Solid-phase simple synthesis method for peptidol

Abstract

A 3,4-diaminobenzoic acid (Dbz) group on a resin is activated by means of isoamyl nitrite to form benzotriazole (Bt), and a reduction reaction is then performed by using sodium borohydride (NaBH4) as a reducing agent. A peptidol can be obtained by means of a rapid reaction and only a simple filtering method; and the generation of by-products accompanying with the reaction can be reduced by changing conditions.

Description

A Convenient Solid-Phase Synthesis Method for Peptide Alcohols Technical Field

[0001] The present invention relates to a solid phase synthesis method for preparing peptide alcohols (peptidol) and their derivatives. Background Art

[0002] Peptides play numerous roles within organisms, forming hormones, neurotransmitters, growth factors, signaling agents, and antibodies. Compared to proteins and antibodies, peptides are easier to prepare and exhibit high target specificity and selectivity, holding great potential for development in drug development and materials applications. Due to their unlimited conformational variability and the ability to modify diverse functional groups, peptides can exhibit a range of diverse biological activities, compared to steroids, which have more limited conformational and functional group variations, and fatty acids, which also possess conformational mobility but cannot be modified with functional groups.

[0003] After carbon-terminal reduction, the previously negatively charged carbon-terminal carboxylic acid functional group is modified, neutralizing the negative charge of the carboxylic acid structure at physiological pH. This removal of the repulsive force created by the negative charge allows the peptide to aggregate more readily, increasing its hydrophobicity and, in turn, altering its receptor binding. This subtle structural change can significantly alter the peptide's folding, target binding, and drug dynamics. For example, enkephalin, an endogenous opioid pentapeptide responsible for neurotransmission and pain regulation, exhibits thirty-fold greater binding to mouse brain membranes after its carbon-terminal modification with a hydroxyl group compared to its original carbon-terminal carboxylic acid structure, demonstrating significant and long-lasting analgesic activity.

[0004] Chemical peptide preparations typically have carboxylic acids or amides at the carbon termini. However, peptides with other carbon-terminal modifications are often found in natural products, exhibiting diverse biological activities and potential as therapeutic drugs. Previous research on the chemical synthesis of peptide alcohols has lacked versatile, convenient, and rapid synthesis methods, necessitating further development in the chemical synthesis of peptide alcohols. Current methods for preparing peptide alcohols typically involve linking the peptide to an amino alcohol on a resin, followed by the modification of the amino acid to the amino alcohol in the liquid phase. This necessitates modifications to the linker, leading to long reaction times for the coupling and cleavage of the amino alcohol. This exposes the prepared peptide to strong acid and base conditions for extended periods, increasing the risk of racemization.

[0005] In solid-phase peptide synthesis (SPPS), a resin is used as a solid support. A stopper is placed inside the container to block the resin. First, a deprotection reagent is used to remove the protecting groups on the resin's backbone. Excess reagents are then removed by washing. The amino acid to be synthesized is dissolved in a coupling reagent and coupled to the resin. After the reaction, a simple wash is required to filter out excess reagents and other byproducts, improving liquid-phase yield and synthesis efficiency. Its convenience and high efficiency make it an advantage in subsequent chemical peptide preparation. In solid-phase peptide synthesis, the resin is used as a solid support, connected to a linker, and then coupled to the amino acid's carbon terminus, exposing the amino terminus for subsequent reactions.

[0006] Amino acids are coupled to resins and then modified. This method is applicable to various peptide C-terminus modifications. For example, starting with Rink amide resin, acid cleavage of the peptide after synthesis can yield a C-terminus amide peptide. The functional group provided by the linker is then attached to the peptide, resulting in a functional group modification at the C-terminus.

[0007] The difficulty in carbon-terminal modification during solid-phase peptide synthesis is due to the need for the carbon terminus to be fixed to the resin. Linking the resin via a side chain functional group or amino terminus leaves the carbon terminus exposed, making carbon-terminal modification less difficult. In 1971, Kenner's team proposed the safety-catch linker method, which uses the carbon terminus to link to the resin. Unactivated, the linker remains stable and unreactive during solid-phase peptide synthesis. Upon activation, the linker forms a leaving group, allowing nucleophilic substitution reactions to modify the peptide carbon terminus with the desired functional group and simultaneously cleave from the solid phase.

