interesting were his studies on the effect of disulfide bonds in cystine on the hypoglycemic effect of insulin (lowering blood sugar). Work in the field of insulin also stimulated a new direction of research - the study of pituitary hormones.
An important direction of his work at the George Washington University was the study of the mechanism of conversion of methionine to cystine in living organisms. In subsequent years, it was these studies that led him to the problem of studying biological transmethylation (the transfer of methyl groups from one molecule to another).
In 1938, the scientist was invited to the Medical College of Cornell University. Here he continued to study insulin and launched research on the hormones of the posterior pituitary gland.
During the Second World War, these studies had to be interrupted for a while. The scientist and his collaborators worked on the synthesis of penicillin. At the end of the war, Du Vignot was able to return to his previous studies. He was particularly intensive in his work on the isolation of a number of hormones from commercially available extracts of the pituitary gland and tissues of the pituitary gland of a bull and a pig.
The posterior lobe of the pituitary gland produces a number of hormones, two of which had by then been isolated in pure form. One of them is oxytocin, which stimulates the smooth muscles of the uterus, the other is vasopressin, a hormone that contracts peripheral arterioles and capillaries, thereby causing an increase in blood pressure. These hormones have proven to be very difficult to distinguish because they have similar physical properties. Because of this, until the mid-1920s. physicians and biochemists considered them to be one substance with a wide spectrum of biological activity. Thanks to the improvement of methods of chemical analysis, in
in particular fractional precipitation, chromatography and electrophoresis, by the 1940s. these hormones were partially separated.
In 1949, Du Vignot, using the "countercurrent distribution" method for a commercial extract with an oxytocin activity of 20 U/mg, obtained a drug with an activity of 850 U/mg. This prompted the scientist to attempt to study the structure of matter. To this end, he carried out the fragmentation of the polypeptide chain. As a result of the complete hydrolysis of the oxytocin preparation and the analysis of its amino acid composition by Du Vignot, the presence of eight different amino acids in an equimolecular ratio was established. The amount of released ammonia corresponded to three amide groups of the type
–CONH 2 , molecular weight – to monomeric octapeptide. One of the eight amino acid residues has been identified as cystine. Experiments on the oxidation of cystine in oxytocin showed that the disulfide bridge in cystine, previously discovered by Du Vignot, is part of the oxytocin ring system.
The sequence of eight amino acids in oxytocin was finally established by Du Vigneau and his coworkers only in 1953. It should be noted that in parallel with Du Vigneau's group, Professor Hans Tuppi (University of Vienna) worked on the same problems in Vienna, who also in 1953 independently of Du Vigneau established the sequence of amino acids in oxytocin using the Sanger method in his work.
Du Vigno went a slightly different way. He and his collaborators relied not primarily on the analysis of terminal amino acids, but on the identification of the components of a large number of lower peptides. They also studied the reaction of oxidized oxytocin with bromine water, which resulted in the formation of a heptapeptide and a brominated peptide. The study of the structure of the latter showed that the sequence of amino acids in the corresponding dipeptide: cystine - tyrazine.
Further, by the dinitrophenyl method, it was found that the N-terminal amino acid in the heptapeptide is isoleucine. Du Vignot concludes that the N-terminal sequence in oxidized oxytocin is:

HO 3 S - cis - tyr - izl.

Amino acids from the hormone oxytocin

Of the thirteen peptides listed below, the first four were obtained by partial hydrolysis of the heptapeptide, the second group, by hydrolysis of oxytocin (in this case, cysteine ​​residues were converted into alanine residues). Then the neutral fraction was separated and treated with bromine water to oxidize the cysteine ​​unit to the cysteic acid unit; the resulting acidic peptide was separated from the neutral peptide on ion exchange resins. The third group of peptides was obtained by hydrolysis of oxytocin desulfurized on Raney nickel. In the formulas below, if the sequence of amino acids in the peptides is known, the amino acid symbols are separated by a dash; if the sequence is unknown, then the characters are separated by a comma.

