PROCESS FOR SOLID-PHASE PEPTIDE SYNTHESIS AND DEVICE

Abstract

The invention relates to a method for carrying out solid-phase peptide synthesis, to an automated parallel solid-phase peptide synthesis, and to a device designed to carry out such a method.

According to the invention, ultrasound with a frequency of more than 25 kHz acts at least intermittently during the method on the reaction medium in which the solid-phase peptide synthesis takes place.

Claims

1. A method for carrying out solid-phase peptide synthesis comprising the steps of a) binding an amino acid protected at the N-terminus by a protecting group to a solid support material via a C-terminus of the amino acid, b) splitting off the protecting group, c) performing at least one peptide propagation and d) terminating the reaction by splitting off the peptide from the support material, wherein steps a) to d) take place in a liquid reaction medium and, at least during one of the steps, ultrasound with a frequency in the range of 25 to 2000 kHz acts at least intermittently on the reaction medium.

2. The method according to claim 1, wherein the ultrasound acts on the reaction medium with a frequency in the range of more than 40, in particular more than 75, preferably more than 100 kHz, particularly preferably more than 110 kHz.

3. The method according to claim 1, wherein the ultrasound acts on the reaction medium with a frequency in the range of not more than 1000 kHz, preferably not more than 500 kHz.

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

5. The method according to claim 1, further comprising a washing step W.sub.b) taking place after step b), a washing step W.sub.c) taking place after step c) and/or a washing step W.sub.d) taking place after step d), wherein ultrasound also acts on the reaction medium during at least one of these steps.

6. The method according to claim 1, wherein the amino acid is protected at the N-terminus by a base-labile protecting group, in particular a protecting group which can be split off by means of secondary amines, in particular fluorenylmethoxycarbonyl (Fmoc).

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

8. The method according to claim 1, wherein ultrasound acts on the reaction medium in exactly one step, in particular in step c).

9. The method according to claim 5, wherein during all steps a) to d) and/or W.sub.b), W.sub.c) and W.sub.d) ultrasound acts on the reaction medium is acted upon without interruption and/or with the same frequency.

10. The method according to claim 5, where when ultrasound acts on the reaction medium in several steps, the ultrasonic frequency varies between the steps, in particular between reaction steps a) to d) and washing steps W.sub.b-d).

11. The method according to claim 5, wherein the frequency during at least one of the washing steps is in the range of 25 to 2000 kHz, preferably in the range of more than 40, in particular more than 75, preferably more than 100 kHz, more preferably more than 110 kHz.

12. The method according to claim 4, wherein the ultrasonic bath is controlled to a temperature range of from 20 to 100° C., preferably from 20 to 70° C., more preferably from 40 to 60° C.

13. The method according to claim 1, wherein the method is carried out semi-automated/automated and/or in parallel.

14. The method according to claim 13, wherein step d) comprises a dosing step, a washing step and a filtering step and, in the case of semi-automated performance, the dosing step is performed manually and the further steps are performed in automated performance.

15. Automated parallel solid-phase peptide synthesis comprising a method according to claim 1.

16. A device for carrying out solid-phase peptide synthesis designed to carry out a method according to claim 1.

17. The device according to claim 16, comprising a means for receiving a synthesis vessel, in particular a synthesis plate, a plurality of synthesis cylinders, a reaction flask or reactor with at least one opening for filling in reactant media, and an ultrasonic bath comprising a liquid, wherein the synthesis vessel can be arranged in the ultrasonic bath in such a way that the synthesis vessel is wetted to a minimum height with the liquid of the ultrasonic bath.

18. The device according to claim 16, wherein the ultrasonic bath is arranged to be adjustable in height and/or temperature.

19. A peptide produced by a method according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0107] FIG. 1 a schematic representation of the device according to the invention for the synthesis of peptides,

[0108] FIG. 2 the plan view of the working region of the device according to FIG. 1,

[0109] FIG. 3 a schematic representation of the synthesis pen for separate feeding, dosing and reagent storage in a preferred embodiment of the invention,

[0110] FIG. 4 a longitudinal section through the synthesis pen according to FIG. 3,

[0111] FIG. 5 section A-A of FIG. 2 through a synthesis plate with the reaction chamber formed according to the invention,

[0112] FIG. 6 a schematic representation of a longitudinal section of a synthesis pen in a further preferred embodiment of the invention,

[0113] FIG. 7 a schematic representation of the sequence of a method for solid-phase peptide synthesis according to a preferred embodiment of the invention,

[0114] FIG. 8 a graphical representation of the stability of tryptophan with the method according to the invention on the basis of the synthesis of endomorphin,