[0008] Among peptide alcohol preparations, the amino alcohol condensation method has seen the most development. This indicates that the development of new resin linkers for hydroxyl group attachment is often the focus of solid-phase peptide alcohol synthesis. However, resin linkers often suffer from long coupling times and low resin loading rates. Excessive coupling times require the amino acid to remain in an alkaline environment for extended periods, increasing the risk of peptide racemization. Furthermore, the preparation of amino alcohols is a significant challenge. Nucleophilic substitution methods are less extensively developed than amino alcohol condensation methods, with only a few publications addressing solid-phase carbon-terminal modification with reducing agents. However, these methods exhibit suboptimal yields, require long reduction times or microwave energy, and also present racemization issues. The only methods with shorter reaction times suffer from severe hydrolysis to form peptides, but these methods are applicable to common amino acids and do not require prior amino alcohol modification. Finally, non-carboxylic acid-linked resin methods offer solid-phase attachment sites beyond the carbon terminus, thereby reducing the challenges of solid-phase peptide carbon-terminal modification. Currently, no comprehensive method for preparing peptide alcohols has been established. Therefore, a robust method for synthesizing peptide alcohols is urgently needed to address these challenges.

[0009] Summary of the Invention

[0010] Other features and advantages of the present invention will be revealed from the following non-limiting detailed description and examples.

[0011] As used herein, “a / an,” “said,” “the,” “at least one,” and “one or more” can be used interchangeably.

[0012] In order that the present invention may be more readily understood, certain terms are first defined, and additional definitions are set forth throughout the detailed description.

[0013] The present invention utilizes common solid-phase peptide synthesis methods to produce a solid-phase peptide. A safety-catch linker is then activated to form a leaving group. A reducing agent is then used to reduce the linker-amino acid junction, simultaneously cleaving the resin to produce the peptide alcohol. This method is applicable to a variety of amino acids, making it more versatile than the previously mentioned methods. In previous solid-phase peptide synthesis, direct reaction of a reducing agent with the resin to produce a peptide alcohol was rare. Therefore, the present invention utilizes a reducing agent for reductive cleavage, allowing the peptide alcohol to be directly obtained without the need for complex post-processing and purification.

[0014] Specifically, the present invention utilizes a safety-catch method to activate 3,4-diaminobenzoic acid (Dbz) resin with isoamyl nitrite to form a benzotriazole (Bt) resin. Sodium borohydride (NaBH4) is then used as a reducing agent for the reduction reaction. This rapid reaction eliminates the need for tedious purification steps and allows the peptide alcohol to be obtained through simple filtration. Hydrolysis and alcoholysis byproducts (e.g., ethyl esters) may be generated after the reaction. Hydrolysis byproducts (e.g., acidic compounds) can be removed by replacing the reducing solvent with dimethylformamide (DMF) to reduce the water content of the solvent and thereby remove the hydrolysis byproducts. Alcoholysis byproducts can be reduced to the peptide alcohol through repeated reduction. Following the reduction of aspartic acid, lactonization is likely to occur during acidic removal of the protecting group. Therefore, the acidic removal step must be performed in an ice bath to inhibit lactonization.

[0015] Therefore, the present invention has developed a simple and rapid method for preparing peptide alcohols, which is applicable to common amino acids. Through the design of the linker, only a mild reducing agent is required to remove the peptide carbon end from the resin and simultaneously form the peptide alcohol. In solid-phase peptide synthesis, 4-amino-3-nitrobenzoic acid (ANB) is connected to Rink amide resin, and the nitro group is reduced to an amino group using stannous dichloride dihydrate and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The linker becomes 3,4-diaminobenzoic acid (Dbz), which is then coupled to the amino acid. After obtaining the target sequence through solid-phase peptide synthesis, the Dbz resin was converted to benzotriazole (Bt) resin through isoamyl nitrite reaction. Considering the swelling of the resin and the good solubility of sodium borohydride (NaBH4) in alcohols, DCM:EtOH = 3:1 was subsequently selected as the solvent system, and NaBH4 was used for the reduction reaction. The conditions for preparing peptide alcohols were an ice bath for 1 hour and reaction at room temperature for 0.5 hours to obtain peptide alcohols.

[0016] Therefore, the present invention provides a method for synthesizing a peptide alcohol, comprising:

[0017] (a) reacting a compound of formula (Ia) with nitrite and acetic acid in a polar solvent to obtain a compound of formula (IIa) having a benzotriazole structure,

[0018] M is the solid support material;

[0019] X is a peptide;

[0020] Y and Y' are protecting groups for protecting the N-terminus of X; and

[0021] (b) reacting the compound of formula (IIa) with sodium borohydride in a mixed solution containing a polar aprotic solvent and an alcohol solvent to reduce the carbon end of the peptide to obtain a peptide alcohol compound.

[0022] In one embodiment, the solid support material comprises a resin.

[0023] In another embodiment, the polar solvent comprises dimethylformamide (DMF).

[0024] In another embodiment, the nitrite comprises isoamyl nitrite.

