FMOC group cleavage method

Abstract

The present invention provides a method for cleaving Fmoc groups, characterized by using a solution containing 3-(diethylamino)propylamine. In particular, the present invention provides a method for preparing solid-phase peptides, wherein Fmoc-protected amino acids are used and the Fmoc groups are cleaved with a solution containing 3-(diethylamino)propylamine.

Description

Technical Field

[0001] The present invention relates to the field of amino function protecting chemicals. In particular, the present invention relates to a method for cleaving Fmoc amino protecting groups. More particularly, the present invention relates to a method for cleaving Fmoc amino protecting groups in peptide synthesis, such as in solid-phase peptide synthesis (SPPS). Background Art

[0002] The Fmoc group is one of the most commonly used amino protecting groups. The most commonly used method for removing the Fmoc group is to treat the protected compound with 1,1-dimethylformamide (DMF) containing piperidine solution. The Fmoc group is one of the most commonly used α-amino protecting groups in polypeptide synthesis. It is particularly common to use the Fmoc group as the α-amino protecting group in sequence elongation in SPPS. The standard procedure for removing the Fmoc group after each amide bond formation cycle in stepwise peptide synthesis is to treat the growing peptide with a solution of 20% piperidine in DMF.

[0003] However, piperidine is a controlled substance because it is used as a precursor in the illegal synthesis of internationally controlled narcotic drugs and psychotropic substances such as fentanyl and phencyclidine (PCP, also known as "angel dust"). For this reason, it is included in the red list of the International Narcotics Control Board (INCB, see the 17th edition in January 2020).

[0004] In addition, the piperidine that requires a large excess to cleave Fmoc is a highly toxic compound. The acute oral toxicity in many types of test animals is high. The LD 50 values for mice, rabbits and rats are 30, 145 and 400 mg / kg, respectively.

[0005] Therefore, there is a need for new synthetic routes to address the disadvantages of piperidine use, which are suitable for application in the manufacture of industrial-scale chemicals, especially peptides, and have no restrictions on piperidine use. Summary of the Invention

[0006] The problem is solved by the present invention, which provides a method for cleaving Fmoc from one or more Fmoc-protected amino groups, wherein the method comprises the step of contacting the Fmoc-protected amino group with a solution containing 3-(diethylamino)propylamine (also known as DEAPA).

[0007] The present invention further provides a method for cleaving Fmoc amino protecting groups by using a solution containing DEAPA in peptide synthesis, especially in solid-phase peptide synthesis.

[0008] In addition, the present invention provides a method for preparing a peptide by Fmoc-based solid-phase peptide synthesis, wherein the method comprises contacting an Fmoc-protected amino group with a solution comprising 3-(diethylamino)propylamine, thereby cleaving Fmoc from one or more Fmoc-protected amino groups.

[0009] Unexpectedly, the method provides a peptide with improved overall purity.

[0010] In one embodiment, the present invention provides a method for preparing a peptide using solid-phase peptide synthesis, wherein the peptide comprises at least one aspartic acid amino acid, and the method is characterized by using a solution comprising DEAPA to cleave the Fmoc amino protecting group. More particularly, the method is characterized by comprising the step of contacting an Fmoc-protected amino group with a solution comprising DEAPA.

[0011] In another embodiment, the present invention provides a method for preparing degarelix in solid-phase polypeptide synthesis, and the method is characterized by using a solution comprising DEAPA to cleave one or more Fmoc amino protecting groups.

[0012] In a preferred embodiment, the method of the present invention is characterized by using a 10% DEAPA solution to cleave the Fmoc amino protecting group. Even more preferably, the solution is DMF containing 10% DEAPA.

[0013] In particular, the present invention provides a method for preparing a peptide in solid-phase polypeptide synthesis, and the method is characterized by using DMF containing a 10% DEAPA solution to cleave the Fmoc amino protecting group. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : After 20% DEAPA deprotection of DMF-d6 containing Fmoc-Gly-Trt-PS resin 1 1H NMR (Example 1): The peak indicated by * corresponds to the H of the DBF-DEAPA adduct; the peak indicated by # corresponds to the H of DBF; the signal at 7.5 - 8.0 ppm refers to a mixture of the aromatic protons of DBF and DBF-DEAPA. The calculated ratio of DBF / DBF-DEAPA adduct is 1 / 1.8. 1 The signal; the peak indicated by # corresponds to the H of DBF 2 signal; the signal at 7.5 - 8.0 ppm refers to a mixture of the aromatic protons of DBF and DBF-DEAPA. The calculated ratio of DBF / DBF-DEAPA adduct is 1 / 1.8.

