Dipropylamine as a base used for Fmoc deprotection in solid-phase peptide synthesis

Abstract

The present invention relates to a method for preparing peptides via solid-phase peptide synthesis, and in particular, to a method for deprotecting R-AA-(AA) n -PF, which is an Fmoc-protected amino acid building block linked to a resin.

Description

Technical Field

[0001] The present invention relates to an optimized method for fluorenylmethoxycarbonyl (Fmoc) deprotection of peptides in peptide solid-phase synthesis (SPPS).

Background Art

[0002] Peptide solid-phase synthesis (SPPS) is a method widely used for peptide synthesis, in which amino acids are covalently linked to a solid support, and peptide growth occurs stepwise using a selective protecting group strategy. Compared with conventional liquid-phase synthesis, SPPS is characterized by high efficiency and high throughput, as well as improved simplicity, speed, and yield. Amino acids form amino acid building blocks and are protected at all reactive functional groups present, and the order of reactions for each functional group can be controlled by selective deprotection. In the basic method of SPPS, the first amino acid building block is linked to the resin at either its C-terminus or N-terminus, usually at its C-terminus. The two most commonly used strategies are fluorenylmethoxycarbonyl (Fmoc)-SPPS and tert-butyloxycarbonyl (Boc)-SPPS, where the N-terminus is protected by Fmoc or Boc. In the next step, the N-terminus of the building block is deprotected in a deprotection step, resulting in a free amine. Formation of a peptide bond requires a carboxylic acid as a reaction partner for the free amine. Thus, the building block to be attached to the free amine is protected at its N-terminus, and the C-terminus needs to undergo an activation step. Then, the free amine linked to the resin and the activated carboxylic acid form a peptide bond in a coupling step, resulting in an N-terminus protected dipeptide in the case of a single amino acid building block. The N-terminus protected dipeptide is then deprotected again to be coupled with another C-terminus activated amino acid building block. This cycle is repeated to form the desired peptide. Once the desired peptide length is reached, all side chains are deprotected and the peptide is cleaved from the resin (Figure 1).R1

[0003] Solid-phase peptide synthesis (SPPS) using Fmoc as the α-amino protecting group for amino acid building blocks is currently the most powerful synthetic method for peptide research and production. Piperidine (PPR) functions as the optimal Fmoc removal reagent in SPPS because it acts both as an efficient base to induce the β-elimination of carbamic acid and as a nucleophile to quench the reactive dibenzofulvene byproduct (Figure 2a). However, PPR is expensive, has a strong odor, and is strictly regulated due to its use in illegal drug manufacturing. PPR can be replaced by a mixture of piperazine (PZ) as a nucleophilic quencher and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base, which has no strong odor but is still expensive (Figure 2b). However, both PPR and PZ / DBU induce the formation of aspartimide, which can hydrolyze to α-peptide or β-peptide in some sequences containing aspartic acid (Figure 2c). The addition of a weak acid such as formic acid or ethyl cyanohydroxyiminoacetate (oxima) to moderate the basicity of the PPR solution reduces the formation of aspartimide, but does not solve the problems of the cost, bad odor, and availability of PPR. In attempts to overcome the limitations of PPR or PZ / DBU, several reagents or aspartate side-chain protecting groups have been reported, but none of them combine low cost, convenient use, high yield, and low aspartimide formation. R2~R8 SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0004] Based on the above prior art, an object of the present invention is to provide a means and method having low cost and excellent availability for using optimized reaction conditions in the deprotection of Fmoc-protected peptides. This object is achieved by the subject matter of the independent claims of this specification, and further advantageous embodiments are described in the dependent claims, examples, drawings, and general description of this specification.

Means for Solving the Problem

[0005] A first aspect of the present invention is a method for preparing a peptide via solid-phase peptide synthesis, R-AA-(AA) which is an Fmoc-protected amino acid building block linked to a resin n -PF (wherein, - R is a resin, - AA is an amino acid building block, - PF is an Fmoc protecting group, - n is the number of coupling cycles) is deprotected using dibutylamine or dipropylamine in a deprotection step before the coupling step to give R-AA-(AA) n is brought about, in the coupling step, another amino acid building block AA-P is coupled to R-(AA)-(AA) end up to n n final coupling cycles to give R-AA-(AA) n+1 -P, wherein P is a protecting group at the N-terminus of the amino acid building block AA or PF, relates to a method.

[0006] Terms and Definitions For the purpose of interpreting this specification, the following definitions apply and, as appropriate, terms used in the singular include the plural and vice versa. If any of the definitions described below conflict with any document incorporated herein by reference, the described definitions shall prevail.

[0007] As used herein, the terms "having", "comprising", "containing", "including" and other similar forms, and their grammatical equivalents are intended to be equivalent in meaning and are intended to be open-ended in the sense that the items following any one of these words are not meant to be limited to only the items recited as an exhaustive listing of such items. For example, an article "comprising" components A, B and C may consist of (i.e., may contain only) components A, B and C, or may contain not only components A, B and C but also one or more other components. Thus, "comprising" and its similar forms, and their grammatical equivalents, are intended and understood to include the disclosure of embodiments of "consisting essentially of" or "consisting of".

[0008] Where a range of values is provided, each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, is included in the scope of the present invention, unless the context clearly dictates otherwise, and any other specified value or intervening value within the specified range is included in the scope of the present invention, unless the specified range has specific excluded limits. Where the specified range includes one or both of the limits, ranges excluding either or both of these included limits are also included in the present invention.

[0009] References herein to "about" a value or parameter include (and describe) variations that are directed to that value or parameter itself. For example, a reference to "about X" includes a reference to "X".

[0010] As used herein, the singular forms "a", "or" and "the", including in the appended claims, include plural referents unless the context clearly dictates otherwise.