[0025] In a specific embodiment, the polar aprotic solvent includes dichloromethane (DCM) or tetrahydrofuran (THF), and the alcohol solvent includes methanol, ethanol, propanol, butanol, etc. In the mixed solution of the polar aprotic solvent and the alcohol solvent, the ratio of the polar aprotic solvent to the alcohol solvent is 10:1 to 1:10, preferably 5:1 to 1:5, and more preferably 3:1 to 1:3.

[0026] In another more specific embodiment, the reaction conditions of step (b) are first ice-bathing for 1 hour and then reacting at room temperature for 0.5 hour.

[0027] In another more specific embodiment, step (c) is further included after step (b), wherein step (c) comprises removing the protecting group on the peptidol compound.

[0028] If the synthesized peptide alcohol molecule is too large (such as branched peptide alcohol, the efficiency of reductive cleavage is poor and the yield is extremely low), a resin with lower steric hindrance (Tenta gel Resin) or a linker (6-amino-hexanoic acid) must be used to improve the yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 relates to peptide alcohol synthesis products using NH2-AQETAol as an example.

[0030] Figure 2 is an HPLC chromatogram of a peptide alcohol synthesis product, exemplified by AQETV. The horizontal axis represents retention time, and the vertical axis represents signal intensity.

[0031] Figure 3 shows an HPLC chromatogram of a peptide alcohol synthesis product with a high proportion of ethyl ester by-products. The horizontal axis is retention time, and the vertical axis is signal intensity.

[0032] Figure 4 shows an HPLC chromatogram of a peptide alcohol synthesis product with a high proportion of hydrolysis by-products. The horizontal axis is retention time, and the vertical axis is signal intensity.

[0033] Figure 5 relates to HPLC chromatograms of products synthesized using peptide alcohols suitable for aspartic acid and asparagine. The horizontal axis represents retention time, and the vertical axis represents signal intensity.

[0034] Figure 6 relates to an HPLC chromatogram of a product synthesized using a peptide alcohol suitable for branched peptide synthesis. The horizontal axis represents retention time, and the vertical axis represents signal intensity.

[0035] Figure 7 relates to an HPLC chromatogram of a product synthesized using a peptide alcohol suitable for the synthesis of Peptaibols. The horizontal axis represents retention time, and the vertical axis represents signal intensity.

[0036] Figure 8 relates to an HPLC chromatogram of a peptide alcohol synthesis product suitable for small molecule library synthesis. The horizontal axis is retention time and the vertical axis is signal intensity. DETAILED DESCRIPTION

[0037] The following examples are non-limiting and merely represent various aspects and features of the present invention.

[0038] This invention establishes several different carbon-terminal reduction methods. Method 1: Using dichloromethane:ethanol as a solvent system for the carbon-terminal reduction reaction is suitable for the preparation of peptide alcohols without severe hydrolysis and ethanolysis. If these severe hydrolysis occurs, methods 2 or 3 must be used instead. Method 2: Using dichloromethane:ethanol as a solvent system for the secondary carbon-terminal reduction reaction. This method can reduce the ethanolysis product to the peptide alcohol. However, peptide alcohols with severe hydrolysis cannot be corrected by this method and must be replaced by method 3. Method 3: Using dimethylformamide as a solvent system for the carbon-terminal reduction reaction can effectively reduce the formation of hydrolysis products, thereby improving the efficiency of peptide alcohol preparation. Method 4: Changing the subsequent acid cleavage side chain protecting group to an ice bath for 1 hour. This method can reduce the proportion of side chain functional groups such as aspartic acid that are attacked by the carbon-terminal hydroxyl group to form lactone derivatives, but it cannot completely prevent the formation of lactone derivatives. The carbon-terminal reduction method has been successfully applied to 20 different amino acids.

[0039] Weigh 0.3333 g (0.1 mmol) of Rink amide resin (loading rate 0.3 mmol / g) and add DMF (4 mL). Soak and shake for 1 hour. Add 20% piperidine dissolved in DMF and react for 10 minutes to remove the Fmoc residue. Drain and repeat. Wash three times with DCM and DMF. Add ANB (0.0728 g, 0.4 mmol, 4 eq) and hexafluorophosphate benzotrizole tetramethyl uranium (HBTU) (0.1516 g, 0.4 mmol, 4 eq). Add DMF (5% NMM, 3 mL) and react for 2 hours. Wash three times with DCM and DMF. The resin was soaked in DMF (4 mL), and tin(II) chloride dihydrate (2.256 g, 98%, 2.5 M, 11.89 mmol, 119 eq) and DBU (300 mL, 2 mmol, 20 eq) were added and allowed to react for 6 hours. The mixture was washed five times with DCM and DMF. The first amino acid (4 eq.) and benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBop) (0.2080 g, 0.4 mmol, 4 eq) were added and reacted in DMF (5% NMM, 3 mL) for 2 hours. The mixture was drained and the reaction was repeated once. The mixture was washed three times with DCM and DMF.