[0015] Figure 2 : After 20% piperidine deprotection of DMF-d6 containing Fmoc-Gly-Trt-PS resin 1 1H NMR (Example 1): The peak indicated by * corresponds to the H of the DBF-piperidine adduct 1Signal; the peak indicated by # corresponds to the H of DBF 2 Signal; the signal at 7.5 - 8.0 ppm refers to a mixture of the aromatic protons of DBF and DBF - piperidine. The calculated ratio of the DBF / DBF - piperidine adduct is 1 / 7.7.

[0016] Figure 3 : Superimposed HPLC curves of degarelix at t0, t1, t2, and t3 (Test 1, Example 7).

[0017] Figure 4 : Superimposed HPLC curves of degarelix at t0, t2, and t3 (Test 2, Example 7). Detailed implementation mode

[0018] Fmoc, namely 9 - fluorenylmethoxycarbonyl, is also referred to as the Fmoc group herein and is used as a protecting group for amino functional groups. For decades, the standard reagent for Fmoc cleavage has been piperidine, and although its toxicity and its status as a listed substance (controlled substance) have always been considered disadvantageous, to date, none of the bases proposed for Fmoc cleavage has become a new standard.

[0019] The terms "amino functional group" and "amino group" refer to any primary or secondary amine, including aliphatic - acyclic and cyclic - and aromatic amines, which can be protected by the Fmoc group. Preferably, such amino functional groups are aliphatic primary amines, more preferably the α - amino group of an amino acid or a peptide.

[0020] In particular, in peptide synthesis, in liquid - phase peptide synthesis (LPPS) and in SPPS, the Fmoc group is used as a protecting group for the α - amino group of amino acids, which are used as building blocks. The chemical conditions required for Fmoc group cleavage are generally orthogonal to the chemical conditions required for cleavage of amino acid side - chain protecting groups. The latter are usually removed after all the elongation steps of the peptide chain are completed. This generally occurs simultaneously with the cleavage of the peptide chain from the solid support when synthesizing a peptide in SPPS.

[0021] The solid - phase peptide preparation can be carried out as step - by - step or fully Fmoc - based SPPS, in which amino acids are coupled one by one to the growing peptide sequence attached to a solid support; or as Fmoc - based convergent SPPS (CSPPS), in which at least two independently prepared peptide fragments are coupled together to form an amide bond and longer peptide fragments until the final sequence is finally obtained, where one of the two fragments participating in the coupling reaction is attached to a solid support.

[0022] The term "Fmoc - based SPPS" or "Fmoc - based peptide synthesis" refers to peptide synthesis in which amino acids are used, where the α - amino group is protected by the Fmoc group.

[0023] The Fmoc group is also used as a protecting group for the amino functional group in the side chain in Boc-based SPPS, i.e., when an amino acid is employed, its α-amino group is protected by the Boc group. In this case, since Boc cleavage requires acidic conditions, protecting the side chain with Fmoc provides the required orthogonality for α-amino deprotection without removing the side chain protection. Also in this case, DEAPA can be used to deprotect the side chain amino functional group.

[0024] As used herein, the terms "peptide fragment" and "fragment" describe an amino acid partial sequence having a minimum length of 2 amino acids relative to a target peptide sequence. Peptide fragments are generally protected at the side chain and at the N-terminal α-amino group that is not involved in the coupling reaction. The N-terminal α-amino group is preferably protected by the Fmoc group.

[0025] The terms "amine deprotecting agent" and "Fmoc cleavage agent" are used herein as synonyms and refer to the reagent used in the present disclosure to cleave the Fmoc amino protecting group.

[0026] The terms "cleavage" and "removal" and the verbs "cleave" and "remove" are used herein as synonyms and refer to the chemical bond breakage that occurs during deprotection of the Fmoc-protected amino group.

[0027] Accordingly, the present invention provides a method for preparing a peptide or a pharmaceutically acceptable salt thereof by using solid-phase peptide synthesis, which is characterized by using a solution containing DEAPA to cleave the Fmoc amino protecting group from the Fmoc-protected α-amino group.

[0028] DEAPA has unexpectedly proven to be suitable for cleaving the Fmoc amino protecting group. It has been shown that DEAPA is superior to the use of piperidine in peptide synthesis, particularly in SPPS, in which piperidine can be used in both liquid and solid phases. DEAPA is a less toxic and more environmentally friendly chemical and has proven to be a fast, safe and efficient reagent.