[0011] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Edition, John Wiley & Sons, Inc.) as well as for chemical methods.

[0012] In the context of this specification, the term, resin R, relates to a solid support. Resins are typically small spherical beads containing a polymer and are of two different sizes, namely, 100 - 200 mesh (75 - 150 microns) and 200 - 400 mesh (35 - 75 microns). The most common resin in solid-phase chemistry is polystyrene (PS), frequently supplemented with divinylbenzene (PS-DVB). Other commonly used resins are polyamine resins and polyethylene glycol-polystyrene (PEG-PS) resins.

[0013] Resins are further functionalized with linkers. For Fmoc-SPPS, common linkers are Rink amide, Wang, hexamethylphosphoramide (HMPA), hexamethylenebisacetamide (HMBA), 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB), 2-chlorotrityl, super acid-sensitive resin (SASRIN), Rink acid, hydrazine, or sulfonamide. For Boc-SPPS, commonly used linkers are Merrifield, PAM, or MBHA. R9

[0014] The term, amino acid building block AA, in the context of this specification, relates to a single amino acid, or a short-chain peptide comprising two to three amino acids, protected at its amino acid side chain with any common protecting group used for amino acids. R10

[0015] The term, n, in the context of this specification, relates to the number of coupling cycles in peptide solid-phase synthesis. The minimum number of coupling cycles is 1. The maximum number of coupling cycles is not defined.

[0016] The term, coupling cycle, in the context of this specification, relates to the step of coupling a deprotected, resin-linked amino acid building block R-AA-(AA) n to an activated amino acid building block AA-P.

[0017] The term, n end relates to the final coupling cycle in solid-phase synthesis, which is achieved when the desired peptide length is reached. When the final desired peptide length is reached, the peptide is, if desired, fully deprotected at its N-terminus and side chains and cleaved from the resin and purified.

[0018] As used in the context of this specification, the term "P" relates to a protecting group at the N-terminus of an amino acid building block, which protecting group may be a common protecting group used in peptide synthesis generally known to those skilled in the art, including benzylamine (NBn), N-carboxybenzyl (Cbz), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), methyltrityl (Mtt), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl (ivDde), or fluorenylmethoxycarbonyl (Fmoc) (see Clayden, Greeves, Warren and Wothers, Organic Chemistry, 2001, page 657).

[0019] As used in the context of this specification, the term "PF" relates to the fluorenylmethoxycarbonyl (Fmoc) protecting group. Fmoc is a base-labile and acid-stable protection commonly used in peptide synthesis. Fmoc is usually introduced via 9-fluorenylmethyl chloroformate (Fmoc-Cl) (see Clayden, Greeves, Warren and Wothers, Organic Chemistry, 2001, pages 656 - 658).

[0020] As used in the context of this specification, the term "polypeptide" relates to a molecule consisting of 50 or more amino acids forming a straight chain linked by peptide bonds. The amino acid sequence of the polypeptide can be the amino acid sequence of an entire protein (found physiologically) or a fragment thereof. The terms "polypeptide" and "protein" are used interchangeably herein and include proteins and their fragments. Polypeptides are disclosed herein as amino acid residue sequences.

[0021] As used in the context of this specification, the term "peptide" relates to a molecule consisting of up to 50 amino acids, particularly up to 30 amino acids, and particularly up to 15 amino acids forming a straight chain linked by peptide bonds.

[0022] The amino acid residue sequence is shown from the amino terminus to the carboxyl terminus. Capital letters at the sequence positions refer to L-amino acids in one-letter notation (Stryer, Biochemistry, 3rd edition, page 21). Lowercase letters at the amino acid sequence positions refer to the corresponding D-amino acids or (2R)-amino acids. The sequence is described from left to right in the direction from the amino terminus to the carboxyl terminus. According to the standard nomenclature, the amino acid residue sequence is represented either in three-letter notation or one-letter notation as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0023] Detailed Description of the Invention The first aspect of the present invention is a method for preparing a peptide via peptide solid-phase synthesis, R-AA-(AA) n -PF (wherein, - R is a resin, - AA is an amino acid building block, - PF is an Fmoc protecting group, - n is the number of coupling cycles) is deprotected using dibutylamine or dipropylamine in the deprotection step before the coupling step to give R-AA-(AA) n is brought about, In the coupling step, another amino acid building block AA-P is n endUp to the final coupling cycle of the loop, R-(AA)-(AA) n is coupled to give R-AA-(AA) n+1 -P, where P is a protecting group at the N-terminus of the amino acid building block AA or PF, relates to a method.

[0024] The term, amino acid building block AA, is a single amino acid or a short-chain peptide containing two to three amino acids, protected at its amino acid side chain with any of the common protecting groups used in peptide synthesis.

[0025] In SPPS, the first amino acid building block AA-(AA) n -PF is linked to the resin R which is a solid phase, giving R-AA-(AA) n PF. In the first amino acid building block, n is 0. The first amino acid building block R-AA-PF is then deprotected using dibutylamine or dipropylamine, giving the deprotected amino acid building block R-AA.

[0026] In certain embodiments, at least one AA comprises aspartic acid.

[0027] Aspartic acid is the amino acid responsible for the formation of aspartimide and subsequent hydrolysis to an α-peptide or β-peptide during SPPS (Figure 2c). The base present in the Fmoc deprotection step of the peptide deprotonates the adjacent secondary amine to aspartic acid, which then reacts in a ring closure to form aspartimide. Hydrolysis of aspartimide occurs via water present during synthesis, forming either the α-peptide or β-peptide of the resulting peptide.

[0028] A method described in the present invention that is different from existing methods for SPPS because the use of dibutylamine or dipropylamine significantly reduces the formation of aspartimide and results in a better peptide yield.