[0040] After obtaining the target solid-phase peptide through the SPPS process, the peptide alcohol synthesis was carried out using four different methods. Method 1 was used first. If the by-product ratio was high, methods 2, 3, and 4 were used.

[0041] Example 1

[0042] Fmoc-AQETV-Dbz-Rink amide resin (0.05 mmol) was added to DMF (4 mL) and shaken for 1 hour. Isoamyl nitrite (60 eq, 400 uL) was mixed with acetic acid (1.8 eq, 5.14 uL) and mixed for 30 minutes. The resin was then added and activated for 2 hours. The resin was washed five times with DCM and DMF, alternating between DCM and DMF. The resin was placed in a 25 mL round-bottom flask and filled with DCM:EtOH (3:1) (4 mL). The flask was placed in an ice bath. NaBH₄ (5 eq, 9.4 mg) was added and allowed to react on ice for 1 hour. After removing the ice bath and continuing the reaction for another 30 minutes, the filtrate was collected, as shown in Figure 1. The organic layer was washed twice with saturated brine and dehydrated with anhydrous MgSO₄. The mixture was filtered and concentrated to dryness under reduced pressure. 20% piperidine dissolved in DMF was added and reacted for 30 minutes to remove the Fmoc residue. The mixture was then dried under reduced pressure. Add TFA:TIS:H2O=95:2.5:2.5 (v / v / v) (2 mL) and react for 30 minutes. Add icy ether for precipitation, centrifuge, and lyophilize.

[0043] Figure 2 is a HPLC chromatogram of the peptide alcohol synthesis product of this example. The horizontal axis is the retention time and the vertical axis is the signal intensity.

[0044] Example 2: Method suitable for high proportion of ethyl ester by-product

[0045] Fmoc-AQETL-Dbz-Rink amide resin (0.05 mmol) was added to DMF (4 mL) and shaken for 1 hour. Isoamyl nitrite (60 eq, 400 uL) was mixed with acetic acid (1.8 eq, 5.14 uL) and mixed for 30 minutes. The resin was then added and activated for 2 hours. The resin was washed five times with DCM and DMF, alternating between the two. The resin was placed in a 25 mL round-bottom flask, and a 3:1 ratio of DCM:EtOH (4 mL) was added. The flask was placed in an ice bath. NaBH₄ (5 eq, 9.4 mg) was added and allowed to react on ice for 1 hour. After removing the ice bath, the reaction continued for another 30 minutes. The filtrate was collected. After draining, the flask was again filled with a 3:1 ratio of DCM:EtOH (4 mL). The flask was placed in an ice bath. NaBH₄ (5 eq, 9.4 mg) was added and allowed to react on ice for 1 hour. The reaction continued for another 30 minutes. The filtrate was collected. The organic layer was washed twice with saturated brine and dehydrated with anhydrous MgSO₄. The mixture was filtered and concentrated under reduced pressure to dryness. 20% piperidine dissolved in DMF was added and reacted for 30 minutes to remove the Fmoc residue. The mixture was then dried. TFA:TIS:H₂O = 95:2.5:2.5 (v / v / v) (2 mL) was added and reacted for 30 minutes. The mixture was precipitated by adding glacial ether, centrifuged, and lyophilized.

[0046] Figure 3 shows the HPLC chromatogram of a peptide alcohol synthesis product with a high proportion of ethyl ester byproducts. The horizontal axis is retention time, and the vertical axis is signal intensity.

[0047] Example 3: Method suitable for high proportion of hydrolysis by-products

[0048] Fmoc-AQETA-Dbz-Rink amide resin (0.05 mmol) was added to DMF (4 mL) and shaken for 1 hour. Isoamyl nitrite (60 eq, 400 μL) was mixed with acetic acid (1.8 eq, 5.14 μL) and mixed for 30 minutes. The resin was then added and activated for 2 hours. The resin was washed five times with DCM and DMF, alternating between the two. The resin was placed in a 25 mL round-bottom flask and placed in an ice bath. DMF (4 mL) and NaBH₄ (5 eq, 0.0094 g) were added, and the resin turned orange-red. The reaction was continued in an ice bath for 1 hour. The ice bath was removed and the reaction continued for another 30 minutes. The filtrate was collected. The organic layer was washed twice with saturated brine and dehydrated with anhydrous MgSO₄. The mixture was filtered and concentrated under reduced pressure to dryness. 20% piperidine dissolved in DMF was added and reacted for 30 minutes to remove the Fmoc residue and drain. Add TFA:TIS:H2O=95:2.5:2.5 (v / v / v) (2 mL) and react for 30 minutes. Add icy ether for precipitation, centrifuge, and lyophilize.