[0115] FIG. 9 a graphical representation of the stability of an acyl carrier protein (ACP) with the method according to the invention,

[0116] FIG. 10 a graphical representation of the stability of an acyl carrier protein (ACP) using a prior art comparison method,

[0117] FIG. 11 a graphical representation of the comparison of the average synthesis quality of the ACP peptide taking into account the synthesis strategy,

[0118] FIG. 12 a graphical representation of the quality of the syntheses according to FIG. 11,

[0119] FIG. 13 a graphical representation of the testing of stock solutions on amino acids of different solution durations,

[0120] FIG. 14 a graphical representation of the comparison of single and double coupling,

[0121] FIG. 15 a graphical representation of the comparison of single and double coupling and also amino acid excess, and

[0122] FIG. 16 a graphical representation of the comparison of the average synthesis quality of a peptide using different ultrasonic frequencies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0123] The synthesis device 1 shown schematically in FIG. 1 is based on a laboratory pipetting robot and has a gripper arm 2 that can be moved in the x, y and z axes. In the working region 3 there is a synthesis vessel, in particular synthesis plates 5, which with regard to the grid and the arrangement of the reaction chambers 9 are derived from microtiter plates known per se and have a 6, 12, 24, 48, 96, 384, 1536 or 3456 grid of the reaction chambers 9, whereby a high degree of parallelisation of the synthesis is achieved. The synthesis plates 5 are placed on a valve block 6 and have a membrane 28 of a porous material on the bottom side for sucking the used reagents and rinsing liquids out of the reaction chambers 9 into a waste by means of the valve block 6, which is connected to a suction pump.

[0124] The synthesis plate 5 is arranged together with the valve block 6 and the sample plate 27 in an ultrasonic bath 50, in particular one that is adjustable in height and can be switched on and off in a controllable manner. The ultrasonic bath 50 has a vessel with a liquid transmission medium in which the synthesis vessel 5 is arranged. Depending on the position of the ultrasonic bath 50, a meniscus of the synthesis vessel 5 of a reaction medium is at least up to half, preferably at least up to three quarters, in particular completely below a filling level of the transmission medium of the ultrasonic bath 5. The ultrasonic bath 50 is designed to transmit ultrasound with frequencies in the range of at least 25 kHz to 2 MHz via the transmission medium.

[0125] The synthesis device 1 is further equipped with one or more rinsing combs 8, which are connected to the corresponding rinsing agent reservoir via the rinsing agent supply lines 10. In order to rinse the samples located in the reaction chambers 9, to which a synthesis building block has been coupled, after the reaction time has elapsed and the spent reaction solution has been drawn off, the rinsing comb 8 with the required rinsing agent is picked up by the gripper arm 2 and moved over the reaction chambers 9 of the synthesis plates 5 for the metered delivery of the rinsing liquid. After rinsing, another rinsing comb 8 is used to supply the solution required for splitting off the temporary protecting group of the coupled synthesis building block, as described above. After an incubation time has elapsed, the splitting-off solution is drawn off via the valve block 6 with the aid of the suction pump 7 and the sample is washed. After washing, a new synthesis cycle starts, with which another synthesis building block is coupled.

[0126] According to the present invention, separate synthesis pens 11 are provided for each synthesis building block, in which pens the reagents 20 are placed in a closed space and can be coated with an inert gas 21. The individual synthesis pens 11 with the corresponding synthesis building block are provided in a holder 4 of the synthesis device 1 and brought to the reaction chamber 9 of the synthesis plates 5 by the gripper arm 2, which grips the synthesis pens 11 at the gripper arm holder 30, for metered delivery of the reagents.

[0127] The synthesis pen 11 used according to the invention consists of a hollow-cylindrical main body 12 with a mouthpiece 14 at the foot end and a screw closure 13 which tightly closes the cylinder space. In the mouthpiece 14 there is an outlet opening which is closed by a valve needle 15 and a stop valve 16 which, in the closed position, rests on a seal 29. The valve needle 15 and stop valve 16 are guided by a piston 18 via a piston rod 17. The required closing pressure 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 for the presentation of the relevant synthesis building block 20, which is advantageously coated with an inert gas 21. In this way, highly reactive reagents can be kept stable over long periods of time under an inert gas atmosphere, which significantly improves the quality of the synthesis products.