[0029] In certain embodiments, the deprotection step is carried out using dipropylamine.

[0030] The use of dipropylamine as a base for deprotecting Fmoc-protected amino acid building blocks or Fmoc-protected peptides solves some of the problems that exist within the scope of the use of bases generally used for Fmoc-deprotecting Fmoc-protected amino acid building blocks or Fmoc-protected peptides, piperidine and piperazine. Both dibutylamine and dipropylamine are non-regulated, inexpensive, odorless, and readily available. Fmoc deprotection using either dibutylamine or dipropylamine results in a good peptide yield and reduces the formation of aspartimide. As shown in Examples 1 to 3 of the present invention, the use of dipropylamine results in a slightly better yield than the use of dibutylamine.

[0031] In certain embodiments, the amount of dipropylamine used is from 10 to 50% (v / v).

[0032] In certain embodiments, the amount of dipropylamine used is from 20 to 40% (v / v).

[0033] In certain embodiments, the amount of dipropylamine used is from 25 to 35% (v / v).

[0034] Dipropylamine was found to act optimally when used as 25% (v / v) in N,N-dimethylformamide (DMF). The addition of an oxime, which is known to be an additive to carbodiimide in SPPS and reduces aspartimide formation, does not further reduce aspartimide formation when dipropylamine is used at the above volume percentage, which simplifies the procedure and saves costs.

[0035] In certain embodiments, the method is carried out at 10 to 90 °C.

[0036] In certain embodiments, the method is carried out at 65 to 90 °C.

[0037] The use of dibutylamine or dipropylamine in SPPS enables the implementation of SPPS at higher temperatures of 65 to 90 °C. The higher temperature enables the use of dibutylamine or dipropylamine having a high boiling point. The high temperature reduces peptide aggregation and resin swelling. Furthermore, the increase in reaction rate shortens the reaction time and results in a higher peptide yield.

[0038] In certain embodiments, the method is carried out at 75 to 90 °C.

[0039] In certain embodiments, AA-P is activated in the activation step prior to the coupling step.

[0040] Other amino acid building blocks AA-P are R-AA-(AA) in the coupling step. nis coupled. To perform the coupling step of AA-P (where P is a protecting group or PF at the N-terminus of the amino acid building block AA), the amino acid building block AA needs to be activated in the activation step. Activation occurs at the C-terminus of the amino acid building block using a common coupling reagent. Common coupling reagents include N,N'-diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-hexafluorophosphate (HATU), tetramethylfluoroformamidinium hexafluorophosphate (TFFH), or benzotriazol-1-yl-oxytripyrrolidinophosphonium-hexafluorophosphate (PyBOP).

[0041] In certain embodiments, the deprotection step and the coupling step can be repeated until a desired peptide length of 60 amino acids or less is reached.

[0042] In certain embodiments, n end is the final coupling cycle to reach the desired peptide length.

[0043] Since the amino acid building block can contain from 1 to 3 amino acids, n end is not the number of amino acids in the peptide.

[0044] In certain embodiments, the method includes a final deprotection step after n end coupling cycles, where R-AA-(AA) nend -P is deprotected to give R-AA-(AA) nend .

[0045] In the final deprotection step, all protecting groups present at the N-terminus and side chains are removed, resulting in a fully deprotected peptide that remains coupled to the resin.

[0046] In certain embodiments, the method includes a cleavage step after n end coupling cycles, where R-AA-(AA) nend is cleaved from resin R to give AA-(AA) nend being obtained.

[0047] In certain embodiments, the method includes a purification step, where AA-(AA) nend is purified.

[0048] The peptide is separated from impurities during the purification step. Common methods for peptide purification are RP-HPLC, flash chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, size exclusion chromatography, hydrophilic interaction chromatography, and solid phase extraction. R11、R12

[0049] In certain embodiments, P is PF.

[0050] The formation of asparagines by aspartic acid can occur in all subsequent deprotections throughout peptide synthesis. Thus, it is advantageous to use only Fmoc as the N-terminal protecting group throughout peptide synthesis. Furthermore, the use of Fmoc as the N-terminal protecting group in all building blocks facilitates the synthesis, particularly with respect to side chain protecting group chemistry. A consistent N-terminal protecting group in all AA-P throughout peptide synthesis allows for the selection of amino acid side chain protecting groups that are stable throughout the synthesis. For example, since Fmoc is base-labile, base-stable protecting groups can be used at the amino acid side chains.

[0051] The present invention is further illustrated by the following examples and drawings from which further embodiments and advantages can be drawn. These examples are intended to illustrate the invention and are not intended to limit its scope.

Brief Description of the Drawings

[0052]

Figure 1

Figure 2

Modes for Carrying Out the Invention

Examples

[0053] [Example 1] Synthesis of first-generation and second-generation peptide dendrimers, G1KL and G2KL, via SPPS Using oxime and N,N'-diisopropylcarbodiimide (DIC), where DIC acts as a coupling reagent, oxime acts as an acidic additive to suppress base-induced side reactions, and DMF acts as a solvent, in the initial goal of developing a high-temperature (60 °C) protocol for solid-phase peptide synthesis of G1KL, the inventors identified dipropylamine (DPA) as an alternative base to replace piperidine. DPA (pKa = 10.9) is slightly less basic than piperidine (pKa = 11.1) and only slightly less volatile (bp(DPA) = 110 °C; bp(piperidine) = 106 °C). The synthesis of the first-generation peptide dendrimer, which requires Fmoc removal of both α-amino and side-chain amino groups, resulted in a similar yield (65%) using 25% DPA compared to the use of piperidine (73%) of the prior art reagents and a much better yield compared to the use of piperazine and DBU (26%) (Table 1). The difference in yield between 20% and 25% DPA was significant at 30%.

