Solid-phase peptide synthesis via side chain linkage

Abstract

To provide a novel method of synthesizing peptides.SOLUTION: The present invention discloses high-purity peptides and peptaibols obtained by solid-phase peptide synthesis using, as a starting resin, a polymer having hydroxyamino acid, hydroxyamino acid amide, hydroxyamino alcohol or a hydroxyamino acid-containing small peptide attached via their side chains.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to the synthesis of peptides. [Overview of the Initiative]

[0002] summary High-purity peptides and peptaibols were obtained by solid-phase peptide synthesis using small peptides containing hydroxyamino acids, hydroxyamino acid amides, hydroxyamino alcohols, or hydroxyamino acids attached to polymers via side chains as the starting resin. [Modes for carrying out the invention]

[0003] Definitions and Abbreviations "Hya" or "hydroxyl amino acid" refers to an amino acid that contains a hydroxyl (-OH) group. The N-terminus or amino-terminus is the first amino acid in a peptide chain. The C-terminus or carboxyl terminus is the last amino acid in the peptide chain and is represented as follows:

[0004] [ka]

[0005] "P," "solid support," or "resin" refers to an insoluble material containing functional groups suitable for reacting with or linking with amino acids or peptides. Such solid supports or resins are well known in the industry.

[0006] "Alkyl", for example C 1-10 -Alkyl or C 1-6-Alkyl refers to a branched or unbranched, well-saturated acyclic hydrocarbon group (i.e., composed of carbon and hydrogen, without double or triple bonds). In some embodiments, alkyl may be substituted or unsubstituted. Alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and in some embodiments, each of these may be optionally substituted. Alkyl substituents include C 1-3 -Alkoxy, halogens (F, Cl, Br, or I), nitro, amino, -SH, and -OH are included but not limited to these.

[0007] "Attachment" refers to the linking of amino acids, peptides, or peptide derivatives to an insoluble support. "Hse" stands for homoserine, and "Hnv" stands for hydroxylnorvaline. "SPPS" or "solid-phase peptide synthesis" means the synthesis of peptides using resins as described herein. "pNA" stands for 4-nitroanilide. "DME" stands for dimethoxyethane.

[0008] "Acid-sensitive resins" refer to insoluble materials or resins containing functional groups suitable for reaction or linkage with amino acids or peptides, which can be cleaved from peptides by acid treatment. An "acid-sensitive protecting group" refers to a protecting group that can be cleaved from an amino acid, peptide, or peptide derivative by acidic treatment or under acidic conditions. "Peptaibol" refers to a peptide that contains an amino alcohol at its C-terminal position, rather than an amino acid or amino acid amide.

[0009] "Step-by-step" refers to a peptide synthesis method in which each amino acid in the peptide chain is introduced individually and sequentially. This method may or may not include an intermediate purification step. A "protected peptide" refers to a peptide in which all functional groups are protected or blocked by protecting groups. A "partially protected peptide" means a peptide in which at least one functional group is protected or blocked by a protecting group.

[0010] Solid-phase peptide synthesis is traditionally performed by attaching the C-terminal amino acid to a suitable solid support via its α-carboxyl functional group, and extending the peptide chain toward the amino terminus by sequentially adding amino acid residues in a gradually growing peptide chain. Hundreds of thousands of publications and patents describe this method and its application to the manufacture of peptide pharmaceuticals.

[0011] Conversely to the attachment of C-terminal carboxyl functional groups, the attachment of amino acids and peptides to suitable resins via amino acid side chains and their applications in SPPS have been described very briefly, particularly in fewer than 30 publications and patents. Most of these publications describe the attachment of amino acids via Asp and Glu side-chain carboxyl functional groups.

[0012] To the knowledge of the present inventors, side-chain attachment of amino acids via side-chain hydroxyl functional groups and their applications in peptide synthesis are limited: side-chain attachment of Fmoc-Hya-pNA (Formula A1) [A. Bernhardt, M. Drewello and M. Schutkowski, The solid-phase synthesis of side-chain-phosphorylated peptide-4-nitroanilides J. Peptide Res. 50, 1997. 143-152] and their use for the synthesis of short nitroanilide substrates, synthesis of Fmoc-Hya-O-allyl ester on 2-chlorotrityl resin with microwave assistance for applications in cyclic peptide preparation (Formula A2 [L. Rizzi, K. Cendic, N. Vaiana, S. Romeo, Alcohols immobilization onto 2-chlorotritylchloride resin under microwave irradiation, Tetrahedron Letters 52 (2011) 2808-2811]), and synthesis of Fmoc-Tyr-O-methyl ester attached on a benzyl-type resin by Mitsunobu redox alkylation of Tyr-phenoxy functional groups (Formula A3 [C. Cabrele, M. Langer and A. G. Beck-Sickinger, Amino Acid Side Chain Attachment Approach and Its Application to the Synthesis of Tyrosine-Containing Cyclic Peptides, J. Org. Chem. 1999, 64, 4353-4361]) and their applications for the synthesis of short cyclic peptides.

[0013] To the knowledge of the present inventors, side-chain attachment of Hse and Hyp has not been disclosed. Furthermore, no application of Hya side-chain-attached onto acid-labile resins for the solid-phase synthesis of protected peptides, protected peptide fragments, as well as protected peptide amides and peptaibols has been reported.

[0014]

Chem.

[0015] Solid-phase peptide synthesis In one embodiment, improved synthesis of pharmaceutically interesting peptide acids, peptide amides, and peptaibols is provided.

[0016]

Chem.

[0017] In one aspect of the present invention, peptides are produced very effectively in high yield and purity by attaching a hydroxy amino acid via its amino acid side chain, or a small peptide containing a hydroxy amino acid in the sequence, onto a trityl or benzhydryl type resin to form the amino acid-resin conjugate or peptide resin conjugate of Formulas I to IV. Here, in Formulas I to IV, P is selected from the supports used in solid-phase peptide synthesis, Pr 1 is H or an amino protecting group selected from Fmoc, Boc, Trt, Dde and Alloc, Pr 2 is an acid-sensitive hydroxy protecting group selected from Trt, Clt, Mmt, Mtt, Dpm and tBu, Hya is a hydroxy amino acid selected from D- or L-Ser, Thr, Tyr, Hse, Hyp, Hnv and the like, and A is an acid-sensitive alkoxy group selected from OH, OTrt, OClt, OMmt, OMtt, ODpm and OtBu, NH2, NHR 1 , NR 1 R 2 (wherein R 1 and R 2 are independently an alkyl group or a protected or semi-protected peptide comprising 1 to 10 amino acids in the sequence.