[0128] In order to reliably exclude cross-contamination in the event of direct contact of the mouthpiece 14 with the sample, in accordance with the invention the reaction chambers 9, in which the samples or the solid phase 26, for example a synthetic resin, are located, are covered on the opening side with a permeable material 25, for example a frit. To couple a synthesis building block 20 to the sample or to the synthetic resin, the mouthpiece 14 of the synthesis pen 11 is placed on the permeable material 25 closing off the reaction chamber, whereby the valve needle 15 is displaced inwardly against the closing pressure of the compression spring 19 and the stop valve 16 is released. After this, the reagent solution can flow out freely, with the dosage of the solution flowing out being determined by the period of time for which the mouthpiece 14 is placed on the material 25.

[0129] With the splitting off of the last temporary protecting group and washing of the samples, the splitting off of the synthesis building blocks 20 coupled to the solid phase 26 takes place. For this purpose, a splitting-off solution is added to the samples by means of a rinsing comb 8 and a splitting-off reaction is initiated. After the incubation time has elapsed, the valve block 6 is switched in such a way that the compounds dissolved in the splitting-off solution are passed into the receiving chambers of a sample plate 27 which, according to a further feature of the invention, is arranged below the valve block 6 and is connected to an extraction system. The sample plates 27 correspond to the synthesis plates 5 with regard to their construction and design. With the transfer of the compounds dissolved from the solid phase 26 into the sample plate 27, the synthesis is completed.

[0130] FIG. 6 shows a further preferred embodiment of the synthesis pen 11, where the same reference signs 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, which is preferably arranged centered in the lid, in particular pressed in, leads through the entire synthesis pen into a dosing cylinder 31. The dosing cylinder 31 is closed downwards, for example with a non-return valve 32. By pressing the lid 13a, a defined quantity of reagent is dispensed by means of a piston. A return means installed in the synthesis pen 11, for example a spring 33, 33a, 33b, returns the piston. At the same time or downstream, the dosing cylinder is filled again 34. A suitable adjusting means in the foot-side mouthpiece 14, for example a non-return valve, ensures that solution can only be dosed by active delivery. This design of the synthesis pen 11 allows contact-free dispensing into the reaction chamber. The closed design of the synthesis pen 11 with a closed reagent reservoir ensures high reagent stability.

[0131] FIG. 7 shows a schematic representation of the method according to the invention.

[0132] The method according to the invention is part of a solid-phase peptide synthesis, as is or can be carried out by the device according to the invention. For this purpose, the N-terminus of an amino acid is protected from undesired reactions by a protecting group. The amino acid protected in this way is bound to a solid support material via its C-terminus (I). Subsequently, the N-terminus is deprotected (II) in order to bind another amino acid protected at the N-terminus to the N-terminus of the previous amino acid by means of peptide propagation (III). Steps II to III are repeated until the desired chain length of amino acids is reached. When the chain length is reached, the reaction is terminated in a step IV by splitting off the peptide from the support material. At least lastly, the peptide is washed with a suitable solvent (step V). Optionally, pre-swelling (step O) of the solid support material, usually a resin, is carried out at the beginning of the method. According to the invention, at least one of said steps is at least temporarily ultrasound-assisted (X). This is to be understood to mean that, at least temporarily, ultrasound (X) of a frequency of at least than 25 kHz is applied to the reaction medium in which the synthesis takes place. It has been found that an ultrasonic bath in which the reaction medium is introduced by means of a vessel is particularly well suited for transmission. Both preparatively and with respect to the synthesis time, it has further been found to be advantageous if ultrasound (X) acts on the reaction medium over several steps, preferably without switching off between the steps. In particular with regard to steps Ito IV, the ultrasound carried out according to the invention can bring about a reduction in the synthesis time in the region of an order of magnitude.

[0133] Table 1 compares the synthesis times of the individual steps of the repeat units for a prior art method without ultrasonic action and according to the invention with ultrasonic action in the range of 50 to 150 kHz. It is clear that the method according to the invention is ten times faster than a comparable method without ultrasound.

TABLE-US-00001 TABLE 1 Comparison of the required synthesis times of methods according to the prior art and according to the invention. Method according Step Prior art to the invention Deprotection (Step II) 3 x 5 min 2 x 1 min Washing (IV) 5 x 1 min 3 x 30 sec Peptide propagation (III) 2 x 30 min 2 x 3 min Total 1 h 20 min 8 min 30 sec

[0134] In addition to the protecting groups bound to the N- or C-terminus, the amino acids can have further protecting groups to block reactive side chains. Attention must be paid here to the requirements with regard to the chemical and physical environment during peptide synthesis, such as ultrasound and base or acid stability. Suitable protecting groups for reactive side chains for use in the method according to the invention are, for example, acid-labile protecting groups, for example S-2,4,6-trimethoxybenzyl (Tmob), triphenylmethyl (Trt), tert-butyl (tBu), tert-butyloxycarbonyl (Boc) and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf).