From heptapeptide:

1. (asp - cis - SO 3 H).
2. (cis - SO 3 H, pro).
3. (cis - SO 3 H, pro, leu).
4. (cis - SO 3 H, pro, leu, gly).

From oxytocin:

5. (lei, gli, pro).
6. (tire, cis - S - S - cis, asp, glu, ley, izl).
7. (tyr, cis - S - S - cis, asp, glu).
8. (cis - S - S - cis, asp, glu).
9. (cis - SO 3 H, asp, glu).

From desulfurized oxytocin:

10. (ala, asp).
11. (ala, asp, glu).
12. (glue, izl).
13. (ala, asp, glu, lei, izl).

Taking into account the structure of the resulting peptides and using the overlay of individual components of the peptides, Du Vignot and co-workers deduced the following amino acid sequence in oxytocin:

cystine - tyrazine - isoleucine - glutamine - NH 2 - asparagine - NH 2 - cystine - proline - leucine - glycine - NH 2.

The structure of oxytocin established by them is shown in fig. one.

It should be noted that, simultaneously with Du Vignot's oxytocin, the structure of another hormone of the posterior pituitary gland, vasopressin, was determined.
The structure of the hormone oxytocin was confirmed by its chemical synthesis in 1954, which was the first complete synthesis of natural peptides. The synthesis included the condensation of N-carbobenoxy-S-benzyl dipeptide (I) with heptapeptide triamide (II) using tetraethylpyrophosphite. After removing the carbobenzoxy and benzyl groups that protected the amino and sulfhydryl groups in both peptides, respectively, the resulting nonapeptide was oxidized with air, resulting in oxytocin (Fig. 2).
Thus, the first structural analysis and the first synthesis of a polypeptide hormone were carried out - an outstanding achievement in biochemistry and medicine. The era of chemical synthesis of biologically active natural peptides began in science with the works of Du Vigneau.

Fig.2. General scheme for the synthesis of oxytocin according to Du Vignot

As is known, in 1955 Du Vigneau was awarded the Nobel Prize in Chemistry "for his work with biologically active compounds, and above all for the first synthesis of a polypeptide hormone."

Synthesis of insulin in the cell

Insulin- a hormone of peptide nature, is formed in the beta cells of the islets of Langerhans of the pancreas. It has a multifaceted effect on the metabolism in almost all tissues. The main action of insulin is to lower the concentration of glucose in the blood.

Insulin increases the permeability of plasma membranes for glucose, activates the key enzymes of glycolysis, stimulates the formation of glycogen from glucose in the liver and muscles, and enhances the synthesis of fats and proteins. In addition, insulin inhibits the activity of enzymes that break down glycogen and fats. That is, in addition to the anabolic action, insulin also has an anti-catabolic effect.

Impaired insulin secretion due to destruction of beta cells - absolute insulin deficiency - is a key link in the pathogenesis of type 1 diabetes mellitus. Violation of the action of insulin on tissues - relative insulin deficiency - has an important place in the development of type 2 diabetes mellitus.

Post-translational modifications of insulin. 1) Preproinsulin (L - leader peptide, B - region 1, C - region 2, A - region 3) 2) Spontaneous folding 3) Formation of a disulfide bridge between A and B 4) Leader peptide and C are cut off 5) Final molecule

The synthesis and release of insulin is a complex process that includes several steps. Initially, an inactive hormone precursor is formed, which, after a series of chemical transformations, turns into an active form during maturation. Insulin is produced throughout the day, not just at night.

The gene encoding the primary structure of the insulin precursor is located on the short arm of chromosome 11.

On the ribosomes of the rough endoplasmic reticulum, the precursor peptide, preproinsulin, is synthesized. It is a polypeptide chain built from 110 amino acid residues and includes sequentially located: L-peptide, B-peptide, C-peptide and A-peptide.