[0018] In another embodiment, the inventors have selected amino alcohols of formulas III to VI (wherein P, X, V, Z and Pr) from amino alcohols derived from natural hydroxyamino acids. 1 As defined above, R 3 and R 4 is an alkyl, aryl, or aralkyl group, and Pr 2 This document discloses that peptraibols, such as octreotide, were obtained by solid-phase synthesis using resin-bonded amino alcohols (where is a trityl, benzhydryl, or benzyl type acid-sensitive protecting group).

[0019] [ka]

[0020] Furthermore, the inventors first disclose that peptides prepared by the application of resins of formulas I-IV, when mounted on a trityl-type resin via the hydroxyl functional group of the hydroxyamino acid side chain, can be cleaved from the resin by mild acidic treatment, in which case the tBu and benzyl-type side chain protecting groups remain intact. In one view, cleavage from the resin is achieved by treatment with a 1-3% acid solution, e.g., TFA, a diluted HCl solution, and possibly by adding a scavenger in a solvent. In another view, cleavage can be carried out in a solvent, e.g., DCM or acetone. Such partially protected peptides have been found to be useful in the synthesis of longer peptides by fragment condensation in solution or on a solid phase. The methods of the present invention expand the versatility of the applications of the resins described in this specification, and also result in a significant improvement in the purity of the resulting pharmaceutical peptides, while simultaneously substantially reducing the cost of their synthesis.

[0021] Several peptides of pharmaceutically interesting interest were prepared by step-by-step methods in solution and on a solid phase, fragment condensation, or combinations thereof, as representative of the novel methods described in this specification. The following examples are representative and do not limit the application to other peptides.

[0022] Lanreotide : In one embodiment, lanreotide was produced by solid-phase synthesis using a Thr-amide bonded to a resin, as shown below.

[0023] [ka]

[0024] Human insulin B chain : Optionally, human insulin B chains were synthesized by SPPS. In one respect, the synthesis begins with Thr-t-butyl bonded to a resin, as described in the examples, using a 4-methoxybenzhydryl resin. Optionally, the synthesis can also be carried out on a solid phase by condensation of a 1-8 partially protected Boc-Phe-Val-Asn(Trt)-Gln(Trt)-His(Trt)-Leu-Cys(Trt)-Gly-OH fragment bonded to a resin with a 9-30 fragment, or by cleavage of the partially protected 9-30 fragment from the resin followed by condensation of the 1-8 and 9-30 fragments in solution.

[0025] [ka]

[0026] Salmon Calcitonin : Optionally, salmon calcitonin can be produced by initiating synthesis from Fmoc-Thr-Pro-NH2 bound to a resin. The peptide chain is then extended using Fmoc-amino acids.

[0027] [ka]

[0028] Optionally, salmon calcitonin bound to the resin can be produced by fragment condensation using 2 to 4 fragments, either on the resin as shown above or in solution as shown below.

[0029] [ka]

[0030] Octreotide : In another embodiment, octreotides were efficiently synthesized by attaching Fmoc-threoninol-OTrt to a 4-methoxybenzhydryl resin via the side chain of threoninol, followed by assembly of the octreotide chain using Fmoc-amino acids, and finally cleavage of the octreotide from the resin using sequential or simultaneous Cys-oxidation, as shown below. Fmoc-threoninol-OTrt is much easier to produce than Fmoc-Thr(tBu)-ol, which can be attached to a resin via the hydroxymethyl group of threoninol on a suitable resin. This is because H-Thr(OtBu)-ol, used as a starting material for the production of Fmoc-Thr(tBu)-ol, is much more difficult to produce than Fmoc-threoninol-OTrt, which is used for attaching threoninol to a resin via its side chain.

[0031] [ka]

[0032] Exenatide : In another example, Fmoc-Ser-NH2 is attached to a trityl resin via its side chain and used for the synthesis of exenatide. This synthesis can be carried out in a step-by-step manner in solution or on a solid phase, or by fragment condensation, after cleavage of a partially protected exenatide fragment from the resin by mild acid treatment, as described below. This method completely avoids most of the impurities typically generated during the synthesis of peptides containing many Pro and Gly residues, and yields high-purity peptides. This method also allows for the complete avoidance of impurities resulting from peptide cleavage from peptide amide linkers using other methods known in the art. The above-mentioned methods known in the art significantly reduce the yield and purity of the peptides.

[0033] [ka]

[0034] In one respect, exenatide can be produced by cleaving partially protected peptides 12-39 from a resin and then condensing them with partially protected fragments 1-11 in solution, as shown below. Alternatively, protected exenatide can be obtained by condensation using fragments 1-13 and 14-39.

[0035] [ka]

[0036] Pramlintide : This method is also very effective in the production of amylin peptides. In one respect, the side chain can be attached using one of the C-terminal Ser, Thr, or Tyr residues of amylin or its derivatives, such as plumrintide. Synthesis can be carried out in a step-by-step manner in solution or on a solid phase, or by fragment condensation. By introducing pseudoprolines (see Ψ, Mutter et al, Peptide Res. (1995 8, 145)) into the growing peptide chain, the synthesis is accelerated and the purity of the resulting peptide is improved.

[0037] [ka]

[0038] Alternatively, the synthesis of plumlintide can be carried out in liquid phase with similar success in terms of the purity and yield of plumlintide. In one embodiment, a resin-bound protected peptide via the Fmoc-Tyr-NH2 side chain can be quantitatively cleaved from the resin at various positions in the peptide chain using mild acid treatment, while retaining the tBu-type side chain protecting group. In one example, as shown below, partially protected 1-10 fragments prepared on a 2-chlorotrityl resin were successfully condensed in a step-by-step manner with partially protected 11-37 fragment amides.

[0039] Pramlintide : [ka]

[0040] Tetracosactide (ACTH 1-24) : In other examples, ACTH 1-24 was effectively prepared by a step-by-step method starting from resin-bonded Fmoc-Tyr-Pro-OtBu, or by condensing partially protected 1-10 fragments with 11-24 fragments or resin-bonded 11-24 fragments in solution, as shown below.

[0041] [ka]

[0042] Bivalirudin : In another example, as shown below, bivalirudin was produced in high yield and high purity by starting with Fmoc-Tyr-Leu-OtBu bound to a resin, extending the peptide chain step-by-step with Fmoc-amino acids, finally deprotecting it, and then cleaving the peptide from the resin.

[0043] Alternatively, bivalirudin was obtained by condensing a protected fragment on a resin, or by cleaving a partially protected peptide containing 4-15 amino acid residues from the resin and then condensing it in solution with a bivalirudin fragment containing 5-16 amino acids. The synthesis of bivalirudin by fragment condensation on a resin of partially protected 1-10 bivalirudin fragments and partially protected 11-20 bivalirudin fragments bound to a resin is described below.