[0054] The synthesis of the second-generation analog G2KL using 25% DPA at room temperature also resulted in a similarly good yield of 46%. The increase in DPA from 20% to 25% did not result in a significant improvement in yield in this case (Table 1).

[0055] DPA resulted in a similar yield as the prior art reagents, while diisopropylamide (DIPA) did not result in the formation of any desired product and was found to be an inappropriate base for deprotection.

[0056] [Example 2] Synthesis of hexapeptides 1 to 7 Next, the inventors investigated the SPPS of aspartimide prone hexapeptides 1 to 7 (Table 1). The use of piperidine for Fmoc deprotection of the peptide resulted in 17% aspartimide (hexapeptide 1), while 20% DPA resulted in only 5%, a slightly lower aspartimide formation than when an oxime was added to piperidine, which led to 6% aspartimide formation (Table 1, hexapeptide 1). The crude product NMR was compared to the independently synthesized β-peptide of VKDGYI (hexapeptide 1; SEQ ID NO: 3), VKD(β)GYI, which would be formed upon hydrolysis of the VKDGYI aspartimide. However, the β-peptide was not detected, and a 49% crude peptide yield (hexapeptide 1) was obtained using 20% DPA at 60°C. In comparison, the yield using prior art conditions containing 20% PPR at 60°C was 47%.

[0057] For hexapeptide 1, deprotection using only 2% DBU was accompanied by 25% aspartimide formation.

[0058] The inventors further investigated Fmoc deprotection using dibutylamine and diisobutylamine in the SPPS of VKDGYI (SEQ ID NO: 3). However, deprotection did not occur using diisobutylamine. Dibutylamine resulted in a similar crude peptide yield as dipropylamine at 60°C (52% (DBA) vs. 53% (DPA)).

[0059] The yield of hexapeptide VKEGYI (hexapeptide 7; SEQ ID NO: 9) was similar to that of VKDGYI (hexapeptide 1; SEQ ID NO: 3), with a 44% crude yield using 20% DPA at 60°C, and no aspartimide formation was observed (Table 1).

[0060] For comparison, hexapeptides 2 to 6 were deprotected using 25% DPA at 60 °C and 20% piperidine at 60 °C. For hexapeptides 2, 3 and 4, when using DPA instead of piperidine, the yield increased and aspartimide formation was reduced by about 25 - 100%. The yields of hexapeptides 4 and 5 were slightly lower when using DPA instead of piperidine, and aspartimide formation remained the same (Table 1).

[0061] In comparison, hexapeptide 1 was obtained at 90 °C with 78% crude purity and 11% aspartimide formation, compared to 96% crude purity and 4% aspartimide formation at 60 °C, indicating that deprotection at 60 °C yields a peptide with as high purity as at 90 °C.

[0062] [Example 3] Synthesis of the peptide drug vibralidine Peptide vibralidine consists of 20 amino acids. The synthesis of this peptide using 25% DPA in DMF at 60 °C gave an isolated yield of 39%, compared to 46% when using piperidine (Table 1). However, this result indicates that DPA is a valuable alternative to piperidine even for more complex peptides.

[0063]

Table 1

[0064]

Table 2

[0065] Materials and methods DMF (N,N-dimethylformamide) was purchased from Thommen-Furler AG, pure oxyma (ethyl hydroxyiminocyanoacetate) was purchased from SENN AG, DIC (N,N'-diisopropylcarbodiimide) was purchased from Iris BIOTECH GMBH, piperidine was purchased from Acros Organics, piperazine, butanol and DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) were purchased from Alfa Aesar, dipropylamine, diisopropylamine, diethylamine, dibutylamine, diisobutylamine, DMAP (4-dimethylaminopyridine), HOBt (hydroxybenzotriazole), DIPEA (N,N-diisopropylethylamine), and DODT (2,2'-(ethylenedioxy)diethanethiol) were purchased from Sigma Aldrich, triisopropylsilane and TFA (trifluoroacetic acid) were purchased from Fluorochem Ltd, and formic acid was purchased from Fluka Analytical. For amino acids, Fmoc-Nle-OH was purchased from Iris BIOTECH GMBH, Fmoc-Asp-OtBu and Fmoc-Glu-OtBu were purchased from Novabiochem, and all other amino acids were purchased from Shanghai Space Peptides Pharmaceuticals Co., Ltd. Chemicals were used as supplied, and solvents were of industrial grade. Amino acids were used as the following derivatives: Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Lys(Fmoc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Asp-OtBu, Fmoc-Glu(tBu)-OH, Fmoc-Glu-OtBu, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ser-OH, Fmoc-Nle-OH, Fmoc-His(Trt)-OH, Fmoc-D-Phe-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH and Fmoc-Asn(Trt)-OH.The Rink amide AM LL resin was purchased from Novabiochem. The Wang resin was purchased from Iris BIOTECH GMBH.

