Solid-phase synthesis or solid-phase technology, which is often called ceramic, is the most common in the production of inorganic materials for various branches of science and industry. These include nuclear fuel, materials for space technology, radio electronics, instrumentation, catalysts, refractories, high-temperature superconductors, semiconductors, ferro- and piezoelectrics, magnets, various composites, and many others.
Solid-phase synthesis is based on chemical reactions in which at least one of the reactants is in the form of a solid. Such reactions are called heterogeneous or solid phase. Solid-phase interaction, in contrast to reactions in a liquid or gaseous medium, consists of two fundamental processes: from the chemical reaction itself and the transfer of matter to the reaction zone.
Solid-state reactions involving crystalline components are characterized by the limited mobility of their atoms or ions and a complex dependence on many factors. These include such as the chemical structure and associated reactivity of reacting solids, the nature and concentration of defects, the state of the surface and morphology of the reaction zone, the contact area of interacting reagents, preliminary mechanochemical activation, and a number of others. All of the above determines the complexity of the mechanisms of heterogeneous reactions. The study of heterogeneous reactions is based on solid state chemistry, chemical physics and physical chemistry of the surface of solids, on the laws of thermodynamics and kinetics.
Often, the mechanism of solid-state reactions is judged only on the basis that experimental data on the degree of interaction as a function of time are best described by any particular kinetic model and the corresponding kinetic equation. This approach can lead to incorrect conclusions.
Processes in solid-state materials have a number of important differences from processes in liquids or gases. These differences are associated, first of all, with a significantly (by several orders of magnitude) lower diffusion rate in solids, which prevents the averaging of the concentration of components in the system and, thus, leads to spatial localization of the occurring processes. Spatial localization, in turn, leads to the fact that both the specific rate of the process (or the diffusion coefficient) and the geometry of the reaction zone contribute to the observed kinetics of the processes. Such features of solid-phase processes determined by geometric factors are called topochemical. In addition, since the transformations under discussion are spatially localized, their rate can be determined both by the processes themselves at the phase boundary (reaction control) and by the rate of supply of any of the components to this interface or removal of the product (s) (diffusion control). These cases for simple systems, for which the model assumptions are satisfied, can be identified experimentally by the form of the time dependence of the degree of conversion. Another feature of phase transformations in solids is related to the fact that the formation of a new phase nucleus in a solid matrix causes the appearance of elastic stresses in the latter, the energy of which in some cases must be taken into account when considering the thermodynamics of these transformations.
A large number of factors affecting the kinetics of solid-phase processes and the microstructure of the materials obtained in this way also determines the multiplicity of types of classification of these processes. Thus, considering the stability of a system with respect to fluctuations of various types, heterogeneous (in the case of systems that are stable to small fluctuations in the occupied volume and unstable to large ones) and homogeneous (in the case of systems that are unstable to small fluctuations) processes are distinguished. For heterogeneous processes, as an example, one can cite transformations that proceed according to the mechanism of formation and growth of nuclei, for homogeneous processes, some order-disorder transitions and spinodal decomposition of solid solutions.
It is necessary to distinguish heterogeneous and homogeneous nucleation from heterogeneous and homogeneous processes in the case of heterogeneous processes. Heterogeneous nucleation refers to the formation of nuclei on structural defects (including point defects, dislocations and phase boundaries); homogeneous nucleation - the formation of nuclei in a defect-free volume of the solid phase.
Analyzing the product of a solid-phase transformation, one distinguishes between single-phase and multiphase nuclei. In the case of multiphase nuclei, the product of the process is a multiphase colony with a characteristic microstructure determined by the surface energy of the boundary of the formed phases; processes of this type are called discontinuous, in contrast to continuous processes in the case of the formation and growth of single-phase nuclei.
Another way to classify solid phase transformations is based on a comparison of the composition of the initial phase and the composition of the reaction product. If they coincide, they speak of diffusion-free processes, and if the composition changes, they speak of diffusion. Moreover, it is useful to distinguish from non-diffusion processes cooperative processes (for example, martensitic transformation) that occur by means of a simultaneous slight displacement of atoms in a large volume of the initial phase.
Diffusionless phase transformations can differ in the type of their thermodynamic characteristics changing during the process.
Transformations of the first kind are processes in which there is a change in the derivatives of the chemical potential with respect to temperature or pressure. This implies an abrupt change during the phase transition of such thermodynamic parameters as entropy, volume, enthalpy, and internal energy. During transformations of the second kind, the first derivatives of the chemical potential with respect to intensive parameters do not change, but the derivatives of higher orders change (starting from the second). In these processes, with continuous entropy and volume of the system, there is an abrupt change in the quantities expressed in terms of the second derivatives of the Gibbs energy: heat capacity, thermal expansion coefficient, compressibility, etc.
Solid-phase reactions between two phases (contacts between three or more phases are unlikely, and the corresponding processes can be represented as combinations of several two-phase reactions) are diffusion processes and can be either heterogeneous or homogeneous, with both heterogeneous and homogeneous nucleation. Homogeneous processes and processes with homogeneous nucleation in such reactions are possible, for example, in the case of the formation of a metastable solid solution with its subsequent decomposition (the so-called internal reactions). An example of such processes can be internal oxidation.
The condition for thermodynamic equilibrium in a solid-state transformation, as in any other chemical transformation, is the equality of the chemical potentials of the components in the starting materials and reaction products. When two solid phases interact, this equality of chemical potentials can be realized in different ways: 1) redistribution of components in the initial phases with the formation of solid solutions; 2) the formation of new phases with a different crystal structure (which, in fact, is usually called a solid-phase reaction), and since the chemical potential of the component in various phases of a multiphase system does not depend on the amount of each phase, equilibrium can be achieved only with complete transformation of the initial phases. The most reliable information about the mechanism of solid-phase reactions is obtained with complex use, which makes it possible to simultaneously observe several parameters of the reacting system, including phase composition, thermal effects, mass changes, and more.
The thermodynamic theory of solid-state reactions was proposed by Wagner and further developed by Schmalzried using the example of addition reactions.
To date, there is no unified classification of a wide variety of heterogeneous reactions. This is due to the difficulty of choosing a criterion as the basis for such a universal classification. According to chemical criteria, reactions are divided into reactions of oxidation, reduction, decomposition, combination, exchange, etc. Along with the indicated criterion, it is widely used as the main criterion for the physical state of reagents:
A characteristic feature of all heterogeneous reactions is the existence and localization at the phase boundary of the reaction zone. The reaction zone, as a rule, of small thickness separates two regions of space occupied by substances of different composition and with different properties. The reasons for the formation of the reaction zone are usually divided into two groups: the relative slowness of diffusion processes and chemical reasons. The last group is due to the high reactivity of atoms or molecules located on the surface of a solid reagent or on the interface between two existing phases. It is known that the surface of a solid or liquid substance has properties that differ from the bulk properties of a compact sample. This makes the interface properties specific. It is here that a significant rearrangement of the crystal packing takes place, the stresses between the two crystal lattices decrease, and the chemical composition changes.
Since mass transfer is carried out by diffusion, and the diffusion mobility of solid particles depends on the defectiveness of its structure, one can expect a significant effect of defects on the mechanism and kinetics of solid-phase reactions. This stage precedes the chemical stage of the transformation of the reactants at the interfacial interface. Thus, the kinetics of heterogeneous reactions is determined both by the nature of the course of the chemical reaction itself and by the method of delivering the substance to the reaction zone. In accordance with the above, the reaction rate will be limited by the chemical stage (chemical kinetics) or diffusion (diffusion kinetics). Such a phenomenon is observed in reality.
According to Wagner, diffusion and, consequently, reaction in solids is carried out mainly due to the mobility of ions and electrons, due to the nonequilibrium state of the lattice. Different ions of the lattice move in it at different speeds. In particular, the mobility of anions in the vast majority of cases is negligible compared to the mobility of cations. Therefore, diffusion and, accordingly, the reaction in solids is carried out due to the movement of cations. In this case, the diffusion of unlike cations can go in the same direction or towards each other. In the case of differently charged cations, the electroneutrality of the system is preserved due to the movement of electrons. Due to the difference in the rates of movement of differently charged cations, an electric potential arises in the system. As a result, the rate of movement of more mobile ions decreases and, conversely, for less mobile? increases. Thus, the resulting electric potential regulates the ion diffusion rates. The latter and the rate determined by it of the entire solid-state transformation process can be calculated on the basis of electronic conductivity and transfer numbers. Obviously, directed diffusion of ions is possible only in an electric field or in the presence of a concentration gradient in the system.