[0044] [ka] [Examples]

[0045] Example 1. Preparation of Fmoc-Thr(4-methoxybenzhydrylpolystyrene)-OtBu [ka]

[0046] 30 mmol of Fmoc-Thr-OtBu, prepared by a conventional reaction between H-Thr-OtBu and Fmoc-OSu, was reacted with 20 g (30 mmol) of 4-methoxybenzhydryl polystyrene resin (product of CBL-Patras) and 60 mmol of DIPEA in 250 ml of THF for 10 hours at room temperature. Next, 60 mmol of methanol was added to the mixture, and the mixture was shaken for a further 4 hours. The resin was filtered and washed three times with THF / MeOH / DIPEA (85:10:5), six times with DMF, four times with IPA, and three times with DEE, and then vacuum-dried to a fixed weight. 29 g of resin-bound Fmoc-Thr-OtBu was obtained with a resin load of 0.95 mmol / g.

[0047] Example 2. Fmoc-Thr(4-methoxybenzhydrylpolystyrene)-O-Clt [ka]

[0048] 30 mmol of Trt-Thr-OMe, prepared by a conventional reaction of H-Thr-OMe with Trt-Cl / Me3SiCl and DIPEA, was reacted with 20 g (30 mmol) of 4-methoxy-4'-polystyrene benzhydryl bromide resin (product of CBL-Patras) and 60 mmol of DIPEA in 250 ml of THF for 10 hours at room temperature. Next, 60 mmol of methanol was added to this mixture, and the mixture was shaken for a further 4 hours. The resin was filtered and washed three times with THF / MeOH / DIPEA (85:10:5), three times with DCM, three times with 1% TFA in DCM, four times with THF, three times with 1N-LiOH in THF / water / methanol (70:15:15), three times with THF / water (75:25), and four times with DMF. Next, it was reacted with 60 mmol of Fmoc-OSu and 30 mmol of DIPEA at room temperature for 2 hours, washed three times with DMF, and three times with DCM. Next, it was reacted with 50 mmol of Trt-Cl and 50 mmol of DIPEA at room temperature for 3 hours, washed four times with DMF and six times with DEE, and then vacuum-dried to a constant weight. 32.3 g of resin-bound Fmoc-Thr-OtBu was obtained with a resin load of 0.78 mmol / g.

[0049] Example 3. Fmoc-Throl(4-methoxybenzhydrylpolystyrene)-O-Clt A) Departure from Fmoc-Sleoninoor [ka]

[0050] 50 mmol of commercially available Fmoc-threoninol (CBL-Patras) in 350 ml of DCM was reacted with 55 mmol of monomer Clt-Cl and 55 mmol of DIPEA at room temperature for 4 hours. The resulting mixture was extracted with water as usual, and the DCM phase was dried over anhydrous sodium sulfate and filtered. To the resulting solution, 30 g of 4-methoxy,4-polystyrenebenzhydryl bromide (CBL-Patras) and 50 mmol of DIPEA were added, and the resulting mixture was stirred at room temperature for 4 hours. The resin was filtered and washed 6 times with DMF, 4 times with IPA, and 4 times with DEE, and then vacuum-dried to a fixed weight. 38.4 g of resin-bound Fmoc-threoninol was obtained at a loading of 0.82 mmol / g.

[0051] B) Departure from Trt-Thr(Resin)-OMe [ka]

[0052] 30 mmol of Trt-Thr-OMe, prepared by the reaction of H-Thr-OMe with Trt-Cl / Me3SiCl and DIPEA according to a conventional method, was reacted with 20 g (30 mmol of 4-methoxy-4'-polystyrene benzhydryl bromide resin (product of CBL-Patras) and 60 mmol of DIPEA) in 250 ml of THF at room temperature for 10 hours. Next, 60 mmol of methanol was added to this mixture, and the mixture was stirred for a further 4 hours. The resin was filtered and washed three times with THF / MeOH / DIPEA (85:10:5) and five times with THF, and then reacted with 30 mmol of LiBH4 in THF.

[0053] Next, the resin was filtered and washed 6 times with THF, 4 times with DCM, 6 times with 1% TFA in DCM, and 3 times with DMF / DIPEA (97:3). Then, it was reacted with 60 mmol of Fmoc-OSu and 30 mmol of DIPEA at room temperature for 2 hours, washed 3 times with DMF and 3 times with DCM, and then reacted with 50 mmol of Clt-Cl and 50 mmol of DIPEA at room temperature for 3 hours, washed 4 times with DMF, 6 times with IPA and 6 times with DEE, and then vacuum-dried to a certain weight. With a resin load of 0.74 mmol / g, 34.7 g of resin-bound Fmoc-Throl-O-Clt was obtained.

[0054] Example 4. Fmoc-Ser(trityl resin)-NH 2 50 mmol of Fmoc-Ser-NH2, prepared according to a standard procedure known in the industry, was dissolved in 0.5 liters of DCM. To this suspension, 30 g of trityl chloride resin (36 mmol) and 65 mmol of DIPEA were added, and the mixture was stirred at room temperature for 6 hours. Subsequently, 25 ml of methanol and 30 mmol of DIPEA were added, and the mixture was stirred for a further 2 hours at room temperature. Next, the resin was filtered and washed three times with DCM / MeOH / DIPEA (90:5:5), five times with DMF, four times with IPA, and four times with DEE, and then vacuum-dried to a fixed weight. 41.1 g of Fmoc-Ser-NH2-containing resin was obtained at a loading of 0.71 mmol / g.

[0055] Example 5. Fmoc-Tyr(2-chlorotrityl resin)-NH 2 Following the procedure described above, 43.7 g of resin was obtained from 50 mmol of Fmoc-Tyr-NH2 and 30 g of 2-CTC chloride resin, with a loading of 0.81 g Tyr / g resin.

[0056] Example 6. Fmoc-Hyp(4-methylbenzhydryl resin)-NH 2 Following the procedure described above, 39.8 g of resin was obtained from 50 mmol of Fmoc-Hyp-NH2 and 30 g of 4-methylbenzhydryl bromide resin, with a load of 0.49 g Hyp / g resin.

[0057] Example 7. Fmoc-Thr(4-methoxybenzhydryl resin)-Pro-NH 2 50 mmol of Fmoc-Thr-Pro-NH2, prepared by coupling Fmoc-Thr(tBu)-OH with H-Pro-NH2 according to a standard procedure known in the industry, was dissolved in 0.5 liters of DME. To the resulting solution, 30 g of 4-methoxybenzhydryl bromide resin (45 mmol) and 65 mmol of DIPEA were added, and the mixture was stirred at room temperature for 6 hours. Next, 25 ml of methanol and 50 mmol of DIPEA were added, and the mixture was stirred at room temperature for 2 hours. The resin was then filtered and washed three times with DME / MeOH / DIPEA (90:5:5), five times with DMF, four times with IPA, and four times with DEE, and then vacuum-dried to a constant weight. 44.5 g of Fmoc-Thr-Pro-NH2-containing resin was obtained at a loading of 0.77 mmol / g.