[0029] The chemical structure of 3-(diethylamino)propylamine (I) is depicted as follows:

[0030]

[0031] Comparison of DEAPA / Other Bases for Fmoc Cleavage

[0032] The deprotection reaction rates of DEAPA and piperidine in the removal of Fmoc from model amino acids such as Fmoc-Phe-OH were compared in two different solvents, DMF and NBP (i.e., N-butyl-1-pyrrolidone), and at two different concentrations, 10% and 20%. Other amines, namely 1,1,3,3-tetramethylguanidine (TMG) and tert-butylamine (TBA), were also tested. The results demonstrated that DEAPA is as suitable as the other tested amines for Fmoc cleavage.

[0033] The Fmoc cleavage mechanism is depicted as follows:

[0034] Scheme 1

[0035]

[0036] The dibenzofulvene (DBF) formed in the first step is the reactive species and can further react with the amine deprotectant to form a DBF-amine adduct (DBF-A). This prevents possible side reactions in which DBF reacts with the free α-amino group of the peptide being prepared or with any other reactive species that may be present in the reaction mixture. Unexpectedly, it was observed that piperidine and DEAPA are able to form the DBF-amine adduct, while TMG and TBA do not form the DBF adduct. Thus, DEAPA is able to act as a scavenger for DBF, thereby preventing side reactions caused by DBF. In this regard, DEAPA unexpectedly reacts in the same manner as piperidine, which is the standard cleavage agent for Fmoc.

[0037] Any effect of DEAPA on racemization in Fmoc-based SPPS was further tested. It is known in the art that cysteine is particularly prone to racemization during peptide synthesis. First, H-Phe-L-Cys-Gly-OH and H-Phe-D-Cys-Gly-OH were prepared as standards as described in Example 2. Then, the Fmoc-based SPPS of H-Phe-L-Cys-Gly-OH was carried out in parallel by removing the Fmoc group (30% base solution) using either DEAPA or piperidine as described in Example 3. No significant difference in the racemization ratio was observed between DEAPA and piperidine, as the D / L ratio % (or the racemization ratio %) was below 0.1 in both experiments.

[0038] Hereinafter, the preparation of a representative peptide by Fmoc-based SPPS using DEAPA as the Fmoc cleavage agent is described.

[0039] For example, linear octreotide (II), i.e., H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (SEQ ID NO: 1), was prepared in three different parallel experiments by cleaving the Fmoc group using DMF containing 10% DEAPA, DMF containing 20% piperidine, or NBP containing 20% piperidine. As described in detail in Example 4, the final product obtained by using DEAPA showed increased purity.

[0040]

[0041] Other peptides such as glucagon, exenatide, etelcalcetide, etc. can be prepared by a similar SPPS procedure using the method of the present invention.

[0042] One of the problems in peptide synthesis is the formation of aspartic acid by-products.

[0043] The basic conditions necessary for Fmoc group cleavage can also favor the formation of unwanted aspartimide derivatives during Fmoc-based SPPS, and the appearance of such derivatives generally increases with the number of Fmoc cleavage cycles after the introduction of an aspartic acid residue (Asp) into the peptide chain. Then, as shown in the following scheme, the aspartimide ring in the by-product can be opened by nucleophilic attack with the formation of other unwanted chemicals:

[0044] Scheme 2

[0045]

[0046] The α-peptide can correspond to the target peptide, but racemization may occur at Asp.

[0047] To analyze the applicability of DEAPA in this case, a model peptide was prepared and then contacted with piperidine or another suitable amine or with DEAPA under basic conditions for an extended period of time.

[0048] The model hexapeptide H-Ala-Lys-Asp-Gly-Tyr-Ile-OH (III, SEQ ID NO: 2, Scheme 3) was prepared by Fmoc-based SPPS using DMF containing 20% piperidine, and the content of aspartimide impurity (IV, Scheme 3) in the final compound was determined and found to be 2.6% (HPLC, Example 5).

[0049] Scheme 3

[0050]

[0051] Then, a stress test is performed to subject the model peptide to alkaline conditions to mimic repeated α-amino deprotection cycles. The resin-bound H-Ala-Lys-Asp-Gly-Tyr-Ile-OH (still carrying side chain protecting groups) is treated in parallel with 20% piperidine, 10% DEAPA, 5% TMG or 20% TBA solution in DMF or NBP at room temperature for 4 hours.

[0052] As shown in Scheme 2 above, after the formation of the aspartimide by-product, the aspartimide ring can be subjected to nucleophilic attack, accompanied by the formation of other by-products such as amine adducts (amine-mediated ring opening) and α-peptides and β-peptides (water-mediated ring opening).

[0053] The hexapeptide sample obtained after the treatment with the Fmoc cleavage solution is analyzed by HPLC, and the results are shown in Table 1 (for the detailed experimental procedure, see Example 6).

