Application of dipropylamine as base in Fmoc deprotection in solid phase peptide synthesis

Abstract

The invention relates to a method for preparing peptides by solid phase peptide synthesis, in particular to a method for deprotecting Fmoc protected amino acid blocks linked to resin R-AA-(AA) n-PF.

Description

[0001] The present invention relates to an optimized method for deprotecting the fluorenylmethoxycarbonyl (Fmoc) group of peptides in solid phase peptide synthesis (SPPS). Background Art

[0002] Solid phase peptide synthesis (SPPS) is a widely used peptide synthesis method in which amino acids are covalently bound to a solid support material and peptide growth occurs stepwise using a selective protecting group strategy. SPPS is characterized by high efficiency and high throughput as well as improved simplicity, speed and yield compared to conventional liquid synthesis. Amino acids are protected at all reactive functional groups present to form amino acid building blocks, whereby the reaction order of each functional group can be controlled by selective deprotection. In the basic method of SPPS, the first amino acid building block is attached to the resin at its C-terminus or N-terminus, usually its C-terminus. The two most commonly used strategies are fluorenylmethyloxycarbonyl (Fmoc) SPPS and tert-butyloxycarbonyl (Boc) SPPS, in which the N-terminus is protected with Fmoc or Boc. In the next step, the N-terminus of the building block is deprotected in a deprotection step to produce a free amine. The formation of a peptide bond requires a carboxylic acid as a reactive partner for the free amine. Therefore, the building block to be attached to the free amine is protected at its N-terminus and the C-terminus must undergo an activation step. The resin-bound free amine and activated carboxylic acid then form a peptide bond in a coupling step, producing an N-terminally protected dipeptide in the case of a single amino acid building block. The N-terminally protected dipeptide is then deprotected again for coupling to another C-terminally activated amino acid building block. The 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] SPPS with Fmoc as the α-amino protecting group of the amino acid building block is currently the main synthetic method for peptide research and production. Piperidine (PPR) is used as the best reagent for removing Fmoc in SPPS because it acts as an effective base to initiate the β-elimination of carbamate and as a nucleophile to quench the reactive dibenzofulvene byproduct ( Figure 2 a). However, PPR is expensive, smelly, and is highly regulated due to its use in illicit drug production. 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 is less smelly but still expensive ( Figure 2 b). However, both PPR and PZ / DBU induce the formation of asparagine in some aspartate-containing sequences, which can be hydrolyzed to either α-peptide or β-peptide ( Figure 2c). Adding weak acids such as formic acid or ethyl cyanohydroxyaminoacetate (Oxyma) to adjust the alkalinity of the PPR solution reduces the formation of asparagine, however this does not address the cost, odor and availability issues of PPR. Several reagents or aspartic acid side chain protecting groups have been reported in an attempt to overcome the limitations of PPR or PZ / DBU, however none of them combine low cost and ease of use with high yield and low asparagine. R2-R8

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

[0005] A first aspect of the invention relates to a method for preparing a peptide by solid phase peptide synthesis, wherein

[0006] Fmoc protected amino acid building blocks attached to the resin R–AA–(AA) n –PF, where

[0007] –R is resin

[0008] –AA is the amino acid building block

[0009] –PF is Fmoc protecting group

[0010] –n is the number of coupling cycles,

[0011] Deprotection using dibutylamine or dipropylamine in the deprotection step before the coupling step yields R–(AA)–(AA) n ,in

[0012] In the coupling step, another amino acid building block AA-P is coupled to R-(AA)-(AA) n , producing R–AA–(AA) n+1 –P, until the final coupling cycle n 末 , wherein P is the protecting group of the amino acid building block AA at the N-terminus or PF.

[0013] Terms and Definitions

[0014] For purposes of interpreting this specification, the following definitions will apply and, whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event of a conflict between any definition set forth below and any document incorporated herein by reference, the set forth definition will control.

[0015] As used herein, the terms "comprising," "having," "containing," and "including," and other similar forms and grammatical equivalents thereof are intended to be equivalent in meaning and to be open ended, in that one or more items following any of these words are not meant to be an exhaustive list of the one or more items, or to be limited to the listed one or more items. For example, something that "comprising" components A, B, and C may consist of components A, B, and C (i.e., contain only), or may contain not only components A, B, and C, but may also include one or more other components. Thus, it is intended and understood that "comprising" and its similar forms and grammatical equivalents thereof encompass disclosure of embodiments that "consist essentially of" or "consist of."

[0016] Where a range of values ​​is provided, it is understood that each intervening value to the tenth of the unit of the lower limit, and any other stated or intervening value in that range, is encompassed within the invention, subject to any specifically excluded limits in that range, unless the context clearly dictates otherwise. Where the range includes one or two limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0017] Reference herein to "about" a value or parameter includes (and describes) variations involving the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0018] As used herein, including in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art (e.g., in 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, 4 th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al., Short Protocols in Molecular Biology (2002) 5 th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0020] In the context of this specification, the term resin R relates to a solid support. Resins are usually small, spherical beads, of two different sizes: 100–200 mesh (75–150 microns) and 200–400 mesh (35–75 microns), including polymers. The most common resin in solid phase chemistry is polystyrene (PS), which is often supplemented with divinylbenzene (PS-DVB). Other commonly used resins are polyamines and polyethylene glycol-polystyrene (PEG-PS) resins.