[0058] Example 8. Fmoc-Tyr(2-chlorotrityl resin)-Pro-OtBu Following a standard procedure known in the industry, 50 mmol of Fmoc-Tyr-Pro-OtBu was prepared and dissolved in 0.5 liters of DCM. To the resulting solution, 30 g of 2-chlorotrityl chloride resin (48 mmol) and 65 mmol of DIPEA were added, and the mixture was stirred at room temperature for 12 hours. Next, 25 ml of methanol and 50 mmol of DIPEA were added, and the mixture was stirred at room temperature for a further 2 hours. The resin was then filtered and washed three times with DCM / MeOH / DIPEA (90:5:5), five times with DMF, four times with IPA, and four times with DEE, and then vacuum-dried to a fixed weight. 44.5 g of Fmoc-Tyr-Pro-OtBu-containing resin was obtained at a loading of 0.64 mmol / g.

[0059] Example 9. Fmoc-Tyr(2-chlorotrityl resin)-Leu-OtBu 50 mmol of Fmoc-Tyr-Leu-OtBu, prepared according to a standard procedure known in the industry, was dissolved in 0.5 liters of THF. To the resulting solution, 30 g of 2-CTC chloride resin (48 mmol) and 65 mmol of DIPEA were added, and the mixture was stirred at 60°C for 12 hours. Next, 25 ml of methanol and 50 mmol of DIPEA were added, and the mixture was stirred at room temperature for a further 2 hours. The resin was then filtered and washed three times with DCM / MeOH / DIPEA (90:5:5), five times with DMF, four times with IPA, and four times with DEE, and then vacuum-dried to a constant weight. 44.5 g of Fmoc-Tyr-Leu-OtBu-containing resin was obtained at a loading of 0.64 mmol / g.

[0060] Example 10. Solid-phase synthesis of peptides and protected peptide segments General procedure A1. Preparation of loaded 2-chlorotrityl resin: General procedure 2-chlorotrityl chloride resin (CTC-Cl) (100 g; load 1.6 mmol / g) (CBL-Patras) is placed in a 2 L peptide synthesis reactor and swelled with 700 mL of dichloromethane (DCM):dimethylformamide (DMF) 1:1 at 25°C for 30 minutes. The resin is filtered and a solution of 100 mmol of Fmoc-amino acid and 300 mmol of diisopropylethylamine (DIEA) in 500 mL of DCM is added. This mixture is stirred under nitrogen at 25°C for 2 hours.

[0061] Next, the remaining active site of the 2-CTC resin is neutralized by adding 10 mL of methanol (MeOH) and reacting for 1 hour. The resin is filtered and washed twice with 400 mL of DMF. The resin is filtered and washed twice for 30 minutes with 500 mL of 25% by volume piperidine in DMF. The resin is then treated four times with 500 mL of DMF. The resin is deswelled by washing three times with 500 mL of isopropanol (IPA). The resin is dried to a fixed weight. 70-95% of the mmol of the amino acids used were bound to this resin.

[0062] B. Solid-phase synthesis general protocol As described in Part A or Example 1, solid-phase synthesis was carried out at 24°C using 1.0 g of amino acids or peptides esterified or attached via side chains to a trityl or benzhydryl type resin. The following protocol was used for the synthesis.

[0063] B1. resin swelling The resin was placed in a 15 ml reactor, treated twice with 7 ml of NMP, and then filtered. B2. Amino acid activation 3.0 equivalents of amino acids and 4.0 equivalents of 1-hydroxybenzotriazole were weighed out and dissolved in 2.5 volumes of NMP in a reactor, and then cooled to 0°C. Next, 3.0 equivalents of DIC were added, and the mixture was stirred for 15 minutes.

[0064] B3. Coupling Next, the solution prepared in B2 was added to the reactor in B1. The reactor was washed once with 1 volume of DCM and then added back into the reactor, which was stirred at 25°C to 30°C for 1 to 3 hours. The completion of the reaction was determined by performing a Kaiser test on the sample. If the coupling reaction was not complete after 3 hours (positive Kaiser test), the reaction mixture was filtered and re-coupled using a fresh solution of activated amino acids. After the coupling was complete, the reaction mixture was filtered and washed four times with NMP (5 volumes per wash).

[0065] B4. Removal of the Fmoc- group The resin obtained in B3 was filtered and then treated with a 5 mL solution containing 25% by volume of piperidine for 30 minutes. The resin was then washed three times with 5 mL of NMP.

[0066] B5. Peptide chain extension After introducing each amino acid, steps B1 to B5 were repeated until the peptide chain was complete. The following Fmoc-amino acids were used to introduce each individual amino acid: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn-OH, Fmoc-Asn(Trt)-OH, Fmoc-D-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-D-Phe-OH, Fmoc-Phe-OH, F moc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(Trt)-OH, Fmoc-D-Trp-OH, Fmoc-Trp-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Trp(Boc)-OH , Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(Clt)-OH, Fmoc-Val-OH, Boc-D-Cys(Trt)-OH, Boc-His(Trt)-OH, Boc-Lys(Boc)-OH, Boc-D-2-Nal-OH, Boc-D-Phe-OH, Boc-Ser(tBu)-OH.

[0067] C. General method for acidic cleavage of peptides containing an Fmoc- or Boc- group at the N-terminus or protected peptide segments from CTC resin. The resin-bound peptide or peptide segment, prepared as described in B1 to B5 above, was completely removed of any residual NMP or other basic components by washing it four times with 5 mL of NMP, three times with 5 mL of IPA, and finally five times with 7 mL of DCM. The resin was then cooled to 0°C, filtered from DCM, and treated twice with 10 mL of 1-2% TFA / DCM solution at 5°C. The mixture was then stirred at 0°C for 20 minutes and filtered. The resin was then washed three times with 10 mL of DCM.

[0068] Next, pyridine (1.3 equivalents relative to TFA) was added to the filtrate to neutralize the TFA. Then, the cleavage solution in the DCM was mixed with an equal volume of water. The resulting mixture was distilled under reduced pressure to remove the DCM (350 torr at 28°C). After the removal of the DCM, the peptide or peptide segment precipitated. Next, the resulting peptide was washed with water and dried at 30-35°C under a vacuum of 15 Torr.

[0069] Example 11. Synthesis of a resin-bound protected peptide by condensation of an N-terminally protected fragment and a resin-bound C-terminally protected fragment. General procedure To a solution of an N-terminus-protected peptide fragment in DMSO / DCM (95:5) at a concentration of 0.15 mmol / ml, 0.2 mmol of HOBt was added, and the resulting solution was cooled to 5°C. Next, 0.14 mmol of DIC was added, and the mixture was stirred at 15°C for 20 minutes. Then, 0.1 mmol of the resin-bound C-terminus fragment was added, and the mixture was stirred at room temperature for a further 6 hours. The completion of the condensation reaction was checked by a Kaiser test. If the Kaiser test remained blue, a second condensation was performed to complete the condensation.