[0054] Table 1. HPLC purity (A%) of H-Ala-Lys-Asp-Gly-Tyr-Ile-OH (III, SEQ ID NO: 2)

[0055]

[0056] a Sum of α-peptide and β-peptide

[0057] When piperidine is used, the formation of piperidine (V) is observed.

[0058]

[0059] The use of DEAPA causes only trace amounts of the corresponding by-products to form, and these by-products are not listed in Table 1.

[0060] Cleavage with TMG causes the formation of β-peptides, which co-elute with the α-peptides and correspond to the target peptide (III).

[0061] In Table 1, Asp indicates the amount of additional total aspartimide impurities (including piperidine by-products) generated by the stress test relative to the initial aspartimide amount (2.6%). This value was shown to be lower when using DEAPA in the corresponding solvent compared to other test amines.

[0062] Therefore, in the sample treated with the DEAPA solution, the purity of the target peptide (III) is higher.

[0063] Accordingly, the present invention provides a method for cleaving an Fmoc amino protecting group, characterized by using a solution containing 3-(diethylamino)propylamine.

[0064] The present invention also provides a method for preparing a peptide, characterized by using a solution containing 3-(diethylamino)propylamine for cleaving the Fmoc amino protecting group.

[0065] Furthermore, the present invention provides a method for preparing a peptide by solid-phase peptide synthesis, characterized by using a solution containing 3-(diethylamino)propylamine for cleaving the Fmoc amino protecting group.

[0066] Preferably, the concentration range of DEAPA is from 5% to 30%, more preferably from 10% to 20%, and most preferably, the range is 10%. Preferably, the solvent is a polar aprotic solvent, more preferably, it is selected from the group consisting of: DMF, NBP, NMP (i.e., 1-methyl-2-pyrrolidone) or similar solvents or mixtures thereof. In the most preferred embodiment, the solution is DMF containing 10% concentration of DEAPA.

[0067] Wherein, throughout the present disclosure, the concentration values are given in % units, and these concentration values refer to vol%.

[0068] In a particularly preferred embodiment, the present invention provides a method for cleaving the Fmoc amino protecting group, characterized by using DMF containing a 10% DEAPA solution.

[0069] The use of DEAPA has also been shown to be advantageous in the synthesis of degarelix (VI). Degarelix is identified by the following sequence:

[0070]

[0071] Wherein, the numbers indicate the amino acid (aa) positions from the N-terminal aa (D-Nal) to the C-terminal aa (D-Ala).

[0072]

[0073] One of the main problems in the preparation of degarelix is the high sensitivity of the (L)-dihydroorotic acid (designated as Hor) moiety of the Aph(Hor) residue in position 5 of the sequence in the presence of an alkaline aqueous solution. Under these conditions, the 6-membered Hor ring undergoes a rapid rearrangement, accompanied by the formation of a 5-membered hydantoin ring.

[0074] The stability of degarelix (VI) against hydantoin rearrangement was tested in a DEAPA solution to monitor any formation of its hydantoin impurity (VII).

[0075]

[0076] Degarelix was maintained in a solution of 10% DEAPA in DMF for 24 hours to simulate conditions corresponding to those used for removing the Fmoc group during full SPPS. The samples were analyzed by HPLC at four checkpoints for up to 24 hours, and the four HPLC curves obtained as shown in Figure 3 and Figure 4 were compared (for the detailed experimental procedure, see Example 7).

[0077] No significant degradation of degarelix was observed. In particular, no increase in hydantoin impurity (VI) was detected.

[0078] Accordingly, the present invention provides a method for preparing degarelix by solid-phase peptide synthesis, characterized by using a solution containing DEAPA for cleaving the Fmoc amino protecting group, wherein the solid-phase synthesis is full SPPS or CSPPS.

[0079] Preferably, in the preparation of degarelix, the concentration range of DEAPA is from 5% to 30%, more preferably from 10% to 20%, and most preferably, the range is 10%. Preferably, the solvent is a polar aprotic solvent, more preferably DMF, NBP, NMP (i.e., 1-methyl-2-pyrrolidone), etc. or a mixture thereof.

[0080] In a particularly preferred embodiment, the present invention provides a method for synthesizing degarelix, characterized by using DMF containing a 10% DEAPA solution for cleaving the Fmoc amino protecting group.