[0135] With reference to the method according to the invention, for example, Fmoc amino acids selected from the following group, which is noted as a single-letter code for the amino acid, are particularly suitable for peptide propagation: 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, F moc-N(Trt)-OH, Fmoc-P-OH, Fmoc-Q-Trt OH, Fmoc-R-PbfOH, Fmoc-S-tBuOH, Fmoc-T-tBuOH, Fmoc-V-OH, Fmoc-W(Boc)-OH, Fmoc-Y-(tBu)-OH, Fmoc-Gln(Tmob)-OH, Fmoc-Asn(Tmob)-OH (TMOB =2,4,6-trimethoxy-benzyl).

[0136] The Fmoc amino acids can be present in both the L and D forms.

[0137] With these, the synthesis time could be reduced more than tenfold compared to the prior art without yield losses.

[0138] Table 2 shows the typical course of synthesis of the method according to the invention in a preferred embodiment on the basis of a pipetting scheme using the example of endomorphin. This basically comprises the steps mentioned above (O-V), which are, however, described in more detail and with sub-steps in the following example. A first step is the pre-swelling of the resin (O), followed by deprotection of the resin, for example with 20% piperidine in DMF, subsequent washing with a solvent, for example DMF or DCM, coupling of the 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.

[0139] The cycle sequence is always the same here. After the last amino acid (AA) has been coupled, it is deprotected, washed, and rinsed with solvent, for example DMF or DCM. During the individual cycles, ultrasound with frequencies in the range of 25 kHz to 2 MHz acts on the reaction medium in the shown embodiment. In the present example, the ultrasound (X) is also not interrupted between the steps. Alternatively, the ultrasound can be interrupted between the steps or during individual steps. However, in the tested frequencies in the range of 40 kHz to 2 MHz and in particularly in the range of 50 kHz to 200 kHz, continuous ultrasound was shown to be particularly advantageous.

[0140] In the example shown, the steps of pre-swelling and final rinsing with solvent, preferably dichloromethane (DCM), are carried out without ultrasound. However, this is only a preferred embodiment, and therefore ultrasound may indeed be provided over all steps.

TABLE-US-00002 TABLE 2 Synthesis sequence of a solid-phase peptide synthesis with the method according to the invention in a preferred embodiment. Pipetting scheme t Repe- Ultra- Volume (ml) (min) tition sound 1. AA, Phe, F 1 Pre-swelling 2  2 2 no 2 Deprotection 1  1 2 yes 3 Washing 1 30 s 3 yes 4 Coupling F in HCTU, 250 μl  3 2 yes DIPEA, 153.3 μl 5 Washing 1 30 s 3 yes 2. AA, Trp, W 6 Deprotection 1  1 2 yes 7 Washing 1 30 s 3 yes 8 Coupling W in HCTU, 250 μl  3 2 yes DIPEA, 153.3 μl 9 Washing 1 30 s 3 yes 3. AA, Pro, P 10 Deprotection 1  1 2 yes 11 Washing 1 30 s 3 yes 12 Coupling P in HCTU, 250 μl  3 2 yes DIPEA, 153.3 μl 13 Washing 1 30 s 3 yes 4. AA, Tyr, Y 14 Deprotection 1  1 2 yes 15 Washing 1 30 s 3 yes 16 Coupling Y in HCTU, 250 μl  3 2 yes DIPEA, 153.3 μl 17 Washing 1 30 s 3 yes 18 Deprotection 1  1 2 yes 19 Washing 1 30 s 3 yes 36 Flushing with 1  1 3 no DCM

[0141] The method according to the invention can be carried out as a so-called short-term or long-term synthesis. The difference between the two is shown as an example in Table 3:

TABLE-US-00003 TABLE 3 Description of short- and long-term synthesis Short-term synthesis Long-term synthesis Deprotection with ultrasound Deprotection with ultrasound Deprotection duration: 30 s Deprotection duration: 1 min Number of deprotection steps: 1 Number of deprotection steps: 2 Washing without ultrasound Washing with ultrasound 30 s each Per washing step: wash 5x Per washing step: wash 3x Single coupling of the AA Double coupling of the AA

[0142] Short-term synthesis differs from long-term synthesis basically in that the duration of deprotection is halved. Furthermore, the number of washing and deprotection steps is reduced. Despite the fact that the long-term synthesis requires a longer synthesis time, it also provides a reduced synthesis time of one tenth compared to the prior art.