[0070] Example 12. Synthesis of partially protected peptides by condensation in solution of an N-terminus protected fragment and a C-terminus protected fragment. General procedure To a solution of the N-terminated fragment at 0.15 mmol / ml in DCM, add 0.2 mmol of HOBt and cool the resulting solution to 5°C. Next, add 0.15 mmol of EDAC and stir the mixture at 15°C for 20 minutes. Then, add 0.15 mmol of the C-terminated fragment and stir at room temperature for a further 2–5 hours. Check for completion of the condensation reaction by HPLC. If incomplete condensation is observed, add an additional 0.015 mmol of EDAC and continue the reaction at room temperature for a further time.

[0071] Example 13. Simultaneous cleavage and deprotection of peptides from resins General method The 1.00 g protected resin-bound peptide prepared as described above is treated with 20 mL of TFA / DTT / water (90:5:5) at 5°C for 3 hours and at 15°C for 1 hour. Next, the resin is washed three times with a cleavage solution, and then the combined filtrate is vacuum concentrated. The crude peptide is precipitated by adding ether, washed several times with ether, and then vacuum dried on KOH to a certain weight.

[0072] Example 14. Peptide deprotection General method 1.00 g of the protected peptide prepared as described above was treated with 20 mL of TFA / DTT / water (90:5:5) at 5°C for 3 hours and at 15°C for 1 hour. The resulting solution was vacuum concentrated, and the deprotected peptide was then precipitated by the addition of diisopropyl ether and washed three times with 10 mL of diisopropyl ether. The resulting solid was vacuum dried to a fixed weight under KOH (25°C, 15 Torr).

[0073] Example 15. Purification of crude peptides; isolation of peptides. General procedure The peptide solution obtained as described above was vacuum concentrated, and ice water and ether were added. After separating the organic phase, the remaining aqueous peptide solution was extracted twice more with ether, and nitrogen or helium was blown into the resulting solution, filtered, and directly added to a 10x25 cm, Lichrospher 100, RP-18, 12 micron (Merck) semi-preparation column; Phase A = 1% TFA in acetonitrile, Phase B = 1% TFA in water; or Kromasil. The HPLC fraction containing the purified peptide was vacuum concentrated to remove as much contaminating acetonitrile as possible, and then freeze-dried using a standard freeze-drying program.

