Method and apparatus for solid phase peptide synthesis

Abstract

The present invention relates to methods for performing solid phase peptide synthesis, automated parallel solid phase peptide synthesis, and devices designed to perform such methods. According to the invention, ultrasound at a frequency greater than 25 kHz is applied at least intermittently during the process to the reaction medium in which the solid phase peptide synthesis is carried out.

Description

[Technical Field]

[0001] The present invention relates to a method for performing solid phase peptide synthesis, an automated parallel solid phase peptide synthesis, and an apparatus adapted for performing such a method. [Background technology]

[0002] Solid-phase peptide synthesis (SPPS, also known as Merrifield synthesis), introduced in 1962 by Nobel laureate Robert Bruce Merrifield, is a peptide synthesis method using an insoluble polymer support. Linear peptides are constructed by the stepwise attachment of sequence-specific, temporarily protected amino acids to the C-terminus of a growing polypeptide chain, which is covalently bound to a synthetic resin support. To ensure that the reaction is controlled and side reactions are avoided, the reactive functional side chains of the amino acids must be blocked with suitable protecting groups. The α-amino groups of the amino acids being connected need only be protected during the actual coupling reaction; the permanent side-chain protecting groups are only cleaved from the peptide after synthesis is complete. In contrast to ribosomal protein biosynthesis, peptide chain elongation occurs from the C-terminus to the N-terminus. Copolymers of polystyrene and 1–2% 1,4-divinylbenzene have proven to be suitable polymer supports. Resin beads, 20–100 μm in diameter, obtained by bead polymerization swell in the solvent used for synthesis, thereby becoming permeable to reagents. The tert-butyloxycarbonyl (Boc) and fluorenyl-9-methoxycarbonyl (Fmoc) groups are primarily used as intermediate α-amino protecting groups. The Boc group is stable to catalytic hydrogenation and alkaline hydrolysis and can be cleaved by mild acidolysis, e.g., with 50% trifluoroacetic acid (TFA). Successive acid deblocking reactions after each coupling step result in partial deblocking of the side-chain protecting groups and slight hydrolysis of the anchor bond to the polymer support. The Fmoc group has the advantage of being cleavable by treatment with a suitable base, such as morpholine, 2-aminoethanol, or piperidine. The use of acid-labile, base-resistant groups as anchor groups to the polymer support and to protect the third functional group of the corresponding amino acid building blocks advantageously allows the intermediate and permanent protecting groups to be cleaved independently of each other.

[0003] The coupling reaction (also called condensation or peptide elongation) is a crucial step in synthesis, as complete conversion is a fundamental requirement for the homogeneity of the final product. Typically, reagents are used in excess, preferably anhydrides, activated esters, or so-called in-situ activators, which form an intermediate activated ester derivative. The continuous repetition of the reaction steps of cleavage of the α-amino protecting group and attachment of the next Nα-protected amino acid (coupling reaction, condensation) has enabled widespread automation of the synthesis process, and the construction of peptide synthesizers, most of which operate according to the flow-through principle. Here, the resin is placed at the bottom of a fritted column, through which reagents and solvents are automatically fed, mixed with the support material, and subsequently extracted. The process is repeated until the desired length of the constructed peptide is reached. Finally, the synthesized peptide is released from the polymer support. Release from the resin matrix is ​​achieved using reagents that selectively cleave the anchor bond between the C-terminal amino acid and the support, depending on the protecting group scheme selected, or synchronously cause partial or complete deblocking of the synthesized peptide. Several peptide syntheses have been developed from solid-phase peptide synthesis (Non-patent Document 1, Non-patent Document 2).

[0004] A major challenge in automated peptide synthesis is avoiding cross-contamination, since in known procedures and automated instruments, reagents pass through the same tubing and cannulae. To prevent cross-contamination, in known equipment configurations, the entire system is rinsed with large amounts of rinsing agent.

[0005] Here, Patent Document 1 proposes an apparatus that allows automated simultaneous multiple parallel synthesis, which can eliminate cross-contamination, resulting in a significant reduction in synthesis time.

[0006] Another approach to shortening the synthesis time of peptide synthesis is to expose the reagents to microwave radiation during synthesis. This can reduce synthesis time by a factor of 10. However, microwave-assisted reactions must be carried out in a special protected space. This limits this method to relatively small reactors and therefore low throughput volumes. Furthermore, it has been found that not all common protecting groups are stable to microwave radiation, which can reduce yields and produce more impurities.

[0007] In 1977, an attempt to use ultrasound to assist synthesis was published in US Pat. No. 5,623,297. However, in later years it became clear that this method, at least in the form shown, did not produce the desired results: in the general method, no reproducible acceleration of synthesis time was observed. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] DE10131088B4 [Patent Document 2] CA1019324 [Non-patent literature]

[0009] [Non-Patent Document 1] E. Atherton and RC Sheppard Solid-Phase Synthesis - A Practical Approach, Oxford University Press, 1989 [Non-patent document 2] H.-D. Jakubke Peptides: Chemistry and Biology, published by Spektrum Akademischer Verlag Heidelberg, 1996 Summary of the Invention

[0010] The object of the present invention is to further accelerate the synthesis time of solid phase peptide synthesis while maintaining or improving yield and purity, which method is particularly desirable for application in automated parallel processes.

[0011] This object is achieved by a method for carrying out automated parallel solid-phase peptide synthesis and by an apparatus for carrying out said method having the features of the independent claims.

[0012] Thus, a first aspect of the present invention relates to a method for carrying out solid phase peptide synthesis (hereinafter also referred to as synthesis or peptide synthesis), the method according to the invention comprising: a) attaching an amino acid N-terminally protected by a protecting group to a solid support material via the C-terminus of the amino acid; b) cleaving the protecting group; c) performing at least one peptide elongation; and d) terminating the reaction by cleaving the peptide from the carrier material; Steps a) to d) are carried out in a liquid reaction medium, and during at least one of the steps, ultrasound waves having a frequency in the range of >25 to 2000 kHz are at least intermittently applied to the reaction medium.

[0013] It has been found that ultrasound has an accelerating effect on the reaction in question in solid-phase peptide synthesis only at frequencies above 40 kHz. This allows the method according to the present invention to reproducibly shorten the synthesis time of solid-phase peptide synthesis to at least within the range of that of microwave-assisted peptide synthesis. However, advantageously, no special safety precautions need to be taken. Furthermore, the required equipment is relatively inexpensive to acquire and maintain. This means that the method can be used in almost any synthesis facility, especially in parallel and / or automatable synthesis facilities.

[0014] Step a) is understood herein to mean that a functional group is directly or indirectly attached to a suitable carrier material, such as a preloaded or non-preloaded resin or amide resin for solid-phase peptide synthesis. This functional group may be protected, in particular with an Fmoc protecting group. Here, preloaded or non-preloaded refers to the fact that at least the first and optionally at least one subsequent amino acid, i.e., the first amino acid of the amino acid sequence to be synthesized, are already attached to the carrier material.

[0015] Frequencies above 40 kHz, preferably above 50 kHz, particularly above 75 kHz, and especially above 100 kHz have proven particularly suitable, since significantly shorter synthesis times can be achieved at higher frequencies. The formation of cavities has been found to be important for the positive effect on peptide synthesis, particularly in improving quality. The associated cavitation increases in intensity with increasing frequency, starting at 40 kHz. In the frequency range from 20 to 40 kHz, only vibrational excitation occurs.

[0016] Preferably, the ultrasound frequency of the method according to the invention does not exceed 2 MHz, in particular 1 MHz. Further description of preferred frequencies follows below.

[0017] The ultrasonically assisted solid phase peptide synthesis (USPS) described herein belongs to the category of sonochemistry in chemical synthesis.