Synthesis Scheme for Double Coupling in Ultrasonic Synthesis (Single Frequency)

[0143]

TABLE-US-00004 TABLE 4 Step Chemical Fmoc-AA V [ml] t [min] deprotection 20% 1 1 suction piperidine deprotection 20% 1 1 suction piperidine washing DMF 1 0.5 suction washing DMF 1 0.5 suction washing DMF 1 0.5 suction addition AA AA + 1 ml G 2 3 (+ HCTU, HCTU + DIPEA) 1 ml DIPEA suction addition AA AA + 1 ml G 2 3 (+ HCTU, HCTU + DIPEA) 1 ml DIPEA suction washing DMF 1 0.5 suction washing DMF 1 0.5 suction washing DMF 1 0.5 suction

[0144] Total duration of a cycle with double coupling: 11 min

[0145] This cycle is repeated until the entire sequence has been synthesised (example ACP: H-VQAAIDYING-NH2.fwdarw.10 amino acids.fwdarw.10 cycles).

[0146] The preparatory steps such as pre-swelling and washing, as well as the final washing steps are not listed here.

Synthesis Scheme for Single Coupling in Ultrasonic Synthesis (Single Frequency)

[0147]

TABLE-US-00005 TABLE 5 Step Chemical Fmoc-AA V [ml] t [min] deprotection 20% 1 1 suction piperidine deprotection 20% 1 1 suction piperidine washing DMF 1 0.5 suction washing DMF 1 0.5 suction washing DMF 1 0.5 suction addition AA AA + 1 ml G 2 3 (+ HCTU, HCTU + DIPEA) 1 ml DIPEA suction washing DMF 1 0.5 suction washing DMF 1 0.5 suction washing DMF 1 0.5 suction

[0148] Total duration of a cycle with single coupling: 8 min

[0149] This cycle is repeated until the entire sequence has been synthesised (example ACP: H-VQAAIDYING-NH2.fwdarw.10 amino acids.fwdarw.10 cycles).

[0150] The preparatory steps such as pre-swelling and washing, as well as the final washing steps are not listed here.

Synthesis Scheme for Single Coupling in Ultrasonic Synthesis (Several Frequencies, for Example 132 kHz and 470 kHz)

TABLE-US-00006 TABLE 6 Fmoc- Volume Time Ultra- F Step Chemical AA [ml] [min] sound [kHz] deprotection 20% 1 2*0.5 yes 132 + 470 piperidine suction deprotection 20% 1 2*0.5 yes 132 + 470 piperidine suction washing DMF 1 0.5 yes 132 suction washing DMF 1 0.5 yes 132 suction washing DMF 1 0.5 yes 132 suction addition AA AA + 1 ml I 2 1 + 2 yes 132 + 470 (+HCTU, HCTU + 1 ml DIPEA) DIPEA suction washing DMF 1 0.5 yes 132 suction washing DMF 1 0.5 yes 132 suction washing DMF 1 0.5 yes 132 suction

[0151] Deprotection: 2*0.5 yes 132+470.fwdarw.0.5 min at 132 kHz and then 0.5 min at 470 kHz addition AA (+HCTU, DIPEA):

[0152] 1+2 yes 132+470.fwdarw.1 min at 132 kHz and then 2 min at 470 kHz

[0153] Total duration of a cycle with single coupling: 8 min

[0154] This cycle is repeated until the entire sequence has been synthesised (example: H-PYLFWLAAI-NH2.fwdarw.9 amino acids 9 cycles).

[0155] The preparatory steps such as pre-swelling and washing, as well as the final washing steps are not listed here.

Synthesis Scheme for LIPS Synthesis (3-Fold Coupling)

[0156]

TABLE-US-00007 TABLE 7 Step Chemical Time Number Total time deprotection 20%  2 min 5  10 min suction piperidine washing DMF 10 sec 5  50 sec suction addition AA distribution of approx.. 3 105 min the pens 20 min activation NMM 15 min suction 10 sec washing DMF 10 sec 3  30 sec suction acetylation capping  2 min 2  4 min suction solution washing DMF 20 sec 5  2 min suction

[0157] Total duration of a cycle with 3-fold coupling: approx. 122 min

[0158] This cycle is repeated until the entire sequence has been synthesised (example: H-VQAAIDYING-NH2.fwdarw.10 amino acids.fwdarw.10 cycles).

[0159] The time needed to distribute the pens depends on several factors, therefore only approximate values are given here.