[0049] Figure 4 shows the HPLC chromatogram of a peptide alcohol synthesis product containing a high proportion of hydrolysis by-products. The horizontal axis represents retention time, and the vertical axis represents signal intensity.

[0050] Example 4: Method applicable to aspartic acid

[0051] Fmoc-AQETD-Dbz-Rink amide resin (0.05 mmol) was added to DMF (4 mL) and shaken for 1 hour. Isoamyl nitrite (60 eq, 400 uL) was mixed with acetic acid (1.8 eq, 5.14 uL) and mixed for 30 minutes. The resin was then added and activated for 2 hours. The resin was washed five times with DCM and DMF, alternating between them. The resin was placed in a 25 mL round-bottom flask and placed in an ice bath. DCM:EtOH = 3:1 (4 mL) was added, along with NaBH4 (5 eq, 0.0094 g). The reaction was allowed to proceed under ice bath for 1 hour. After removing the ice bath and continuing for another 30 minutes, the filtrate was collected. The organic layer was washed twice with saturated brine and dehydrated with anhydrous MgSO4. The mixture was filtered and concentrated to dryness under reduced pressure. 20% piperidine dissolved in DMF was added and shaken for 30 minutes to remove the Fmoc residue. The mixture was then dried under ice bath. Add TFA:TIS:H2O=95:2.5:2.5 (v / v / v) (2 mL) and react under ice bath for 1 hour. Add icy ether for precipitation, centrifuge and freeze-dry.

[0052] Figure 5 is an HPLC chromatogram of the peptide alcohol synthesis products of aspartic acid and asparagine. The product with the higher signal is the desired product. The horizontal axis is the retention time and the vertical axis is the signal intensity.

[0053] Example 5: Methods for branched peptide synthesis

[0054] Weigh 0.4166 g (0.1 mmol) of TentaGel resin (loading rate 0.24 mmol / g) and add DMF (4 mL). Soak and shake for 1 hour. Add 20% piperidine dissolved in DMF and react for 10 minutes to remove Fmoc. Drain and repeat. Wash three times with DCM and DMF. Add Fmoc-6-carbamic acid-Dbz (0.1948 g, 0.4 mmol, 4 eq) and HBTU (0.1516 g, 0.4 mmol, 4 eq). Add DMF (5% NMM, 3 mL) and react for 2 hours. Wash three times with DCM and DMF. Add 20% piperidine dissolved in DMF and react for 10 minutes to remove Fmoc. Drain and repeat. Wash three times with DCM and DMF. Add Fmoc-L-Lys(Fmoc)-OH (0.1040 g, 0.175 mmol, 3.5 eq) and HBTU (0.1326 g, 0.175 mmol, 3.5 eq) in DMF (5% NMM, 2 mL) and allow to react for 2 hours. Wash the mixture three times with DCM and DMF. Add 20% piperidine dissolved in DMF and allow to react for 10 minutes to remove the Fmoc residue. Drain and repeat the process. Wash the mixture three times with DCM and DMF. Add Fmoc-L-Lys(Fmoc)-OH (0.2060 g, 0.35 mmol, 3.5 eq) and HBTU (0.2652 g, 0.35 mmol, 3.5 eq) in DMF (5% NMM, 2 mL) and allow to react for 1 hour. Wash the mixture three times with DCM and DMF. Add 20% piperidine dissolved in DMF and react for 10 minutes to remove Fmoc, drain, and repeat. Wash with DCM and DMF three times, alternating between. Add Fmoc-L-Lys(Fmoc)-OH (0.4130 g, 0.7 mmol, 3.5 eq) and HBTU (0.5304 g, 0.7 mmol, 3.5 eq) in DMF (5% NMM, 2 mL) and react for 2 hours. Wash with DCM and DMF three times, alternating between. Add 20% piperidine dissolved in DMF and react for 10 minutes to remove Fmoc, drain, and repeat. Wash with DCM and DMF three times, alternating between. Add Boc-L-Lys(Boc)-OH (0.8260 g, 1.4 mmol, 3.5 eq) and HBTU (1.0608 g, 1.4 mmol, 3.5 eq) in DMF (5% NMM, 4 mL) and react for 2 hours. The mixture was washed alternately with DCM and DMF three times to obtain Boc-K8(Boc)K4K2K-Dbz-TentaGel resin.