[0081] In a preferred embodiment, the synthesis of degarelix of the present invention is carried out by using at least one compound selected from the group consisting of: Fmoc-Aph(Hor)-OH, Fmoc-Aph(PG)-OH, Fmoc-Phe(NO 2 )-OH, Fmoc-D-Phe(NO 2 )-OH and a peptide fragment containing one or more of Aph(Hor), Phe(NO 2 ) and D-Phe(NO 2 ). PG is an amino protecting group selected from the group consisting of: tert-butoxycarbonyl, formyl, allyloxycarbonyl and benzyloxycarbonyl. In particular, the present invention provides a method for preparing degarelix by Fmoc-based full SPPS following the means described in WO2017103275, Example 2, page 34, characterized by using a solution containing DEAPA instead of piperidine to cleave the Fmoc amino protecting group.

[0082] In another embodiment, the present invention provides the use of 3-(diethylamino)propylamine for cleaving Fmoc amino protecting groups. In particular, the present invention provides the use of 3-(diethylamino)propylamine in peptide synthesis, preferably in solid-phase peptide synthesis, for cleaving Fmoc amino protecting groups.

[0083] Compared to piperidine, which has been the standard cleavage agent for Fmoc for decades, DEAPA has lower toxicity (rat LD 50 = 830 mg / kg). DEAPA has shown mechanical comparability with piperidine, thus allowing the applicability of this method to be extended to general Fmoc cleavage. In specific examples, DEAPA has shown superiority in terms of side reactions, resulting in higher purity products. DEAPA is not on the list of controlled substances, and even its price is lower than that of piperidine.

[0084] Abbreviations

[0085] Aph p-aminophenylalanine

[0086] h hour

[0087] min minute

[0088] GnRH gonadotropin-releasing hormone

[0089] SPPS solid-phase peptide synthesis

[0090] Fmoc-Aph(Hor)-OH 9-fluorenylmethoxycarbonyl-N(4)-(L-hydroxythreonyl)-4-aminophenylalanine

[0091] Fmoc-Phe-OH 9-fluorenylmethoxycarbonyl-L-phenylalanine

[0092] Aph(Hor) N(4)-(L-hydroxythreonyl)-4-aminophenylalanine

[0093] Hor dihydroxythreonyl moiety

[0094] Hor-OH (L) dihydroxyorotic acid

[0095] Fmoc 9-fluorenylmethoxycarbonyl

[0096] Boc tert-butoxycarbonyl

[0097] HPLC high performance liquid chromatography

[0098] DIPEA / DIEA diisopropylethylamine

[0099] DEAPA 3-(diethylamino)propylamine

[0100] TFA trifluoroacetic acid

[0101] DMF N,N - dimethylformamide

[0102] DMA N,N - dimethylacetamide

[0103] NMP 1 - methyl - 2 - pyrrolidone

[0104] NBP 1 - butyl - 2 - pyrrolidone

[0105] ACN Acetonitrile

[0106] DCM Dichloromethane

[0107] DBF Dibenzofulvene

[0108] DIC Diisopropylcarbodiimide

[0109] TIS Triisopropylsilane

[0110] OxymaPure Ethyl 2 - cyano - 2 - hydroxyiminoacetate

[0111] RRT Relative retention time

[0112] RT Room temperature

[0113] TM Target molecule

[0114] Trt - PS resin Polystyrene trityl resin

[0115] Examples

[0116] The following examples provide detailed experimental conditions of the method of the present invention and are intended to illustrate rather than limit all possible embodiments thereof.

[0117] Unless otherwise indicated, all materials, solvents and reagents are obtained from commercial suppliers, of the highest grade, and used without further purification.

[0118] Solid - phase synthesis of peptides is carried out manually or using common peptide synthesizers, such as Biotage Syrowave instrument (automatic synthesis) and Biotage MultiSynTech (semi - automatic synthesis).

[0119] HPLC method

[0120] Examples 1 - 6: The HPLC - MS analysis was performed as follows: On an Agilent 1260 Infinity II system coupled with an electrospray ionization mass spectrometer (positive ion mode, m / z = 100 - 1500, fragmentation voltage 30 V), using a chromatographic column Agilent Zorbax - SB - C18 5 μm, 250×4.6 mm or Phenomenex Luna C18 5 μm, 250×4.6 mm; temperature: 25 °C; injection volume: 10 μL, UV: 220 nm, mobile phase: H 2 O + 0.08% TFA (A) and CH 3 CN + 0.08% TFA (B), flow rate: 0.5 mL / min or 1.0 mL / min.