[0021] The resin is further functionalized with a linker. For Fmoc-SPPS, commonly used linkers are Rink amide, Wang, hexamethylphosphoric triamide (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

[0022] In the context of the present specification, the term amino acid building block AA relates to a single amino acid or a short peptide comprising two to three amino acids which are protected at their amino acid side chains with any common protecting groups for amino acids. R10

[0023] The term n in the context of this specification relates to the number of coupling cycles in solid phase peptide synthesis. The minimum number of coupling cycles is 1. The maximum number of coupling cycles is not defined.

[0024] In the context of this specification, the term coupling cycle refers to the step in solid phase peptide synthesis where the deprotected, resin-bound amino acid building block R-(AA)-(AA) n Coupling with activated amino acid building blocks AA–P.

[0025] The term n in the context of this specification 末 Involves a final coupling cycle in solid phase synthesis, wherein once the desired peptide length is reached, the final coupling cycle is reached. Once the final desired peptide length is reached, the peptide is fully deprotected at its N-terminus and side chains, cleaved from the resin, and purified if necessary.

[0026] In the context of the present specification, the term P relates to a protecting group at the N-terminus of an amino acid building block, wherein the protecting group may be any common protecting group generally known to those skilled in the art for peptide synthesis, including benzylamine (NBn), N-carboxybenzyl (Cbz), tert-butyloxycarbonyl (Boc), allyloxycarbonyl (Alloc), methyltrityl (Mtt), 1-(4,4-dimethyl-2,6-dioxyylidenecyclohex-1-ylidene)ethyl (Dde), 1-(4,4-dimethyl-2,6-dioxyylidenecyclohexylidene)-3-methylbutyl (ivDde) ​​or fluorenylmethoxycarbonyl (Fmoc) (see Clayden, Greeves, Warren and Wothers, Organic Chemistry, 2001, p. 657).

[0027] The term PF in the context of this specification refers to the fluorenylmethoxycarbonyl (Fmoc) protecting group. Fmoc is a base-labile and acid-stable protecting agent commonly used in peptide synthesis. Fmoc is usually introduced via 9-fluorenylmethoxychloride (Fmoc-Cl) (see Clayden, Greeves, Warren and Wothers, Organic Chemistry, 2001, p.656-658).

[0028] In the context of this specification, the term polypeptide refers to a molecule consisting of 50 or more amino acids, which forms a straight chain in which the amino acids are linked by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or a fragment thereof. The terms "polypeptide" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as sequences of amino acid residues.

[0029] In the context of the present specification, the term peptide relates to a molecule consisting of up to 50 amino acids, in particular up to 30 amino acids, more in particular up to 15 amino acids, forming a linear chain wherein the amino acids are linked by peptide bonds.

[0030] The amino acid residue sequences are given from the amino to the carboxyl terminus. Capital letters at sequence positions refer to the single-letter codes for L-amino acids (Stryer, Biochemistry, 3 rded.p.21). Lowercase letters at amino acid sequence positions refer to the corresponding D- or (2R)-amino acid. Sequences are written from left to right in the direction from the amino terminus to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are represented by three-letter or one-letter codes 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). DETAILED DESCRIPTION

[0031] A first aspect of the invention relates to a method for preparing a peptide by solid phase peptide synthesis, wherein

[0032] Fmoc protected amino acid building blocks attached to the resin R–AA–(AA) n –PF, where

[0033] –R is resin

[0034] –AA is the amino acid building block

[0035] –PF is Fmoc protecting group

[0036] –n is the number of coupling cycles,

[0037] Deprotection using dibutylamine or dipropylamine in the deprotection step before the coupling step yields R–(AA)–(AA) n ,in

[0038] In the coupling step, another amino acid building block AA-P is coupled to R-(AA)-(AA) n , producing R–AA–(AA) n+1 –P, until the final coupling cycle n 末 , wherein P is the protecting group of the amino acid building block AA at the N-terminus or PF.

[0039] The term amino acid building block AA relates to a single amino acid or a short peptide comprising two to three amino acids, which are protected at their amino acid side chains with any of the usual protecting groups used in peptide synthesis.

[0040] In SPPS, the first amino acid building block AA–(AA) n–PF is linked to the resin R (which represents the solid phase), producing R–AA–(AA) n PF. In the first amino acid building block, n is equal to 0. The first amino acid building block R-AA-PF is then deprotected using dibutylamine or dipropylamine to obtain a deprotected amino acid building block R-AA.

[0041] In certain embodiments, at least one AA comprises aspartic acid.