In the synthesis of substances in the solid state, it is often necessary to control not only the chemical (elemental and phase) composition of the resulting product, but also its microstructural organization. This is due to the strong dependence of both chemical (for example, activity in solid-phase reactions) and many physical (magnetic, electrical, optical, etc.) properties on the characteristics of the structural organization of a solid at various hierarchical levels. The first of these levels includes the elemental composition of a solid and the method of mutual arrangement of the atoms of elements in space - the crystal structure (or features of the nearest coordination environment of atoms in amorphous solids), as well as the composition and concentration of point defects. As the next level of the structure of a solid body, we can consider the distribution of extended defects in a crystal, which determines the sizes of regions in which (corrected for the existence of point defects) translational symmetry in the arrangement of atoms is observed. Such regions can be considered perfect microcrystals and are called regions of coherent scattering. Speaking about the regions of coherent scattering, it must be remembered that, in the general case, they are not equivalent to compact particles forming a solid-phase material, which may contain a significant number of extended defects, and, consequently, regions of coherent scattering. The coincidence of regions of coherent scattering with particles (which in this case are called single-domain) is usually observed only for sufficiently small (less than 100 nm) sizes of the latter. Subsequent structural levels can be associated with the shape and size distribution of the particles forming the powder or ceramic material, their aggregation, aggregation of primary aggregates, etc.
Different applications of solid phase materials have different, often conflicting requirements for the structural characteristics listed above and therefore require different synthetic methods. Therefore, it is more correct to speak about the methods of synthesis not of solid-phase substances, but of solid-phase materials and, in each case, choose a synthesis method taking into account the field of subsequent application of the resulting product.
In the general case, methods for the synthesis of solid-phase materials can be classified according to their distance from the thermodynamically equilibrium conditions for the flow of chemical processes used. In accordance with the general laws, under conditions corresponding to a state as far as possible from equilibrium, a significant excess of the nucleation rate over the growth rate of the formed nuclei is observed, which obviously leads to obtaining the most dispersed product. In the case of carrying out the process near thermodynamic equilibrium, the growth of already formed nuclei occurs faster than the formation of new ones, which in turn makes it possible to obtain coarse-grained (in the limiting case, single-crystal) materials. The growth rate of crystals is also largely determined by the concentration of extended (nonequilibrium) defects in them.
Combinatorial synthesis can be carried out not only in solution (liquid-phase synthesis), but also on the surface of a solid chemically inert phase. In this case, the first starting material is chemically "sewn" to the functional groups on the surface of the polymer carrier (most often an ester or amide bond is used) and treated with a solution of the second starting material, which is taken in a significant excess so that the reaction goes to completion. There is a certain convenience in this form of reaction, since the technique for isolating products is facilitated: the polymer (usually in the form of granules) is simply filtered off, thoroughly washed from the remains of the second reagent, and the target compound is chemically cleaved from it.
In organic chemistry, there is not a single reaction that in practice provides quantitative yields of target products in any case. The only exception is, apparently, the complete combustion of organic substances in oxygen at high temperature to CO 2 and H 2 O. Therefore, purification of the target product is always an indispensable, and often the most difficult and time-consuming task. A particularly difficult task is the isolation of products of peptide synthesis, for example, the separation of a complex mixture of polypeptides. Therefore, it is in peptide synthesis that the method of synthesis on a solid polymer substrate, developed in the early 1960s by R.B. Merifield, has become most widely used.
The polymer carrier in the Merrifield method is a granular cross-linked polystyrene containing chloromethyl groups in benzene rings, which are linkers that bind the support to the first amino acid residue of the polypeptide. 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-stage cycle (deprotection - aminoacylation) can, in principle, be repeated as many times as required to build a polypeptide chain of a given length.
Further development of Merifield's ideas was directed, first of all, to the search for and creation of new polymeric materials for substrates, the development of methods for the separation of products and the creation of automated installations for the entire cycle of polypeptide synthesis.
The effectiveness of the Merifield method has been demonstrated by the successful synthesis of a number of natural polypeptides, in particular insulin. Especially clearly its advantages have been demonstrated on the example of the synthesis of the enzyme ribonuclease. So, for example, at the cost of considerable efforts, over the course of several years, Hirschman and 22 employees performed the 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 a total of 11,931 different operations, including 369 chemical reactions, was performed by two participants (Gatte and Merrifield) in just a few months.
Merrifield's ideas served as the basis for the creation of various methods for the combinatorial synthesis of libraries of polypeptides of various structures.
So in 1982, an original strategy for multi-stage parallel synthesis of peptides on the solid phase was proposed, known as the “split method” ( split- splitting, separation) or the “mix and split” method (Fig. 3). Its essence is as follows. Let's say that from three amino acids (A, B and C) you need to get all possible combinations of tripeptides. To do this, the granules of a solid polymer carrier (P) are divided into three equal portions and treated with a solution of one of the amino acids. In this case, all amino acids are chemically bound to the surface of the polymer by one of their functional groups. The obtained polymers of three grades are thoroughly mixed, and the mixture is again divided into three parts. Then each part, containing all three amino acids in equal amounts, is again treated with one of the same three amino acids and nine dipeptides are obtained (three mixtures of three products each). Another mixing, division into three equal parts and treatment with amino acids gives the desired 27 tripeptides (three mixtures of nine products) in just nine stages, while obtaining them separately would require a synthesis of 27 × 3 = 81 stages.
"Biologist. magazine Armenia, 1 (65), 2013 SOLID-PHASE SYNTHESIS OF CARDIAC-ACTIVE PEPTIDE ISOLATED FROM PIG ATRIUM G.S. CHAILYAN Institute of Biochemistry. Bunyatyan NAS RA..."
Experimental and theoretical articles
Experimental and theoretical articles
Biologist. magazine Armenia, 1 (65), 2013
SOLID-PHASE SYNTHESIS OF CARDIAC PEPTIDE,
ISOLATED FROM PIG ATRIUM
G.S. CHAILYAN
Institute of Biochemistry. Bunyatyan NAS RA
In order to continue research acad. Galoyan, we carried out a series of experiments to isolate, purify and determine the biological orientation of newly isolated peptide compounds from the atria and auricular parts of the pig's heart. To conduct biotests, it was necessary to obtain preparative amounts of the studied samples. To do this, we used the method of solid-phase synthesis of peptides with its further modification. The purity and identity of the synthesized preparations were checked by high performance liquid chromatography and mass spectral analysis.
Solid phase synthesis - fmoc-amino acids - HPLC - phenylisothiocyanate - mass spectral analysis:
fmoc- – – For further studies established by Galoyan, a series of experiments on the isolation, purification and determination of biological direction of peptides isolated from pigs atria were carried out. For fulfilling the biotests the preparative amount of samples was required. A modified method of solid phase peptide synthesis was used. The synthesized peptide purity and identity were defined by HPLC and mass-spectral analysis.
Solid phase synthesis – high performance liquid chromatography – mass spectrometry – fmoc-aminoacids – cardiopeptides – atria Galoyan et al. studied the ways of regulation and mechanisms of action of hypothalamic neurohormones on various processes in the body.
Confirmation of the idea of the interconnected, interdependent, integral functioning of such a system as the hypothalamus - pituitary gland - adrenal glands was a turning point in endocrinology. Replenishment of this concept SOLID-PHASE SYNTHESIS OF A CARDIAC ACTIVE PEPTIDE ISOLATED FROM THE PIG ATRIUM of the tual triad put forward by Galoyan regarding the interaction of the hypothalamus - pituitary gland
– heart, is a huge scientific achievement. Subsequently, a new tissue-target-heart was discovered, the ability of this organ to control the functioning of specific peptides was shown, as well as the existence of a feedback mechanism between the hypothalamus and the heart through these peptides.
The discovery of cardioactive compounds - the neurohormone K, C, G and a number of others in the hypothalamus of various animals served as the beginning of work not only on the study of the molecular mechanisms of action of these neurohormones, but also on the search for similar compounds in the heart. The basis for a comprehensive study of the biochemical and physicochemical properties of cardioactive principles was the data on the presence of 2 cardioactive compounds in the heart muscle. The participation of the neurohormone “C” in the regulation of glycolytic processes and the level of cyclic nucleotides was established through inhibition of PDE cAMP, cAMP-dependent protein kinase, etc. This compound has been shown to be low molecular weight and belongs to the glycopeptides.
In the Laboratory of Analytical Chromatography and Peptide Synthesis, we carried out work on the isolation and purification of peptide compounds from the atria and auricular areas of the pig's heart. During the separation of peptide fractions by preparative HPLC, we isolated and purified to a homogeneous state 20 compounds of peptide nature. To determine the biological focus, all preparations were tested for changes in ECG components in rats. The results of the experiments showed that 7 compounds have versatile factors of influence on certain components of the ECG.
Peptide No. 7 showed the greatest activity in changing the amplitude of the R component, the duration of the QRS complex, the S amplitude, and other parameters.
To study the biological mechanisms of action of this drug, it became necessary to have a large amount of the test sample. Due to the fact that the process of isolation and purification of biological products is extremely inefficient, laborious, time-consuming and cannot provide good reproducibility, it has become extremely important to carry out the chemical synthesis of this drug. Analyzing the world literature in the field of chemical synthesis of peptides, we came to the conclusion that the most optimal for us is the method of solid-phase synthesis using fmoc-protected amino acids. Discovered in 1984, Solid phase peptide synthesis has many advantages over conventional synthesis in terms of efficiency, as well as convenient processing and purification.