[0018] The chemical effects of ultrasound cannot be a direct effect of the sound field, since typical frequencies are orders of magnitude too low to excite even simple rotational motions.

[0019] The positive effects are speculated to be directly related to the cavitation induced by the ultrasound and the resulting pressure pulses. Cavitation occurs in the frequency range of 40 kHz to 2 MHz.

[0020] Three sonochemical reactions are postulated. 1. Reactions with radical or radical-ionic intermediates in homogeneous systems. Extreme pressure and high temperature in cavitation bubbles can cause reactions such as OH radicals in the aqueous phase. · and H · Radicals are generated, which leads to the formation of, among other things, H2O2 within the bubbles. 2. Reactions based on ionic reactions in heterogeneous systems. These are primarily assisted by the mechanical effects of cavitation in the solvent. Asymmetric bubbles form on solid particles. The implosion of the asymmetric bubbles on the particles creates a liquid jet that shoots out in one direction towards the imploding bubble. This aids in the absorption of the solvent and dissolved substances into the porous material, while the other liquid phases undergo phase mixing. 3. Reactions that also involve radical reactions in heterogeneous systems. The radical pathway may produce different products compared to the ionic pathway, such as in the Kornblum-Russell reaction.

[0021] Cavitation bubbles are more likely to form in the lower frequency range and then become larger and more asymmetric, which leads to more intense but less uniform mixing, whereas at higher frequencies, a greater number of smaller, more symmetric bubbles are generated, resulting in more radical exchange between the cavitation bubbles and the environment.

[0022] Cavitation is "the formation, growth, and implosive collapse of gas bubbles in a liquid. Cavitational collapse produces locally high temperatures (about 5000 K), high pressures (about 1000 atm), very large heating and cooling rates (>109 K / sec)" and liquid jets (about 400 km / hr). Cavitation bubbles are vacuum bubbles (Suslick 1998). The vacuum is created by the fast-moving surface and inert liquid. The resulting pressure difference overcomes the cohesive and adhesive forces within the liquid.

[0023] At frequencies above 110 kHz, especially above 125 kHz, and preferably above 130 kHz, an acceleration of the reaction process and a corresponding reduction in reaction time and improvement in yield can be observed. It was found that, compared to standard systems, it is not necessary to work with a 40-fold excess of amino acid; the same result can be achieved with a 4-fold excess. This in turn significantly reduces the reactant amounts, thus leading to considerable cost savings. Furthermore, the yield is increased, since no racemization was observed at frequencies in the 110-500 kHz range.

[0024] In a preferred embodiment of the method according to the present invention, ultrasound is assumed to be transmitted to the reaction medium via an external liquid bath. This is clearly different from methods that transmit ultrasound to the reaction medium directly or exclusively via solid-state transmission means. Transmission via at least one liquid medium has been found to result in more consistent, reproducible, and gentle synthesis results. The synthesis time required using a liquid-containing transmission medium exhibits less variability than synthesis times using, for example, a probe immersed in the reaction medium. Furthermore, test equipment is much simpler than when using a probe. Probe immersion inevitably leads to probe contamination, necessitating periodic probe cleaning, which equals or at least significantly reduces the gain in synthesis time.

[0025] The amount of energy converted into cavitation depends on several factors describing the motion transferred from the cavitation generator to the liquid. The intensity of the acceleration is one of the most important factors affecting the efficient conversion of energy into cavitation. The greater the acceleration, the greater the pressure difference that occurs. This increases the likelihood of creating vacuum bubbles in the liquid rather than waves. This means that the greater the acceleration, the greater the proportion of energy that is converted into cavitation. In the case of (ultrasonic) transducers, the intensity of the acceleration is determined by the amplitude of the vibrations. In addition to the intensity of the ultrasound waves, it is also important that the liquid is accelerated in such a way that losses due to turbulence, friction and wave generation are as low as possible. A unidirectional path of movement is optimal for this.

[0026] Therefore, the selection of the conduction medium is very important for the effectiveness of peptide synthesis. In addition to the selection of the conditions, the material of the conduction medium must also be optimized. In addition to water as the conduction medium, organic solvents, especially lower and medium alcohols such as ethanol, propanol, and butanol, are also preferably used as the conduction medium.

[0027] It is advantageous to select different conductive media in combination. The test facility should be understood as a bath within a bath. In other words, the reaction takes place in a reaction medium arranged in a (reaction) vessel. This reaction vessel is then arranged in a vessel containing a first conductive medium, which is then arranged in a further conductive medium. Thus, the ultrasound is transmitted via the further conductive medium to the first conductive medium and from there to the reaction medium.

[0028] Particularly advantageously, the first conductive medium is constituted by lower and medium alcohols, in particular of the type mentioned above, and / or the further conductive medium is constituted by water.

[0029] At high ultrasonic frequencies, the temperature in the liquid bath increases as expected. However, at frequencies up to 500 kHz, this effect can be very well controlled, as the resulting temperature increase can be easily compensated for by cooling devices such as continuous coolers (cryostats) or cooling of the water bath with Peltier elements, so that the yield is not compromised by the potentially resulting racemization.

[0030] When using frequencies significantly greater than 500 kHz, up to 1000 kHz, it has been found to be reasonable to counter the temperature increase, for example by cooling the bath, in order to guarantee quality.

[0031] The ultrasonic bath is preferably subjected to temperature control, more particularly to a temperature range of 20 to 100°C, preferably 20 to 70°C, particularly preferably 40 to 50°C.

[0032] In a further preferred embodiment of the present invention, it is assumed that the amino acid is protected at the N-terminus by a temporary (initial) protecting group, particularly fluorenylmethoxycarbonyl (Fmoc), which is base-labile and can be cleaved using a secondary amine. Compared to the protecting groups used in the boc method, these protecting groups have been shown to be particularly stable to ultrasound at the frequencies used in the present invention, and therefore particularly high yields and purities may be achieved. Deprotection is preferably carried out using a suitable base, as described above or in experimental results. Preferably, 20% piperidine in DMF (dimethylformamide) is used.

[0033] Until now, the advantages of ultrasonically assisted peptide synthesis have only been confirmed for BocOC synthesis. It has been found that ultrasonic assistance is not applicable to Fmoc-based synthesis because the resins used are disrupted under ultrasound. Due to the variety of reaction media and side-chain protecting groups used, it was assumed that only the coupling to the resin could be ultrasonically assisted. Surprisingly, however, advantages in terms of reaction time and yield can be demonstrated even for ultrasonically assisted Fmoc-based peptide synthesis during the individual synthesis steps according to the method of the present invention within the frequency range described.

[0034] The reactive side chains of the peptides synthesized using the method according to the invention are preferably also protected by a (second) protecting group. Depending on the functional group to be protected, acid-stable protecting groups selected in particular from the group consisting of S-2,4,6-trimethoxybenzyl (Tmob), triphenylmethyl (Trt), tert-butyl (tBu), tert-butyloxycarbonyl (Boc), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) have been shown to be particularly stable in the peptide synthesis according to the invention using ultrasound in the mentioned frequency range.

[0035] With reference to the method according to the invention, for example, amino acids are listed by one-letter or three-letter code: Fmoc-A-OH, Fmoc-C(Trt)-OH, Fmoc-D(OtBu)-OH, Fmoc-E(OtBu)-OH, Fmoc-F-OH, Fmoc-G-OH, Fmoc-H(Trt)-OH, Fmoc-I-OH, Fmoc-K(Boc)-OH, Fmoc-L-OH, Fmoc-M-OH, Fmoc-N ... Particularly suitable for peptide elongation are Fmoc amino acids selected from the group: -N(Trt)-OH, Fmoc-P-OH, Fmoc-Q-Trt-OH, Fmoc-R-Pbf-OH, Fmoc-S-tBu-OH, Fmoc-T-tBu-OH, Fmoc-V-OH, Fmoc-W(Boc)-OH, Fmoc-Y-(tBu)-OH, Fmoc-Gln(Tmob)-OH, Fmoc-Asn(Tmob)-OH. This refers to both the L- and D-forms of the amino acids.