[0066] Analytical RP-HPLC was performed using an Ultimate 3000 Rapid Separation LC-MS system (DAD-3000RS diode array detector) with an Acclaim RSLC 120 C18 column (2.2 μm, 120 Å, 3 × 50 mm, flow rate 1.2 mL / min) from Dionex. Data recording and processing were carried out using Dionex Chromeleon management system version 6.80 (Analytical RP-HPLC). All RP-HPLC used HPLC grade acetonitrile and Milli-Q deionized water. The eluents were A: MilliQ deionized water containing 0.05% TFA; D: MilliQ deionized water / acetonitrile (10:90, v / v) containing 0.05% TFA (see item 3 below, except for the analysis of Fmoc deprotection). Preparative RP-HPLC was performed using a Waters automatic Prep LC controller system containing the following four modules: Waters 2489 UV / Vis detector, Waters 2545 pump, Waters Fraction Collector III, and Waters 2707 autosampler. A Reprospher column (C18-DE, 100 × 30 mm, particle size 5 μm, pore size 100 Å, flow rate 40 mL / min) from Dr. Maisch GmbH was used. Compounds were detected by UV absorption at 214 nm using a Waters 248 tunable absorbance detector. Data recording and processing were carried out using Waters ChromScope version 1.40 from Waters Corporation. All RP-HPLC used HPLC grade acetonitrile and Milli-Q deionized water. The eluents were A: MilliQ deionized water containing 0.1% TFA; D: MilliQ deionized water / acetonitrile (10:90, v / v) containing 0.1% TFA. The MS spectra recorded on a Thermo Scientific LTQ Orbitrap XL were provided by the MS analysis service of the Department of Chemistry, University of Bern (group of PD Dr. Stefan Schurch).

[0067] Peptide solid-phase synthesis (SPPS) SPPS of G1KL All peptide dendrimers were synthesized using standard 9-fluorenylmethoxycarbonyl (Fmoc) peptide solid-phase synthesis. The synthesis of peptide dendrimers was all carried out at 60 °C (or room temperature) under nitrogen bubbling. All peptide dendrimers were synthesized using Rink amide LL resin (0.26 - 0.29 mmol / g). The branching point consisted of Fmoc-Lys(Fmoc)-OH for two free amines (main chain and side chain) after Fmoc deprotection.

[0068] The resin was first deprotected twice, for 1 minute and 4 minutes, using the corresponding deprotection cocktail. For each amino acid, double coupling (twice for 8 minutes) was performed using, per coupling, 3 mL of 0.2 M corresponding Fmoc-protected amino acid in DMF, 1.5 mL of 0.5 M oxyma in DMF, and 2 mL of 0.5 M DIC in DMF. The deprotection steps (1 minute and 4 minutes) were achieved using the corresponding deprotection solution.

[0069] After SPPS, the peptide dendrimers were cleaved from the resin at room temperature for 3 hours using a corresponding mixture of 7 mL of trifluoroacetic acid / triisopropylsilane / mQ water (TFA / TIS / H2O) in a ratio of 94 / 5 / 1. Then, the peptide was precipitated using approximately 25 mL of cold tert-butylmethyl ether and centrifuged at 4400 rpm for 10 minutes. The supernatant was removed, and the peptide was dried with argon and then lyophilized. All peptides were obtained as TFA salts.

[0070] SPPS of G2KL The synthesis of G2KL was carried out at room temperature using a mechanical stirrer with the same reagents as above, and branching was carried out using Fmoc-Lys(Fmoc)-OH. Double deprotection was carried out for 2 x 10 minutes. Double coupling was carried out for 2 x 1 hour for the first three amino acids and the first generation, and 3 x 1 hour for the second generation. The branching point consisted of Fmoc-Lys(Fmoc)-OH for the two free amines (main chain and side chain) after Fmoc deprotection. The same conditions as above were used for cleavage.

[0071] Solid-phase peptide synthesis of linear peptides All peptides were synthesized using standard 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis. The synthesis of linear peptides was all carried out at 60 °C (or 90 °C) under nitrogen bubbling. Except for the use of Wang resin (1.2 mmol / g) to obtain a carboxylic acid functional group at the C-terminus, all peptides were synthesized using Rink amide LL resin (0.26 - 0.29 mmol / g).

[0072] The resin was first deprotected twice for 1 minute and 4 minutes using the corresponding deprotection cocktail. For each amino acid, double coupling (2 x 8 minutes) was carried out using 3 mL of 0.2 M of the corresponding Fmoc-protected amino acid in DMF, 1.5 mL of 0.5 M of oxyma in DMF, and 2 mL of 0.5 M of DIC in DMF for each coupling. The deprotection steps (1 minute and 4 minutes) were achieved using the corresponding deprotection solution.

[0073] For synthesis at 90 °C, the coupling time was 2 x 4 minutes and the deprotection time was 0.5 minute and 2.5 minutes.

[0074] For bibalyzine, due to the carboxyl C-terminus, the first amino acid coupling was carried out using DMAP (0.2 equivalent in DMF) as the coupling reagent.

[0075] After SPPS, the peptide was cleaved from the resin using a corresponding ratio of 94 / 5 / 1 of 7 mL of a mixture of trifluoroacetic acid / triisopropylsilane / mQ water (TFA / TIS / H2O) at room temperature for 3 hours, and for hexapeptide 5, using a corresponding ratio of 94 / 2.5 / 2.5 / 1 of 7 mL of a mixture of TFA / TIS / DODT / H2O. The peptide was then precipitated using approximately 25 mL of cold tert-butyl methyl ether and centrifuged at 4400 rpm for 10 minutes. The supernatant was removed and the peptide was dried with argon before being lyophilized and / or purified. All peptides were obtained as TFA salts.

[0076] Fmoc Deprotection in Solution 50 mg of Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH or Fmoc-PEG-OH was dissolved in a total volume of 500 μL of the corresponding deprotection conditions. The deprotection conditions used in DMF were 20% v / v piperidine, 25% v / v dipropylamine, 5% w / v piperazine + 2% v / v DBU, 2% v / v DBU, 25% v / v dipropylamine + 3% w / v piperazine, 25% v / v diethylamine, 25% v / v diisopropylamine, and 25% diisobutylamine. The reaction mixture was stirred at room temperature for 30 minutes. After the reaction, 10 μL for each condition was diluted in MeCN at a final volume of 1 mL.

[0077] All samples were analyzed by analytical RP-HPLC-MS using solvents B (100% mQ water + 0.1% formic acid) and C (90% MeCN + 10% mQ water + 0.1% formic acid) with a gradient from 100% B to 100% C over 7 minutes.