Almost immediately after synthesis in the ER (endoplasmic reticulum-endoplasmic reticulum), a signal (L) peptide is cleaved from this molecule - a sequence of 24 amino acids that are necessary for the passage of the synthesized molecule through the hydrophobic lipid membrane of the ER. Proinsulin is formed (a polypeptide produced by the beta cells of the islets of Langerhans of the pancreas.

Proinsulin is a precursor in the process of insulin biosynthesis. It consists of two chains present in the insulin molecule (A-chain and B-chain), connected by a C-peptide or (C-chain, connecting chain), which is cleaved off during the formation of insulin from the proinsulin molecule), which is transported to the Golgi complex , further in the tanks of which the so-called maturation of insulin occurs.

Maturation is the longest stage of insulin formation. In the process of maturation, a C-peptide, a fragment of 31 amino acids connecting the B-chain and the A-chain, is cut out from the proinsulin molecule with the help of specific endopeptidases. That is, the proinsulin molecule is divided into insulin and a biologically inert peptide residue.

In secretory granules, insulin combines with zinc ions to form crystalline hexameric aggregates.

Insulin has a complex and multifaceted effect on metabolism and energy. Many of the effects of insulin are realized through its ability to act on the activity of a number of enzymes.

Insulin is the only hormone lowering blood glucose, this is implemented via:

Increased absorption of glucose and other substances by cells;

activation of key enzymes of glycolysis;

An increase in the intensity of glycogen synthesis - insulin boosts the storage of glucose by liver and muscle cells by polymerizing it into glycogen;

decrease in the intensity of gluconeogenesis - the formation of glucose in the liver from various substances decreases

Anabolic effects:

enhances the absorption of amino acids (especially leucine and valine) by cells;

enhances the transport of potassium ions, as well as magnesium and phosphate into the cell;

enhances DNA replication and protein biosynthesis;

enhances the synthesis of fatty acids and their subsequent esterification - in adipose tissue and in the liver, insulin promotes the conversion of glucose into triglycerides; with a lack of insulin, the opposite occurs - the mobilization of fats.

Anti-catabolic effects:

· inhibits the hydrolysis of proteins - reduces the degradation of proteins;

Reduces lipolysis - reduces the flow of fatty acids into the blood.

Conclusion

Indeed, the first complete synthesis of a peptide, the hormone oxytocin (1953), containing only 8 amino acid residues, was considered an outstanding achievement that brought its author, V. du Vignot, the Nobel Prize in 1955. However, in the next twenty years, the synthesis of polypeptides of this complexity turned into into routine, so that at present the synthesis of polypeptides consisting of 100 or more amino acid residues is no longer considered an insurmountable task. The use of new methods made it possible to synthesize the hormone insulin and other hormones. In this work, we got acquainted with the concept of "polypeptides", analyzed and explained what caused such dramatic changes in the field of polypeptide synthesis. We got acquainted with the synthesis of peptides and their solid-phase synthesis.

Literature

1 Plane R. Interview with Vincent du Vigneaud. Journal of Chemical Education, 1976, v. 53, no. 1, p. 8–12;
2. Du Vigneaud V. A Trail of Research in Sulfur Chemistry and Metabolism and Related Fields. Ithaca, New York: Cornell University Press, 1952;
3. Bing F. Vincent du Vigneaud. Journal of Nutrition, 1982, v. 112, p. 1465–1473;
Du Vigneaud V., Melville D.B., Gyo..rgy P., Rose K.S. Identity of Vitamin H with Biotin. Science, 1940, v. 92, p. 62–63; Nobel Prize Winners. 4. Encyclopedia. Per. from English. T. 2. M.: Progress, 1992

5. http://ru.wikipedia.org/wiki/%D0%98%D0%BD%D1%81%D1%83%D0%BB%D0%B8%D0%BD#.D0.A1.D0. B8.D0.BD.D1.82.D0.B5.D0.B7_.D0.B8.D0.BD.D1.81.D1.83.D0.BB.D0.B8.D0.BD.D0.B0_. D0.B2_.D0.BA.D0.BB.D0.B5.D1.82.D0.BA.D0.B5

6. http://www.chem.isu.ru/leos/base/comb/comb03.html

what is the mass of the part of the DNA molecule encoding the insulin molecule, if it is known that this molecule contains 51 amino acids, and the average

the molecular weight of one nucleotide is 345 a.u. eat.?

photosensitive protein (opsin) of the visual pigment of rods of the retina of vertebrates and visual cells of invertebrates - rhodopsin consists

of 348 amino acid residues. determine the relative molecular weight of this protein, assuming that the average mass of one amino acid residue is 116

Task number 1.