Using several different methods: hydrolysis of this drug, modification of amino acids with phenylisothiocyanate, we obtained the amino acid composition of peptide No. 7. Using the data of mass spectral and NMR analyzes, Edmon degradation, we were able to obtain not only the amino acid composition, but also the amino acid sequence of peptide No. 7.
Phe-Val-Pro-Ala-Met-Gly-Ile-Arg-Pro An efficient solid phase synthesis process largely depends on the correct choice of various conditions for its implementation, such as the choice of resin, solvent and synthesis kinetics. These variables affect the degree of swelling of the resin and its association with amino acids, the number of binding sites, which ultimately affects the synthesis of the peptide as a whole. We adapted the process of solid-phase synthesis in relation to our studied peptides, taking into account the peculiarities of their amino acid sequence.
G.S. CHAILYAN Material and methods. All reagents, solvents, resins used are from Advanced Chem Techcompany. We used fmoc groups to protect the N-terminus of amino acids during the synthesis and dimethylformamide (DMF) as a solvent during the entire synthesis. We used acid-labile 2-chlorotrityl resin as a substrate. The removal of the protective fmoc groups was carried out using a solution of piperidine in DMF.
Very important in the synthesis process is the landing of the first amino acid on the resin. Two grams of 2Cl-Trt resin was poured into a 10 ml syringe. DMF was drawn into the syringe, the resin was allowed to swell for 15 minutes. After that, the DMF was washed out. Then, a solution of the first amino acid (fmoc-Pro) and a reaction activator (DIPEA) were drawn into the syringe in a ratio of 1 RESIN/1.2 FMOC-PRO/4DIPEA. The reaction of planting the first amino acid lasted 3 hours. A very important condition for the synthesis is the absence of free linkers after planting the first amino acid, therefore, after binding to the first amino acid, the resin was treated with a mixture of methylene, DIPEA (diisopropylethylamine) and DMF in the ratio 80DMF/15MEOH/5DIPEA to block possibly remaining free ends. After that, the resin was washed with DMF 5 times for 5 minutes. Then, amino acids were deblocked with a 30% solution of piperidine in DMF 8 times for 5 minutes. After that, the resin was washed with DMF 5 times for 5 minutes. This cycle was repeated throughout the synthesis of the peptide. After each amino acid addition and deblocking step, the progress of the reaction was monitored by the Kaiser test, which is the reaction of ninhydrin with a free amino group to form a characteristic dark blue color. Thanks to this test, it became possible to step-by-step control of reactions of loading and deblocking of amino acids.
Purification and control of the synthesized peptide no. 7 were carried out on a 2-component preparative HPLC system from Waters (USA). A Rheodyne injector with a loop volume of 500 µl was used to inject the sample. Detection was carried out in the range of 190–360 nm. We used a “Symmetry Si-100 C18” (4.6x250 mm) column for reverse phase HPLC. The flow rate was 50 ml/min. A gradient eluent system H2O/ACN/TFA (98/2/0.1) / (0.100.0.1) was used. Analysis time 15 min. Rechromatography was performed on a Knauer HPLC analytical system. An XbridgeC18 column (2.6x150 mm) was used. Detection was carried out at 214 nm.
To confirm the obtained data, the synthesized preparation was subjected to mass spectral analysis on the CSU "Analitycal spectrometry".
Results and discussion. The data obtained on the chromatogram suggest that the purity of the synthesized peptide No. 7 after purification is more than 99.6% (Fig. 1).
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We carried out a comparative chromatographic analysis of native peptide No. 7 on an X-bridgeC18 column under the same conditions as its synthesized analogue. The comparison results are presented (Fig. 2).
Solid-Phase Synthesis of a Cardioactive Peptide Isolated from Pig Atria
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Rice. Fig. 4. Spectrograms of synthesized (A) and native (B) preparations.
G.S. CHAILYAN As can be seen from the comparison of chromatograms, the synthesized analog and native peptide No. 7 are identical in mass and release time, which indicates the identity of their structures and amino acid sequence. Thus, using the method of solid-phase peptide synthesis and taking into account the structural features of the studied peptide, we managed to obtain a homogeneous and identical to the native peptide consisting of 9 amino acids. In the future, having a sufficient amount of the drug, we plan to conduct a series of biotests to identify not only the ways of regulating cardiac activity by this peptide, but also the mechanisms of action on other organs and systems.
LITERATURE
Popova T.V., Srapionyan R.M., Galoyan A.A. Detection and identification in the heart of a bull new 1.
cardioactive proteins. Question. honey. Chemistry, 37, 2, p. 56-58, 1991.
Srapionyan R.M., Sahakyan S.A., Sahakyan F.M., Galoyan A.A. Isolation and characterization 2.
cardioactive tryptic fragment of neurohormone C carrier protein. Neurochemistry, 2, 3, p. 263-271, 1983.
Srapionyan, R.M. Misiryan, S.S. Separation of low molecular weight corona active compounds 3.
cardiac muscle by a combination of gel filtration and polyacrylamide gel electrophoresis. Biologist. magazine Armenia, 27, 10, 102-104, 1974.
4. Srapionyan, R.M. Popova, T.V. Galoyan, A.A. Distribution of cardioactive protein complexes in the heart of various animals. Biologist. magazine Armenia, 40, 7, 588-590, 1987.
5. Galoyan A. The Regulation of Neurosecretion and Hormones of Hypothalamo-Neurohypophyseal System, USSR, 1963.
6. Galoyan A.A. Biochemistry of Novel Cardioactive Hormones and Immunomodulators of the Functional System Neurosecretory Hypothalamus, Endocrine Heart. Science Publ. p. 240, 1997.
7. Galoyan A.A., Besedovsky H. Handbook of Neurochemistry and molecular Neurobiology, 3rd edition, Springer Publishers, 500 p., 2008.
8. Galoyan A.A., Brain Neurosecretorycytokins: Immune Response and Neuronal Survival, VIII, 188 p., 2004.
9. Galoyan A.A., Srapionyan R.M. The purification of coronarodilatory proteins isolated from the hypothalamus. Dokl. Akad. NaukArm.SSR, 42, 4, p. 210-213, 1966.
10. March J., Smith M. March's advanced organic chemistry. Published by John Wiley & Sons, Inc., Hoboken, New Jersey, p. 133, 2007.
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The invention relates to a solid phase method for the synthesis of a peptide of the formula H-D--Nal--Thr-NH 2 , which uses both Boc-protected and Fmoc-protected amino acids and a chloromethylated polystyrene resin. 10 z.p. f-ly.
The field of technology to which the invention belongs
The present invention relates to a method for preparing a peptide containing three or more amino acid residues, having an N-terminal amino acid, a penultimate amino acid adjacent to the N-terminal amino acid, and a C-terminal amino acid.
Prior Art
Solid phase peptide synthesis was introduced in 1963 to overcome many of the problems of intermediate purification steps associated with solution synthesis of peptides (Stewart et. al. Solid Phase Peptide Synthesis. Pierce Chemical Co., 2nd ed., 1984). In solid phase synthesis, amino acids are assembled (eg, joined) into a peptide in any desired sequence, while one end of the chain (eg, C-terminus) is attached to an insoluble carrier. Once the desired sequence has been assembled on the carrier (support), the peptide is then released (ie cleaved) from the carrier. The two standard protecting groups for the α-amino groups of amino acids to be linked are Boc, which is removed with a strong acid, and Fmoc, which is removed with a base. The present invention relates to a convenient method for the production of peptides using a combination of both of these protections for α-amino groups in one synthesis on an inexpensive resin from chloromethylated polystyrene.
When designing solid phase peptide synthesis using any of the above α-amino protection schemes, it is important that any reactive "side groups" of the amino acids that make up the peptide are protected from unwanted chemical reactions during chain assembly. It is also desirable that the chemical groups chosen to protect the various side groups are not removed by the reagents used to deprotect the .beta.-amino groups. Thirdly, it is important that the bond of the growing peptide chain to the resin particle is resistant to the reagents used in the chain assembly process to remove any type of α-amino protection. In the case of an α-amino protection scheme using Fmoc, the side group protection must be resistant to the alkaline reagents used to remove the Fmoc. In practice, these side chain protecting groups are usually removed with mildly acidic reagents after the assembly of the peptide chain has been completed. If a β-amino group protection scheme using Boc is used, the side group protection must be resistant to the weakly acidic reagent used to remove the Boc group in each cycle. In practice, these side chain protecting groups in the β-amino protection scheme with Boc are usually removed with anhydrous HF after peptide chain assembly is complete. Thus, in practice, the commonly used side chain protection groups in the α-amino protection scheme with Fmoc are not stable under the conditions used to deprotect the α-amino groups with Boc. Therefore, two types of protection schemes for α-amino groups are not combined during the assembly of the peptide chain in solid-phase peptide synthesis. In addition, although the cheapest polymer resin used in peptide synthesis (chloromethylated polystyrene or "Maryfield resin") is widely used together with amino acids protected by Boc groups, it has been concluded in the literature that it is not applicable in the case of protection of α-amino groups with Fmoc groups due to its instability under alkaline conditions (see Stewart et. al. Solid Phase Peptide Synthesis. Pierce Chemical Co., 2nd ed., 1984). The present invention is directed to a method for co-using certain peptides in solid phase synthesis of both Boc-protected and Fmoc-protected amino acids on a Merifield resin.