[0042] Aspartic acid is the main amino acid that forms asparagine during SPPS and is subsequently hydrolyzed to α-peptide or β-peptide ( Figure 2 c). The base present in the Fmoc deprotection step of the peptide deprotonates the adjacent secondary amine to aspartic acid, which in turn reacts in a ring closure to form asparagine. Asparagine is hydrolyzed by the water present in the synthesis to form the alpha or beta peptide of the resulting peptide.

[0043] The method of the present invention, in which the use of dibutylamine or dipropylamine greatly reduces the formation of asparagine, thereby making the yield of peptides higher, is different from the existing SPPS process.

[0044] In certain embodiments, the deprotection step is performed with dipropylamine.

[0045] The use of dipropylamine as a base for the deprotection of Fmoc-protected amino acid building blocks or Fmoc-protected peptides solves several problems in the use of conventional bases for Fmoc deprotection of Fmoc-protected amino acid building blocks or Fmoc-protected peptides, piperidines and piperazines. Both dibutylamine and dipropylamine are unregulated, cheap, non-smelly, and readily available. Fmoc deprotection with dibutylamine or dipropylamine results in good peptide yields and reduced formation of asparagine. As shown in Examples 1 to 3 of the present invention, the use of dipropylamine still produces slightly better yields than the use of dibutylamine.

[0046] In certain embodiments, dipropylamine is used in an amount of 10 to 50% (v / v).

[0047] In certain embodiments, dipropylamine is used in an amount of 20 to 40% (v / v).

[0048] In certain embodiments, dipropylamine is used in an amount of 25 to 35% (v / v).

[0049] It was found that dipropylamine worked best when used in a 25% (v / v) solution in N,N-dimethylformamide (DMF). When dipropylamine was used at that volume percentage, Oxyma, an additive used in carbodiimide in SPPS and known to reduce asparagine formation, did not further reduce asparagine formation, further simplifying the process and saving costs.

[0050] In certain embodiments, the method is performed at 10 to 90°C.

[0051] In certain embodiments, the method is performed at 65 to 90°C.

[0052] The use of dibutylamine or dipropylamine in SPPS enables SPPS to be performed at higher temperatures of 65 to 90°C. Higher temperatures enable the use of dibutylamine or dipropylamine, which have high boiling points. High temperatures reduce the aggregation of peptides and the swelling of the resin. In addition, the reaction time is reduced by increasing the reaction rate, resulting in higher peptide yields.

[0053] In certain embodiments, the method is performed at 75 to 90 °C.

[0054] In certain embodiments, AA-P is activated in an activation step prior to the coupling step.

[0055] In the coupling step, another amino acid building block, AA–P, is coupled to R–(AA)–(AA) n . In order for the coupling step AA–P to occur, where P is a protecting group or PF at the amino acid building block AA at its N-terminus, the amino acid building block AA needs to be activated in an activation step. Activation occurs at the C-terminus of the amino acid building block using common coupling reagents. Commonly used coupling reagents are N,N′-diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate (HATU), tetramethylfluoroformamidine hexafluorophosphate (TFFH) or benzotriazol-1-yl-oxytripyrrolidine hexafluorophosphate (PyBOP).

[0056] In certain embodiments, the deprotection step and the coupling step can be repeated until the desired peptide length is reached, not to exceed 60 amino acids.

[0057] In certain embodiments, n 末 is the final coupling cycle to achieve the desired peptide length.

[0058] n 末 It is not equal to the number of amino acids in a peptide, since the amino acid building blocks can contain from 1 to 3 amino acids.

[0059] In certain embodiments, the method comprises 末 A final deprotection step after a coupling cycle, in which R-AA-(AA) n末 –P deprotection to produce R–AA–(AA) n末 .

[0060] 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.

[0061] In certain embodiments, the method comprises 末 The cleavage step after the coupling cycle, in which R–AA–(AA)– n末 Cleavage from resin R to produce AA–(AA) n末 .

[0062] In certain embodiments, the method comprises a purification step, wherein AA-(AA) n末 purification.

[0063] In the purification step the peptide is separated from the impurities. 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. R11R12

[0064] In certain embodiments, P is PF.

[0065] The formation of asparagine from aspartic acid occurs throughout the peptide synthesis and in all subsequent deprotections. Therefore, it is advantageous to use only Fmoc as the N-terminal protecting group throughout the peptide synthesis. In addition, the use of Fmoc as the N-terminal protecting group in all building blocks facilitates synthesis, especially in terms of side chain protecting group chemistry. A consistent N-terminal protecting group on all AA–Ps throughout the peptide synthesis allows the selection of amino acid side chain protecting groups that are stable throughout the synthesis. For example, Fmoc is base labile, so pure base stable protecting groups can be used on the amino acid side chains.

[0066] The present invention is further illustrated by the following examples and figures, from which other embodiments and advantages can be derived. These examples are intended to illustrate the present invention but not to limit its scope. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 : Solid phase peptide synthesis (SPPS) cycle, with a) deprotection step, b) activation step, c) coupling step, d) final deprotection step, e) cleavage step; AA–P can be different amino acid building blocks in each cycle;.