The mRNA chain fragment has the nucleotide sequence: CCCACCCAGUA. Determine the nucleotide sequence on DNA, tRNA anticodons, and amino acid sequence in a protein fragment using the genetic code table.

Task number 2. A fragment of a DNA chain has the following nucleotide sequence: TACCTCCACCTG. Determine the nucleotide sequence on the mRNA, the anticodons of the corresponding tRNA, and the amino acid sequence of the corresponding fragment of the protein molecule using the genetic code table.

Task #3
The nucleotide sequence of the DNA chain fragment is AATGCAGGTCACTCCA. Determine the sequence of nucleotides in i-RNA, amino acids in the polypeptide chain. What happens in a polypeptide if, as a result of a mutation in a gene fragment, the second triplet of nucleotides falls out? Use the gen.code table
Workshop-solving problems on the topic "Protein biosynthesis" (Grade 10)

Task #4
The gene section has the following structure: CHG-AGC-TCA-AAT. Specify the structure of the corresponding section of the protein, information about which is contained in this gene. How will the removal of the fourth nucleotide from the gene affect the structure of the protein?
Task number 5
Protein consists of 158 amino acids. How long is the gene encoding it?
The molecular weight of the protein X=50000. Determine the length of the corresponding gene. The molecular weight of one amino acid is on average 100.
Task number 6
How many nucleotides does the gene (both strands of DNA) contain, in which the insulin protein of 51 amino acids is programmed?
Task number 7
One of the DNA strands has a molecular weight of 34155. Determine the amount of protein monomers programmed in this DNA. The molecular weight of one nucleotide is on average 345.
Task number 8
Under the influence of nitrous acid, cytosine is converted to guanine. How will the structure of the synthesized tobacco mosaic virus protein with the amino acid sequence: serine-glycine-serine-isoleucine-threonine-proline change if all cytosine nucleotides have been exposed to acid?
Task number 9
What is the molecular weight of a gene (two strands of DNA) if a protein with a molecular weight of 1500 is programmed in one strand? The molecular weight of one amino acid is on average 100.
Task number 10
A fragment of the polypeptide chain is given: val-gli-phen-arg. Determine the structure of the corresponding t-RNA, i-RNA, DNA.
Task number 11
A fragment of the DNA gene is given: CCT-TCT-TCA-A ... Determine: a) the primary structure of the protein encoded in this region; b) the length of this gene;
c) the primary structure of the protein synthesized after the loss of the 4th nucleotide
in this DNA.
Task number 12
How many codons will there be in i-RNA, nucleotides and triplets in the DNA gene, amino acids in the protein, if 30 t-RNA molecules are given?
Task number 13

It is known that all types of RNA are synthesized on a DNA template. The fragment of the DNA molecule, on which the central loop region of tRNA is synthesized, has the following nucleotide sequence: ATAGCTGAACGGACT. Set the nucleotide sequence of the t-RNA section that is synthesized on this fragment, and the amino acid that this t-RNA will transfer during protein biosynthesis, if the third triplet corresponds to the t-RNA anticodon. Explain the answer. To solve the problem, use the table of the genetic code.