Lanreotide®, which is a somatostatin analog, is known to inhibit growth hormone release and also inhibit insulin, glucagon, and exocrine pancreatic secretion.
US Patent No. 4,853,371 discloses and claims Lanreotide®, a process for its preparation, and a method for inhibiting the secretion of growth hormone, insulin, glucagon, and exocrine pancreatic secretion.
US Patent No. 5147856 discloses the use of Lanreotide® for the treatment of restenosis.
US Patent No. 5411943 discloses the use of Lanreotide® for the treatment of hepatoma.
US Patent No. 5073541 discloses the use of Lanreotide® for the treatment of lung cancer.
US Patent Application No. 08/089410, filed July 9, 1993, discloses the use of Lanreotide® for the treatment of melanoma.
US Pat. No. 5,504,069 discloses the use of Lanreotide® to inhibit accelerated solid tumor growth.
US Patent Application No. 08/854941, filed May 13, 1997, discloses the use of Lanreotide® for weight loss.
US Patent Application No. 08/854,943, filed May 13, 1997, discloses the use of Lanreotide® for the treatment of insulin resistance and Syndrome X.
US Patent No. 5688418 discloses the use of Lanreotide® to prolong the viability of pancreatic cells.
PCT Application No. PCT/US 97/14154 discloses the use of Lanreotide® for the treatment of fibrosis.
US Patent Application No. 08/855311, filed May 13, 1997, discloses the use of Lanreotide® for the treatment of hyperlipidemia.
US Patent Application No. 08/440061, filed May 12, 1995, discloses the use of Lanreotide® for the treatment of hyperamylinemia.
US Patent Application No. 08/852221, filed May 7, 1997, discloses the use of Lanreotide® for the treatment of hyperprolactinemia and prolactinomas.
The essence of the invention
The present invention provides a method for preparing a peptide containing three or more amino acid residues, having an N-terminal amino acid, a penultimate amino acid adjacent to the N-terminal amino acid, and a C-terminal amino acid, said method comprising the following steps:
(a) attaching the first amino acid to the solid support resin by an ether bond to form the first coupling product, which includes (i) reacting an aqueous solution of cesium carbonate with an alcoholic solution of the first amino acid to form a cesium salt of the first amino acid, (ii) obtaining a solvent-free cesium salt of the first amino acid, (iii) reacting the solid support resin with the cesium salt of the first amino acid in a dry (anhydrous) polar aprotic solvent to form the first addition product,
where the first amino acid corresponds to the C-terminal amino acid of the peptide, the amino group of the non-side (main) chain of the first amino acid is blocked by Boc, and the first amino acid does not have a functional group in the side chain that requires protection, and the solid support - resin - is a resin of chloromethylated polystyrene;
(b) deprotection (deblocking) Boc from the product of the first accession with an acid to form a deblocked product of the first accession;
(c) Optionally, attaching the next amino acid to the deblocked first attachment product, which includes reacting the next amino acid with the deblocked first attachment product in an organic solvent containing a peptide growth reagent to obtain a blocked (protected) next attachment product, wherein the next amino acid has in the main chain an amino group blocked by Boc, and if the next amino acid has one or more functional groups in the side chain, then the functional groups in the side chain do not require protection or the functional groups in the side chain have protective groups that are resistant to the acid or alkaline reagents used to remove the protection, respectively, Boc and Fmoc;
(d) deprotecting Boc from the blocked next adduct, which includes reacting the blocked next adduct with an acid to obtain a deprotected next adduct;
(e) optionally, repeating steps (c) and (d), with each cycle generating a released product of the (X+1)th next attachment, where X is the number of the required repetition of the cycle;
(f) adding the next amino acid to the released first attachment product from step (b) or, optionally, to the released (X+1)th next attachment product from step (e), which involves reacting the next amino acid with said first attachment product or with the specified deblocked product of the (X+1)-th next accession in an organic solvent containing a reagent for growing the peptide to obtain a blocked (protected) product of the next accession, and the next amino acid has a blocked Fmoc amino group of the main chain, provided that if the next amino acid has one or more functional groups in the side chain, then the functional groups in the side chain do not require protection, or the functional groups in the side chain have protective groups that are resistant to the alkaline reagents used to deprotect Fmoc;
(g) deprotecting Fmoc from the blocked next adduct, which includes reacting the blocked next adduct with a primary or secondary amine to obtain a deprotected next adduct;
(h) optionally, repeating steps (e) and (g), with each cycle generating a deblocked product of the (X+1)th next addition, where X is the number of the necessary repetition of the cycle, until the penultimate one is included in the peptide and deblocked amino acid;
(i) attaching the N-terminal amino acid to the deprotected (X+1)th next accession product, which includes reacting the N-terminal amino acid with the deprotected (X+1)th next accession product in an organic solvent containing a peptide growth reagent , to obtain a blocked end product, wherein the N-terminal amino acid has a backbone amino group blocked by Boc or Fmoc;
(j) deprotecting Boc or Fmoc from the blocked completed addition product, comprising reacting the blocked completed addition product with an acid in the case of Boc or a base in the case of Fmoc to form the completed peptide product on the resin;
(j) if the completed peptide product on the resin has side chain functional groups, then optionally deprotecting the side chain functional groups of the completed peptide product on the resin, which includes reacting the completed peptide product on the resin with suitable deprotecting reagents to obtain the completed deprotected peptide product on resin; And
(k) cleaving the peptide from the solid resin carrier of the completed peptide product on resin or the completed peptide product on deprotected resin to obtain a peptide, which comprises reacting the completed peptide product on resin or the completed peptide product on deprotected resin with ammonia, primary amine or secondary amine to the practical completion of the cleavage of the peptide from the resin;
with the proviso that steps (e) and (g) in the synthesis of the peptide must be carried out at least once.
Preferred is the process according to the present invention wherein the ammonia, primary amine or secondary amine in step (k) is in a solvent containing an alcohol and optionally an aprotic polar solvent,
Preferred is the method according to the present invention, where step (l) further comprises the following steps:
precipitation of the cleaved peptide from the solvent;
separating by filtration the solid resin support and the precipitated peptide, and
extracting the peptide with an acidic solution to isolate the peptide.
Preferred is the method according to the present invention, where the first amino acid is Boc-L-Thr.
Preferred is the method according to the present invention, wherein the first amino acid is the cesium salt of Boc-L-Thr, yielding Boc-L-Thr resin as the first coupling product, and the deblocked first coupling product is H-L-Thr resin.
Preferred is the process of the present invention wherein the acid used to remove the Boc protecting group in step(s) is trifluoroacetic acid (TFA).
The preferred method, related to the immediately preceding process, is where the organic solvent is methylene chloride, chloroform, or dimethylformamide and the peptide growth reagent is diisopropylcarbodiimide, dicyclohexylcarbodiimide, or N-ethyl-N"-(3-dimethyl- aminopropyl)carbodiimide.
The preferred method, related to the immediately preceding method, is a method comprising carrying out steps (e) and (g) six times after the formation of a deblocked product of the first attachment of the formula H-L-Thr-resin, where subsequent amino acids are attached in the order: Fmoc-L-Cys( Acm), Fmoc-L-Val, Fmoc-L-Lys(Boc), Fmoc-D-Trp, Fmoc-L-Tyr(O-t-Bu) and Fmoc-L-Cys(Acm) to form the product H-Cys( Acm)-Tyr(O-t-Bu)-D-Trp-Lys(Boc)-Val-Cys(Acm)-Thr-resin.
The preferred method, relating to the immediately preceding method, is a method comprising the addition of Boc-D--Nal to H-Cys(Acm)-Tyr(O-t-Bu)-D-Trp-Lys(Boc)-Val-Cys(Acm) -Tnr-resin according to step (c) to obtain Boc-D--Nal-Cys(Acm)-Tyr(O-t-Bu)-D-Trp-Lys(Boc)-Val-Cys(Ast)-Thr-resin.