[0068] Figure 2: Shown are (a) the mechanism of Fmoc (1) deprotection and capture of dibenzofulvene (2). (b) Structural formulas of reagents used to remove Fmoc; PPR (3), PZ (4), DBU (5), Oxyma (6), DBA (7). (c) Mechanism of asparagine (9) formation and its hydrolysis to α-peptide (10) or β-peptide (11) during SPPS; 8 = tBuOH, B = base, Nu = nucleophile.

[0069] Example

[0070] Example 1: Synthesis of first and second generation peptide dendrimers G1KL and G2KL by SPPS

[0071] In the initial goal of developing a high temperature (60°C) protocol for solid phase peptide synthesis of G1KL, using Oxyma and N,N′-diisopropylcarbodiimide (DIC) as coupling agent, Oxyma as acidic additive to suppress base-induced side reactions, and DMF as solvent, the inventors identified dipropylamine (DPA) as an alternative base to piperidine. DPA (pKa=10.9) is only slightly less basic than piperidine (pKa=11.1) and slightly less volatile (bp(DPA)=110°C; bp(piperidine)=106°C). The synthesis of the first generation of peptide dendrimers required the removal of the alpha amino group and the side chain amino Fmoc, and the use of 25% DPA gave similar yields (65%) as the prior art reagent piperidine (73%) and much better yields than the use of piperazine and DBU (26%) (Table 1). The difference in yield between 20% and 25% DPA was significant, reaching 30%.

[0072] The synthesis of the second generation analogue G2KL resulted in an equally good yield of 46% using 25% DPA at room temperature. In this case, the increase in DPA from 20% to 25% did not lead to a significant improvement in yield (Table 1).

[0073] While DPA produced similar yields as prior art reagents, diisopropylamide (DIPA) did not result in any formation of the desired product, making it an unsuitable base for deprotection.

[0074] Example 2: Synthesis of Hexapeptides 1 to 7

[0075] The inventors then investigated the SPPS of the asparagine-biased hexapeptides 1 to 7 (Table 1). Fmoc deprotection of the peptide with piperidine gave 17% asparagine (hexapeptide 1), while only 5% was obtained with 20% DPA, which was slightly lower than the asparagine formation with the addition of Oxyma to piperidine, which resulted in 6% asparagine formation (Table 1, hexapeptide 1). The crude product NMR was compared with the independently synthesized VKDGYI (hexapeptide 1; SEQ ID NO: 3), the β-peptide of VKD(β)GYI, which is formed upon hydrolysis of the VKDGYI asparagine. However, no β-peptide was detected, and a 49% crude peptide yield (hexapeptide 1) was obtained with 20% DPA at 60°C. In contrast, using prior art conditions, including 20% ​​PPR at 60°C, the yield was 47%.

[0076] For hexapeptide 1, deprotection was accompanied by 25% asparagine formation using only 2% DBU.

[0077] The present inventors also studied Fmoc deprotection with dibutylamine and diisobutylamine in SPPS of VKDGYI (SEQ ID NO: 3). However, deprotection did not occur when diisobutylamine was used. At 60°C, dibutylamine produced a crude peptide yield similar to that of dipropylamine (52% (DBA) vs. 53% (DPA)).

[0078] 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 crude yield of 44% at 60°C with 20% DPA, and no asparagine formation was observed (Table 1).

[0079] Hexapeptides 2 to 6 were deprotected with 25% DPA at 60° C. and 20% piperidine at 60° C. as a comparison. Using DPA instead of piperidine, the yields of hexapeptides 2, 3 and 4 increased and the formation of asparagine was reduced by 25 to 100%. If the formation of piperidine and asparagine remained fairly similar, the yields of hexapeptides 4 and 5 were slightly lower when DPA was used instead (Table 1).

[0080] In contrast, hexapeptide 1 obtained at 90°C had a crude purity of 78% with 11% asparagine formed, compared to 96% crude purity and 4% asparagine at 60°C, indicating that deprotection at 60°C as well as 90°C resulted in highly pure peptides.

[0081] Example 3: Synthesis of the peptide drug Bivalirudin

[0082] Bivalirudin peptide consists of 20 amino acids. The peptide was synthesized at 60°C using 25% DPA in DMF with an isolated yield of 39% and 46% when piperidine was used (Table 1). However, the results show that DPA is a valuable alternative to piperidine even with more complex peptides.

[0083] Table 1: Extended SPPS yields of peptide dendrimers and linear peptides using various Fmoc deprotection conditions.