[0036] Additionally, special side-chain protected post-translationally modified amino acids, e.g. Regarding Ser / Thr phosphorylation Fmoc-Ser(PO(OBzl)OH), Fmoc-Thr(PO(OBzl)OH), Fmoc-Tyr(PO(OMe)2), Fmoc-Tyr(PO(OBzl)OH), Fmoc-Tyr(PO(OBzl)2)-OH, Fmoc-Tyr(PO3H2)-OH, Fmoc-Tyr(PO(NMe2) 2) , Fmoc-Tyr(PO(NMe2)2), Fmoc-Ppa(Bzl)-OH, Fmoc-Pmp-OH, Fmoc-F2Pmp-OH, Regarding sulfation of Tyr Fmoc-Tyr(SO3nP)-OH, Fmoc-Tyr(SO3DCV)-OH, Regarding Arg methylation Fmoc-Arg(Me,Pbf)-OH, Fmoc-ADMA(Pbf)-OH, Fmoc-SDMA(Boc2)-ONa, Regarding Lys methylation Fmoc-Lys(Me,Boc)-OH, Fmoc-Lys(Me2)-OH, Fmoc-Lys(Me3Cl)-OH, Regarding citrullination Fmoc-citrulline-OH, Regarding Asn glycosylation Fmoc-Asn(β-DGlcNAc(Ac)3)-OH, Fmoc-Asn(β-DGlcNAc(Ac)3-(1-4)-β-DGlcNAc(Ac)2)-OH, Regarding Ser / Thr glycosylation Fmoc-Ser / Thr(α-DGlnNAc(Ac)3)-OH, Fmoc-Ser / Thr(β-DGal(Ac)4-(1-3)α-DGlnNAc(Ac)2)-OH, Fmoc-Ser / Thr(sialylOMe(A c) 4-(1-6)-α-D-GlnNAc(Ac)2)-OH, Fmoc-Ser / Thr(sialylOMe(Ac)4-(1-3)-β-D-Gal(Ac)3-(1-3)α-DGlnNAc(Ac)2)-OH can also be used.

[0037] Likewise, synthetic building blocks developed for the synthesis of complex peptide sequences, such as Fmoc pseudoproline dipeptides, so-called Dmb building blocks, e.g. Fmoc-(Dmb)Gly-OH, or Hmb building blocks FmocHmbXaa as the dipeptide FmocXaaDmbGly, as well as Hmsb building blocks, Hnb building blocks, Mmsb building blocks, unnatural amino acids, e.g. Naphthylalanine, Fmoc-L-2Nal-OH Ornithine, Fmoc-L-Orn(Aloc)-OH and methylated variants, Polyethylene glycol, Fmoc-O1Pen-OH, Fmoc-AEEP, Fmoc-TTDS-OH and all other Fmoc-protected aminopolyethylene glycol acids, In particular, derivatized amino acids, e.g. Fmoc-Lys(biotin)-OH, FMOC-Lys(Cy5)-OH can be used.

[0038] Generally, all building blocks that have a temporarily protected amine function, preferably Fmoc protected, and a carboxylic acid function that can be converted to an active ester or amine reactive group can be used for USPS.

[0039] Advantageously, ultrasound acts on the reaction medium in only one step, in particular in step c). Alternatively or additionally, ultrasound acts on the reaction medium in at least one further step, preferably in steps a), b) and / or d). The reaction-accelerating properties of ultrasound according to the invention can be observed in each of the steps mentioned.

[0040] Here, it is particularly preferred that the action of the ultrasound is not interrupted between or during the individual steps, since this could result in a reduction in yield.

[0041] The greatest reduction in synthesis time and high yields have been achieved up to now when ultrasound is applied to the reaction medium throughout the synthesis, i.e., during washing, deprotection, condensation and coupling. On the other hand, ultrasound is not necessarily advantageous in the preliminary step of pre-swelling, nor in the final washing.

[0042] Solid-phase peptide synthesis involves several washing steps that can be distinguished from one another. The individual types of washing steps may be distinguished by their respective upstream reactions. Thus, at least one washing after coupling of the first amino acid to the resin (initial washing), a washing after decoupling of the protecting group in step b) (hereinafter referred to as step W) b ), washing after coupling of amino acids to extend the peptide chain (hereinafter referred to as step W c ), and step d) the final wash after cleavage of the final temporary protecting group of the completed peptide from the carrier material (hereinafter referred to as step W d ) can be mentioned.

[0043] During the individual washing steps, ultrasound also favorably influences the reaction, significantly reducing the required rinse agent and rinsing time. Thus, washing with only one ultrasonic rinse step already achieves the same results as the four rinses typically required in standard synthesis. Washing Step W c , i.e., washing after step c) is particularly important for improving yield and quality. Studies have shown that the washing step W bIt has been shown that the steps can be omitted entirely. Preferably, however, all washing steps are carried out in the method according to the invention.

[0044] In particular, in this cleaning step, ultrasonic frequencies in the range of 100 to 500 kHz, preferably in the range of 100 to 200 kHz, and especially in the range of 120 to 140 kHz are preferred. In either of these ranges, the amount of solvent, e.g., DMF, required for cleaning or rinsing within each individual rinse cycle of the cleaning step is b The amount of water may be reduced in such a way that only one rinse step is required per

[0045] Particularly advantageously, the ultrasound is applied throughout the washing step W, in particular without any interruptions. b , W c and W d It is assumed that all steps a) to d) of peptide synthesis, including the above, act on the reaction medium within the aforementioned frequency range.

[0046] Depending on the peptide to be synthesized, the individual steps a) to d) and especially step W) may be modified to improve the quality and shorten the reaction time. b , c and d It has been found that different frequencies are optimal for the reaction, i.e., the frequency exhibits a more beneficial effect. Therefore, it is preferred that the ultrasound acts on the reaction medium at different frequencies in the individual steps. In particular, it is preferred that the frequency is changed between steps and / or that ultrasound waves of different frequencies are superimposed on each other.

[0047] The carrier materials are essentially materials known to those skilled in the art for peptide synthesis. These are artificial / synthetic resins, Knorr Amid Resin LS 1% DVB, Wang Resin, Chlorotrityl Resin, PRG Resin, Tentagel Resin, Chemmatrix Resin, Generally, resins from the group of preloaded or non-preloaded and / or functionalized resins for solid phase synthesis are particularly advantageous.

[0048] Non-paramagnetic synthetic resins are preferred because magnetic separation of synthesized peptides is significantly more complicated than separation by filtration, and paramagnetic resins have been shown to be disrupted by low frequency (up to 40 kHz) ultrasound to form very fine particles, clogging the filter material during this process.

[0049] In the context of the present invention, the solvents preferably used are DMF (N,N-dimethylformamide), NMP (N-methyl-2-pyrrolidone) or DMA (N,N-dimethylacetamide).

[0050] The base used to catalyze the condensation reaction is preferably NMP, 4-methylmorpholine or diisopropylethylamine in DIPEA, DMF or another solvent.

[0051] The solution for cleaving the temporary Fmoc protecting group is preferably 20% piperidine in DMF. Other cleavage methods are known in principle to those skilled in the art.

[0052] The coupling reagent used is preferably HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), HCTU (2-(6-chloro-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate), PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), DCC, dicyclohexylcarbodiimide, DIC, diisopropylcarbodiimide, EDC, or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0053] All amino acids / reagents are dissolved in the solvent used, thereby obtaining a reaction medium / reactant medium / reagent solution. When reagents are mentioned in the context of the methods and apparatus, it is also understood to include solutions of reagents.