The preferred method, related to the immediately preceding method, involves the simultaneous removal of the Boc group protecting D--Nal, the O-t-Bu group protecting Tyr, and the Boc group protecting Lys in Boc-D--Nal-Cys(Acm)-Tyr( O-t-Bu)-D-Trp-Lys(Boc)-Val-Cys(Acm)-Thr-resin according to step (i), to obtain a completed peptide product on the resin of the formula H-D- -Nal-Cys(Acm)-Tyr- D-Trp-Lys-Val-Cys(Acm)-Thr-resin.
The preferred method, related to the immediately preceding method, comprises cleavage of the H-D-β-Nal-Cys(Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr peptide from the solid resin by carrying out the reaction H-D-βNal-Cys (Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-resin with ammonia in a solvent containing an alcohol and optionally an aprotic polar solvent to substantially complete elimination to give H-D--Nal-Cys (Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-NH 2 .
The preferred process, related to the immediately preceding process, is where the alcohol is methanol and the polar aprotic solvent is dimethylformamide.
The preferred method, related to the immediately preceding method, involves the simultaneous removal of the Acm groups protecting Cys and cyclization of the resulting deprotected Cys residues in the completed peptide product of the formula H-D--Nal-Cys(Acm)-Tyr-D-Trp-Lys-Val -Cys(Acm)-Thr-NH 2 by carrying out the reaction of H-D- -Nal-Cys(Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-NH 2 with a solution of iodine in alcohol to almost complete deprotection and cyclization to give H-D--Nal--Thr-NH 2 .
The preferred method, related to the immediately preceding method, is the method where the peptide is H-D--Nal--Thr-NH 2 .
A preferred method, related to the immediately preceding method, is one wherein the peptide is a somatostatin analog.
The terms used in the description of the present invention are defined as follows:
"first amino acid": encompasses any amino acid in which the amino group in the main chain (not in the side) is protected by Boc, which is a commercial product or can be synthesized according to methods known to a person of ordinary skill in the art, for example Boc-L-Thr;
"first attachment product": describes a product that is attached to a solid carrier resin that results from the addition of a first amino acid to a solid carrier resin, eg Boc-L-Thr resin;
"deblocked first coupling product": describes the product resulting from the removal or removal of the Boc group from the first coupling product - for example, H-L-Thr-resin, where "H" is the available hydrogen of the amino group of the main chain, resulting from the deprotection step;
"next amino acid": describes any amino acid in which the amino group in the main chain is protected by Boc or Fmoc, which is commercially available or can be synthesized according to methods known to one of ordinary skill in the art. Since step (c) and step (e) may be included in a repeating cycle where step is performed more than once, each time step (c) or step (e) is performed, the "next amino acid" may be independently selected from a group known or likely to be synthesized amino acids in which the amino group in the main chain is protected by Boc or Fmoc;
"blocked product of the (X+1)-th next accession": describes the product attached to the solid support resin, which is the result of the connection of the next amino acid with the "deblocked product of the next accession". Since steps (c) and (d) and steps (e) and (g) can be included in a repeating cycle where the following amino acids can be attached, the term "blocked product of the (X+1)th next attachment" refers to the product obtained as a result of each of the previous cycles of accession;
"unblocked product of the (X+1)th next accession": describes the product resulting from the removal of the Fmoc group from the "blocked product of the (X+1)th next accession";
"completed peptide product on resin": describes a peptide product attached to a solid support resin after an N-terminal amino acid has been attached to the peptide chain and after the amino group of the N-terminal amino acid backbone has been deprotected or deblocked, but which still has any protecting groups on the functional groups of the side chains, not removed by the reaction, carrying out the removal of the protective group from the main chain of the N-terminal amino acid; And
"completed peptide product on a deprotected resin": describes a peptide product attached to a solid resin support where all the protecting groups have been removed or deprotected from the functional groups of the amino acid side chains.
Examples of acids that can be used to deprotect Boc are trifluoroacetic acid (TFA), methanesulfonic acid, and organic solutions containing HCl.
Examples of primary and secondary amines that can be used to deprotect Fmoc are 4-(aminomethyl)piperidine, piperidine, diethylamine, DBU and tris(2-aminoethyl)amine.
Examples of non-nucleophilic bases that can be used to neutralize TFA salts of freed amino groups (RNH 3 + CF 3 COO - these salts must be converted to "free" amines (NH 2) before or during the addition of the next amino acid, otherwise the addition will not take place) are diisopropylethylamine (DIEA) and triethylamine (TEA).
Examples of organic solvents that can be used in amino acid addition reactions are methylene chloride, chloroform, dichloroethane, dimethylformamide, diethylacetamide, tetrahydrofuran, ethyl acetate, 1-methyl-2-pyrrolidone, acetonitrile, or a combination of these solvents.
Examples of peptide extenders include substituted carbodiimides such as: diisopropylcarbodiimide, dicyclohexylcarbodiimide, or N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide.
Carboxyl groups and amino groups that participate in the formation of a peptide amide bond are referred to as a "side chain" carboxyl group or amino group, respectively. On the other hand, any amino acid functional groups that do not participate in the formation of a peptide amide bond are referred to as "side chain" functional groups.
The term "base-resistant group" refers to protecting groups used to protect amino acid functional groups that (1) are base-resistant, e.g., cannot be removed by bases such as 4-(aminoethyl)piperidine, piperidine, or tris(2 -aminoethyl)amine, which are bases commonly used to remove the Fmoc protecting group, and (2) can be removed with an acid such as trifluoroacetic acid or by another method such as catalytic hydrogenation.
The symbols "Fmoc" and "Boc" are used here and in the accompanying formula to denote 9-fluorenylmethoxycarbonyl and t-butyloxycarbonyl, respectively.
The method described above can be used to prepare peptides, preferably somatostatin analogues, such as Lanreotide® octapeptide, which has the following formula: H-D--Nal--Thr-NH 2 . If H-D--Nal--Thr-NH 2 is to be synthesized, the base-resistant protecting groups used to protect the Cys, Lys and Tyr side chain functional groups can be acetamidomethyl (Acm), Boc and tert-butyl, respectively. Asm is preferred over Cys.
By somatostatin analog is meant a peptide that exhibits a biological activity similar (ie, agonist) or opposite (ie, antagonist) to that of somatostatin.
In the formula H-D--Nal--Thr-NH 2 , each of the usual three-letter amino acid symbols (eg, Lys) refers to a structural amino acid residue. For example, the symbol Lys in the above formula represents -NH-CH((CH 2) 4 NH 2)-CO-. The symbol D- -Nal- represents the amino acid residue D-2-naphthylalanilyl. The brackets denote a disulfide bond linking the free thiols of two Cys residues in the peptide, indicating that the amino acids of the peptide inside the brackets form a cycle.
Based on the description given here, a person skilled in the art will be able to most fully use the present invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. In addition, all publications, patent applications, patents and other references cited herein are incorporated herein by reference to them.
The peptide can be prepared in accordance with the method of the present invention according to the following procedure.
A solution of 0.5 molar equivalents of cesium carbonate in water is slowly added to a solution of 1 molar equivalents of Boc-AA 1 (Bachem California, Torrance, CA) where AA 1 corresponds to the C-terminal amino acid dissolved in alcohol, preferably methanol. The resulting mixture was stirred for about 1 hour at room temperature, then all alcohol and all water were removed under reduced pressure to give a dry powder of Boc-AA 1 cesium salt. Merifield resin, 1.0 equivalents (chlor-methylated polystyrene, 200-400 mesh, chloride ion incorporation 1.3 meq/g, Advanced ChemTech, Louisville, Kentucky or Polymer Laboratories, Church Stretton, England) is washed with a chlorinated solvent, preferably dichloromethane ( DCM), an alcohol, preferably methanol, and a polar aprotic solvent, preferably dimethylformamide (DMF). Cesium salt Boc-AA 1 powder is dissolved in an anhydrous (dry) polar aprotic solvent, preferably DMF, and the solution is combined with the previously washed resin. The slurry is stirred gently at about 45°-65°C, preferably at 50°-60°C, for about 48 to 106 hours, preferably 85 to 90 hours, under an inert atmosphere such as nitrogen. The resin is separated by filtration and washed thoroughly with a polar aprotic solvent, preferably DMF, water and finally an alcohol such as MeOH. Boc-AA 1 resin is dried under reduced pressure.
Boc-AA 1 -resin is introduced into a glass reactor with a filter bottom made of coarse fused glass. The resin is washed with a chlorinated solvent such as DCM, deblocked with an organic acid, preferably 25% TFA in DCM, briefly washed with a chlorinated solution such as DCM and an alcohol such as MeOH, neutralized with an organic base, preferably triethylamine in DCM, and washed again with DCM and a polar aprotic solvent such as DMF to give a deprotected AA 1 resin.