Synthesis Scheme for ABI Synthesis (1-Fold Coupling) Without Capping (Acetylation)

[0160]

TABLE-US-00008 TABLE 8 Programme Duration (approx. module Process from the manual) Comment B deprotection  15 min 2× at least A dissolving the   8 min amino acid in cartridge D washing 2.5 min duration varies depending on the number of cycles performed 5× at least E transfer of the 2.1 min dissolved amino acid into reaction vessel F coupling  15 min D washing 2.5 min duration varies depending on the number of cycles performed 5× at least

[0161] Total duration of a cycle with 1-fold coupling: approx. 80 min.

[0162] The times can only be given with approximate values, as the individual modules can have different lengths, which in turn depends on the sequence to be synthesised. In addition, internal sensors measure the proportion of deprotected Fmoc groups during deprotection.

[0163] This cycle is repeated until the entire sequence has been synthesised (example: H-VQAAIDYING-NH2.fwdarw.10 amino acids.fwdarw.10 cycles).

[0164] Within the modules, additional washing steps are included, and therefore these are not shown separately.

Synthesis Scheme for ABI Synthesis (2-Fold Coupling) with Capping (Acetylation)

TABLE-US-00009 TABLE 9 Programme Duration (approx. module Process from the manual) Comment B deprotection  15 min 2× at least A dissolving the   8 min amino acid in cartridge D wash 2.5 min duration varies depending on the number of cycles performed 5× at least E transfer of the 2.1 min 2× dissolved amino acid into reaction vessel A dissolving the   8 min amino acid in cartridge D wash 2.5 min duration varies depending on the number of cycles performed 5× at least E transfer of the 2.1 min 2× dissolved amino acid into reaction vessel F coupling  15 min 2× C coupling 9.5 min D wash 2.5 min duration varies depending on the number of cycles performed 5× at least

[0165] Total duration of a cycle with 2-fold coupling: approx. 130 min.

[0166] The times can only be given with approximate values, as the individual modules can have different lengths, which in turn depends on the sequence to be synthesised. In addition, internal sensors measure the proportion of deprotected Fmoc groups during deprotection.

[0167] This cycle is repeated until the entire sequence has been synthesised (example: H-VQAAIDYING-NH2.fwdarw.10 amino acids.fwdarw.10 cycles.

[0168] Within the modules, additional washing steps are included; these are not shown separately. [0169] ACP H-VQAAIDYING-NH2 M=1063.2 Da [0170] Synthesis scale: 25 μmol [0171] Synthetic resin: Knorr Amid Resin LS 1% DVB [0172] Activator: HCTU [0173] Base: DIPEA [0174] Amino acids used:

TABLE-US-00010 Amino acid Permanent (L-amino acids) protecting group Fmoc-Ala-OH — Fmoc-Asp(tBu)-OH Tert. Butyl Fmoc-Gly-OH — Fmoc-Ile-OH — Fmoc-Asn(Trt)-OH Trityl Fmoc-Gln(Trt)-OH Trityl Fmoc-Val-OH — Fmoc-Tyr(tBu)-OH Tert. Butyl

[0175] Ratio of free amino function resin:amino acid:activator:base; 1:4:3.9:8

[0176] FIG. 11 compares the average synthesis quality of the ACP peptide H-VQAAIDYING-NH.sub.2, taking into account the synthesis strategy:

TABLE-US-00011 660-SL3 LIPS: ACP in microtiter plate (MTP) LIPS (standard protocol) robot 3-fold coupling duration: 22.5 h USPS 132 kHz 50% power: ACP in ultrasound, 132 kHz 1-fold batches) coupling (average of 2 duration: 2.5 h USPS 470 kHz 50% power: ACP in ultrasound, 470 kHz 1-fold batches) coupling (average of 2 duration: 2.5 h USPS 1000 kHz 60% power: ACP in ultrasound, 1000 kHz 1-fold batches) coupling (average of 2 duration. 2.5 h

[0177] It can be seen that the level of frequency increases the product quality.

[0178] FIG. 12 shows graphically the qualities of the syntheses of H-VQAAIDYING-NH.sub.2 25 μmol according to FIG. 11.

TABLE-US-00012 Ultrasound Ultrasound Ultrasound Frequency Ultrasound during during during Coupling Batch number [kHz] power [%] washing? deprotection? coupling? number A-USPS_H_1 132 50 yes yes yes 2 A-USPS_H_2 132 50 yes yes yes 2 B-USPS_H_1 132 50 no yes yes 2 B-USPS_H_2 132 50 no yes yes 2 C-USPS_H_1 132 50 no no yes 2 C-USPS_H_2 132 50 no no yes 2 D-USPS_H_1 470 100 yes yes yes 2 D-USPS_H_2 470 100 yes yes yes 2

[0179] As a result, it can be stated that a permanent sounding increases the product quality and that an increase in frequency also increases the product quality.