1. A blue-eyed man whose parents had brown eyes married a brown-eyed woman whose father had blue eyes and whose mother

brown. What offspring can be expected from this marriage, if it is known that the brown eye gene dominates the blue eye gene?
2. There were two brothers in the family. One of them, a patient with hemorrhagic diathesis, married a woman also suffering from this disease. All three of their children (2 girls and 1 boy) were also sick. The second brother was healthy and married a healthy woman. Of their four children, only one had hemorrhagic diathesis. Determine which gene determines hemorrhagic diathesis.
3. In a family where both parents had normal hearing, a deaf child was born. Which trait is dominant. What are the genotypes of all members of this family?
4. A man suffering from albinism marries a healthy woman whose father suffered from albinism. What kind of children can be expected from this marriage, given that albinism is inherited in humans as an autosomal recessive trait?

1. What is a pair of alternative features? Which sign from the pair is called

recessive?
2. One form of schizophrenia is inherited as a recessive trait. Determine the probability of having a child with schizophrenia from healthy parents, if it is known that the grandmother on the paternal side and the grandfather on the maternal side suffered from this disease.
3. What is an analysis cross?
4. In cattle, polledness (lack of horns) dominates over hornedness.
Polled bull was crossed with three cows. From crossing with one horned cow
a horned calf was born, from crossing with another - a horned calf, from crossing with a horned cow a horned calf was born. What are the genotypes of all the animals involved in crossbreeding?
5. If in wheat the gene that determines the short spike length does not completely dominate the gene responsible for the appearance of the longer spike, then what length of the spikes can appear when two plants with medium-length spikes are crossed?
6. Andalusian (blue) chickens are heterozygotes that usually appear when crossing
white and black chickens. What plumage will have offspring obtained from crossing
white and blue hens, if the gene for black plumage in hens is known to be an incomplete dominance gene (with respect to the recessive gene responsible for
formation of white plumage)?
7. The mother has a second blood group and is heterozygous. My father has the fourth blood group. What blood groups are possible in children?
8. Formulate the second law of Mendel and the law of purity of gametes.
9. What cross is called dihybrid? Which polyhybrid?
10. A tomato plant with red pear-shaped fruits is crossed with a plant with red spherical fruits. 149 plants with red spherical fruits and 53 plants with yellow spherical fruits were obtained. Determine dominant and
recessive traits, genotypes of parents and offspring.
11. It is known that cataracts and red hair in humans are controlled by dominant genes located in different pairs of chromosomes (autosomal). A red-haired, non-cataracted woman married a blond-haired man who recently had cataract surgery. Determine what children can be born to these spouses, if we keep in mind that the man's mother has the same phenotype as his wife, that is, she is red-haired and does not have cataracts.
12. What is the peculiarity of the inheritance of sex-linked traits?
14. What interaction of non-allelic genes is called epigenesis (epistasis)
15. In horses, the action of the genes of the black suit (C) and the red suit (c) is manifested only in the absence of the dominant gene D. If it is present, then the color is white. What offspring will be obtained when horses with the CcDd genotype are crossed?

• Activities and pedagogical views of Jan Amos Comenius
• The rate constant is calculated by the formula Rate constant value
• Phase diagrams of two-component systems "polymer - solvent" Phase diagrams of two-component systems "polymer - solvent"
• Fine and hyperfine structure of optical spectra
• How to learn chemistry on your own from scratch: effective ways
• Properties and characteristics of alkenes
• Characteristics of a student from the place of internship
• Characteristics for a student who had an internship at an enterprise: writing rules
• Biography of Faraday. Great scientists. Michael Faraday. discovery of electromagnetic rotation
• Modern innovations in education

• Substituents on the benzene ring are divided into two groups Reactions of the benzene ring
• Types of chemical reactions in organic chemistry Electrophilic addition reactions
• Methods for separating heterogeneous mixtures
• Polymethacrylic acid
• General characteristics of the elements of the VI A subgroup Elements 6a of the subgroup
• Theoretical foundations of physics
• Graph theory in chemistry. graph theory. Basic definitions and theorems of graph theory
• Algebra and harmony in chemical applications
• Tests on the course "Ecology and nature management
• WWF Russia Director Igor Chestin: Yakutia is among the leaders I know that you travel a lot… Why do you need this