Any desired number of amino acids is then optionally attached to the deprotected AA 1 resin. If the next amino acid has an α-amino group with Fmoc protection (Fmoc-AA x), then the side chain group either does not require protection (for example, Fmoc-Gly, Fmoc-Ala, Fmoc-Phe, or Fmoc-Thr) or the side chain does. protect with a base-resistant group. A molar excess of Fmoc-AA x (where x is the position number of the amino acid in the peptide, counted from the C-terminus) is attached for approximately 60 minutes to the deprotected AA 1 resin with a peptide growth reagent such as diisopropylcarbodiimide (DIC), in a DCM/DMF mixture. The addition resin was washed with DMF, alcohol and DCM to give a Fmoc-AA x -AA 1 resin. Attachment can be checked with the Kaiser ninhydrin method. Then, the Fmoc-AA x -AA 1 resin is washed once with DMF and then deblocked with a solution of a base in an organic solvent such as piperidine in DMF to obtain an AA x -AA 1 resin. The AA x -AA 1 resin is then washed with DMF, followed by washing several times with both an alcohol such as MeOH and DCM. The AA x -AA 1 resin is then washed once with DMF for about 3 minutes, three times with isopropanol, preferably for about 2 minutes each time, and three times with DCM, preferably for about 2 minutes each time. The resin is then ready for further attachment of either an Fmoc-protected amino acid as described above or a Boc-protected amino acid as described below.
Similarly, if any subsequent amino acid to be attached to the deprotected AA 1 resin is selected with a protected Boc-amino group (Boc-AA x), then either no protection is required for the side chain group (this can be Boc-Gly, Boc- Ala, Boc-Phe or Boc-Thr), or the side chain must be protected with a group resistant to removal by both acid and base, which may be Boc-Cys(Acm). If Boc-AA x is selected, it is attached using the same reagents and solvents as described above for Fmoc-amino acids, and completeness (completion) of attachment can be checked by the Kaiser ninhydrin method. Thereafter, the Boc-AA x -AA 1 resin is deprotected with an acid solution in an organic solvent such as TFA in DCM to give a CF 3 CO - H + -AA x -AA 1 resin. This resin is then washed several times with a chlorinated solvent such as DCM, an alcohol such as MeOH, and neutralized with a non-nucleophilic base such as triethylamine in DCM, and then washed several more times with a chlorinated solvent such as DCM to give AA x -AA 1 - resin. The resin is then ready for further attachment of the protected Boc or Fmoc amino acid as described above.
Depending on the desired sequence of the peptide and the type of α-amino protected amino acid used (either Fmoc protected or Boc protected), a suitable combination of the above attachment procedures is used, depending on which amino acid is to take place in the peptide sequence - side chain , having a protective group that can be removed with either the base necessary to remove Fmoc from the α-amino group, or the acid necessary to remove Boc from the α-amino group. Such a protected amino acid may be N-β-Boc-N″-β-Fmoc-lysine or N-β-Fmoc-N″-β-Boc-lysine. If this is the case, all selectable protecting groups for the α-amino groups of subsequent amino acids, up to the N-terminal amino acid, must be compatible with the side group protection selected for that position. This means that the side chain protecting groups must be resistant to the deblocking agent used to deprotect the α-amino groups of subsequent amino acids. For the N-terminal amino acid, either Boc or Fmoc can be used as the α-amino protection, since deprotection of the N-terminal amino acid can simultaneously deprotect some of the protected side chains without undesirably affecting the peptide synthesis strategy, since no amino acids are available anymore. are added.
The completed peptide chain, which is still attached to the resin, must be deprotected and released. To remove all base-resistant protecting groups and the α-amino blocking group of the N-terminal amino acid, if applicable, the peptide on the resin is treated with an acid in an organic solvent such as TFA in DCM. To remove any acid-resistant protecting groups and the α-amino blocking group of the N-terminal amino acid, if applicable, the peptide on the resin is treated with an organic base such as piperidine in DMF. Alternatively, the acid-resistant groups may be retained until removed upon subsequent cleavage of the peptide with ammonia or an amine base. The peptide on the deprotected resin is then washed with a chlorinated solvent such as DCM, an alcohol such as MeOH and dried to constant weight under reduced pressure.
The peptide is cleaved from the resin and the C-terminus is converted to the amide by suspending the peptide on the resin in 3:1 MeOH/DMF. The slurry is cooled to a temperature below about 10° C. under a nitrogen atmosphere and anhydrous ammonia gas is introduced under the surface of the solvent until the solution is saturated with it, while the temperature is maintained below about 10° C. The slurry is gently stirred for about 24 hours while allowing the temperature to rise to about 20°C. The degree of completion of the reaction is checked by the disappearance of the methyl ester intermediate in HPLC under suitable conditions depending on the type of peptide. The reaction mixture is cooled and the required amount of anhydrous ammonia is added until the peak area corresponding to methyl ester on HPLC is less than 10% of the peak area of the desired product. The slurry is cooled to below about 10° C. and stirring is continued overnight to precipitate the peptide. The precipitate and resin are separated by filtration and washed with cold MeOH. The precipitate and resin are dried under reduced pressure, the product is extracted from the resin with an aqueous solution of acetic acid.
If the peptide contains protected Cys residues in its sequence, the thiol groups can be deprotected and the residues cyclized according to the following procedure. The peptide containing the protected Asm groups of the Cys is dissolved in an aqueous solution of acetic acid under a nitrogen atmosphere. The solution is stirred rapidly and a solution of iodine in alcohol is added in one portion. The mixture is stirred and checked by HPLC for complete deprotection. Then the reaction is stopped by titration with 2% sodium thiosulfate solution until the color of the solution disappears. The crude mixture was purified by preparative chromatography on a C8 cartridge with a gradient of acetonitrile in 0.1 ammonium acetate buffer, desalted on a C8 cartridge with a gradient of acetonitrile in 0.25 N acetic acid, and lyophilized to give the target peptide.
An exemplary embodiment of the invention
The following example is provided to illustrate the method of the present invention and should not be construed as limiting its scope.
Example 1. H 2 -D- -Nal--Thr-NH 2
A) Boc-L-Thr-resin
A solution of 2.58 g of cesium carbonate in 2.5 ml of water was slowly added to a solution of 3.48 g of Boc-L-threonine (Bachem California, Torrance, CA) dissolved in 7 ml of methanol. The resulting mixture was stirred for approximately 1 hour at room temperature, then all methanol and all water were removed under reduced pressure to give a dry powder of Boc-L-threonine cesium salt. 10 g of Maryfield resin (chloromethylated polystyrene, 200-400 mesh, chlorine incorporation 1.3 meq/g, Advanced ChemTech, Louisville, Kentucky) was washed with dichloromethane (DCM), methanol (MeOH) and dimethylformamide (DMF) (each 2 times 70 ml). Boc-L-threonine cesium salt powder was dissolved in 60 ml of dry DMF and the solution was combined with the resin washed as above. The slurry was gently stirred at a temperature of approximately 50°-60°C for approximately 85 to 90 hours under nitrogen. The resin was separated by filtration and washed thoroughly with DMF, deionized water and finally with MeOH. The Boc-threonine resin was dried under reduced pressure at approximately 40°C. The inclusion of threonine was 0.85±0.15 meq/g dry resin.
B) H-D- -Nal-Cys(Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-resin
2.0 g of the Boc-threonine resin from step (A) was introduced into a 50 ml glass reactor with a coarse fused glass filter bottom (load 1.74 mmol). The resin was washed 2 times with DCM (20 ml), each time for approximately 5 minutes, deblocked with 25% TFA in DCM (30 ml) - the first time for approximately 2 minutes and the second time for approximately 25 minutes, washed 3 times for approx. 2 min DCM (20 ml), isopropanol (20 ml) and DCM (20 ml), neutralized twice for approx. 5 min with 10% triethylamine in DCM (20 ml), washed 3 times for approx. 2 min with DCM and washed once with DMF (20 ml) for about 5 min.
To the deblocked resin was added 1.8 g (4.35 mmol, 2.5 eq.) of Fmoc-L-cysteine(Acm) (Bachem, CA) and 683 μl (4.35 mmol, 2.5 eq.) of diisopropyl- carbodiimide (DIC) in 14 ml of 2:1 DCM/DMF for approximately 1 hour. 2 min DXM (20 ml). Binding was checked by the Kaiser nihydrin method.
After attachment, the resin was washed 1 time with DMF and then deblocked with a solution of piperidine in DMF. The deblocked resin was then washed with DMF and washed several times simultaneously with MeOH and DCM. The coupling resin was washed 1 time for about 3 minutes with DMF (20 ml), 3 times for about 2 minutes with isopropanol (20 ml) and 3 times with DCM (20 ml) for about 2 minutes each time. Binding was tested by the Kaiser ninhydrin method.
Each of the following protected amino acids was attached to the washed resin using DIC in DMF/DCM and released as described above in the following sequence: Fmoc-L-valine, Fmoc-L-lysine(Boc), Fmoc-D-tryptophan, Fmoc-L-tyrosine (O-t-Bu) and Fmoc-L-cysteine (Acm) (all from Bachem California), Boc-D-2-naphthylalanine (Synthethech, Albany, OR).