[0180] FIG. 13 shows the testing of stock solutions on amino acids of different solution durations.

[0181] With the different stock solutions, syntheses of the ACP peptide are carried out using ultrasound at 1000 kHz with different solution times for the amino acids used.

[0182] It can be seen that a shorter dissolution time of the amino acids used increases the product quality.

[0183] FIG. 14 is a graphical representation of the comparison of yield and quality in terms of coupling number at different frequencies H-VQAAID.

TABLE-US-00013 Synthesis number (number of Coupling Frequency approaches) number ultrasound USPS_H (2) 1 132 kHz 50% USPS_H (2) 2 132 kHz 50% USPS_H (2) 1 470 kHz 100% USPS_H (2) 2 470 kHz 100%

[0184] It can be seen that at low frequencies (132 kHz) the LCMS quality increases with an increase in the coupling number.

[0185] At high frequencies (470 kHz) there is hardly any difference in the LCMS quality.

[0186] Frequency-independently, however, the yield increases with an increase in the coupling number.

[0187] For the experiment (FIG. 15), a different peptide is chosen than for the previous tests. The sequence is PYLFWLAAI-NH2

[0188] This is also a difficult peptide to synthesise. [0189] ACP H-PYLFWLAAI-NH2 M=1092.6 Da [0190] Synthesis scale: 25 μmol [0191] Synthetic resin: Knorr Amid Resin LS 1% DVB [0192] Activator: HCTU [0193] Base: DIPEA

[0194] Amino acids used:

TABLE-US-00014 Amino acid Permanent (L-amino acids) protection group Fmoc-Ala-OH — Fmoc-Phe-OH — Fmoc-Ile-OH — Fmoc-Leu-OH — Fmoc-Pro-OH — Fmoc-Trp(Boc)-OH Butyloxycarbonyl Fmoc-Tyr(tBu)-OH Tert. Butyl

[0195] Ratio of free amino function resin:amino acid:activator:base; 1:4:3.9:8

[0196] FIG. 15 shows graphically the comparison of single and double coupling and amino acid excess for this peptide.

TABLE-US-00015 Synthesis number (number of Frequency batches) Coupling number ultrasound 150819USPS_H (2) 1 (single coupling) 470 kHz 50% 241019USPS_H (2) 2 (double Coupling) 470 kHz 50%

[0197] At the same frequency, there is no significant difference in terms of synthesis quality.

[0198] When the coupling number is increased, however, there is a clear increase in the relative yield.

[0199] FIG. 16 shows a graphical representation of the comparison of the average synthesis quality of a peptide using different ultrasonic frequencies.

[0200] Shown are the syntheses of the peptide PYLFWLAAI-NH2, which were synthesised using single coupling at different ultrasonic frequencies.

Without ultrasound: [0201] Conventional ABI synthesis with 40-fold excess of amino acid. [0202] Conventional ABI synthesis with 4-fold excess of amino acid.

[0203] The lower the excess of amino acid, the poorer the LCMS quality in a conventional ABI synthesis.

Simple Ultrasonic Frequencies 4-fold Excess of Amino Acid [0204] Frequencies: 40 kHz, 132 kHz, 470 kHz

[0205] With increasing frequency, the LCMS quality increases.

Coupled Ultrasonic Frequencies (Deprotection, Coupling), Washing Exclusively at Lower Frequency

[0206] Coupled frequencies: 40 kHz+470 kHz and 132 kHz+470 kHz

[0207] Switching between frequencies leads to a significant deterioration of the synthesis quality.

Ultrasound vs. Conventional ABI Synthesis

[0208] To achieve good to very good LCMS quality in conventional ABI synthesis, very high excesses of amino acid are necessary (40-fold).

[0209] With the help of ultrasound, a 4-fold excess of amino acid is sufficient. Here, the higher the frequency used, the higher the LCMS quality.

[0210] Equivalent results showed the following parameters:

[0211] ABI (40x excess) and 470 kHz (4x excess).

[0212] By means of the ultrasonic synthesis, at least equivalent and usually better results can be achieved in a shorter time and with reduced use of solvents and amino acids.

[0213] FIGS. 8 to 10 each show the composition of a peptide synthesised by solid-phase peptide synthesis. The peptides shown in FIGS. 8 to 9 were produced by means of the method according to the invention, while FIG. 10 is based on a peptide synthesised according to the prior art by means of Tetras. All methods were carried out with the device according to the invention.