[0084]

[0085]

[0086] a) Amino acids are indicated by single letter codes, D-amino acids are lowercase, italic K indicates branched L-lysine, and the C-terminus is a carboxamide except for bivalirudin which is a carboxyl group. b) SPPS was performed in DMF using Oxyma / DIC as coupling reagent and the indicated bases were used to remove Fmoc. PPR = piperidine, PZ = piperazine, DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene, DPA = dipropylamine, DIPA = diisopropylamine, DBA = dibutylamine, DIBA = diisobutylamine. Percentages (%) are expressed as w / v for PZ and v / v for the rest. c) The crude purity of hexapeptides 1–7 is as follows: % desired product (% asparagine or glutarimide / % other byproducts). The crude product after resin cleavage was precipitated, washed, dried, and analyzed by analytical HPLC to determine the percentage of desired product, asparagine, and other byproducts. d) Crude yields were calculated relative to the amount of resin, its indicated loading and crude purity. e) Isolated yields were calculated after preparative RP-HPLC purification based on the amount of resin and its indicated loading. nd = not determined.

[0087] Materials and methods

[0088] DMF (N,N-dimethylformamide) was purchased from Thommen-Fuller AG, Oxyma Pure (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, 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 Shengpu Zetai Pharmaceutical Technology Co., Ltd. The supplied chemicals were used, while the solvents were of technical grade. The amino acids used are 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, Fm oc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH and Fmoc-Asn(Trt)-OH. Rink Amide AM LL resin was purchased from Novabiochem. Wang resin was purchased from Iris BIOTECH GMBH. An Acclaim RSLC 120C18 column (2.2 μm, 3×50 mm, flow rate 1.2 mL / min), analytical RP-HPLC was performed using an Ential 3000 rapid separation LC-MS system (DAD-3000RS diode array detector). Data recording and processing were performed using the Dionex Chromeleon Management System version 6.80 (analytical RP-HPLC). All RP-HPLC used HPLC grade acetonitrile and Milli-Q deionized water. The elution solutions were: A: MilliQ deionized water containing 0.05% TFA; D: MilliQ deionized water / acetonitrile (10:90, v / v) containing 0.05% TFA (except for the analysis of Fmoc deprotection in solution, see Section 3 below). Preparative RP-HPLC was performed using a Waters AutoPrep 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 Dr. Maisch GmbH Repropher column (C18-DE, 100×30 mm, particle size 5 μm, pore size 100, flow rate 40 mL / min) was used. Compounds were detected by UV absorption at 214 nm using a Waters 248 tunable absorbance detector. Data recording and processing were performed using Waters ChromScope 1.40 from Waters. HPLC grade acetonitrile and Milli-Q deionized water were used for all RP-HPLC. The elution solutions were: A: MilliQ deionized water containing 0.1% TFA; D: MilliQ deionized water / acetonitrile (10:90, v / v) containing 0.1% TFA. MS spectra, recorded on a Thermo Scientific LTQ OrbitrapXL, were provided by the MS Analysis Service of the Department of Chemistry, Biochemistry and Pharmaceutical Sciences at the University of Bern (Professor Dr Stefan Schürch's group).

[0089] Solid Phase Peptide Synthesis (SPPS)

[0090] G1KL’s SPPS

[0091] All peptide dendrimers were synthesized using standard 9-fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis. All syntheses of peptide dendrimers were performed at 60°C (or room temperature) under nitrogen bubbling. All peptide dendrimers were synthesized using RinkAmide LL resin (0.26-0.29 mmol / g). The branch point consisted of Fmoc-Lys(Fmoc)-OH, and two free amines (main chain and side chain) were obtained after Fmoc deprotection.

[0092] First, the resin was deprotected twice with the corresponding deprotection mixture, one minute and four minutes. Each amino acid was double coupled (twice for eight minutes), using 3 mL of a 0.2 M DMF solution of the corresponding Fmoc-protected amino acid, 1.5 mL of a 0.5 M Oxyma DMF solution, and 2 mL of a 0.5 M DIC DMF solution for each coupling. The deprotection step was achieved with the corresponding deprotection solution (one minute and four minutes).

[0093] After SPPS, the peptide dendrimers were cleaved from the resin with 7 mL of a mixture of trifluoroacetic acid / triisopropylsilane / mQ water (TFA / TIS / H2O) in a relative ratio of 94 / 5 / 1 at room temperature. The peptides were then precipitated with about 25 mL of cold tert-butyl methyl ether and centrifuged at 4400 rpm for 10 minutes. The supernatant was removed and the peptides were dried with argon before lyophilization. All peptides were obtained in the form of TFA salts.

[0094] G2KL’s SPPS

[0095] The synthesis of G2KL was performed at room temperature with the same reagents as above with a mechanical stirrer and branching with Fmoc-Lys(Fmoc)-OH. Double deprotection was performed during 2×10 min. Double coupling was performed during 2×1 h for the first three amino acids and the first generation and during 3×1 h for the second generation. The branch point consisted of Fmoc-Lys(Fmoc)-OH, and two free amines (main chain and side chain) were obtained after Fmoc deprotection. Cleavage was performed using the same conditions as above.

[0096] SPPS of linear peptides

[0097] All peptides were synthesized using standard 9-fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis. All syntheses of linear peptides were performed at 60°C (or 90°C) under nitrogen bubbling. All peptides were synthesized using Rink Amide LL resin (0.26-0.29 mmol / g), except for bivalirudin, which used Wang resin (1.2 mmol / g) to obtain the C-terminal carboxylic acid functional group.