The completed peptide chain was deblocked and protected twice with 75:20:5 DCM/TFA/anisole (30 ml) for about 2 minutes and about 25 minutes, washed 3 times for about 2 minutes each time with DCM (20 ml), isopropanol (10 ml) and DCM (20 ml), neutralized 2 times for about 5 min with 10% triethylamine in DCM (20 ml) and washed 3 times for about 2 min with DCM (20 ml) and MeOH (20 ml) . The resin was dried under reduced pressure. The dry weight was 3.91 g (103% of theoretical yield).
B) H-D- -Nal-Cys(Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-NH 2
2.93 g of the peptide-loaded resin from step (B) (1.3 mmol-eq.) was suspended in 50 ml of a 3:1 MeOH/DMF mixture. The slurry was cooled to a temperature below about 10° C. under a nitrogen atmosphere and dry ammonia gas was purged until the solution was saturated with it, while the temperature was maintained below about 10° C. The slurry was gently stirred for about 24 hours, allowing the temperature to rise to about 20°C. The degree of completion of the reaction was checked by the disappearance of the methyl ether intermediate using HPLC (VYDAC® sorbent, grain size 5 μm, pore size 100 Å, C18, isocratic elution with 26% CH 3 CN in 0.1% TFA, speed 1 ml /min, recording at 220 mm; under these conditions, the retardation time Rt ~ 14 min for the methyl ester and ~ 9.3 min for the amide product). The reaction mixture was cooled and an excess of anhydrous ammonia was added until the peak area corresponding to methyl ester on HPLC was less than 10% of the peak area of the desired product. The slurry was cooled to a temperature below approximately 10°C, stirring was continued overnight to precipitate the peptide. The precipitate and resin were separated by filtration and washed with 15 ml of cold MeOH. The precipitate and resin were dried under reduced pressure, the product was extracted from the resin with 50% aqueous acetic acid solution (3 x 30 ml). HPLC analysis showed 870 mg (0.70 mmol) of the title product in the mixture (96% pure in isocratic HPLC system).
D) H-D- -Nal--Thr-NH 2
500 mg (0.40 mmol) of the peptide from step (B) was dissolved in 300 ml of 4% acetic acid and heated to about 55° C. under nitrogen. The solution was stirred rapidly and a 2% w/v solution of iodine in 7.7 ml of MeOH (0.60 mmol) was added in one portion. The mixture was stirred for approximately 15 min, then the reaction was stopped by titration with 2% sodium thiosulfate solution until the color disappeared (~2 ml). The mixture was cooled to room temperature and filtered. The mixture was purified by preparative chromatography on a C8 column (YMC, Inc., Wilmington, NC) with a gradient of acetonitrile in 0.1 M ammonium acetate, desalted on a C8 YMC column with a gradient of acetonitrile in 0.25 N acetic acid, and lyophilized to give 350 mg of target peptide in 99% purity.
Based on the above description, a person skilled in the art can easily recognize the essential features of the present invention and, without departing from its spirit and scope, make various changes and modifications to the invention to adapt it to various applications and conditions. Thus, other embodiments of the invention are also covered by the claims.
CLAIM
1. A method for preparing a peptide of the formula H-D--Nal--Thr-NH 2 , said method comprising the following steps:
(a) attaching a first amino acid to a solid support resin by an ether bond to form a "first coupling product", which includes (i) reacting an aqueous solution of cesium carbonate with an alcoholic solution of the first amino acid to form the cesium salt of the first amino acid, (ii) obtaining a solvent-free cesium salt of the first amino acid, (iii) reacting the solid support resin with the cesium salt of the first amino acid in an anhydrous polar aprotic solvent to form a "first addition product",
where the first amino acid is Boc-L-Thr, which corresponds to the C-terminal amino acid of this peptide, and the solid media resin is a resin of chloromethylated polystyrene;
(b) deprotecting the Boc from the first addition product with an acid to form a "deprotected first addition product";
(c) Optionally, adding to the “deblocked first attachment product” the “next amino acid”, which includes reacting the “next amino acid” with the “deblocked first attachment product” in an organic solvent containing the peptide growth reagent to obtain the “blocked next amino acid product”. addition", whereby the "next amino acid" has a Boc-blocked amino group in the main chain, and if this "next amino acid" has one or more functional groups in the side chain, then the functional groups in the side chain do not require protection or these functional groups in the side chain have protecting groups that are stable to acidic or alkaline deprotection agents, respectively, Boc and Fmoc;
(d) deprotecting Boc from the "blocked next product" which includes reacting the "blocked next product" with an acid to obtain a "deblocked next product";
(e) optionally, repeating steps (c) and (d), with each cycle producing a "deblocked product of the (X+1)th next attachment", where X is the number of cycles desired to be repeated;
(e) adding the "next amino acid" to the "deblocked first link product" from step (b) or, optionally, to the "deblocked product of the (X+1)th next link" from step (e), which includes carrying out the reaction " next amino acid" with the specified "deblocked product of the first attachment" or with the specified "deblocked product of the (X + 1)th next attachment" in an organic solvent containing a reagent for growing the peptide to obtain a "blocked next attachment product", and this "next amino acid "has an Fmoc blocked main chain amino group, provided that if that "next amino acid" has one or more functional groups in the side chain, then the functional groups in the side chain do not require protection, or the functional groups in the side chain have protecting groups that are resistant to alkaline reagents used to deprotect Fmoc;
(g) deprotecting the "blocked next product" Fmoc, which includes reacting the "blocked next product" with a primary or secondary amine to produce a "deblocked next product";
(h) optionally, repeating steps (e) and (g), with each cycle producing a "deblocked product of the (X+1)th next attachment", where X is the desired number of cycle repetitions until they are included in the peptide and the penultimate amino acid is released;
(i) adding an N-terminal amino acid to the "deblocked product of the (X+1)th next accession", which includes reacting the N-terminal amino acid with the "deblocked product of the (X+1)th next accession" in an organic solvent containing a reagent for extending a peptide to form a "blocked complete attachment product" wherein the "N-terminal amino acid" has a backbone amino group blocked by Boc or Fmoc;
(j) deprotecting Boc or Fmoc from the "blocked completed product" comprising reacting the "blocked completed product" with an acid in the case of Boc or a base in the case of Fmoc to form the completed peptide product on the resin;
(j) if the "resin-terminated peptide product" has side chain functional groups, then optionally deprotecting the "resin-terminated peptide product" side chain functional groups, which includes reacting the "resin-terminated peptide product" with the appropriate deprotecting reagents to produce a "complete peptide product on a deprotected resin"; And
(k) cleaving the peptide from the solid resin carrier of the "finished peptide product on resin" or "finalized peptide product on deprotected resin" to obtain a peptide, which comprises reacting "finished peptide product on resin" or "finished peptide product on resin"; deprotected resin" with ammonia, a primary amine, or a secondary amine until cleavage of the peptide from the resin is nearly complete;
provided that steps (e) and (g) in the synthesis of the peptide are carried out six times after the formation of the "deblocked product of the first attachment" of the formula H-L-Thr-resin, where subsequent amino acids are attached in the order: Fmoc-L-Cys(Acm), Fmoc -L-Val, Fmoc-L-Lys(Boc), Fmoc-D-Trp, Fmoc-L-Tyr(O-t-Bu) and Fmoc-L-Cys(Acm) to form H-Cys(Acm)-Tyr (O-t-Bu)-D-Trp-Lys(Boc)-Val-Cys(Acm)-Thr-resin.
2. The method according to claim 1, wherein the ammonia, primary amine or secondary amine in step (k) is in a solvent containing an alcohol and optionally an aprotic polar solvent.
3. The method according to claim 1, where step (l) further comprises the following steps:
(i) precipitating the cleaved peptide from the solvent;
(ii) filtering off the solid resin support and the precipitated peptide, and
(iii) extracting the peptide with an acidic solution to isolate the peptide.
4. The method according to any one of claims 1 to 3, wherein the first amino acid is the cesium salt of Boc-L-Thr, yielding Boc-L-Thr resin as the first coupling product, and the "deblocked first coupling product" is H-L-Thr -resin.
5. The method according to claim 4, wherein the acid used to remove the Boc protecting group in step (i) is trifluoroacetic acid (TFA).
6. The method according to claim 5, wherein the organic solvent is methylene chloride, chloroform, or dimethylformamide, and the peptide growth reagent is diisopropylcarbodiimide, dicyclohexylcarbodiimide, or N-ethyl-N"-(3-dimethyl-aminopropyl)carbodiimide.
7. Method according to claim 6, comprising attaching Boc-D--Nal to H-Cys(Acm)-Tyr(O-t-Bu)-D-Trp-Lys(Boc)-Val-Cys(Acm)-Thr-resin according to step (i) to obtain Boc-D--Nal-Cys(Acm)-Tyr(O-t-Bu)-D-Trp-Lys(Boc)-Val-Cys(Acm)-Thr-resin.