[0121] Example 7: The HPLC analysis was performed as follows: On an Agilent 1260 Infinity II system, using a chromatographic column Waters Cortecs C18 2.7 μm, 4.6×150 mm; temperature: 30 °C; injection volume: 10 μL, UV: 245 nm; pH 5.5 phosphate buffer: 25 mM potassium phosphate pH 5.5; pH 3.5 phosphate buffer: 25 mM potassium phosphate pH 3.5; mobile phase: 75% pH 5.5 buffer: 25% CH 3 CN (A); 65% pH 3.5 buffer: 35% CH 3 CN (B); flow rate: 1.0 mL / min, sample concentration 0.5 mg / ml; gradient: 0 - 5 minutes 0% B, 5 - 45 minutes 0 - 100% B, 45 - 46 minutes 100 - 0% B, 46 - 60 minutes 0% B.

[0122] Example 1: Monitoring the formation of DBF-amine adducts

[0123] 50 mg of dry resin Fmoc - Gly - Trt - PS was swollen in 2 mL of DMF for 30 minutes. The resin was filtered, and 0.75 mL of a 20% base solution (DEAPA, piperidine, TBA, or TMG) in DMF - d6 was added to the resin and stirred for 30 minutes. The resin was filtered, and direct analysis of the filtrate was performed by 1 1H NMR spectroscopy to reveal the presence of individual DBF or the formation of DBF - amine adducts (Scheme 1).

[0124] 1 The 1H NMR spectrum showed the formation of the DBF - DEAPA adduct ( Figure 3 ) in a DBF / DBF - DEAPA ratio of 1 / 1.8 and the formation of the DBF - piperidine adduct ( Figure 4 ) in a DBF / DBF - piperidine ratio of 1 / 7.7 after 30 minutes.

[0125] No base-DBF adducts were observed in the case of TBA or TMG.

[0126] Example 2: Fmoc-based SPPS of H-Phe-L-Cys-Gly- OH and H-Phe-D-Cys-Gly-OH in DMF as reference compounds for Cys racemization testing

[0127] Synthesis was carried out using Fmoc-Gly-Trt-PS resin (200 mg, loading 1.1 mmol / g). After swelling the resin in 2 mL of DMF, the Fmoc protecting group was removed by DMF containing 20% piperidine (2 × 2 mL, 15 minutes each), and the resin was washed with DMF (4 × 2 mL). Fmoc-L-Cys(Trt)-OH (or Fmoc-D-Cys(Trt)-OH) and Fmoc-Phe-OH (more than three-fold with respect to the resin loading) were pre-activated with DIC and OxymaPure (more than three-fold of the reagent with respect to the resin loading) for 3 minutes and coupled to the resin within 60 minutes. After each coupling step, the Fmoc protecting group was removed by treating the peptide resin with DMF containing 20% piperidine (2 × 2 mL, 15 minutes each), and the resin was washed with DMF (4 × 2 mL). After Fmoc cleavage of the N-terminal α-amino group, the peptide resin was washed with DMF (3 × 2 mL) and DCM (3 × 2 mL). The dried peptide resin was suspended in 5 mL of a mixture of TFA / TIS / H 2 O / 1-dodecanethiol (92.5 / 2.5 / 2.5 / 2.5 v / v / v / v) and stirred for 2 hours. The resin was filtered off, and diisopropyl ether (20 mL) cooled to 4 °C was added to the solution. The peptide was filtered and dried in vacuo to give the crude H-Phe-L-Cys-Gly-OH or H-Phe-D-Cys-Gly-OH as a reference compound for the racemization test.

[0128] HPLC-MS analysis gradient: 0 - 30 minutes 0 - 60% B; flow rate: 0.5 mL / minute.

[0129] Example 3: Cys racemization testing during Fmoc-based SPPS of H-Phe-L-Cys-Gly-OH in DMF using DEAPA and piperidine as deprotecting agents Example 4: Fmoc-based SPPS of H-D-Phe-Cys-Phe-D-Tm-Lys-Thr-

[0130] Two SPPS of H-Phe-L-Cys-Gly-OH were as reported above in Example 2, but carried out using the following conditions for parallel cleavage of the Fmoc group: DMF containing 30% DEAPA or piperidine, for 60 minutes.

[0131] HPLC-MS analysis gradient: 0 - 30 minutes 0 - 60% B; flow rate: 0.5 mL / minute.

[0132] The racemization ratio (D / L %) in the preparation of H-Phe-Cys-Gly-OH was determined by the HPLC area % (A%) of the two diastereoisomers and calculated as (H-Phe-D-Cys-Gly-OH A%) / (H-Phe-L-Cys-Gly-OH A%) × 100. In both experiments, D / L % was <0.1.