[0054] The concentration of amino acid used always depends on the scale of the synthesis and its solubility in the solvent used, such as DMF, NMP or DMA.

[0055] To achieve good synthesis results, the amino acid (AA) to be coupled and the reagent for forming the activated ester (activator) are preferably used in at least equimolar amounts relative to the scale of synthesis. However, it is common to use equimolar amounts of AA and activator together, with both in excess relative to the scale of synthesis. The excess ranges from 4-fold to 100-fold. To achieve quality comparable to the method of the present invention using prior art solid-phase peptide synthesis, at least a 40-fold excess is required, depending on the peptide sequence. This is because a high excess of reactants favors the formation of the product, i.e., an extended peptide chain. The concentration of the amino acid here depends primarily on the solubility of the protected AA in the solvent used and is preferably between 0.2M and 0.6M.

[0056] In the experiments described in this application, amino acids were used at a concentration of 0.4M.

[0057] Here, too, the ultrasound is advantageously performed without any interruption between the successive (ultrasound-assisted) steps a) through d), with only the frequency varying. Advantageously, the method is envisioned to be performed semi-automatically / automatically and / or in parallel. The combination of parallel automated methods and ultrasound-assisted techniques according to the present invention allows for the generation of unique peptides, i.e., peptides tailored to specific individuals, at a scale and throughput that makes specific forms of neoantigen-based cancer therapy available to a large number of patients. Neoantigens are mutation-induced changes in tumor cell proteins. They can be identified using next-generation sequencing (NGS) techniques. Typically, the number of different neoantigens ranges between 100 and 200. A corresponding number of synthetic peptides can mimic these neoantigens in vitro and be used for tumor-specific immunization of patients. The number, composition, and amino acid sequence of neoantigens are unique to each patient. Therefore, the corresponding peptide copies must also be adapted to the individual, i.e., personalized. To fully commercialize this promising treatment, it must be possible to test it on a large number of patients under the same conditions within a reasonable timeframe. This requires the availability of large numbers of personalized antigens in a very short time. The described form of personalized cancer therapy therefore places high demands on speed, parallelism, and quality of peptide synthesis. The method according to the present invention may meet these requirements.

[0058] The process according to the invention is particularly suitable for large-scale applications, the term "large-scale applications" meaning batch sizes of 1 l to 50 l, in particular 100 to 500 l.

[0059] Therefore, a further aspect of the present invention is an automated parallel solid-phase peptide synthesis comprising the method according to the invention in one of the described embodiments.

[0060] The process according to the invention is particularly advantageously carried out at room temperature or at moderately elevated temperatures. Particularly preferred are reaction temperatures in the range from 20 to 100° C., preferably in the range from 20 to 70° C., in particular in the range from 40 to 60° C. Temperature control is preferably carried out at least in steps a), b) and / or d).

[0061] The method according to the invention has been found to be particularly suitable for large scale production of litraglutide and semaglutide, especially on scales above 100 g of product.

[0062] A further aspect of the present invention relates to an apparatus for carrying out solid-phase peptide synthesis, designed to carry out the method according to the invention in one of the described embodiments. For this purpose, the apparatus according to the invention comprises an ultrasonic transducer which transmits ultrasonic waves, preferably at a frequency in the range of 25 kHz to 2 MHz, in particular 40 kHz to 1 MHz, preferably in the range of 100 to 500 kHz, to a reaction medium via a liquid conducting medium.

[0063] The device preferably comprises means for receiving one or more synthesis vessels with at least one opening for filling with a reactant medium, in particular a synthesis plate in the form of a microtiter plate having 96, 384, 1536 or 3456 reaction chambers, a synthesis cylinder or a synthesis flask or a synthesis reactor, and an ultrasonic bath containing a conducting liquid, the synthesis vessels may be placed in the ultrasonic bath in such a way that the synthesis vessels are wetted to a minimum height with the conducting liquid of the ultrasonic bath.

[0064] Advantageously, a minimum height is understood to mean a height at which the transmission of ultrasonic waves from the ultrasonic transducer of the ultrasonic device to the reaction medium is ensured in such a way that the sound waves are generated almost exclusively by the liquid conducting medium. For this purpose, the height of the surface of the conducting medium on the outer surface of the synthesis vessel corresponds to at least half the height of the meniscus of the reaction medium in the synthesis vessel, preferably between half and the full height, preferably between three-quarters and the full height.

[0065] The synthesis vessels are preferably designed as synthesis plates, e.g., as microtiter plates, in particular as side-by-side and juxtaposed synthesis cylinders, e.g., in the form of beakers or syringes, or as synthesis flasks, e.g., round-bottom flasks or reactors (e.g., according to Hochst or Syringe). In particular, the design of the synthesis vessel as a round-bottom flask or reactor is preferred for use in large-scale semi-automated / automated solid-phase peptide synthesis, since it allows handling of volumes of 1-50 liters in flasks or 100-500 liters in reactors.

[0066] On the other hand, the use of microtiter plates or parallel synthesis cylinders is particularly preferred for parallel and automated solid-phase peptide synthesis, although the aforementioned are more commonly used for large-scale applications of individual peptides such as litraglutide and semaglutide. The present invention proposes an apparatus that operates without any dilution device and tubing for feeding and metering the synthesis building blocks. A separate synthesis pen is provided for each synthesis building block, is mounted in a holder of the synthesis device for synthesis, is gripped by the gripper arm of the device, is removed from this holder and delivers a metered amount of the building block to a synthesis vessel, in particular a carrier material located in the reaction chamber of a synthesis plate.

[0067] Thus, the reagent reservoir and dosing device form a self-contained unit, eliminating all rinsing processes and the attendant drawbacks previously required for synthesis building block changes and reagent delivery.

[0068] An area is defined for parallel synthesis (Figure 2). The working area is dimensioned so that a synthesis pen, moved by a gripper arm, can contact all points of the working area. Preferably, up to 10 synthesis stations are arranged in this working area, particularly symmetrically. The dimensions of the synthesis stations are preferably based on standard microtiter plates. The synthesis stations may be of modular design and may include a base with connections for solvent extraction and an exposed frame for holding the synthesis plates. Depending on the compartments of the synthesis plate used, for example, 6, 12, 24, 48, 96, 384, 1536, or 3456 individual syntheses may be carried out in parallel on one synthesis plate. At larger synthesis scales, synthesis cylinders or syringe bodies may be used in special receptacles.

[0069] According to a further feature of the invention, the reaction chamber of the synthesis plate may be closed on the open side with a permeable material, for example a frit. A sample plate may be placed below the frame to receive the dissolved peptides after the cleavage reaction. With the proposed device for solid-phase synthesis, both the synthesis and the cleavage of the resulting compounds from the support material, the synthetic resin, are possible without manual intervention.

[0070] The sample plate is equipped with individual holding chambers whose arrangement and design correspond to the grid of reaction chambers of the synthesis plate. This ensures easy and error-free assignment of specific compounds after their cleavage from the carrier material or synthetic resin. Therefore, the reaction products can be easily transferred directly to high-throughput screening lines.

[0071] The synthesis pen (Figures 3 and 4) has a hollow cylindrical body (reagent reservoir) that may be closed by a screw closure, and a mouthpiece at its bottom end with an outlet opening that fits over the free opening of the reaction chamber of the synthesis plate. The outlet opening is closed by a valve needle and stop valve, which are guided by a piston rod and the body's piston and releasably fixed in their closed position by a compression spring acting on the piston. The cylindrical space below the piston is used to hold the single synthesis building blocks and inert gas. Metered delivery of reagent is achieved by simply placing the mouthpiece on a permeable material covering the open side of the reaction chamber. At the same time, depressing the valve needle releases the stop valve from its valve seat, freeing the outlet opening. The amount of reagent dispensed is determined by the length of time the mouthpiece is placed on the permeable material. When the mouthpiece is lifted, the outlet opening automatically closes again.