8. The method according to claim 7, including the simultaneous removal of the Boc group blocking D- -Nal, the O-t-Bu group protecting Tyr, and the Boc group protecting Lys in Boc-D- -Nal-Cys(Acm)-Tyr(O-t -Bu)-D-Trp-Lys(Boc)-Val-Cys(Acm)-Thr-resin, according to step (d) to obtain a completed peptide product on the resin of formula H-D--Nal-Cys(Acm)-Tyr-D -Trp-Lys-Val-Cys(Acm)-Thr-resin.
9. The method according to claim 8, comprising cleaving the peptide H-D--Nal-Cys(Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr from the solid resin by carrying out the reaction H-D--Nal-Cys( Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-resin with ammonia in a solvent containing an alcohol and optionally an aprotic polar solvent until substantially complete elimination to give H-D--Nal-Cys (Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-NH 2 .
10. The process according to claim 9, wherein the alcohol is methanol and the polar aprotic solvent is dimethylformamide.
11. The method according to claim 10, comprising simultaneously removing the Acm groups protecting Cys and cyclizing the resulting deprotected Cys residues in the "complete peptide product on the resin" of the formula H-D--Nal-Cys(Acm)-Tyr-D-Trp- Lys-Val-Cys(Acm)-Thr-NH 2 by carrying out the reaction of H-D- -Nal-Cys(Acm)-Tyr-D-Trp-Lys-Val-Cys(Acm)-Thr-NH 2 with a solution of iodine in alcohol to substantially complete deprotection and cyclization to give H-D--Nal--Thr-NH 2 .
Solid phase peptide synthesis was proposed by R. B. Merrifield of the Rockefeller University (Nobel Prize 1984). This method is based on the assembly of a peptide on an insoluble polymer support by sequential addition of amino acid residues with protected α-amino and side groups. 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.
The term solid-phase rather refers to the physical characteristics of the substance on the carrier, since the chemical reaction on the polymer carrier proceeds in one phase - in solution. In a suitable solvent, the polymer swells, turning into a low-viscosity but highly structured gel (crosslinked polymers), or dissolves (in the case of non-crosslinked polymers), and the synthesis process occurs at an ultramicroheterogeneous level, in a practically homogeneous system.
Solid-phase organic synthesis requires a polymer base - resin S to which the linker is attached L. At the first stage a substrate molecule is attached to the linker A.Molecule A immobilized (i.e. ceases to be mobile), but retains the ability to react with another reagent IN(stage 2).
Product AB remains on the resin, allowing it to be separated from excess reagent IN(and by-products) by simple washing. (You can add all new reagents, successively complicating the original substrate A, the main thing is that the linker remains unchanged in these reactions). Bifunctional linker L is selected so that its connection with the resin S was more durable than with the substrate A. Then at the last stage the target compound AB can be separated from the resin, destroying its connection with the linker. It is clear that the connection L-AB must be cleaved under mild conditions without damaging either the compound itself (bond A-IN), nor contact of the linker with the resin (bond L-S).
Thus, ideally, by washing the resin after each step and cleaving the bond with the carrier, a pure substance is obtained. It is natural to assume that the use of a large excess of reagents and subsequent separation from the resin in many cases makes it possible to shift the chemical equilibrium towards the formation of the target product and reduce the synthesis time. The disadvantages of solid-phase organic synthesis include the need to use a sufficiently large excess (2–30 equivalents) of reagents, difficulties in identifying intermediate synthesis products, and the relatively high cost of modified polymer supports, which is determined by the cost of the linker.
Introduced by Merrifield into the practice of organic synthesis, chloromethyl polystyrene (crosslinked with a small amount of divinylbenzene), the so-called Merrifield resin, is the most accessible of the polymer carriers.
Methodology and main stages of solid-phase peptide synthesis
The task at hand requires the introduction of a polymer carrier with a grafted amino acid into a reaction with a heterocycle activated for substitution. Let us consider in more detail the methodological aspect of obtaining immobilized amino acids on polymeric carriers.
Stage1. Immobilization of an N-protected amino acid on a polymer carrier.
The first stage of our scheme is the immobilization of the amino acid on a polymer carrier. In order to avoid such side processes as the formation of oligopeptides, the amino acid is preliminarily protected. Typically, N-protected amino acids are used and the resulting bond between the amino acid and the carrier is of the amide or ester type.
The most commonly used amino group protections in solid phase organic synthesis are the carbamate-type protecting groups tert-butoxycarbonyl (Boc) and 9H-fluorenylmethoxycarbonyl protection (Fmoc), X is the protected group:
It should be noted that the choice of protecting group is determined by the type of polymer carrier used. The conditions for immobilization of protected amino acids are different for different types of polymeric carriers. Immobilization of Boc-amino acids on Merrifield resin, which is a chloromethylated polystyrene, is carried out in situ as cesium salts with the addition of a suspension of cesium carbonate in dimethyl phthalate (DMF) and catalytic amounts of potassium iodide. The excess of reagents in relation to the amount of carrier is selected in each case individually and amounts to 1.5-4 equivalents.
The immobilization of Fmoc-amino acids on a Wang (X=O) polymer carrier to form a benzyl-type ester linker is carried out by the carbodiimide method using diisopropylcarbodiimide (DIC) in the presence of 4-(dimethylamino)pyridine (DMAP) as a catalyst. The immobilization reaction with sterically unhindered amino acids proceeds at room temperature. Immobilization of sterically hindered amino acids requires carrying out the reaction at 40–60°C for 2 days and repeating immobilization (Scheme 1). Immobilization of Fmoc - amino acids on a Rink polymer carrier (X=NH) with the formation of an amide linker of the benzhydryl type is carried out in the presence of a Castro reagent (1H-1,2,3-benzotriazol-1-yloxy) tris-(dimethylamino)phosphonium hexafluorophosphate (BOP), diisopropylethylamine base (DIEA) and 1-hydroxybenzotriazole (HOBt), as a catalyst. The reaction proceeds at room temperature for 2 hours for sterically unhindered amino acids and 4-6 hours for sterically hindered amino acids.
Stage 2Deprotection of a Protected Amino Acid on a Polymer Carrier
At the second stage planned by us (after the immobilization of the protected amino acid), it is required to remove the protective group to activate the amino group. The methods for removing Boc- and Fmoc-protection are different. Removal of the Boc protection of the amino acids on the Merrifield resin is carried out with 50% trifluoroacetic acid in dichloromethane for half an hour, under these conditions the Merrifield linker remains intact.
After deprotection, the resin is washed with a solution of triethylamine to remove trifluoroacetic acid. Removal of the Fmoc protection of amino acids on Wang (X=O) and Rink (X=NH) carriers is carried out with a 20% solution of piperidine in DMF for 40–50 min.
A significant reduction in resin weight after removing the Fmoc protection can serve as a basis for gravimetric determination of the degree of immobilization of protected amino acids at the first stage of solid phase synthesis. It is recommended to sequentially treat the resin with a solution of piperidine in dimethyl phthalate, first for 5–10 minutes, then 30 minutes in a fresh solution. After deprotection, the resin is washed at least 4 times with dimethyl phthalate to remove products of destruction of the Fmoc protection. Monitoring the progress of the acylation reaction on the carrier or removing the protective function from the amino group is possible using the Kaiser test.
Stage 3Nucleophilic substitution in heterocycles involving an amino acid immobilized on a support
The next stage planned by us for practical implementation is the aromatic nucleophilic substitution reaction; the grafted amino acid serves as the nucleophile, and the activated heterocycle is in solution. Most reactions of nucleophilic substitution in supports do not differ in performance from reactions in the liquid phase. However, it should be borne in mind that the process temperature should not exceed 120 С, above which the polystyrene base of the carrier begins to break down. Under the conditions of the reaction carried out on the carrier, the linker must also be preserved.
When choosing suitable activated heterocyclic substrates, the nature of the leaving group in the heterocycle should be taken into account.
Stage 4Removal of the target compound from polymeric carriers
Most linkers in solid-phase organic synthesis are cleaved in an acidic environment. The resistance of the linkers to acid drops sharply when going from the Merrifield resin to the Wang and Rink resin. The Rink linker cleaves under milder conditions (10–20% CF3COOH) than the Wang linker (50% CF3COOH). Merrifield resin is passive under these conditions, and transesterification in NaOMe/MeOH solution is used to cleave it, leading to the formation of an acid ester.
We recall once again that the nature of the linker determines the type of terminal function in the formed molecule removed from the substrate. Wang's resin allows you to get acids, and Rink's resin - amides.
The advantages of this scheme of solid-phase peptide synthesis:
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:
The presence of an anchor or linker is a chemical function that ensures the connection of the substrate with the applied compound. It must be covalently bound to the resin. The anchor must also be a reactive functional group in order for substrates to interact with it.
The bond formed between the substrate and the linker must be stable under the reaction conditions.
There must be ways to break the connection of the product or intermediate with the linker.