[0133] Cys-Thr-ol (linear octreotide, II, SEQ ID NO: 1) using DEAPA or piperidine as Fmoc cleavage agents Example 5: Fmoc-based SPPS of H-Ala-Lys-Asp-

[0134] Synthesis was carried out using Fmoc-Thr(tBu)-ol-Trt-PS resin (200 mg, loading 1.1 mmol / g). After swelling the resin in 2 mL of DMF or NBP, the Fmoc protecting group was removed by DMF containing 10% DEAPA (or DMF or NBP containing 20% piperidine) (2 × 2 mL, 15 minutes each), and the resin was washed with DMF or NBP (4 × 2 mL). Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Phe-OH (more than threefold in terms of resin loading) were pre-activated with DIC and OxymaPure for 3 minutes and coupled to the resin within 60 minutes. In the case of the first insertion of Fmoc-Cys(Trt)-OH (Cys in the final sequence) 7 ) the coupling was repeated a second time. After each coupling step, the Fmoc protecting group was removed by treating the peptide resin with DMF containing 10% DEAPA (2 × 2 mL, 15 minutes each) or DMF or NBP containing 20% piperidine (2 × 2 mL, 15 minutes each), and the resin was washed with DMF or NBP (4 × 2 mL). After Fmoc cleavage of the N-terminal amino group, the peptide resin was washed with DMF or NBP (3 × 2 mL) and DCM (3 × 2 mL). The dried peptide resin was suspended in 5 mL of a mixture of TFA / TIS / 1-dodecanethiol (90 / 5 / 5 v / v / v) and stirred for 4 hours. The resin was filtered off and diisopropyl ether (20 mL) cooled to 4 °C was added to the solution. The peptide was filtered and dried in vacuo to obtain crude linear octreotide (II). The HPLC purity reported as the sum of all target molecule adducts is shown in Table 3. It should be noted that all substances reported in Table 3 are not impurities but precursors of the final octreotide (TM = target molecule).

[0135] HPLC-MS analysis gradient: 20 - 40% B from 0 - 15 minutes; flow rate: 0.5 mL / minute.

[0136] Table 2. HPLC Purity of DMF Containing Linear Octreotide (II) with DMF Containing 10% DEAPA or DMF Containing 20% Piperidine or NBP as a Deprotecting Agent

[0137]

[0138] a HPLC purity calculated as the sum of all target product adducts

[0139] Gly-Tyr-Ile-OH (III, SEO ID NO: 2) using piperidine / DMF Fmoc cleavage for aspartimide formation detection Example 6: Stress stability testing of H-Ala-Lys-Asp-Gly-Tyr-Ile-

[0140] Synthesis was carried out using Fmoc-Ile-Trt-PS resin (800 mg, loading 1.1 mmol / g). After swelling the resin in 2 mL of DMF, the Fmoc protecting group was removed by DMF containing 20% piperidine (2 × 2 mL, 15 minutes each), and the resin was washed with DMF (4 × 2 mL). Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH (more than three-fold in terms of resin loading) were pre-activated with DIC and OxymaPure (more than three-fold reagent in terms of resin loading) for 3 minutes and coupled to the resin within 60 minutes. After each coupling step, the Fmoc protecting group was removed by treating the peptide resin with DMF containing 20% piperidine (2 × 2 mL, 15 minutes each), and the resin was washed with DMF (4 × 2 mL). After Fmoc cleavage of the N-terminal amino group, the peptide resin was washed with DMF (3 × 2 mL) and DCM (3 × 2 mL). 100 mg of dry peptide resin was suspended in 3 mL of a mixture of TFA / TIS / H 2 O (90 / 5 / 5 v / v / v) and stirred for 2 hours. The resin was filtered off, and diisopropyl ether (10 mL) cooled to 4 °C was added to the solution. The peptide was filtered and dried under vacuum to obtain crude H-Ala-Lys-Asp-Gly-Tyr-Ile-OH (II) with an HPLC purity of 97.4% and 2.6% aspartimide impurity (IV).

[0141] HPLC-MS analysis gradient: 10 - 40% B from 0 - 30 minutes; flow rate: 0.5 mL / minute.

[0142] OH (II, SEO ID NO: 2) with DMF or NBP containing piperidine, DEAPA, TMG, and TBA Example 7: Stability of degarelix in the presence of DEAPA

[0143] Swell 100 mg of dry resin H-Ala-Lys-Asp-Gly-Tyr-Ile-Trt-PS (prepared as described in Example 2) in 2 mL of DMF or NBP for 30 minutes. Filter the resin and add a 2 mL solution of 10% DEAPA (or 20% piperidine, 5% TMG, 20% TBA) in DMF or NBP to the resin and stir at room temperature for 4 hours. Filter the resin and wash it with DMF or NBP (3 × 2 mL) and DCM (3 × 2 mL). Suspend the dry peptide resin in 3 mL of the mixture TFA / TIS / H 2 O (90 / 5 / 5 v / v / v) and stir for 2 hours. Filter out the resin and add diisopropyl ether (10 mL) cooled to 4 °C to the solution. Filter the peptide and dry it under vacuum to obtain crude H-Ala-Lys-Asp-Gly-Tyr-Ile-OH (II) and asparagine imide impurity (III) in different proportions depending on the conditions used.