[0098] First, the resin was deprotected twice with the corresponding deprotection mixture, one minute and four minutes. Each amino acid was double coupled (twice for eight minutes), using 3 mL of a 0.2 M DMF solution of the corresponding Fmoc-protected amino acid, 1.5 mL of a 0.5 M Oxyma DMF solution, and 2 mL of a 0.5 M DIC DMF solution for each coupling. The deprotection step was achieved with the corresponding deprotection solution (one minute and four minutes).

[0099] For the synthesis at 90°C, the coupling times were 2 x 4 minutes and the deprotection times were 0.5 and 2.5 minutes.

[0100] For bivalirudin, due to the carboxyl C-terminus, DMAP (0.2 equivalents, DMF solution) was used as the coupling reagent for the first amino acid coupling.

[0101] After SPPS, the peptides were cleaved from the resin with 7 mL of a mixture of trifluoroacetic acid / triisopropylsilane / mQ water (TFA / TIS / H2O) in a relative ratio of 94 / 5 / 1 for three hours at room temperature, or for hexapeptide 5, with 7 mL of a mixture of TFA / TIS / DODT / H2O in a relative ratio of 94 / 2.5 / 2.5 / 1. The peptides were then precipitated with about 25 mL of cold tert-butyl methyl ether and centrifuged at 4400 rpm for 10 minutes. The supernatant was removed and the peptides were dried with argon before lyophilization and / or purification. All peptides were obtained in the form of TFA salts.

[0102] Fmoc deprotection in solution

[0103] 50mg Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH or Fmoc-PEG-OH were dissolved in the corresponding deprotection conditions, with a total volume of 500 μL. 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 were diluted in MeCN for each condition, with a final volume of 1 mL.

[0104] 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 of 100% B to 100% C in 7 minutes.

[0105] Analyze the data

[0106] After freeze-drying, a crude white solid G1KL (20% v / v piperidine) (90.5 mg, 72.5%) was obtained. Analytical RP-HPLC: R =2.11 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 42 H 84 N 12 O7 calculated / found 869.66 / 869.66Da[M+H] + .

[0107] After freeze-drying, a crude white solid G1KL (5% w / v piperazine + 2% v / v DBU) (30.4 mg, 26.2%) was obtained. Analytical RP-HPLC: R =1.85 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 42 H 84 N 12 O7 calculated / found 869.66 / 869.66Da[M+H] + .

[0108] After freeze-drying, a crude white solid G1KL (20% v / v diisopropylamine) (0.3 mg, 0.0%, trace amount) was obtained. Analytical RP-HPLC: t R = -min (A / D 100:0 to 0:100 in 7.50 min, λ = 214 nm). HRMS (ESI+): C 42 H 84 N 12 O7 calculated / found 869.66 / 869.66Da[M+H] + .

[0109] After freeze-drying, a crude white solid G1KL (20% v / v dipropylamine) (42.2 mg, 35.2%) was obtained. Analytical RP-HPLC: R =1.79 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 42 H 84 N 12 O7 calculated / found 869.66 / 869.66Da[M+H] + .

[0110] After freeze-drying, a crude white solid G1KL (25% v / v dipropylamine) (82.4 mg, 64.5%) was obtained. Analytical RP-HPLC: R =1.88 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 42 H 84 N 12 O7 calculated / found 869.66 / 869.66Da[M+H] + .

[0111] After freeze-drying, crude white solid G2KL (20% v / v piperidine) (214.4 mg, 64.9%) was obtained. Analytical RP-HPLC: R=2.35 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 102 H 200 N 28 O 17 Calculated / measured 2090.56 / 2090.56Da[M+H] + .

[0112] After freeze-drying, a crude white solid G2KL (5% w / v piperazine + 2% DBU) (189.9 mg, 53.9%) was obtained. Analytical RP-HPLC: R =2.32 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 102 H 200 N 28 O 17 Calculated / measured 2090.56 / 2090.56Da[M+H] + .

[0113] After freeze-drying, crude white solid G2KL (20% v / v dipropylamine) (134.4 mg, 42.2%) was obtained. Analytical RP-HPLC: t R =2.40 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 102 H 200 N 28 O 17 Calculated / measured 2090.56 / 2090.56Da[M+H] + .

[0114] After freeze-drying, crude white solid G2KL (25% v / v dipropylamine) (151.4 mg, 46.4%) was obtained. Analytical RP-HPLC: R =2.38 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 102 H 200 N 28 O 17 Calculated / measured 2090.56 / 2090.56Da[M+H] + .

[0115] Preparative RP-HPLC gave VKDGYI (SEQ ID NO: 3) (20% v / v piperidine) (3.2 mg, 4.5%) as a white solid. Analytical RP-HPLC: R=1.92 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 32 H 52 N8O9 calculated / measured 693.39 / 693.39Da[M+H] + .