[0078] Analytical Data G1KL (20% v / v piperidine) was obtained as a white crude solid after lyophilization (90.5 mg, 72.5%). Analytical RP-HPLC: t R = 2.11 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 42 H84 N 12 Calculated / Measured value of O7: 869.66 / 869.66 Da [M+H] + .

[0079] G1KL (5% w / v piperazine + 2% v / v DBU) was obtained as a white crude solid after lyophilization (30.4 mg, 26.2%). Analytical RP-HPLC: t R = 1.85 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 42 H 84 N 12 Calculated / Measured value of O7: 869.66 / 869.66 Da [M+H] + .

[0080] G1KL (20% v / v diisopropylamine) was obtained as a white crude solid after lyophilization (0.3 mg, 0.0%, trace). Analytical RP-HPLC: t R = - min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 42 H 84 N 12 Calculated / Measured value of O7: 869.66 / 869.66 Da [M+H] + .

[0081] G1KL (20% v / v dipropylamine) was obtained as a white crude solid after lyophilization (42.2 mg, 35.2%). Analytical RP-HPLC: t R = 1.79 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 42 H 84 N 12 Calculated / Measured value of O7: 869.66 / 869.66 Da [M+H] + .

[0082] G1KL (25% v / v dipropylamine) was obtained as a white crude solid after lyophilization (82.4 mg, 64.5%). Analytical RP-HPLC: t R= 1.88 min (7.50 min from 100:0 to 0:100 of A / D, λ = 214 nm). HRMS (ESI+): C 42 H 84 N 12 O7 Calculated / Measured 869.66 / 869.66 Da [M+H] + .

[0083] G2KL (20% v / v piperidine) was obtained as a white crude solid after lyophilization (214.4 mg, 64.9%). Analytical RP-HPLC: t R = 2.35 min (7.50 min from 100:0 to 0:100 of A / D, λ = 214 nm). HRMS (ESI+): C 102 H 200 N 28 O 17 Calculated / Measured 2090.56 / 2090.56 Da [M+H] + .

[0084] G2KL (5% w / v piperazine + 2% DBU) was obtained as a white crude solid after lyophilization (189.9 mg, 53.9%). Analytical RP-HPLC: t R = 2.32 min (7.50 min from 100:0 to 0:100 of A / D, λ = 214 nm). HRMS (ESI+): C 102 H 200 N 28 O 17 Calculated / Measured 2090.56 / 2090.56 Da [M+H] + .

[0085] G2KL (20% v / v dipropylamine) was obtained as a white crude solid after lyophilization (134.4 mg, 42.2%). Analytical RP-HPLC: t R = 2.40 min (7.50 min from 100:0 to 0:100 of A / D, λ = 214 nm). HRMS (ESI+): C 102 H 200 N 28 O 17 Calculated / Measured 2090.56 / 2090.56 Da [M+H] + .

[0086] G2KL (25% v / v dipropylamine) was obtained as a white crude solid after lyophilization (151.4 mg, 46.4%). Analytical RP-HPLC: t R = 2.38 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 102 H 200 N 28 O 17 Calculated / measured 2090.56 / 2090.56 Da [M+H] + .

[0087] VKDGYI (SEQ ID NO: 3) (20% v / v piperidine) was obtained as a white solid after preparative RP-HPLC (3.2 mg, 4.5%). Analytical RP-HPLC: t R = 1.92 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 32 H 52 N8O9 Calculated / measured 693.39 / 693.39 Da [M+H] + .

[0088] VKDGYI (SEQ ID NO: 3) (20% v / v piperidine + 0.5 M oxime) was obtained as a white crude solid after lyophilization (10.8 mg, 16.8%). Analytical RP-HPLC: t R = 1.99 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 32 H 52 N8O9 Calculated / measured 693.39 / 693.39 Da [M+H] + .

[0089] VKDGYI (SEQ ID NO: 3) (5% w / v piperazine + 2% v / v DBU) was obtained as a white crude solid after lyophilization (5.7 mg, 0.0%). Analytical RP-HPLC: t R = - min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 32 H 52Calculated / Measured value of N8O9 693.39 / - Da [M+H] + . (The compound was not observed).

[0090] VKDGYI (SEQ ID NO: 3) (2% v / v of DBU) was obtained as a white crude solid after lyophilization (36.6 mg, 25.7%). Analytical RP-HPLC: t R = 2.05 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 32 H 52 Calculated / Measured value of N8O9 693.39 / 693.39 Da [M+H] + .

[0091] VKDGYI (SEQ ID NO: 3) (20% v / v of dipropylamine) was obtained as a white crude solid after lyophilization (37.3 mg, 49.3%). Analytical RP-HPLC: t R = 1.84 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 32 H 52 Calculated / Measured value of N8O9 693.39 / 693.39 Da [M+H] + .

[0092] VKDGYI (SEQ ID NO: 3) (25% v / v of dipropylamine) was obtained as a white solid after preparative RP-HPLC (11.5 mg, 16.0%). Analytical RP-HPLC: t R = 1.20 min (from 100:0 to 0:100 of A / D in 3.5 min, λ = 214 nm). MS (ESI+): C 32 H 52 Calculated / Measured value of N8O9 693.39 / 693.42 Da [M+H] + .

[0093] VKDGYI (SEQ ID NO: 3) (25% v / v of dipropylamine) was obtained as a white crude solid after lyophilization (39.6 mg, 52.9%). Analytical RP-HPLC: t R= 1.96 min (7.50 min from 100:0 to 0:100 of A / D, λ = 214 nm). HRMS (ESI+): C 32 H 52 N8O9 calculated / measured 693.39 / 693.39 Da [M+H] + .