[0144] Table 1 in the specification lists the results. ΔAsp indicates the difference between the amount of asparagine imide formed after stress testing (including piperidine if present) and the amount produced during the synthesis of the hexapeptide (2.6%, see Example 2).

[0145] HPLC-MS analysis gradient: 10 - 40% B from 0 - 30 minutes; flow rate: 0.5 mL / minute.

[0146] Figure 3

[0147] The degarelix sample is a lyophilized powder with a purity of approximately 99%. It is dissolved in DMF with 10% DEAPA at the following two different concentrations: approximately 170 g / L (Test 1) and approximately 17 g / L (Test 2). The stability at room temperature is monitored at four checkpoints, namely t0, 1 hour (t1), 4 hours (t2), and 24 hours (t3) for 24 hours.

[0148] The overlaid HPLC curves are shown in Figure 4 (Test 1) and ​ (Test 2).

[0149] Test 1: After 24 hours, the curve did not change. Only the specified impurity RRT 0.85 (X, unknown) was absent at t0 and increased to a value of 0.06% at t3 (purity changed from 99.17% to 99.10%).

[0150] The hydantoin impurity (VI) at RRT 1.03 did not increase: it was 0.10% at both t0 and t3.

[0151] Test 2: After 24 h, the curve did not change. Only the specified impurity RRT 0.85 (X, unknown) was not present at t0 and increased to a value of 0.04% at t3 (purity changed from 99.18% to 99.10%).

[0152] The hydantoin impurity (VI) at RRT 1.03 did not increase: it was 0.09% at both t0 and t3.

Claims

1. A method for cleaving Fmoc from one or more Fmoc-protected amino groups, wherein, the method comprises the step of contacting the Fmoc-protected amino group with a solution comprising 3-(diethylamino)propylamine.

2. The method according to claim 1, wherein, the concentration of 3-(diethylamino)propylamine in the solution ranges from 5 vol% to 30 vol%.

3. The method according to any one of the preceding claims, wherein, the concentration of the 3-(diethylamino)propylamine in the solution is 10 vol%.

4. The method according to claim 3, wherein, the solution further comprises a solvent selected from the group consisting of N,N-dimethylformamide, N-methylpyrrolidone, N-butylpyrrolidone, and mixtures thereof.

5. The method according to claim 4, wherein, the solution consists of 10 vol% 3-(diethylamino)propylamine and N,N-dimethylformamide.

6. The method according to claim 5, wherein, the cleavage of the Fmoc is carried out in peptide synthesis.

7. The method according to claim 6, wherein, the peptide comprises at least one aspartic acid amino acid.

8. The method according to claim 6, wherein, the peptide is selected from the group consisting of degarelix, octreofide, exenatide, etelcalcetide, and glucagon.

9. The method according to claim 8, wherein, degarelix is prepared by using protected amino acids selected from the group consisting of Fmoc-Aph(Hor)-OH, Fmoc-Aph(PG)-OH, Fmoc-Phe(NO2)-OH, and Fmoc-D-Phe(NO2)-OH, wherein PG is an amino protecting group selected from tert-butoxycarbonyl, formyl, allyloxycarbonyl, and benzyloxycarbonyl.

10. A method for preparing a peptide by Fmoc-based solid-phase peptide synthesis, wherein, the method comprises the step of contacting an Fmoc-protected amino group with a solution comprising 3-(diethylamino)propylamine to cleave the Fmoc from one or more Fmoc-protected amino groups.

11. The method according to claim 10, wherein, the concentration of 3-(diethylamino)propylamine in the solution ranges from 5 vol% to 30 vol%.

12. The method according to any one of claims 10 and 11, wherein, the solution further comprises a solvent selected from the group consisting of N,N-dimethylformamide, N-methylpyrrolidone, N-butylpyrrolidone, and mixtures thereof.

13. The method according to claim 12, wherein, the peptide comprises at least one aspartic acid amino acid.

14. The method according to claim 12, wherein, the peptide is selected from the group consisting of degarelix, octreofide, exenatide, etelcalcetide, and glucagon.

15. A use of 3-(diethylamino)propylamine for cleaving an Fmoc amino protecting group, wherein, The Fmoc amino protecting group is employed in peptide synthesis.

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