[0055] Boc-K8(Boc)K4K2K-Dbz-TentaGel resin (0.05 mmol) was added to DMF (4 mL) and shaken for 1 hour. Isoamyl nitrite (60 eq, 400 uL) was mixed with acetic acid (1.8 eq, 5.14 uL) and mixed for 30 minutes. The resin was then added and activated for 2 hours. The resin was washed five times with DCM and DMF, alternating between them. The resin was placed in a 25 mL round-bottom flask and filled with DCM:EtOH = 3:1 (4 mL). The flask was placed in an ice bath. NaBH4 (5 eq, 9.4 mg) was added and reacted in an ice bath for 1 hour. The ice bath was removed and the reaction continued for another 30 minutes. The filtrate was collected. The organic layer was washed twice with saturated brine and dried over anhydrous MgSO4. The mixture was filtered and concentrated to dryness under reduced pressure. TFA:TIS:H2O = 95:2.5:2.5 (v / v / v) (2 mL) was added and the reaction continued for 30 minutes. Add icy ether to precipitate, centrifuge and freeze-dry.

[0056] Figure 6 is an HPLC chromatogram of the peptide alcohol synthesis product of branched peptide synthesis. The horizontal axis is retention time and the vertical axis is signal intensity.

[0057] Example 6: Method for the synthesis of Peptaibols

[0058] Weigh 0.3333 g (0.1 mmol) of Rink amide resin (loading rate 0.3 mmol / g) into DMF (4 mL) and let it soak with shaking for 1 hour. Add 20% piperidine dissolved in DMF and react for 10 minutes to remove the Fmoc residue. Drain and repeat. Wash the mixture three times with DCM and DMF. Add ANB (0.0728 g, 0.4 mmol, 4 eq) and HBTU (0.1516 g, 0.4 mmol, 4 eq) and DMF (5% NMM, 3 mL) and let it react for 2 hours. Wash the mixture three times with DCM and DMF. Soak the resin in DMF (4 mL) and add Tin(II) chloride dihydrate (2.256 g, 98%, 2.5 M, 11.89 mmol, 119 eq) and DBU (300 μL, 2 mmol, 20 eq) and let it react for 6 hours. Wash the reaction mixture five times with DCM and DMF. Add Fmoc-Phe-OH (155 mg, 0.4 mmol, 4 eq) and PyBop (0.2080 g, 0.4 mmol, 4 eq) and react in DMF (5% NMM, 3 mL) for 2 hours, drain, and repeat the reaction. Wash the reaction mixture three times with DCM and DMF. The target solid-phase peptide was subsequently obtained through SPPS. The Fmoc-amino acid was dissolved in DMF (0.2 M) and an equivalent amount of Oxyma dissolved in DMF (0.5 M) and two equivalents of DIC dissolved in DMF (1.0 M) were added. The Fmoc protecting group was removed twice using 20% ​​piperidine dissolved in DMF (4 mL), each for 10 minutes. After the reaction was complete, the reaction mixture was washed three times with DCM and DMF. Unless otherwise noted, the coupling time was 1 hour. The order and amount of Fmoc-amino acid addition are detailed below.Fmoc-L-Gln(Trt)-OH (122 mg, 0.2 mmol, 2 eq), Fmoc-L-Glu(OtBu)-OH (85 mg, 0.2 mmol, 2 eq), Fmoc-Aib-OH (131 mg, 0.4 mmol, 4 eq) were coupled for 2 hours, Fmoc-Aib-OH (131 mg, 0.4 mmol, 4 eq) was coupled for 3 hours, Fmoc-L-Val-OH (136 mg, 0.4 mmol) was coupled for 1 hour, and Fmoc-Val-OH (136 mg, 0.4 mmol) was coupled for 1 hour. , 4eq) were coupled for 2 hours, Fmoc-L-Pro-OH (136 mg, 0.4 mmol, 4eq), Fmoc-Aib-OH (131 mg, 0.4 mmol, 4eq) were coupled for 2 hours twice, Fmoc-L-Leu-OH (142 mg, 0.4 mmol, 4eq), Fmoc-L-Gly-OH (120 mg, 0.4 mmol, 4eq) were coupled for 1.5 hours, Fmoc-Aib-OH (131 mg, 0 .4mmol, 4eq), Fmoc-L-Val-OH (136mg, 0.4mmol, 4eq), Fmoc-Aib-OH (131mg, 0.4mmol, 4eq) were coupled for 4 hours, Fmoc-L-Gln(Trt)-OH (245mg, 0.4mmol, 4eq), Fmoc-L-Ala-OH (125mg, 0.4mmol, 4eq) were coupled for 2 hours, Fmoc-Aib-OH (131mg, 0.4 mmol, 4 eq), Fmoc-L-Ala-OH (62 mg, 0.2 mmol, 2 eq), and Fmoc-Aib-OH (131 mg, 0.4 mmol, 4 eq) were coupled for 2 hours. Fmoc-L-Pro-OH (136 mg, 0.4 mmol, 4 eq) and Fmoc-Aib-OH (131 mg, 0.4 mmol, 4 eq) were coupled with ultrasound for 10 minutes each time twice. Finally, acetic anhydride (20 μL) was dissolved in 5% NMM in DMF (2 mL) for 30 minutes.