[0094] VKDGYI (SEQ ID NO: 3) (25% v / v dipropylamine, 90 °C) was obtained as a white crude solid after lyophilization (26.8 mg, 33.5%). Analytical RP-HPLC: t R = 2.01 min (7.50 min from 100:0 to 0:100 of A / D, λ = 214 nm). HRMS (ESI+): C 32 H 52 N8O9 calculated / measured 693.39 / 693.39 Da [M+H] + .

[0095] VKD(β)GYI (SEQ ID NO: 11) was obtained as a white solid after preparative RP-HPLC (6.4 mg, 14.2%). Analytical RP-HPLC: t R = 1.19 min (2.2 min from 100:0 to 0:100 of A / D, λ = 214 nm). MS (ESI+): C 32 H 52 N8O9 calculated / measured 693.39 / 693.42 Da [M+H] + .

[0096] GDGAKF (SEQ ID NO: 4) (20% v / v piperidine) was obtained as a white crude solid after lyophilization (40.6 mg, 40.9%). Analytical RP-HPLC: t R = 1.75 min (7.50 min from 100:0 to 0:100 of A / D, λ = 214 nm). HRMS (ESI+): C 26 H 40 N8O8 calculated / measured 593.30 / 593.30 Da [M+H] + .

[0097] GDGAKF (SEQ ID NO: 4) (25% v / v dipropylamine) was obtained as a white crude solid after lyophilization (38.9 mg, 49.2%). Analytical RP-HPLC: t R = 1.76 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 26 H 40 N8O8 calculated / measured 593.30 / 593.30 Da [M+H] + .

[0098] VKDRYI (SEQ ID NO: 5) (20% v / v piperidine) was obtained as a white crude solid after lyophilization (44.0 mg, 40.3%). Analytical RP-HPLC: t R = 1.99 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 36 H 61 N 11 O9 calculated / measured 792.47 / 792.47 Da [M+H] + .

[0099] VKDRYI (SEQ ID NO: 5) (25% v / v dipropylamine) was obtained as a white crude solid after lyophilization (44.2 mg, 43.4%). Analytical RP-HPLC: t R = 2.00 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 36 H 61 N 11 O9 calculated / measured 792.47 / 792.47 Da [M+H] + .

[0100] GDRAKF (SEQ ID NO: 6) (20% v / v piperidine) was obtained as a white crude solid after lyophilization (44.2 mg, 50.6%). Analytical RP-HPLC: t R = 1.84 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 30 H 48 N 10Calculated / Measured value of O9: 693.36 / 693.39 Da [M+H] + .

[0101] GDRAKF (SEQ ID NO: 6) (25% v / v dipropylamine) was obtained as a white crude solid after lyophilization (52.9 mg, 62.5%). Analytical RP-HPLC: t R = 1.86 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 30 H 48 N 10 Calculated / Measured value of O9: 693.36 / 693.39 Da [M+H] + .

[0102] VKDCYI (SEQ ID NO: 7) (20% v / v piperidine) was obtained as a white crude solid after lyophilization (46.2 mg, 53.1%). Analytical RP-HPLC: t R = 2.23 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 33 H 54 Calculated / Measured value of N8O9S: 739.37 / 739.38 Da [M+H] + .

[0103] VKDCYI (SEQ ID NO: 7) (25% v / v dipropylamine) was obtained as a white crude solid after lyophilization (42.7 mg, 48.0%). Analytical RP-HPLC: t R = 2.23 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 33 H 54 Calculated / Measured value of N8O9S: 739.37 / 739.38 Da [M+H] + .

[0104] VKDAYI (SEQ ID NO: 8) (20% v / v piperidine) was obtained as a white crude solid after lyophilization (42.7 mg, 54.7%). Analytical RP-HPLC: t R= 2.09 min (7.50 min from 100:0 to 0:100 of A / D, λ = 214 nm). HRMS (ESI+): C 33 H 54 N8O9 calculated / measured 707.40 / 707.41 Da [M+H] + .

[0105] VKDAYI (SEQ ID NO: 8) (25% v / v dipropylamine) was obtained as a white crude solid after lyophilization (40.4 mg, 51.3%). Analytical RP-HPLC: t R = 2.09 min (7.50 min from 100:0 to 0:100 of A / D, λ = 214 nm). HRMS (ESI+): C 33 H 54 N8O9 calculated / measured 707.40 / Da [M+H] + .

[0106] VKEGYI (SEQ ID NO: 9) (20% v / v piperidine) was obtained as a white crude solid after lyophilization (34.8 mg, 47.7%). Analytical RP-HPLC: t R = 1.99 min (7.50 min from 100:0 to 0:100 of A / D, λ = 214 nm). HRMS (ESI+): C 33 H 54 N8O9 calculated / measured 707.40 / 707.41 Da [M+H] + .

[0107] VKEGYI (SEQ ID NO: 9) (5% w / v piperazine + 2% v / v DBU) was obtained as a white crude solid after lyophilization (38.2 mg, 52.4%). Analytical RP-HPLC: t R = 1.90 min (7.50 min from 100:0 to 0:100 of A / D, λ = 214 nm). HRMS (ESI+): C 33 H 54 N8O9 calculated / measured 707.40 / 707.41 Da [M+H] + .

[0108] VKEGYI (SEQ ID NO: 9) (20% v / v dipropylamine) was obtained as a white crude solid after lyophilization (32.3 mg, 44.3%). Analytical RP-HPLC: t R = 1.98 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 33 H 54 N8O9 calculated / measured 707.40 / 707.41 Da [M+H] + .