[0116] After freeze-drying, a crude white solid VKDGYI (SEQ ID NO: 3) (20% v / v piperidine + 0.5 M Oxyma) (10.8 mg, 16.8%) was obtained. Analytical RP-HPLC: t R =1.99 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 32 H 52 N8O9 calculated / measured 693.39 / 693.39Da[M+H] + .

[0117] After freeze-drying, a crude white solid VKDGYI (SEQ ID NO: 3) (5% w / v piperazine + 2% v / v DBU) (5.7 mg, 0.0%) was obtained. Analytical RP-HPLC: R = -min (A / D 100:0 to 0:100 in 7.50 min, λ = 214 nm). HRMS (ESI+): C 32 H 52 N8O9 calculated / measured 693.39 / -Da[M+H] + . (No compound observed).

[0118] After freeze-drying, crude white solid VKDGYI (SEQ ID NO: 3) (2% v / v DBU) (36.6 mg, 25.7%) was obtained. Analytical RP-HPLC: R =2.05 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 32 H 52 N8O9 calculated / measured 693.39 / 693.39Da[M+H] + .

[0119] After freeze-drying, a crude white solid VKDGYI (SEQ ID NO: 3) (20% v / v dipropylamine) (37.3 mg, 49.3%) was obtained. Analytical RP-HPLC: R =1.84 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 32H 52 N8O9 calculated / measured 693.39 / 693.39Da[M+H] + .

[0120] Preparative RP-HPLC gave VKDGYI (SEQ ID NO: 3) (25% v / v dipropylamine) (11.5 mg, 16.0%) as a white solid. Analytical RP-HPLC: R =1.20 min (A / D 100:0 to 0:100 in 3.5 min, λ=214 nm). MS (ESI+): C 32 H 52 N8O9 calculated / measured 693.39 / 693.42Da[M+H] + .

[0121] Lyophilized crude VKDGYI (SEQ ID NO: 3) (25% v / v dipropylamine) (39.6 mg, 52.9%): Analytical RP-HPLC: t R =1.96 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 32 H 52 N8O9 calculated / measured 693.39 / 693.39Da[M+H] + .

[0122] After freeze-drying, a crude white solid VKDGYI (SEQ ID NO: 3) (25% v / v dipropylamine, 90° C.) (26.8 mg, 33.5%) was obtained. Analytical RP-HPLC: R =2.01 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 32 H 52 N8O9 calculated / measured 693.39 / 693.39Da[M+H] + .

[0123] Preparative RP-HPLC gave VKD(β)GYI (SEQ ID NO: 11) (6.4 mg, 14.2%) as a white solid. Analytical RP-HPLC: R =1.19 min (A / D 100:0 to 0:100 in 2.2 min, λ=214 nm). MS (ESI+): C 32 H 52 N8O9 calculated / measured 693.39 / 693.42Da[M+H] + .

[0124] After freeze-drying, a crude white solid GDGAKF (SEQ ID NO: 4) (20% v / v piperidine) (40.6 mg, 40.9%) was obtained. Analytical RP-HPLC: R =1.75 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 26 H 40 N8O8 calculated / measured 593.30 / 593.30Da[M+H] + .

[0125] After freeze-drying, a crude white solid GDGAKF (SEQ ID NO: 4) (25% v / v dipropylamine) (38.9 mg, 49.2%) was obtained. Analytical RP-HPLC: R =1.76 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 26 H 40 N8O8 calculated / measured 593.30 / 593.30Da[M+H] + .

[0126] After freeze-drying, a crude white solid VKDRYI (SEQ ID NO: 5) (20% v / v piperidine) (44.0 mg, 40.3%) was obtained. Analytical RP-HPLC: R =1.99 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 36 H 61 N 11 O9 calculated / found 792.47 / 792.47Da[M+H] + .

[0127] After freeze-drying, a crude white solid VKDRYI (SEQ ID NO: 5) (25% v / v dipropylamine) (44.2 mg, 43.4%) was obtained. Analytical RP-HPLC: R =2.00 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 36 H 61 N 11 O9 calculated / found 792.47 / 792.47Da[M+H] + .

[0128] After lyophilization, a crude white solid GDRAKF (SEQ ID NO: 6) (20% v / v piperidine) (44.2 mg, 50.6%) was obtained. Analytical RP-HPLC: R =1.84 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 30 H 48 N 10 O9 calculated / found 693.36 / 693.39Da[M+H] + .

[0129] After freeze-drying, a crude white solid GDRAKF (SEQ ID NO: 6) (25% v / v dipropylamine) (52.9 mg, 62.5%) was obtained. Analytical RP-HPLC: R =1.86 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 30 H 48 N 10 O9 calculated / found 693.36 / 693.39Da[M+H] + .

[0130] After lyophilization, VKDCYI (SEQ ID NO: 7) (20% v / v piperidine) (46.2 mg, 53.1%) was obtained as a crude white solid. Analytical RP-HPLC: R =2.23 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 33 H 54 N8O9S calculated / measured 739.37 / 739.38Da[M+H] + .