[0059] Ac-UPUAUAQUVUGLUPVUUEQF-Dbz-Rink amide resin (0.05 mmol) was added to DMF (4 mL) and shaken for 1 hour. Isoamyl nitrite (60 eq, 400 uL) was mixed with acetic acid (1.8 eq, 5.14 uL) and mixed for 30 minutes. The resin was then added and activated for 2 hours. The resin was washed five times with DCM and DMF, alternating between the two. The resin was placed in a 25 mL round-bottom flask and filled with DCM:EtOH = 3:1 (4 mL). The flask was placed in an ice bath. NaBH₄ (5 eq, 9.4 mg) was added and allowed to react on ice for 1 hour. After removing the ice bath, the reaction continued for another 30 minutes and the filtrate was collected. The organic layer was washed twice with saturated brine and dehydrated with anhydrous MgSO₄. The mixture was filtered and concentrated to dryness under reduced pressure. TFA:TIS:H₂O = 95:2.5:2.5 (v / v / v) (2 mL) was added and allowed to react for 30 minutes. Add icy ether to precipitate, centrifuge and freeze-dry.

[0060] Figure 7 is an HPLC chromatogram of the peptide alcohol synthesis product synthesized by Peptaibols. The horizontal axis is retention time and the vertical axis is signal intensity.

[0061] Example 7: Applicable to small molecule library synthesis

[0062] Weigh 0.3333 g (0.1 mmol) of Rink amide resin (loading rate 0.3 mmol / g) into DMF (4 mL) and let it soak with shaking for 1 hour. Add 20% piperidine dissolved in DMF and react for 10 minutes to remove the Fmoc residue. Drain and repeat. Wash the mixture three times with DCM and DMF. Add ANB (0.0728 g, 0.4 mmol, 4 eq) and HBTU (0.1516 g, 0.4 mmol, 4 eq) and DMF (5% NMM, 3 mL) and let it react for 2 hours. Wash the mixture three times with DCM and DMF. Soak the resin in DMF (4 mL) and add Tin(II) chloride dihydrate (2.256 g, 98%, 2.5 M, 11.89 mmol, 119 eq) and DBU (300 μL, 2 mmol, 20 eq) and let it react for 6 hours. Wash the reaction mixture five times with DCM and DMF. Add Fmoc-Gly-OH (0.1189 g, 0.4 mmol, 4 eq) and PyBop (0.2080 g, 0.4 mmol, 4 eq) and react in DMF (5% NMM, 3 mL) for 2 hours, drain, and repeat the reaction. Wash the reaction mixture three times with DCM and DMF. The target solid-phase peptide was then obtained through SPPS. The desired coupling agent was dissolved in DMF (0.2 M), and an equivalent amount of Oxyma dissolved in DMF (0.5 M) and two equivalents of DIC dissolved in DMF (1.0 M) were added. The Fmoc protecting group was removed twice with 20% piperidine in 4 mL of DMF, each for 10 minutes. Upon completion, the reaction mixture was washed three times with DCM and DMF. The coupling time was 2 hours. The order and equivalents of the coupling agents are detailed below. Terephthalic acid (0.0332 g, 0.2 mmol, 2 eq), Boc-piperazine (0.0373 g, 0.2 mmol, 2 eq), and Boc-piperazine-TPA-G-Dbz-Rink amide resin (0.05 mmol) were added to DMF (4 mL) and shaken for 1 hour. Isoamyl nitrite (60 eq, 400 uL) was mixed with acetic acid (1.8 eq, 5.14 uL) and mixed for 30 minutes. The resin was then added and activated for 2 hours. The resin was washed five times with DCM and DMF, alternating between them. The resin was placed in a 25 mL round-bottom flask and a mixture of DCM:EtOH (3:1) (4 mL) was added. The flask was placed in an ice bath and NaBH4 (5 eq, 9.4 mg) was added. The reaction was allowed to proceed on ice for 1 hour. After removing the ice bath, the reaction continued for another 30 minutes. The filtrate was collected. The organic layer was washed twice with saturated brine, dried over anhydrous MgSO4, filtered, concentrated under reduced pressure and dried, and 20% TFA dissolved in DCM (2 mL) was added and reacted for 30 minutes.The mixture was drained, precipitated by adding icy ether, centrifuged and freeze-dried.