[0072] In a further design of the synthesis pen (Figure 6), the screw closure of the reagent reservoir is replaced by a movable lid with a bayonet closure. A piston rod, preferably located in the center of the pressed-in lid, runs through the entire synthesis pen and into the dosing cylinder. The dosing cylinder is closed at the bottom, for example, by a non-return valve. By pressing the lid, a defined amount of reagent is dispensed by the piston. A return means, for example a spring, installed in the synthesis pen returns the piston. At the same time or subsequently, the dosing cylinder is refilled. A suitable actuator, for example a non-return valve, in the mouthpiece on the lower end side ensures that the solution can only be dispensed by active delivery. This design of the synthesis pen allows contactless dispensing into the reaction chamber. The closed design of the synthesis pen with closed reagent reservoirs ensures high reagent stability.

[0073] Advantageously, the ultrasonic bath of the device according to the invention is liftable, which allows the reaction vessel to be lowered into the ultrasonic bath, preferably to a predetermined height, in multiple steps or continuously.

[0074] Furthermore, ultrasonic baths can be used in a variety of test equipment, especially in a variety of synthesis vessels.

[0075] The ultrasonic bath is also advantageously temperature-controllable, in particular designed to achieve a controlled temperature range of 20-100°C, preferably in the range of 20-70°C, in particular in the range of 40-60°C. In particular, the device has a cooling system to reduce the temperature of the bath due to heating, for example, by high ultrasonic frequencies. This is particularly advantageous when using frequencies from 500 kHz, in particular from 1000 kHz.

[0076] Furthermore, the ultrasonic bath or its ultrasonic generator of the device according to the invention is designed to generate and transmit varying frequencies, in particular at least one in the low frequency range (40-75 kHz) and one in the high frequency range (100-2000 kHz, preferably 100-500 kHz), into the liquid bath. For this purpose, it is advantageous if the various frequencies can be switched alternately or additionally with one another.

[0077] In a preferred embodiment, the ultrasonic bath, or rather the ultrasonic generator of the ultrasonic bath, is provided with a required power with a nominal power in the range of 40-100 W, in particular in the range of 50-70 W, with peaks achieved in the range of 100-300 W, preferably in the range of 170-280 W, and for large-scale applications in the range of 250 W-700 W, in particular in the range of 500-600 W.

[0078] By using a separate synthesis pen for each synthesis building block and covering the open side of the reaction chamber of the synthesis plate, the risk of contamination is significantly reduced and cross-contamination is virtually eliminated: carryover of synthesis building blocks, which often occurs due to inadequate rinsing processes, is no longer possible.

[0079] Elimination of the rinsing process not only significantly reduces the consumption of organic solvents, but also accelerates the synthesis many times over.

[0080] The described embodiments can be advantageously combined with one another, unless stated otherwise in individual cases. Otherwise, the embodiments of the invention apply equally to methods and devices.

[0081] In the following the invention will be explained in detail using practical examples and results which serve for illustrative purposes only. [Brief explanation of the drawings]

[0082] [Figure 1] 1 is a schematic diagram of an apparatus according to the invention for the synthesis of peptides. [Figure 2] FIG. 2 is a plan view of the working area of ​​the device according to FIG. 1; [Figure 3] FIG. 1 is a schematic diagram of a combination pen for separate feeding, dosing, and reagent storage in a preferred embodiment of the present invention. [Figure 4] 4 is a longitudinal section of the composite pen according to FIG. [Figure 5] 3 is a cross section AA of FIG. 2 of a synthesis plate having reaction chambers formed according to the present invention. [Figure 6] 1 is a schematic diagram of a longitudinal section of a synthetic pen in accordance with a further preferred embodiment of the present invention; [Figure 7] 1 is a schematic diagram of a method sequence for solid phase peptide synthesis according to a preferred embodiment of the present invention. [Figure 8] 1 is a graph showing the stability of tryptophan using a method according to the present invention based on the synthesis of endomorphins. [Figure 9] 1 is a graph showing the stability of acyl carrier protein (ACP) using the method according to the present invention. [Figure 10] 1 is a graph depicting the stability of acyl carrier protein (ACP) using a comparative prior art method. [Figure 11] 1 is a graph showing a comparison of the average synthesis quality of ACP peptides taking into account the synthesis strategy. [Figure 12] 12 is a graph showing the quality of the synthesis according to FIG. 11; [Figure 13] 1 is a graph depicting testing of stock solutions for amino acids for various solubilization durations. [Figure 14] 1 is a graph showing a comparison between single coupling and double coupling. [Figure 15] 1 is a graph showing a comparison of single coupling and double coupling, as well as excess amounts of amino acids. [Figure 16] 1 is a graph showing a comparison of the average synthesis quality of peptides using different ultrasonic frequencies. DETAILED DESCRIPTION OF THE INVENTION

[0083] The synthesis device 1, shown diagrammatically in Figure 1, is based on a laboratory pipetting robot and has a gripper arm 2 that can move in the x, y, and z axes. The working area 3 contains a synthesis plate 5, which is derived from a microtiter plate known per se for its grid and arrangement of synthesis vessels, in particular reaction chambers 9, and has a 6, 12, 24, 48, 96, 384, 1536, or 3456 grid of reaction chambers 9, thereby achieving a high degree of parallelization of the synthesis. The synthesis plate 5 is placed in a valve block 6, which has a porous membrane 28 on its bottom side that allows the reagents and rinse solutions used to be aspirated from the reaction chambers 9 and discarded by the valve block 6, which is connected to a suction pump.

[0084] The synthesis plate 5 is placed together with the valve block 6 and the sample plate 27 in an ultrasonic bath 50, in particular a bath that is height-adjustable and can be controllably switched on and off. The ultrasonic bath 50 has a container containing a liquid conducting medium in which the synthesis vessel 5 is placed. Depending on the position of the ultrasonic bath 50, the meniscus of the synthesis vessel 5 with the reaction medium is at least halfway, preferably at least up to three-quarters, and in particular completely below the filling level of the conducting medium of the ultrasonic bath 5. The ultrasonic bath 50 is designed to transmit ultrasonic waves at frequencies ranging from at least 25 kHz to 2 MHz through the conducting medium.

[0085] The synthesis apparatus 1 further comprises one or more rinse combs 8 connected to corresponding rinse agent reservoirs via rinse agent supply lines 10. After the reaction time has elapsed and the used reaction solution has been withdrawn, the rinse comb 8 containing the required rinse agent is picked up by the gripper arm 2 and moved above the reaction chamber 9 of the synthesis plate 5, delivering a metered dose of rinse liquid, in order to rinse the sample located in the reaction chamber 9 to which the synthetic building blocks have been coupled. After rinsing, another rinse comb 8 is used to deliver the solution required for cleaving the temporary protecting groups of the coupled synthetic building blocks as described above. After an incubation time has elapsed, the cleavage solution is withdrawn by means of the suction pump 7 via the valve block 6, washing the sample. After rinsing, a new synthesis cycle begins, in which another synthetic building block is coupled.

[0086] According to the present invention, a separate synthesis pen 11 is provided for each synthesis building block, and reagents 20 are placed in a closed space in the pen, which may be sealed with an inert gas 21. Each synthesis pen 11 with its corresponding synthesis building block is mounted in a holder 4 of the synthesis device 1, and is carried to a reaction chamber 9 of a synthesis plate 5 by a gripper arm 2 that grips the synthesis pen 11 with a gripper arm holder 30, for metered delivery of the reagents.