[0074] The compounds listed below were prepared by following the procedures described in Examples 16-23 below. Example 16. Lanreotide Example 17. Insulin B-chain Example 18. Salmon Calcitonin Example 19. Octreotide Example 20. Exenatide Example 21. Pramlintide Example 22. Tetracosactide (ACTH 1-24) Example 23. Bivalirudin Summary of the Disclosure of This Specification [Section 1] Formula I or II below: [ka] (In the formula, Hya is a residue of a hydroxyamino acid; Pr 1 It is an orthogonal amino protecting group with respect to H or the resin and to other protecting groups; A is a hydroxyl group, or an acid-sensitive hydroxyl protecting group selected from tBu, Trt, and Clt, or NR 1 R 2 (In the formula, R 1 and R 2 is H or C 1-10 It is an alkyl group, or a peptide ester, peptide amide, or peptibol containing 0 to 30 amino acids; X, Y, Z, and V are each independent ortho, meta, or para substituents, and H, Cl, F, C 1-10 Alkyl or C 1-10 Selected from alkoxy; and P is an insoluble support or insoluble linker-resin conjugate suitable for solid-phase peptide synthesis. A resin joint represented by [this]. [Section 2] Equations III to VI below: [ka] (In the formula, Pr 1 It is an orthogonal amino protecting group with respect to H, or the resin and other protecting groups; Pr 2 is an orthogonal hydroxyl protecting group for H or resin; R 3 and R 4 These are, independently, H or C 1-10 It is alkyl; X, Y, Z, and V are each independent ortho, meta, or para substituents, and H, Cl, F, C 1-10 Alkyl or C 1-10 Selected from alkoxy; and P is an insoluble support or insoluble linker-resin conjugate suitable for solid-phase synthesis of peptides. A resin joint represented by [this]. [Section 3] In the method for manufacturing resin bonded bodies of formulas I to VI, Prepare a hydroxyl-containing amino acid or amino alcohol or peptide derivative in which at least one of the side chains of the contained hydroxyl amino acid or amino alcohol is not protected, or selectively deprotect a hydroxyl amino acid or hydroxyl amino alcohol or peptide derivative in which the side chain of the hydroxyl amino acid or hydroxyl amino alcohol is selectively deprotected, and then attach it to a suitable resin by reaction with a resin halide, where the resin is selected from the group consisting of trityl-type resins and linkers, or benzhydryl-type resins, or benzyl-type resins; and Add alcohol or thio alcohol to mask unreacted resin halides; A method that includes a process. [Section 4] A method for producing a monoalkylated Fmoc-aminodialcohol, comprising reacting an Fmoc-diaminoalcohol with an alkyl halide in an organic solvent, such as dichloromethane, in the presence of a base, such as a tert-amine base, wherein the alkyl is selected from trityl, 4-methyltrityl, 4-methoxytrityl, and 2-chlorotrityl. [Section 5] A solid-phase synthesis method for biologically active free or partially protected peptides, cyclic peptides, or peptaibols, comprising using the resin conjugate described in item 1 or 2 as a functionalized resin in solid-phase peptide synthesis. [Section 6] The following formula: ED-2-Nal-Cys(A)-Tyr(C)-D-Trp(F)-Lys(E)-Val-Cys(A)-Thr(Resin)-NH2 (In the formula: The "D-" indicates the chirality of the amino acid that follows the D-amino acid; A is a thiol protecting group independently selected from Trt, Mmt, Acm, or StBu; C is a hydroxy protecting group selected from Clt, Trt, or tBu; F is either H or Boc; E is an amino protecting group selected from Mtt, Mmt, Trt, or Boc; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. A peptide represented by [this symbol]. [Section 7] The peptide according to item 6, wherein the peptide is a lanreotide bound to a resin. [Section 8] In a method for producing lanreotide, the peptide resin conjugate described in item 7 is treated with a mild acid selected from a solution of trifluoroacetic acid containing a scavenger, if desired; and The resulting peptide is oxidized using an oxidizing agent selected from air, hydrogen peroxide, DMSO, or iodine; A method that includes that. [Section 9] A method for producing lanreotide, comprising treating the peptide resin conjugate described in item 7 with a mild acid, such as a solution of trifluoroacetic acid containing iodine. [Section 10] The following formula: E-Phe-Val-Asn(A)-Gln(A)-His(A)-Leu-Cys(B)-Gly-Ser(C)-His(A)-Leu-Val-Glu(C)-Ala-Leu-Tyr (C)-Leu-Val-Cys(A)-Gly-Glu(C)-Arg(D)-Gly-Phe-Phe-Tyr(C)-Thr(C)-Pro-Lys(E)-Thr(Resin)-OC (In the formula, A is a carboxamide or imidazole protecting group selected from H, or Trt, Mtt, or Mmt; B is a Cys protecting group selected from Mmt, Trt, Acm, or StBu; C is a hydroxy-, carboxy-, or phenoxy-protecting group selected from tBu, Trt, or Clt; D is a guanidine protecting group selected from Pbf or Pmc; E is an amino protecting group selected from H, or Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. A peptide represented by [this symbol]. [Section 11] The peptide according to item 10, wherein the peptide is a human insulin B-chain. [Section 12] A method for producing insulin B-chains, comprising contacting the resin-bound peptide described in item 10 with an acidic solution, for example, a solution of trifluoroacetic acid in dichloromethane containing a scavenger, if desired. [Section 13] The following formula: E-Ser(C)-His(A)-Leu-Val-Glu(C)-Ala-Leu-Tyr(C)-Leu-Val-Cys(B)-Gly-Glu(C)-Arg(D)-Gly-Phe-Phe-Tyr(C)-Thr(C)-Pro-Lys(E)-Thr(Resin)-O- (In the formula, A is a carboxamide- or imidazole-protecting group selected from Trt, Mtt, or Mmt; B is a Cys protecting group selected from Mmt, Trt, Acm, or StBu; C is independently selected from tBu, Trt, or Clt, and is a hydroxy-, carboxy-, or phenoxy-protecting group; D is a guanidine protecting group selected from H, Pbf, or Pmc; E is an amino protecting group selected from H, or Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. A peptide represented by [this symbol]. [Section 14] The peptide according to item 13, wherein the peptide corresponds to a partially protected 9-30 fragment of the human insulin B-chain. [Section 15] A method for producing insulin B-chains, comprising condensing the peptide described in item 13 with a protected 1-8 insulin fragment Boc-Phe-Val-Asn(A)-Gln(A)-His(A)-Leu-Cys(B)-Gly-OH: Here, A is a carboxamide- or imidazole-protecting group, each independently selected from Trt, Mtt, and Mmt; and B is a Cys protecting group selected from Mmt, Trt, Acm, or StBu. [Section 16] The following formula: E-Cys(B)-Ser(C)-Asn(A)-Leu-Ser(C)-Thr(C)-Cys(B)-Val-Leu-Gly-Lys(E)-Leu-Ser(C)-Gln(A)-Glu(C)-Leu-His( A)-Lys(E)-Leu-Gln(A)-Thr(C)-Tyr(C)-Pro-Arg(D)-Thr(C)-Asn(A)-Thr(C)-Gly-Ser(C)-Gly-Thr(Resin)-Pro-NH2 (In the formula, A is a carboxamide- or imidazole-protecting group, each independently selected from Trt, Mtt, or Mmt; B is a Cys protecting group independently selected from Mmt, Trt, Acm, or StBu; Each C is independently selected from tBu, Trt, or Clt, and is a hydroxyl-, carboxy-, or phenoxy-protecting group; D is a guanidine protecting group selected from Pbf or Pmc; E is independently an amino protecting group selected from H, or Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. A peptide represented by [this symbol]. [Section 17] The peptide according to item 16, wherein the peptide is salmon calcitonin. [Section 18] In a method for producing salmon calcitonin, The peptide described in item 16 is treated with a mild acid, for example, a solution of trifluoroacetic acid containing a scavenger if desired; The resulting peptide solution is oxidized using a suitable oxidizing agent selected from air, hydrogen peroxide, DMSO, or iodine; Deprotect the peptide; The peptide was purified by chromatography; and Freeze-dry the peptide; A method that includes that. [Section 19] In a method for producing salmon calcitonin, The peptide resin conjugate described in item 16 is treated with a mild acid, such as a solution of trifluoroacetic acid; The peptide is oxidized with iodine; The peptide is deprotected by treating it with an acid, for example, a solution of trifluoroacetic acid or hydrochloric acid containing a scavenger; and Salmon calcitonin peptide is purified by chromatography and then freeze-dried; A method that includes that. [Section 20] A method for producing peptides as described in item 17, comprising condensing partially protected fragments 11-32 of salmon calcitonin represented by the formula:H-Lys(E)-Leu-Ser(C)-Gln(A)-Glu(C)-Leu-His(A)-Lys(E)-Leu-Gln(A)-Thr(C)-Tyr(C)-Pro-Arg(D)-Thr(C)-Asn(A)-Thr(C)-Gly-Ser(C)-Gly-Thr(Resin)-Pro-NH2 with partially protected fragments 1-10 of salmon calcitonin represented by the formula:E-Cys(B)-Ser(C)-Asn(A)-Leu-Ser(C)-Thr(C)-Cys(B)-Val-Leu-Gly-OH: Here, A is a carboxamide- or imidazole-protecting group, each independently selected from Trt, Mtt, or Mmt; B is a Cys protecting group independently selected from Mmt, Trt, Acm, or StBu; Each C is independently selected from tBu, Trt, or Clt, and is a hydroxyl-, carboxy-, or phenoxy-protecting group; D is a guanidine protecting group selected from H, Pbf, or Pmc; E is independently an amino protecting group selected from H, or Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. [Section 21] The following formula: [ka] (In the formula, A is an amino protecting group selected from H, or Fmoc, Boc, Trt, Nps, Mtt, or Mmt; The "D-" indicates the chirality of the amino acid that follows the D-amino acid; B is a thiol protecting group selected from Trt, Mmt, Acm, or StBu; C is either H or Boc; E is a hydroxy protecting group selected from Clt, Trt, or tBu; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. A partially protected peptide represented by [the specified symbol]. [Section 22] The peptide according to item 21, wherein the peptide is an octreotide bound to a resin. [Section 23] In a method for producing octreotide, The peptide resin conjugate described in item 22 is treated with a mild acid, for example, a solution of trifluoroacetic acid containing a scavenger; and The resulting peptide solution is oxidized using a suitable oxidizing agent selected from air, hydrogen peroxide, DMSO, or iodine; A method that includes that. [Section 24] In a method for producing octreotide, The peptide resin conjugate described in item 22 is treated with a mild acid, such as a solution of trifluoroacetic acid containing iodine; and Deprotect, purify, freeze-dry, and