[0131] After freeze-drying, a crude white solid VKDCYI (SEQ ID NO: 7) (25% v / v dipropylamine) (42.7 mg, 48.0%) was obtained. Analytical RP-HPLC: R =2.23 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 33 H 54 N8O9S calculated / measured 739.37 / 739.38Da[M+H] + .

[0132] After lyophilization, VKDAYI (SEQ ID NO: 8) (20% v / v piperidine) (42.7 mg, 54.7%) was obtained as a crude white solid. Analytical RP-HPLC: R =2.09 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 33 H 54 N8O9 calculated / measured 707.40 / 707.41Da[M+H] + .

[0133] After freeze-drying, a crude white solid VKDAYI (SEQ ID NO: 8) (25% v / v dipropylamine) (40.4 mg, 51.3%) was obtained. Analytical RP-HPLC: R =2.09 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 33 H 54 N8O9 calculated / measured 707.40 / Da[M+H] + .

[0134] After freeze-drying, a crude white solid VKEGYI (SEQ ID NO: 9) (20% v / v piperidine) (34.8 mg, 47.7%) was obtained. Analytical RP-HPLC: R =1.99 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 33 H 54 N8O9 calculated / measured 707.40 / 707.41Da[M+H] + .

[0135] After freeze-drying, a crude white solid VKEGYI (SEQ ID NO: 9) (5% w / v piperazine + 2% v / v DBU) (38.2 mg, 52.4%) was obtained. Analytical RP-HPLC: R =1.90 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 33 H 54 N8O9 calculated / measured 707.40 / 707.41Da[M+H] + .

[0136] After freeze-drying, a crude white solid VKEGYI (SEQ ID NO: 9) (20% v / v dipropylamine) (32.3 mg, 44.3%) was obtained. Analytical RP-HPLC: t R=1.98 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 33 H 54 N8O9 calculated / measured 707.40 / 707.41Da[M+H] + .

[0137] After freeze-drying, a crude white solid VKEGYI (SEQ ID NO: 9) (20% v / v dipropylamine + 0.5 M Oxyma) (36.5 mg, 50.0%) was obtained. Analytical RP-HPLC: R =1.93 min (A / D 100:0 to 0:100 in 7.50 min, λ=214 nm). HRMS (ESI+): C 33 H 54 N8O9 calculated / measured 707.40 / 707.41Da[M+H] + .

[0138] Preparative RP-HPLC gave bivalirudin (25% v / v dipropylamine) (93.3 mg, 38.7%) as a foamy white solid. Analytical RP-HPLC: R =1.52 min (A / D 100:0 to 0:100 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] + .

[0139] Preparative RP-HPLC gave bivalirudin (20% v / v piperidine) (111.6 mg, 46.3%) as a foamy white solid. Analytical RP-HPLC: R =1.52 min (A / D 100:0 to 0:100 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] + .

[0140] References

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[0143] R3:Ralhan,K.,KrishnaKumar,VG,&Gupta,S.,RSC Advances,2015,5(126),104417–104425.

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Claims

1. A method for preparing a peptide by solid phase peptide synthesis, wherein Fmoc protected amino acid building blocks, linked to the resin R–AA–(AA) n –PF, where -R is resin -AA is the amino acid building block -PF is Fmoc protecting group -n is the number of coupling cycles, Deprotection using dibutylamine or dipropylamine in the deprotection step before the coupling step yields R–(AA)–(AA) n ,in In the coupling step, another amino acid building block AA-P is coupled to R-AA-(AA) n , producing R–AA–(AA) n+1 –P, until the final coupling cycle n 末 , wherein P is the protecting group of the amino acid building block AA at the N-terminus or PF.

2. The method of claim 1, wherein the at least one AA comprises aspartic acid.

3. A process according to any one of the preceding claims, wherein the deprotection step is carried out with dipropylamine.

4. A process according to any one of the preceding claims, wherein the amount of dipropylamine is 10% to 50% (v / v), in particular 20% to 40% (v / v), more particularly 25% to 35% (v / v).

5. The process according to any one of the preceding claims, wherein the process is carried out at 10 to 90°C, in particular 65 to 90°C, more in particular 75 to 90°C.

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

7. A method according to any one of the preceding claims, wherein the deprotection step and the coupling step are repeated until the desired peptide length, not exceeding 60 amino acids, is reached.

8. A method according to any one of the preceding claims, wherein n 末 is the final coupling cycle to achieve the desired peptide length.

9. A method according to any one of the preceding claims, wherein the method comprises n 末 A final deprotection step after a coupling cycle, in which R-AA-(AA) n末 –P deprotection to give R–AA–(AA) n末 .

10. A method according to any one of the preceding claims, wherein the method comprises n 末 The cleavage step after the coupling cycle, where R–AA–(AA) n末 Cleavage from resin R to produce AA–(AA) n末 .

11. The method according to claim 11, comprising a purification step, wherein AA-(AA) n末 purification.

12. A method according to any preceding claim, wherein P is PF.

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