[0087] The synthesis pen 11 used in accordance with the present invention comprises a hollow cylindrical body 12 with a mouthpiece 14 at its lower end and a screw closure 13 sealing the cylindrical space. Within the mouthpiece 14 is an outlet opening closed by a valve needle 15 and a stop valve 16, which rest on a seal 29 in the closed position. The valve needle 15 and stop valve 16 are guided by a piston 18 via a piston rod 17. The closing pressure required for the stop valve 16 is generated by a compression spring 19, which rests on the piston 18 and is supported against the inner end face of the screw closure 13. The free space below the piston 18 is used to provide the appropriate synthesis building blocks 20, which are advantageously sealed with inert gas 21. In this way, highly reactive reagents can be kept stable under an inert gas atmosphere for extended periods, thereby significantly improving the quality of the synthesis product.

[0088] To ensure that cross-contamination is avoided when the mouthpiece 14 comes into direct contact with the sample, according to the present invention, the reaction chamber 9, in which the sample or solid phase 26, e.g., a synthetic resin, is located, is covered on its open side with a permeable material 25, e.g., a frit. To couple the synthesis building block 20 to the sample or synthetic resin, the mouthpiece 14 of the synthesis pen 11 is placed on the permeable material 25 closing the reaction chamber, which displaces the valve needle 15 inward against the closing pressure of the compression spring 19 and releases the stop valve 16. The reagent solution is then free to flow out, the dosage of which is determined by the duration for which the mouthpiece 14 is placed on the material 25.

[0089] Once the last temporary protecting group has been cleaved and the sample has been washed, cleavage of the synthesis building blocks 20 coupled to the solid phase 26 takes place. For this purpose, a cleavage solution is added to the sample by means of a rinse comb 8, initiating the cleavage reaction. After an incubation period, the valve block 6 is switched so that the compounds dissolved in the cleavage solution pass into the receiving chamber of a sample plate 27, which, according to a further feature of the invention, is placed below the valve block 6 and connected to an extraction system. The sample plate 27 corresponds in construction and design to the synthesis plate 5. The transfer of the dissolved compounds from the solid phase 26 to the sample plate 27 marks the end of the synthesis.

[0090] FIG. 6 shows a further preferred embodiment of the synthesis pen 11; identical reference numerals correspond to each other. The screw closure of the reagent reservoir is replaced in this design by a movable lid 13a with a bayonet closure. A piston rod 17, preferably located in the center of the depressed lid, runs through the entire synthesis pen to a dosing cylinder 31. The dosing cylinder 31 is closed downwards, for example, by a non-return valve 32. By pressing the lid 13a, a defined amount of reagent is dispensed by the piston. A return means, for example, springs 33, 33a, 33b, installed in the synthesis pen 11 returns the piston. Simultaneously or subsequently, the dosing cylinder is refilled 34. Suitable adjustment means, for example, a non-return valve, in the lower mouthpiece 14 ensure that the solution can only be dispensed by active delivery. This design of the synthesis pen 11 allows contactless dispensing into the reaction chamber. The closed design of the synthesis pen 11 with its closed reagent reservoir ensures high reagent stability.

[0091] FIG. 7 shows a schematic diagram of the method according to the invention.

[0092] The method according to the present invention is part of a solid-phase peptide synthesis that is or may be performed by the device according to the present invention. For this purpose, the N-terminus of an amino acid is protected from undesired reactions by a protecting group. The thus protected amino acid is attached to a solid support material via its C-terminus (I). The N-terminus is then deprotected (II), and another N-protected amino acid is attached to the N-terminus of the previous amino acid by peptide elongation (III). Steps II-III are repeated until the desired chain length of the amino acid is reached. Once this chain length is reached, the reaction is stopped by cleaving the peptide from the support material in step IV. At least finally, the peptide is washed with a suitable solvent (step V). Optionally, a pre-swelling of the solid support material, typically a resin (step O), is performed at the beginning of the method. According to the present invention, at least one of the above steps is at least temporarily ultrasonically assisted (X). This is understood to mean that ultrasound (X) at a frequency of at least 25 kHz is applied to the reaction medium in which the synthesis is carried out. Ultrasonic baths, in which the reaction medium is introduced using a vessel, have been found to be particularly well suited for conduction. It has further been found to be advantageous, both preparatory and with regard to synthesis time, if ultrasound (X) is applied to the reaction medium over several steps, preferably without being switched off between steps. In particular with regard to steps I to IV, the ultrasound carried out according to the invention can result in a reduction in synthesis time by an order of magnitude.

[0093] Table 1 shows a comparison of the synthesis times of the individual steps of the repeating unit between the prior art method without ultrasonic action and the method according to the invention using ultrasonic action in the range of 50-150 kHz. It is clear that the method according to the invention is 10 times faster than the comparative method without ultrasonic action.

[0094] [Table 1]

[0095] In addition to the protecting group attached to the N- or C-terminus, the amino acid may have additional protecting groups to block reactive side chains. Here, attention must be paid to the requirements regarding the chemical and physical environment during peptide synthesis, such as ultrasonic and base or acid stability. Protecting groups for reactive side chains suitable for use in the method according to the present invention are, for example, acid-labile protecting groups, such as S-2,4,6-trimethoxybenzyl (Tmob), triphenylmethyl (Trt), tert-butyl (tBu), tert-butyloxycarbonyl (Boc), and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf).

[0096] With reference to the method according to the invention, for example, amino acids are shown as single letter codes: Fmoc-A-OH, Fmoc-C(Trt)-OH, Fmoc-D(OtBu)-OH, Fmoc-E(OtBu)-OH, Fmoc-F-OH, Fmoc-G-OH, Fmoc-H(Trt)-OH, Fmoc-I-OH, Fmoc-K(Boc)-OH, Fmoc-L-OH, Fmoc-M-OH, Fmoc-N(Trt)-OH, Fmoc Fmoc amino acids selected from the group -P-OH, Fmoc-Q-Trt-OH, Fmoc-R-Pbf-OH, Fmoc-S-tBu-OH, Fmoc-T-tBu-OH, Fmoc-V-OH, Fmoc-W(Boc)-OH, Fmoc-Y-(tBu)-OH, Fmoc-Gln(Tmob)-OH, Fmoc-Asn(Tmob)-OH (TMOB=2,4,6-trimethoxybenzyl) are particularly suitable for peptide elongation.

[0097] Fmoc amino acids may exist in both the L and D configurations.

[0098] Their use allows the synthesis time to be reduced by more than 10 times compared to the prior art without any loss of yield.

[0099] Table 2 shows a typical course of synthesis of the method according to the invention in a preferred embodiment based on the pipetting scheme, using the example of an endomorphine. It essentially comprises the steps (O-V) described above, which are described in more detail with substeps in the following examples. The first step is pre-swelling of the resin (O), followed by deprotection of the resin, for example with 20% piperidine in DMF, washing with a solvent, for example DMF or DCM, coupling of an amino acid (I), washing again with a solvent, for example DMF or DCM, deprotection of the amino acid, and finally washing with a solvent, preferably DMF or DCM.

[0100] Here, the sequence of cycles is always the same. Once the last amino acid (AA) has been coupled, it is deprotected, washed, and rinsed with a solvent, such as DMF or DCM. During each cycle, in the embodiment shown, ultrasound is applied to the reaction medium at a frequency ranging from 25 kHz to 2 MHz. In this example, ultrasound (X) is also not interrupted between steps. Alternatively, ultrasound may be interrupted between steps or during the individual steps. However, continuous ultrasound has been shown to be particularly advantageous for test frequencies in the range of 40 kHz to 2 MHz, especially in the range of 50 kHz to 200 kHz.