obtain octreotide with a purity of over 99%. A method that includes that. [Section 25] The following formula: E-His(A)-Gly-Glu(C)-Gly-Thr(C)-Phe-Thr(C)-Ser(C)-Asp(C)-X-Lys(E)-Gln(A)-Met-Glu(C)-Glu(C)-Glu(C)-Ala-Val-Arg (D)-Leu-Phe-Ile-Glu(C)-Trp(F)-Leu-Lys(E)-Asn(A)-Gly-Gly-Pro-Ser(C)-Ser(C)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-NH2 (In the formula, X is Leu-Ser(tBu) or Leu-ΨSer; A is independently a carboxamide- or imidazole-protecting group selected from H, or Trt, Mtt, or Mmt; Each C is independently selected from tBu, Trt, or Clt, and is a hydroxyl-, carboxy-, or phenoxy-protecting group; D is a guanidine protecting group selected from H, Pbf, or Pmc; E is independently an amino protecting group selected from H, or Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; F is either H or Boc; and Resin is a suitable acid-sensitive resin for solid-phase peptide synthesis. A partially protected peptide represented by [the specified symbol]. [Section 26] The peptide according to item 25, wherein the peptide is exenatide. [Section 27] The following formula: Y-Glu(C)-Glu(C)-Ala-Val-Arg(D)-Leu-Phe-Ile-Glu(C)-Trp(F)-Leu-Lys(E)-Asn(A)-Gly-Gly-Pro-Ser(C)-Ser(C)-Gly-Ala-Pro-Pro-Pro-Ser(Resin)-NH2 (In the formula, Y is H, E-Glu(C), E-Met-Glu(C), E-Gln(A)-Met-Glu(C) or E-Lys(E')-Gln(A)-Met-Glu(C); A is a carboxamide protecting group, independently selected from H, or Trt, Mtt, or Mmt; E and E' are each independently H, or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; F is either H or Boc; C is independently selected from tBu, Trt, or Clt, and is a hydroxyl- or carboxyl-protecting group; D is a guanidine protecting group selected from H, or Pbf or Pmc; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. A peptide that is a partially protected exenatide fragment 12-39, 13-39, or 14-39, represented by [the specified formula]. [Section 28] The following formula: E-His(A)-Gly-Glu(C)-Gly-Thr(C)-Phe-Thr(C)-Ser(C)-Asp(C)-XY (In the formula, X is Leu-Ser(tBu) or Leu-ΨSer; E is an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; A is a carboxamide- or imidazole-protecting group selected from H, or Trt, Mtt, or Mmt; C is independently selected from tBu, Trt, or Clt, and is a hydroxyl- or carboxyl-protecting group; Y is OZ, Lys(E)-Gln(A)-OZ, Lys(E)-Gln(A)-Met-OZ, or Lys(E)-Gln(A)-Met-Glu(C)-OZ, where Z is an electrophilic activating group of the carboxyl group, independently selected from H, or Bt, Su, Pfp, Tcp, or Pnp. Partially protected peptides represented by, in particular, exenatide fragments 1-11, 1-13, 1-14 and 1-15. [Section 29] In a method for producing exenatide, Condensing one fragment described in item 27 with one fragment described in item 28 to form a partially protected or resin-bound exenatide sequence, Deprotect or detach from the resin, and Exenatide is deprotected, chromatographically purified, and freeze-dried. A method that includes that. [Section 30] The following formula: E-Lys(E)-Cys(B)-Asn(A)-Thr(C)-Y-Cys(B)-Y-Gln(A)-Arg(D)-Leu-Ala-Asn(A)-Phe-Leu-Val-His(A)-X-Asn( A)-Asn(A)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(C)-Asn(A)-Val-Gly-Ser(C)-Asn(A)-Thr(C)-Tyr(Resin)-NH2 (In the formula, X is either Ser(tBu)-Ser(tBu) or Ser(tBu)-ΨSer; Y is Ala-Thr(tBu) or Ala-ΨThr; A is independently a carboxamide- or imidazole-protecting group selected from H, or Trt, Mtt, or Mmt; B is a Cys protecting group independently selected from Mmt, Trt, Acm, or StBu; Each C is independently selected from tBu, Trt, or Clt, and is a hydroxyl-, carboxyl-, or phenoxy-protecting group; D is a guanidine protecting group selected from H, Pbf, or Pmc; E is an amino protecting group selected from H, or Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. A partially protected peptide represented by [this symbol]. [Section 31] The peptide according to item 30, which is a protected or partially protected plumrintide. [Section 32] The following formula: Sequence Z-Asn(A)-Phe-Leu-Val-His(A)-X-Asn(A)-Asn(A)-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr(C)-Asn(A)-Val-Gly-Ser(C)-Asn(A)-Thr(C)-Tyr(Resin)-NH2 (In the formula, Z is H, or E-Gln(A)-Arg(D)-Leu-Ala, E-Arg(D)-Leu-Ala, E-Leu-Ala, or E-Ala; X is either Ser(tBu)-Ser(tBu) or Ser(tBu)-ΨSer; A is a carboxamide- or imidazole-protecting group selected from H, or Trt, Mtt, or Mmt; C is a hydroxyl-, carboxy-, or phenoxy-protecting group selected from tBu, Trt, or Clt; D is a guanidine protecting group selected from H, Pbf, or Pmc; E is an amino protecting group selected from H, or Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. Protected or partially protected peptides represented by, in particular, protected or partially protected pramlintide fragments 10-38, 11-38, 12-38 and 14-38. [Section 33] The following formula: [ka] A method for producing partially protected and oxidized pramlintide fragments comprising: The protected peptide is assembled on an acid-sensitive resin, such as a 2-CTC resin; Cleave the peptide protected from the resin; The peptide bound to the resin is oxidized by contact with a mild acid, such as dilute trifluoroacetic acid in an organic solvent like dichloromethane, where the solvent contains 2 to 200 molar excess amounts of iodine relative to the fragment; A method that includes that. [Section 34] In a method for producing pramlintide, the fragment described in claim 33 is used in a solution or on a solid phase, one of the fragments described in claim 32 or claim 33, or the following formula: E-Lys(E)-Cys(B)-Asn(A)-Thr(C)-Y-Cys(B)-Y-Gln(A)-Arg(D)-Leu-Ala-OZ, E-Lys(E)-Cys(B)- A method comprising condensing with one of the fragments represented as Asn(A)-Thr(C)-Y-Cys(B)-Y-Gln(A)-Arg(D)-OZ, E-Lys(E)-Cys(B)-Asn(A)-Thr(C)-Y-Cys(B)-Y-Gln(A)-OZ, or E-Lys(E)-Cys(B)-Asn(A)-Thr(C)-Y-Cys(B)-Y-OZ: Here, Y is independently Ala-Thr(tBu) or Ala-ΨThr; Z is a group selected from H, or Bt, Su, Pfp, Tcp, or Pnp; A is independently a carboxamide- or imidazole-protecting group selected from H, or Trt, Mtt, or Mmt; Each C is independently a hydroxy-, carboxy-, or phenoxy-protecting group selected from tBu, Trt, or Clt; D is independently a guanidine protecting group selected from H, or Pbf or Pmc; E is independently an amino protecting group selected from H, or Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; B is a Cys protecting group selected independently from Mmt, Trt, Acm, or StBu; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. [Section 35] The following formula: ED-Phe-Pro-Arg(D)-Pro-Gly-Gly-Gly-Gly-Asn(A)-Gly-Asp(C)-Phe-Glu(C)-Glu(C)-Ile-Pro-Glu(C)-Glu(C)-Tyr(Resin)-Leu-OC (In the formula, A is a carboxamide- or imidazole-protecting group selected from H, or Mtt or Mmt; C is independently a hydroxyl- or carboxyl-protecting group selected from tBu, Trt, or Clt; D is a guanidine protecting group selected from H, Pbf, or Pmc; E is an amino protecting group selected from H, or Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. A protected or partially protected peptide represented by [the specified symbol]. [Section 36] The protected peptide according to item 35, wherein the protected peptide is bivalirudin. [Section 37] The following formula: EX-Tyr(Resin)-Leu-OC (In the formula, X is the bivalirudin peptide sequence; E is H, or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; C is a hydroxyl- or carboxyl-protecting group selected from tBu, Trt, or Clt; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. A protected or partially protected fragment represented by [this symbol]. [Section 38] In a method for producing bivalirudin, the bivalirudin fragment of formula: EX-Tyr(Resin)-Leu-OC described in claim 37 is condensed with the bivalirudin fragment of formula: ED-Phe-Y-OZ in solution or on a solid phase to produce ED-Phe-YX-Tyr(Resin)-Leu-OC; Deprotection, or cleavage and deprotection from the resin, yields bivalirudin; Bivalirudin was purified by chromatography; By freeze-drying, we obtain bivalirudin with a purity of 99% or higher. A method that includes that. Here, X and Y are independently bivalirudin peptide sequences; The "D-" indicates the chirality of the amino acid that follows the D-amino acid; C is a hydroxy protecting group selected from Clt, Trt, or tBu; E is an amino protecting group selected from Fmoc, Mtt, Mmt, Trt, Boc or Nps; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. [Section 39] The following formula: E-Ser(C)-Tyr(C)-Ser(C)-Met-Glu(C)-His(A)-Phe-Arg(D)-Trp(F)-Gly-Lys(E)-Pro- Val-Gly-Lys(E)-Lys(E)-Arg(D)-Arg(D)-Pro-Val-Lys(E)-Val-Tyr(Resin)-Pro-OC (In the formula, A is a carboxamide- or imidazole-protecting group selected from H, or Trt, Mtt, or Mmt; C is independently a hydroxy-, carboxy-, or phenoxy-protecting group selected from tBu, Trt, or Clt; D is an independent guanidine protecting group selected from H, Pbf, or Pmc; E is an independent amino protecting group selected from H, or Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; F is either H or Boc; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. A protected or partially protected peptide represented by [the specified symbol]. [Section 40] A protected or partially protected peptide as described in item 39, which is an ACTH(1-24) peptide. [Section 41] The following formula: EX-Tyr(Resin)-Pro-OC (In the formula, X is the ACTH(1-24) sequence; E is an amino protecting group selected from H, or Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; and C is a hydroxy-, carboxy-, or phenoxy-protecting group selected from tBu, Trt, or Clt; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis. A protected or partially protected fragment represented by [this symbol]. [Section 42] In the method for producing ACTH(1-24), The ACTH fragment of formula:EX-Tyr(Resin)-Pro-OC described in item 37 is condensed with the ACTH fragment of formula:EY-OZ in solution or on a solid phase to produce EYX-Tyr(Resin)-Pro-OC; Deprotection, or cleavage and deprotection from the resin, results in ACTH(1-24); ACTH(1-24) was purified by chromatography; By freeze-drying, ACTH(1-24) with a purity of 99% or higher is obtained. Methods that include: Here, X and Y are each independently ACTH sequences; C is a hydroxy protecting group selected from Clt, Trt, or tBu; E is an amino protecting group selected from Fmoc, Mtt, Mmt, Trt, Boc or Nps; and Resin is an acid-sensitive resin suitable for H or solid-phase peptide synthesis.