[0214] The products obtained from the various methods were separated by HPLC and the individual peaks were assigned by mass spectrometry and UV-vis spectrometry. Equipment with the following parameters was used: [0215] HPLC MS System [0216] Dionex Binary HPLC Pump [0217] Running medium A: water plus 0.1% formic acid [0218] Running medium B: acetonitrile plus 0.1% formic acid [0219] Flow: 0.5 ml/min [0220] Gilson autosampler for up to 4 microtiter plates [0221] Dionex column oven [0222] Temperature: 30° C. [0223] Dionex UV detector [0224] Measurement at 220 nm [0225] Dionex/Thermo Finnigan Surveyor MSQ Single Quadrupole Mass Spectrometer [0226] Ionisation mode: ESI [0227] Sample temperature: 350° C. [0228] Cone voltage: 50 V [0229] HPLC separation column: Merk, Chromolith WP300, RP18, 100-4.6 mm

TABLE-US-00016 Time (min) % Running medium B  0  5  2  5 12 100 14 100 15  5

[0230] FIG. 8 shows that the above-mentioned protecting groups for blocking the reactive side chains are stable in the method according to the invention. For this purpose, the results of a peptide synthesis according to the method of the invention are shown for three of the most common protecting groups.

[0231] FIG. 8 shows the analysis results of an endomorphin synthesis carried out according to the method of the invention on the basis of the long-term synthesis procedure. Theoretical considerations initially suggested that the oxidation-sensitive tryptophan could be oxidised by the ultrasound during the synthesis. However, this was not confirmed. Rather, the synthesis was successful with a purity of 83%. Only a few by-products were identified.

[0232] Methionine, trityl and Tmob protecting groups also proved stable during the method according to the invention in individual tests.

[0233] FIG. 9 shows the synthesis of the acyl carrier protein (ACP) with the sequence VQAAIDYING-OH, produced according to the method of the invention with a long-term synthesis.

[0234] The protecting groups Fmoc-Q(Tmob)-OH and Fmoc-N(Tmob)-OH were used. The synthesised peptide is basically very difficult to prepare due to its strongly hydrophobic character. Nevertheless, it was possible to produce it with a purity of 82% by means of the method according to the invention. Compared to the synthesis of the same peptide with the prior art Tetras method shown in FIG. 10, where only a purity of 79% could be achieved, it can be seen that the method according to the invention can, among other things, achieve an improvement in yields. In addition, the synthesis time of the peptide produced by the method according to the invention was completed in 2.5 h, whereas the comparative method according to the prior art required 25 h. The method according to the invention is thus ten times shorter.

[0235] The use of the method according to the invention and the device according to the invention advantageously lead to a reduction of the synthesis time to a maximum of one tenth of the synthesis time for methods according to the prior art without microwave support. It could be shown that this in no way coincides with a reduction of the yield, rather it could be shown in a direct comparison with a standard method that the method according to the invention produced a higher purity of the target peptide, in particular when using the device according to the invention.

LIST OF REFERENCE SIGNS

[0236] 1 Synthesis device [0237] 2 Gripper arm [0238] 3 Working region [0239] 4 Holder [0240] 5 Synthesis plate [0241] 6 Valve block [0242] 7 Suction pump [0243] 8 Rinsing comb [0244] 9 Reaction chambers [0245] 10 Rinsing agent supply line [0246] 11 Synthesis pen [0247] 12 Main body, hollow cylinder [0248] 13, 13a Closure, screw closure, movable lid with bayonet closure [0249] 13b Locking bayonet closure [0250] 14 Mouthpiece [0251] 15 Valve needle [0252] 16 Stop valve [0253] 17 Piston rod [0254] 18 Piston [0255] 19 Compression spring [0256] 20 Synthesis building block [0257] 21 Inert gas [0258] 23 Outlet opening [0259] 25 Permeable material/frit [0260] 26 Solid phase [0261] 27 Sample plate [0262] 28 Membrane [0263] 29 Seal [0264] 30 Gripper arm receptacle [0265] 31 Dosing cylinder [0266] 32 Outlet valve [0267] 33 Return spring [0268] 33 3a Fastening for return spring [0269] 33b Return spring, screw grub fastening [0270] 34 Gap for cylinder filling [0271] 35 Union nut [0272] 36 Dosing cannula guide [0273] 37 Dosing cannula [0274] 50 Ultrasonic bath [0275] O Pre-swelling [0276] I Binding of an amino acid protected at the N-terminus by a protecting group to a solid support material via a C-terminus of the amino acid [0277] II Splitting-off of the protecting group [0278] III Performing at least one peptide propagation [0279] IV Termination of the reaction by splitting off the peptide from the support material [0280] V Washing [0281] X Ultrasound action