[0101] In the example shown, the steps of pre-swelling and final rinsing with a solvent, preferably dichloromethane (DCM), are carried out without ultrasound, however this is only a preferred embodiment, and therefore ultrasound may in fact be provided throughout all steps.

[0102] [Table 2]

[0103] The method according to the invention may be carried out as a so-called short or long synthesis, the difference between the two being shown by way of example in Table 3.

[0104] [Table 3]

[0105] The short synthesis differs from the long synthesis in that the duration of deprotection is essentially halved. Furthermore, the number of washing and deprotection steps is reduced. Despite the fact that the long synthesis requires a longer synthesis time, it also offers a 10-fold reduction in synthesis time compared to the prior art. [Example]

[0106] Synthesis scheme of double coupling in ultrasonic synthesis (single frequency)

[0107] [Table 4]

[0108] This cycle is repeated until the entire sequence is synthesized (e.g., ACP:H-VQAAIDYING-NH2 → 10 amino acids → 10 cycles).

[0109] Preliminary steps such as pre-expansion and washing, and final washing steps are not listed here.

[0110] Synthesis scheme for single coupling in ultrasonic synthesis (single frequency)

[0111] [Table 5]

[0112] This cycle is repeated until the entire sequence is synthesized (e.g., ACP:H-VQAAIDYING-NH2 → 10 amino acids → 10 cycles).

[0113] Preliminary steps such as pre-expansion and washing, and final washing steps are not listed here.

[0114] Synthesis scheme of single coupling in ultrasonic synthesis (multiple frequencies, e.g., 132 kHz and 470 kHz)

[0115] [Table 6]

[0116] This cycle is repeated until the entire sequence is synthesized (e.g., H-PYLFWLAAI-NH2 → 9 amino acids → 9 cycles).

[0117] Preliminary steps such as pre-expansion and washing, and final washing steps are not listed here.

[0118] LIPS synthesis (triple coupling) synthesis scheme

[0119] [Table 7]

[0120] This cycle is repeated until the entire sequence is synthesized (e.g., H-VQAAIDYING-NH2 → 10 amino acids → 10 cycles).

[0121] The time required to dispense a pen depends on several factors and is therefore only an approximation given here.

[0122] Synthesis scheme of ABI synthesis (single coupling) without capping (acetylation)

[0123] [Table 8]

[0124] The time can only be given as an approximation, as the individual modules may have different lengths, which in turn depend on the sequence being synthesized. Additionally, an internal sensor measures the percentage of deprotected Fmoc groups during deprotection.

[0125] This cycle is repeated until the entire sequence is synthesized (e.g., H-VQAAIDYING-NH2 → 10 amino acids → 10 cycles).

[0126] Further cleaning steps are included within the module and therefore are not shown separately.

[0127] Synthesis scheme of ABI synthesis (double coupling) using capping (acetylation)

[0128] [Table 9]

[0129] The time can only be given as an approximation, as the individual modules may have different lengths, which in turn depend on the sequence being synthesized. Additionally, an internal sensor measures the percentage of deprotected Fmoc groups during deprotection.

[0130] This cycle is repeated until the entire sequence is synthesized (e.g., H-VQAAIDYING-NH2 → 10 amino acids → 10 cycles).

[0131] Additional cleaning steps are included within the module and are not shown separately.

[0132] ACP H-VQAAIDYING-NH2 M=1063.2Da Synthesis scale: 25μmol Synthetic resin: Knorr Amid resin LS 1%DVB Activator: HCTU Base: DIPEA Amino acids used:

[0133] [Table 10]

[0134] FIG. 11 is a comparison of the average synthesis quality of the ACP peptide H-VQAAIDYING-NH2 taking into account the synthesis strategy. 660-SL3 LIPS: ACP LIPS robot in microtiter plates (MTP) Triple coupling (standard protocol) Duration: 22.5 hours USPS 132kHz 50% Output: Ultrasonic ACP, 132kHz Single Coupling (average of 2 batches) Duration: 2.5 hours USPS 470kHz 50% Output: Ultrasonic ACP, 470kHz Single Coupling (average of 2 batches) Duration: 2.5 hours USPS 1000kHz 60% output: ultrasonic ACP, 1000kHz single coupling (average of 2 batches) duration: 2.5 hours

[0135] It can be seen that the level of frequency improves the quality of the product.

[0136] FIG. 12 is a graph showing the quality of the synthesis of 25 μmol of H-VQAAIDYING-NH 2 according to FIG. 11 .

[0137] [Table 11]

[0138] As a result, it can be stated that sustained sonication improves the quality of the product, and that an increase in frequency also improves the quality of the product.

[0139] FIG. 13 shows the testing of stock solutions for amino acids with various solubilization durations.

[0140] The synthesis of ACP peptides is carried out using various stock solutions, using 1000 kHz ultrasound, and using various solubilization times for the amino acids used.

[0141] It is found that the shorter the dissolution time of the amino acid used, the better the quality of the product.

[0142] FIG. 14 is a graph showing a comparison of the yield and quality of H-VQAAID for the number of couplings at various frequencies.

[0143] [Table 12]

[0144] At low frequencies (132 kHz), it can be seen that the quality of LCMS improves with increasing number of couplings.

[0145] At high frequencies (470 kHz), there is little difference in LCMS quality.

[0146] However, regardless of frequency, the yield increases with increasing number of couplings.

[0147] For the experiment (Figure 15), a different peptide is chosen from the previous studies: the sequence is PYLFWLAAI-NH2.

[0148] This is also a difficult peptide to synthesize.

[0149] ACP H-PYLFWLAAI-NH2 M=1092.6Da Synthesis scale: 25μmol Synthetic resin: Knorr Amid resin LS 1%DVB Activator: HCTU Base: DIPEA Amino acids used:

[0150] [Table 13]

[0151] FIG. 15 is a graph showing a comparison of single and double coupling and the excess of amino acids for this peptide.

[0152] [Table 14]

[0153] At the same frequency, there is no noticeable difference in synthesis quality.

[0154] However, the relative yields clearly increase with increasing number of couplings.

[0155] FIG. 16 shows a graph depicting a comparison of the average synthesis quality of peptides using different ultrasonic frequencies.

[0156] The synthesis of the peptide PYLFWLAAI-NH2 is shown, which was synthesized using single coupling at various ultrasonic frequencies.

[0157] No ultrasound: Conventional ABI synthesis using a 40-fold excess of amino acids. Conventional ABI synthesis using a four-fold excess of amino acids.

[0158] In conventional ABI synthesis, the lower the excess of amino acids, the lower the quality of the LCMS.

[0159] Simple ultrasonic frequencies 4x excess amino acids Frequency: 40kHz, 132kHz, 470kHz

[0160] The quality of LCMS improves with increasing frequency.

[0161] Combined ultrasonic frequencies (deprotection, coupling), cleaning only at lower frequencies Combined frequencies: 40kHz+470kHz and 132kHz+470kHz

[0162] Switching frequencies significantly reduces synthesis quality.

[0163] Ultrasonic vs. conventional ABI synthesis To achieve good to very good LCMS quality with conventional ABI synthesis, a very high excess of amino acids is required (40-fold).

[0164] With ultrasound, a four-fold excess of amino acids is sufficient, where the higher the frequency used, the better the quality of the LCMS.

[0165] The equivalent results are ABI (40X excess) and 470kHz (4X excess) The parameters are shown.

[0166] With ultrasonic synthesis, at least comparable, and usually better, results can be achieved in a shorter time and with less solvent and amino acid usage.