[0063] Figure 8 is a HPLC chromatogram of a peptide alcohol synthesis product synthesized by the small molecule library. The horizontal axis is retention time, and the vertical axis is signal intensity.

[0064] Example 8: Optimization of peptide carbon-terminal reduction reaction

[0065] Conditions were optimized using Fmoc-AQETA-Dbz-Rink amide. When discussing the product ratios in the reaction below, unless otherwise noted, the sum of the integrated peak areas of the primary products recorded in SIM mode in the mass spectrum is used as the denominator, and the integrated peak area of ​​the target product is used as the numerator to calculate the proportion of the primary product. The initial consideration is the selection of an appropriate reaction solvent, taking into account resin swelling and the good solubility of sodium borohydride in alcohols. Solvent system testing began with THF:CH3OH. At a THF:CH3OH ratio of 1:3, the crude product contained 93% NH2-AQETA-COOCH3. Results indicated methanolysis as the primary reaction, and the resin was observed to adhere to the walls of the reaction flask, preventing efficient reaction. This is hypothesized to be due to the increased polarity of the solvent due to the increased methanol content. The hydrophobic structure of the resin, however, predisposes it to aggregation in the solvent, preventing effective resin swelling and, consequently, reduced yield. Therefore, the solvent system was changed to THF:CH3OH = 3:1, and the peptide alcohol ratio reached 86%; however, 3% NH2-AQETA-COOCH3 was still observed, as well as 11% hydrolysis byproducts. To avoid methanolysis, the solvent system was changed to DCM:EtOH, but only the main product, NH2-AQETAol, was formed. The solvent ratios were then adjusted. However, under the same conditions, the signal of one byproduct gradually increased with the ethanol ratio. Based on molecular weight comparison, it was inferred that it was the ethanolysis product NH2-AQETA-COOCH2CH3. As the DCM ratio increased, the peptide alcohol ratio also increased, and the ethanolysis and hydrolysis ratios decreased. Even at a DCM:EtOH ratio of 1:3, the ethanolysis ratio was significantly higher than that without methanolysis. Therefore, DCM:EtOH = 3:1 was subsequently selected as the solvent system, as shown in Table 1.

[0066] Table 1. Ratios of various products under different reduction conditions of NH2-AQETAol.

[0067] Product ratio: Set the main product m / z in the SIM mode of the crude product mass spectrum, add the integrated front areas of the main product m / z as the denominator, and calculate the proportion of the main product; ND: Not detected.

[0068] While the invention has been described and illustrated in sufficient detail for those skilled in the art to make and use it, it will be apparent that various alternatives, modifications and improvements will occur without departing from the spirit and scope of the invention.

[0069] Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the foregoing objects and obtain the aforementioned purposes and advantages, as well as those inherent therein. The processes and methods for producing the same represent preferred embodiments and are exemplary and do not limit the scope of the invention. Modifications and other uses thereof will occur to those skilled in the art. Such modifications are within the spirit of the invention and are defined by the scope of the claims.

Claims

1. A method for synthesizing a peptide alcohol, comprising: (a) reacting the compound of formula (Ia) with nitrite and acetic acid in a polar solvent to obtain a compound of formula (IIa) having a benzotriazole structure, M is the solid support material; X is a peptide; Y and Y' are protecting groups for protecting the N-terminus of X; and (b) reacting the compound of formula (IIa) with sodium borohydride in a mixed solution containing a polar aprotic solvent and an alcohol solvent to reduce the carbon end of the peptide to obtain a peptide alcohol compound.

2. The method of claim 1, wherein the solid support material comprises a resin.

3. The method of claim 1, wherein the polar solvent comprises dimethylformamide (DMF), and the nitrite comprises isoamyl nitrite.

4. The method of claim 1, wherein the polar aprotic solvent comprises dichloromethane, and the alcohol solvent comprises ethanol.

5. The method according to claim 1, wherein the reaction conditions of step (b) are first ice bathing for 1 hour and then reacting at room temperature for 0.5 hour.

6. The method of claim 1, further comprising a step (c) after the step (b), wherein the step (c) comprises removing the protecting group on the peptidol compound.

7. The method of claim 1, wherein the step (b) produces hydrolysis by-products or ethanolysis by-products.

8. The method of claim 7, wherein the hydrolysis byproducts can be used to replace the reducing solvent with dimethylformamide (DMF) to reduce the water content of the solvent and remove the hydrolysis byproducts.

9. The method of claim 7, wherein the ethanolysis byproduct is reduced to a peptide alcohol by repeated reduction.

10. The method of claim 1, wherein when the peptide is aspartic acid, after the reduction of aspartic acid, the acid protection group removal step must be carried out in an ice bath to inhibit lactonization reaction.

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