Solid phase synthesis. The structure of peptides. Peptide bond formation reactions
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 seems to be the complete combustion of organic substances in oxygen at high temperature to CO 2 and H 2 O. Therefore, the 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 aimed primarily at finding and creating new polymer materials for substrates, development of methods for separating products and creating automated facilities 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 includes a total of 11,931 different operations, including 369 chemical reactions, was completed by two participants (Gatte and Merrifield) in just a few months.
Merrifield's ideas served as the basis for the creation various methods 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.
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 proceeding 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. In the interaction of two solid phases, the indicated equality of chemical potentials can be realized 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 when complete transformation 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 various 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 rate of reactions 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 electric field or if there is 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 as 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.
Solid-phase synthesis is based on the fact that the first link of the future oligomer is covalently attached to the "anchor" group H., chain extension is carried out with standard protected monomers according to the usual schemes used for synthesis in solutions. To conclude. synthesis stage. the oligomer is cleaved from N. and purified by appropriate methods. Solid-phase synthesis is used in the main. to obtain polypeptides, oligonucleotides and oligosaccharides.
During the synthesis of polypeptides as N. naib. a copolymer of styrene and 1-2% divinylbenzene is widely used, modified by the introduction of a dimethoxybenzyl chloride anchor group to attach the first amino acid (with a protected N H 2 group) at the C-terminus, for example:
After removal of the N-protective group, the extension of the polypeptide chain is carried out by standard methods of peptide synthesis in solution (see Peptides). As condensing agents, Naib. carbodiimides are often used or amino acids are preliminarily converted into activator. ethers.
In the synthesis of oligonucleotides, macroporous glasses or silica gel are used as nucleic acids. The anchor group is a carboxyl group, separated from N. spec. "leg", for example:
B-purine or pyrimidic base
At the first stage, the nucleoside is attached to the carrier at the 3 "-hydroxyl group of deoxyribose, in which the hydroxyl group in position 5" is protected by the dimethoxytrityl group (CH 3 OS 6 H 4) 2 (C 6 H 5) C (DMTr ); the amount of the latter after its splitting off is easily measured spectrophotometrically, which serves as a quantity. characteristic of the loading of the carrier and allows you to evaluate the yields at subsequent stages of building the oligonucleotide chain. After the removal of the DMTr group, the chain is assembled using phosphitamides (fl. I; M. Kaposers, 1980) or phosphonates (hydrophosphonates) (II; R. Stremberg, 1986):
For the implementation of solid-phase synthesis, high yields (at the level of 96-99%) are required at each stage of the district, as well as effective methods purification and isolation of synthesized. connections.
The use of a solid phase makes it possible to significantly simplify and speed up each stage of the chain growth of the oligomer, since the separation of excess components, condensing agents and by-products in the solution is achieved by filtering the reactions. mixtures and washing N. with a suitable set of solutions. Thus, the process of assembling the oligomer chain breaks down into a number of standard operations: deblocking the growing end of the chain, dosing the next protected monomer and condensing agent, feeding this mixture to the N. column for the calculated time, and washing out the N. with a suitable solvent. The cycle of building up a monomeric link m. b. automated.
At the heart of the automatic prom. synths lies common circuit diagram(see fig.). Numerous synthesizer models differ in the design of columns and their number, the method of supplying reagents and solvents, etc. Control and programming is carried out using a built-in or remote computer.
Schematic diagram of the automatic device. prom. synthesizers (electrical control line is indicated by a dotted line): 1 - supply line of monomers (M 1 , M n) and a condensing agent (KA); 2-line for the supply of reagents (eg, oxidizing agents, acylating agents, to-t, etc.) and p-solvents (P 1 , P n); 3 - switching valves; 4-column with media, equipped with distributing. valve; 5-photometric cell; 6-meter; 7-control and programming unit; 8-display.
The potential of solid-phase synthesis was demonstrated by the synthesis of ribonuclease A (R. Merrifield, 1969) and human growth hormone (D. Yamashiro, 1970), 124 and 183 amino acids long, respectively. However, due to the small but constant racemization that occurs during the formation of a peptide bond, the synthesizer. proteins have a low biol. activity, therefore automatic. synthesizers are used by Ch. arr. to obtain short polypeptides (10-30 links), including for preparative
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 completed. 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 attachment in an organic solvent containing a reagent for growing the peptide to obtain a blocked (protected) next attachment product, and the next amino acid has a main chain amino group blocked by Fmoc, 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 deprotection reagents to obtain the completed peptide product on resin removed protection; 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, relating to the immediately preceding method, is a method comprising carrying out steps (e) and (g) six times after the formation of a deblocked first coupling product 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 protecting Cys groups 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 chain) 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. Thereafter, the AA x -AA 1 resin is 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. reaction mixture cool and add the required amount of anhydrous ammonia until the HPLC peak area corresponding to methyl ester is less than 10% of the peak area of the target 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). Powder of cesium salt of Boc-L-threonine 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. After addition, the resin was washed once for about 3 minutes with DMF (20 ml), 3 times for about 2 minutes with 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 ester intermediate using HPLC (VYDAC® sorbent, grain size 5 μm, pore size 100 Å, C18, elution under isocratic conditions 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", and the "next amino acid" has an amino group blocked by Boc 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 desired cycle repetitions;
(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 obtain 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, comprising 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 .