[0109] VKEGYI (SEQ ID NO: 9) (20% v / v dipropylamine + 0.5 M oxime) was obtained as a white crude solid after lyophilization (length="36.5" mg, 50.0%). Analytical RP-HPLC: t R = 1.93 min (from 100:0 to 0:100 of A / D in 7.50 min, λ = 214 nm). HRMS (ESI+): C 33 H 54 N8O9 calculated / measured 707.40 / 707.41 Da [M+H] + .

[0110] Bivalirudin (25% v / v dipropylamine) was obtained as a foamy white solid after preparative RP-HPLC (93.3 mg, 38.7%). Analytical RP-HPLC: t R = 1.52 min (from 100:0 to 0:100 of A / D in 3.50 min, λ = 214 nm). HRMS (ESI+): C 98 H 138 N 24 O 33 calculated / measured 2179.99 / 2179.99 Da [M+H] + .

[0111] Bivalirudin (20% v / v piperidine) was obtained as a foamy white solid after preparative RP-HPLC (111.6 mg, 46.3%). Analytical RP-HPLC: t R = 1.52 min (from 100:0 to 0:100 of A / D in 3.50 min, λ = 214 nm). HRMS (ESI+): C 98 H 138 N 24 O33 Calculated value / Measured value 2179.99 / 2179.99 Da [M+H] + .

[0112] List of references R1: Merrifield, R. B., J. Am. Chem. Soc., 1963, 85(14), 2149-2154. R2: W. Li, N. M. O’Brien-Simpson, M. A. Hossain, J. D. Wade, W. Li, N. M. O’Brien-Simpson, M. A. Hossain and J. D. Wade, Aust. J. Chem., 2019, 73, 271-276. R3: Ralhan, K., KrishnaKumar, V. G., & Gupta, S., RSC Advances, 2015, 5(126), 104417-104425. R4: O. F. Luna, J. Gomez, C. Cardenas, F. Albericio, S. H. Marshall and F. Guzman, Molecules, 2016, 21, 1542. R5: T. Michels, R. Dolling, U. Haberkorn and W. Mier, Org. Lett., 2012, 14, 5218-5221. R6: C.-C. Chen, B. Rajagopal, X. Y. Liu, K. L. Chen, Y.-C. Tyan, F. Lin and P.-C. Lin, Amino Acids, 2014, 46, 367-374. R7: V. Rodriguez, H. Pineda, N. Ardila, D. Insuasty, K. Cardenas, J. Roman, M. Urrea, D. Ramirez, R. Fierro, Z. Rivera and J. Garcia, Int. J. Pept. Res. Ther., 2020, 26, 585-587. R8: G. Martelli, P. Cantelmi, C. Palladino, A. Mattellone, D. Corbisiero, T. Fantoni, A. Tolomelli, M. Macis, A. Ricci, W. Cabri and L. Ferrazzano, Green Chem., 2021, 23, 8096 - 8107. R9: Palomo, J. M.. Rsc Advances, 2014, 4(62), 32658 - 32672. R10: Isidro - Llobet, A., Alvarez, M., & Albericio, F., Chem. Rev., 2009, 109(6), 2455 - 2504. R11: Insuasty Cepeda, D. S., Pineda Castaneda, H. M., Rodriguez Mayor, A. V., Garcia Castaneda, J. E., Maldonado Villamil, M., Fierro Medina, R., & Rivera Monroy, Z. J., Molecules, 2019, 24(7), 1215. R12: Mant, Colin T. et al. "HPLC analysis and purification of peptides. "Peptide Characterization and Application Protocols”. Humana Press, 2007, p. 3 - 55.

Claims

1. A method for preparing a peptide via solid-phase peptide synthesis, comprising: R-AA-(AA)-Fmoc-protected amino acid building blocks linked to the resin ｎ -PF (wherein, - R is a resin, - AA is an amino acid building block, - PF is an Fmoc protecting group, - n is the number of coupling cycles) is deprotected using dibutylamine or dipropylamine in the deprotection step prior to the coupling step to give R-AA-(AA) ｎ is brought about In said coupling step, another amino acid building block AA-P is coupled to R-(AA)-(AA) ｅｎｄ up to the final coupling cycle n ｎ to give R-AA-(AA) ｎ＋１ -P, where P is a protecting group or PF at the N-terminus of the amino acid building block AA, method.

2. The method according to claim 1, wherein at least one AA contains aspartic acid.

3. The method according to claim 1 or 2, wherein the deprotection step is carried out using dipropylamine.

4. The method according to any one of claims 1 to 3, wherein the amount of dipropylamine is 10 to 50% (v / v), particularly 20 to 40% (v / v), particularly 25 to 35% (v / v).

5. The method according to any one of claims 1 to 4, which is carried out at 10 to 90 °C, particularly 65 to 90 °C, particularly 75 to 90 °C.

6. The method according to any one of claims 1 to 5, wherein the AA-P is activated in an activation step prior to the coupling step.

7. The method according to any one of claims 1 to 6, wherein the deprotection step and the coupling step can be repeated until a desired peptide length of 60 amino acids or less is reached.

8. n ｅｎｄ The method according to any one of claims 1 to 7, which is the final coupling cycle in which n reaches the desired peptide length.

9. n ｅｎｄ including a final deprotection step after the ｎｅｎｄ coupling cycle, wherein R-AA-(AA) ｎｅｎｄ -P is deprotected to give R-AA-(AA) and the method according to any one of claims 1 to 8

10. n ｅｎｄ including a cutting step after the nth coupling cycle, wherein R-AA-(AA) ｎｅｎｄ is cleaved from said resin R to give AA-(AA) ｎｅｎｄ The method according to any one of claims 1 to 9, wherein

11. including a purification step, AA-(AA) ｎｅｎｄ The method according to claim 11, wherein is purified.

12. The method according to any one of claims 1 to 11, wherein P is PF.

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