Claims

1. A conjugate of a protected or partially protected ACTH(1-24) peptide and a resin, wherein the conjugate of the peptide and resin is given by the following formula: E-Ser(C)-Tyr(C)-Ser(C)-Met-Glu(C)-His(A)-Phe-Arg(D)-Trp(F)-Gly-Lys(E)-Pr o-Val-Gly-Lys(E)-Lys(E)-Arg(D)-Arg(D)-Pro-Val-Lys(E)-Val-Tyr(resin)-Pro-O-C (In the formula, A is a carboxamide or imidazole protecting group selected from H or Trt, Mtt, or Mmt; Each C is independently a hydroxy, carboxyl, or phenoxy protecting group selected from tBu, Trt, or Clt; Each D is independently a guanidine protecting group selected from H, Pbf, or Pmc; Each E is independently an amino protecting group selected from H or Fmoc, Boc, Trt, Nps, Mtt, Mmt, or Clt; F is either H or Boc; and The resin is an acid-sensitive resin suitable for solid-phase peptide synthesis, and is a trityl-type resin or a benzhydryl-type resin, and in Tyr(resin), Tyr is attached to the resin via its side-chain OH. A conjugate of the protected or partially protected peptide and resin, represented by the above.

2. In a method for producing a sufficiently deprotected ACTH(1-24) peptide, A protected or partially protected ACTH(1-24) peptide-resin conjugate according to claim 1 is prepared by condensing a peptide represented by formula E-X-Tyr(resin)-Pro-O-C and a peptide represented by formula E-Y-OZ in a solution or solid layer, and Deprotection, or cleavage and deprotection from the resin, yields the ACTH(1-24) peptide; By purification by chromatography and freeze-drying, ACTH(1-24) peptide with a purity of 99% or higher is obtained. In a method that includes the following, X and Y are, independently, fragments of the ACTH peptide; In the above formula E-Y-OZ, Z is H, Bt, Su, Pfp, Tcp, or Pnp; C is a hydroxy protecting group selected from Clt, Trt, or tBu; E is an amino protecting group selected from Fmoc, Mtt, Mmt, Trt, Boc or Nps; and The resin is an acid-sensitive resin suitable for solid-phase peptide synthesis, and is either a trityl-type resin or a benzhydryl-type resin; The manufacturing method characterized by the following features.

3. The aforementioned resin is given by the following formula: 【Chemistry 1】 (In the formula, X, Y, Z, and V are each independently located on the ortho, meta, or para positions, and H, Cl, F, C １－１０ Alkyl and C １－１０ Selected from alkoxy; and P is represented as an insoluble solid support or an insoluble linker-resin composite suitable for solid-phase synthesis of peptides. The joint according to claim 1.

Images