[0167] Figures 8-10 show the composition of peptides synthesized by solid-phase peptide synthesis, respectively. The peptides shown in Figures 8-9 were produced using the method of the present invention, while Figure 10 is based on a peptide synthesized by the prior art using Tetras. All methods were performed using the apparatus of the present invention.

[0168] The products obtained from the various methods were separated by HPLC and individual peaks were assigned by mass spectrometry and UV-vis spectroscopy. HPLC-MS system Dionex Binary HPLC Pump Electrophoresis medium A: water and 0.1% formic acid Running medium B: acetonitrile and 0.1% formic acid Flow rate: 0.5ml / min Gilson autosampler for up to 4 microtiter plates Dionex Column Oven Temperature: 30℃ Dionex UV detector Measured at 220 nm Dionex / Thermo Finnigan Surveyor MSQ Single Quadrupole Mass Spectrometer Ionization mode: ESI Sample temperature: 350℃ Cone voltage: 50V HPLC separation column: Merck, Chromolith WP300, RP18, 100-4.6 mm An apparatus with the following parameters was used.

[0169] [Table 15]

[0170] Figure 8 shows that the above-mentioned protecting groups for blocking reactive side chains are stable in the method according to the invention. To this end, the results of peptide synthesis according to the method of the invention are shown for three of the most common protecting groups.

[0171] Figure 8 shows the analytical results of the synthesis of endomorphin, carried out by the method of the present invention based on the long synthesis procedure. Theoretical considerations initially suggested that the oxidation-sensitive tryptophan might be oxidized by ultrasound during the synthesis. However, this was not confirmed. Rather, the synthesis was successful, with a purity of 83%. Only minor by-products were identified.

[0172] Methionine, trityl and Tmob protecting groups have also been shown in separate studies to be stable during the method according to the invention.

[0173] FIG. 9 shows the synthesis of an acyl carrier protein (ACP) having the sequence VQAAIDYING-OH, produced by the methods of the present invention using long-term synthesis.

[0174] The protecting groups Fmoc-Q(Tmob)-OH and Fmoc-N(Tmob)-OH were used. The synthesized peptides are fundamentally very difficult to prepare due to their strong hydrophobicity. Nevertheless, the method of the present invention produced a peptide with a purity of 82%. Compared to the synthesis of the same peptide using the prior art Tetras method shown in Figure 10, which achieved only 79% purity, the method of the present invention can achieve particularly improved yields. Furthermore, the synthesis time for the peptide produced by the method of the present invention was completed in 2.5 hours, compared with 25 hours for the comparative prior art method. Thus, the method of the present invention is 10 times shorter.

[0175] The method according to the invention and the use of the device according to the invention, advantageously without microwave assistance, reduced the synthesis time by up to 10 times compared to the synthesis time of the prior art methods. This could be shown not to correspond to a reduction in yield, but rather, in direct comparison with standard methods, it could be shown that the method according to the invention produced target peptides of higher purity, especially when the device according to the invention was used.

[0176] List of Reference Numbers 1 Synthesizer 2 Gripper Arms 3 Working area 4 holder 5. Synthetic Plates 6 Valve Block 7. Suction pump 8 Linscomb 9. Reaction Chamber 10 Rinse aid supply line 11 Synthetic Pen 12 Body, hollow cylinder 13, 13a Movable lid with closure, screw closure, bayonet closure 14 Mouthpiece 15 Valve needle 16 Stop valve 17 Piston rod 18 Piston 19 Compression spring 20 Synthetic Building Blocks 21 Inert gas 23 Outlet opening 25 Permeable materials / frits 26 Solid phase 27 Sample Plate 28 membrane 29 Seals 30 Gripper arm receiver 31 Dosing Cylinder 32 Outlet valve 33 Return spring 33a Return spring fastening part 33b Return spring, screw grab fastener 34 Cylinder filling gap 35 union nut 36 Administration Cannula Guide 37 Administration Cannula 50 ultrasonic bath O Pre-inflation I. An amino acid whose N-terminus is protected by a protecting group is attached to a solid support material via the C-terminus of the amino acid. II. Cleavage of protecting groups III. Perform at least one peptide elongation IV. Terminating the reaction by cleaving the peptide from the support material V Cleaning X Ultrasonic action

Claims

1. 1. A method for performing solid phase peptide synthesis, comprising: a) attaching an amino acid N-terminally protected by a protecting group to a solid support material via the C-terminus of said amino acid; b) cleaving the protecting group; c) performing at least one peptide elongation; and d) stopping the reaction by cleaving the peptide from the carrier material Including, A process wherein steps a) to d) are carried out in a liquid reaction medium, and during at least one of said steps, ultrasound at a frequency in the range of 100 to 2000 kHz is at least intermittently applied to said reaction medium.

2. 2. The method of claim 1, wherein the ultrasound acts on the reaction medium at a frequency in the range of greater than 110 kHz.

3. 3. The method according to claim 1, wherein the ultrasound acts on the reaction medium at a frequency in the range of up to 1000 kHz.

4. 4. The method according to claim 1, wherein the ultrasound is transmitted to the reaction medium via an external liquid bath.

5. 5. The method according to claim 1, further comprising a washing step Wb) carried out after step b), a washing step Wc) carried out after step c), and / or a washing step Wd) carried out after step d), wherein ultrasound is also applied to the reaction medium during at least one of these steps.

6. 6. The method according to claim 1, wherein the amino acid is N-terminally protected by a base-labile protecting group, a protecting group that can be cleaved with a secondary amine, or fluorenylmethoxycarbonyl (Fmoc).

7. 7. The method according to claim 1, wherein the amino acid comprises a protecting group for protecting a side chain, the protecting group being selected from the group consisting of S-2,4,6-trimethoxybenzyl (Tmob), triphenylmethyl (Trt), tert-butyl (tBu), tert-butyloxycarbonyl (Boc), and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf).

8. 8. The method according to claim 1, wherein ultrasound is applied to the reaction medium in step c).

9. 9. The method according to claim 1, wherein the step in which ultrasound is applied is performed without interruption and / or at the same frequency during the step.

10. 10. The method according to claim 1, wherein when ultrasound is applied to the reaction medium in several steps, the frequency of the ultrasound is varied between the steps.

11. 6. The method of claim 5, wherein said frequency during at least one of said cleaning steps is in the range of 100 to 2000 kHz.

12. The method according to any one of claims 1 to 11, wherein the reaction medium on which the ultrasonic waves act is controlled to a temperature range of 20 to 100°C.

13. Method according to any one of claims 1 to 12, characterized in that it is carried out semi-automatically / automatically and / or in parallel.

14. 14. The method according to any one of claims 1 to 13, wherein step d) comprises the steps of dosing, washing and filtering, and in the case of semi-automated implementation the dosing step is performed manually and further steps are performed in automated implementation.

15. A method for carrying out automated parallel solid phase peptide synthesis, comprising the method of any one of claims 1 to 14.

16. 15. An apparatus (1) for carrying out solid phase peptide synthesis, for carrying out the method according to any one of claims 1 to 14, comprising means for receiving a synthesis vessel (5) with at least one opening for filling the reaction medium, and an ultrasonic bath (50) containing a liquid, the ultrasonic bath (50) comprising an ultrasonic transducer for generating ultrasonic waves in the liquid at a frequency in the range of 100 to 2000 kHz, the apparatus being configured so that the synthesis vessel (5) can be placed in the ultrasonic bath (50) in such a way that the synthesis vessel (5) is wetted to a minimum height with the liquid in the ultrasonic bath (50).

17. 17. Apparatus according to claim 16, characterized in that the ultrasonic bath (50) is configured so that its height and / or temperature are adjustable.

18. An apparatus as described in claim 16 or 17, wherein the ultrasonic bath (50) or the ultrasonic transducer is switchable to at least two frequencies including frequencies in the range.

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